the treatment of infectious ovine keratoconjunctivitis in ...infectious ovine keratoconjunctivitis...

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The treatment of infectious ovine keratoconjunctivitis in pre-export

feedlot sheep in Western Australia.

Fraser Robert Murdoch

BVMS MANZCVS (Sheep Medicine) FHEA MRCVS

This thesis is presented for the degree of Doctor of Philosophy of

Murdoch University.

2016

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I declare that this thesis is my own account of my research and contains as its main

content work which has not been submitted for a degree at any tertiary education

institution.

……………………………………………..

Fraser R. Murdoch

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Abstract Approximately two million sheep are exported from Australia annually, worth in excess of

$170 million to the Australian economy. With the live export industry facing increasing

scrutiny it is essential that the industry take steps to optimise the welfare of those animals

involved.

Infectious ovine keratoconjunctivitis (IOK) is a significant, infectious eye disease of sheep

and is the reason for the rejection of many sheep from the live export chain. Establishing a

practical and effective treatment protocol has the potential to reduce economic losses

associated with rejected stock and to improve the welfare of those animals presenting with

clinical disease.

Injectable oxytetracycline (OTC) has been used as a treatment worldwide and has been

shown to be effective. However, in pre-embarkation feedlots the number of animals is so

large that such individual treatment is not feasible. This research investigated the clinical

efficacy and impact on animal health of OTC given in-feed or in-water.

Oxytetracycline is absorbed from the gastro-intestinal tract following oral administration in

feed or water. This research showed that oral administration of OTC results in changes to

the rumen microflora population.

Although the change in rumen microflora corresponded with reduced feed intakes, feed

intakes returned to normal following cessation of treatment, indicating that any changes to

the ruminal microbiome are transient. Oxytetracycline given in-feed for five days results in a

significant clinical improvement in IOK.

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In-water OTC caused a persistent decrease in feed and water intake rendering it an

unsuitable treatment.

Mild cases of IOK can be successfully treated with in-feed OTC for a five-day period at a dose

of 20 mg/kg bodyweight. Sheep with more severe IOK can be successfully treated with two

intramuscular injections of OTC at 20 mg/kg bodyweight four days apart.

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Ethics Approval All experimental work was approved by the Murdoch University Animal Etchics committee

under permit numbers: R2159/08, R2409/11, R2460/11 and R 2613/03.

The use of a questionnaire was approved by Murdoch University Human Research Ethics

and Integrity Committee under project number 2014/205

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Table of Contents 1 Introduction ...................................................................................................................... 22

2 Literature Review ............................................................................................................. 25

2.1 Infectious keratoconjunctivitis in other species ....................................................... 26

2.2 Nomenclature............................................................................................................ 27

2.3 Clinical presentation .................................................................................................. 27

2.4 Impacts on production .............................................................................................. 28

2.5 Infectious ovine keratoconjunctivitis and the export of live animals ....................... 30

2.6 Risk Factors ................................................................................................................ 31

Season ............................................................................................................................... 31

Housing ............................................................................................................................. 33

Vectors .............................................................................................................................. 33

Breed ................................................................................................................................. 34

Age .................................................................................................................................... 35

Environmental factors ...................................................................................................... 36

Concurrent disease ........................................................................................................... 36

Carrier animals .................................................................................................................. 37

2.7 Microbiological causative agents .............................................................................. 38

Rickettsiae ......................................................................................................................... 38

Mycoplasma sp. ................................................................................................................ 39

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Moraxella sp. .................................................................................................................... 40

Chlamydia sp. .................................................................................................................... 40

2.8 Other causes of keratoconjunctivitis ........................................................................ 41

2.9 Treatment .................................................................................................................. 42

2.10 Antibiotics.................................................................................................................. 45

Topical agents ................................................................................................................... 45

Injectable .......................................................................................................................... 48

Oral treatments ................................................................................................................ 50

3 General Aims .................................................................................................................... 52

4 Epidemiology of infectious ovine keratoconjunctivitis at a pre-export feedlot .............. 53

4.1 Introduction............................................................................................................... 53

4.2 Materials and Methods ............................................................................................. 53

4.3 Results ....................................................................................................................... 55

4.4 Discussion .................................................................................................................. 58

4.5 Conclusions................................................................................................................ 62

5 Identification of the microbiological causes and possible treatment of infectious ovine

keratoconjunctivitis in a pre-export quarantine feedlot. ........................................................ 63

5.1 Materials and methods ............................................................................................. 64

Statistical analysis ............................................................................................................. 66

5.2 Results ....................................................................................................................... 66

5.3 Discussion .................................................................................................................. 70

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5.4 Conclusions................................................................................................................ 77

6 Determination of the efficacy of in-water medication in the treatment of ovine

infectious keratoconjunctivitis................................................................................................. 78

6.1 Introduction............................................................................................................... 78

6.2 Hypothesis ................................................................................................................. 80

6.3 Materials and Methods ............................................................................................. 80

6.4 Results ....................................................................................................................... 83

6.5 Discussion .................................................................................................................. 92

6.6 Conclusion ................................................................................................................. 97

7 Maximising intake of in-water oxytetracycline ................................................................ 98

7.1 Introduction............................................................................................................... 98

7.2 Hypotheses ................................................................................................................ 99

7.3 Materials and methods ............................................................................................. 99

Statistical analysis ........................................................................................................... 100

7.4 Results ..................................................................................................................... 101

7.5 Discussion ................................................................................................................ 104

7.6 Conclusions.............................................................................................................. 106

8 Determination of oral bioavailability of oxytetracycline in sheep. ................................ 107

8.1 Introduction............................................................................................................. 107

8.2 Materials and Methods ........................................................................................... 109

Sample analysis ............................................................................................................... 110

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8.3 Results ..................................................................................................................... 112

8.4 Discussion ................................................................................................................ 114

8.5 Conclusions.............................................................................................................. 124

9 The impact of oral oxytetracycline on rumen health ..................................................... 125

9.1 Introduction............................................................................................................. 125


Treatments ..................................................................................................................... 132

Rumen fluid samples ...................................................................................................... 132

Rumen microbial profiling .............................................................................................. 132

Methodolgy for T-RFLP ................................................................................................... 133

Methodolgy for Pyrosequencing .................................................................................... 134

Blood samples ................................................................................................................. 135

Statistical Analysis .......................................................................................................... 135

9.3 Results ..................................................................................................................... 138

Bodyweight and body condition score ........................................................................... 140

Feed Intake ..................................................................................................................... 140

Water Intake ................................................................................................................... 143

Plasma Beta-Hydroxybutyrate (BHB) concentrations .................................................... 144

Faecal Score .................................................................................................................... 145

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Rumen pH ....................................................................................................................... 146

Plasma OTC concentrations ............................................................................................ 147

Rumen fluid parameters ................................................................................................. 148

9.4 Discussion ................................................................................................................ 155

9.5 Conclusions.............................................................................................................. 159

10 The use of medicated feed to treat infectious ovine keratoconjunctivitis ................ 160

10.1 Introduction............................................................................................................. 160

10.2 Hypothesis ............................................................................................................... 161


Preparation of medicated pellets (in-feed medication). ................................................ 161


10.4 Results ..................................................................................................................... 162

10.5 Discussion ................................................................................................................ 165

11 Clinical efficacy of in-feed OTC ................................................................................... 166

11.1 Introduction............................................................................................................. 166

11.2 Hypothesis ............................................................................................................... 166

11.3 Materials and methods .......................................................................................... 166


Plasma sample analysis .................................................................................................. 168

11.4 Results ..................................................................................................................... 169

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11.5 Discussion ................................................................................................................ 172

11.6 Conclusion ............................................................................................................... 174

12 The treatment of infectious ovine keratoconjunctivitis with in-feed medication in a

pre-embarkation feedlot. ...................................................................................................... 175

12.1 Introduction............................................................................................................. 175

12.2 Hypotheses .............................................................................................................. 175


12.4 Results ..................................................................................................................... 177

12.5 Discussion ................................................................................................................ 183

13 General Discussion ...................................................................................................... 184

14 Conclusions ................................................................................................................. 192

15 Appendix 1 .................................................................................................................. 193

16 Appendix 2 .................................................................................................................. 201

17 Appendix 3 .................................................................................................................. 202

18 References .................................................................................................................. 209

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Table of Figures Figure 5.1 Eye grades over time .............................................................................................. 69

Figure 6.1 Mean feed intake (kg/head/day) over time. Shaded area represents the duration

of Oral treatment. Error bars represent standard errors ........................................................ 85

Figure 6.2 Mean water intake (L/head/day) over time. Shaded area represents the duration

of oral treatment. Error bars represent standard errors......................................................... 87

Figure 6.3 Mean clinical eye grade (average both eyes) over time. Shaded area represents

the duration of Oral treatment. IM OTC treatment given on day 0 and 4. Error bars

represent standard errors. ....................................................................................................... 89

6.4 Mean plasma OTC concentrations at different time points during the experiment. ........ 91

Figure 6.5 Mean plasma OTC concentration during treatment period for the Oral and the IM

treatment groups ..................................................................................................................... 91

Figure 7.1 Change in feed intake (g/head/day) over time. Shaded box represents treatment

period. Error bars represent standard errors ........................................................................ 102

Figure 7.2 Change in water intake (ml/head/day) over time. Shaded box represents

treatment period. Error bars represent standard errors. ..................................................... 102

Figure 7.3 OTC intakes (based on Experiment 1) for different doses of OTC during treatment

period. .................................................................................................................................... 103

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Figure 8.1 Plasma OTC concentrations over time following IV treatment, error bars show

standard error ........................................................................................................................ 112

Figure 8.2 Plasma OTC concentrations over time following medication with in-feed (IF) OTC

with error bars showing standard error. ............................................................................... 113

Figure 8.3 Plasma OTC concentrations over time following medication with in-water (IW)

OTC. Error bars show standard error. .................................................................................... 113

Figure 9.1 Graph of mean feed intake over time for Treatments (11mg/kg and 22mg/kg oral

OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

................................................................................................................................................ 141

Figure 9.2 Graph of mean ME intake over time for Treatments (11mg/kg and 22mg/kg oral


................................................................................................................................................ 142

Figure 9.3 Graph of mean water intake over time for Treatments (11mg/kg and 22mg/kg

oral OTC). Shaded box represents OTC treatment period. Error bars represent standard

errors. ..................................................................................................................................... 143

Figure 9.4 Graph of mean plasma BHB concentrations over time for Treatments (11mg/kg

and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent

standard errors. Black line represents BHB concentration at which sheep are at risk of sub-

clinical ketosis. ....................................................................................................................... 144

Figure 9.5 Graph of mean faecal score over time for Treatments (11mg/kg and 22mg/kg oral


................................................................................................................................................ 145

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Figure 9.6 Graph of mean rumen pH over time for Treatments (11mg/kg and 22mg/kg oral


................................................................................................................................................ 146

Figure 9.7 Graph of mean plasma OTC over time for Treatments (11mg/kg and 22mg/kg oral


................................................................................................................................................ 147

Figure 9.8 nMDS of rumen bacterial communities associated with oral dose of OTC taken on

day 5. Treatments are: 11mg OTC/kg LW/day () and 22mg OTC/kg LW/day (). Each point

in the ordination shows the overall microbial profile of an individual animal. The closer two

points are in the ordination the more similar are their profiles. .......................................... 150

Figure 9.9 Abundance of rumen bacteria classified to level of phyla for individual control and

OTC treated sheep. (Cont) untreated control sheep, (Low) treated with 11 mg OTC/kg live

weight/day and (High) treated with 22 mg OTC/kg live weight/day. ................................... 151

Figure 9.10 Abundance of rumen bacteria classified, where possible, to level of family for

individual control and OTC treated sheep. (Cont) control sheep, (Low) 11 mg OTC/kg live

weight/day and (High) 22 mg OTC/kg live weight/day.......................................................... 153

Figure 10.1 Feed intake (g/head/day) over time. Shaded box represents treatment period.

Error bars represent standard errors..................................................................................... 163

Figure 10.2 Water intake (kg/head/day) over time. Shaded box represents treatment period.

Error bars represent standard errors..................................................................................... 164

Figure 11.1 Feed intake (g/hd/day) of Control vs IF treatments over time. Shaded box

represents treatment period. Error bars represent standard errors. ................................... 171

Figure 11.2 Water intake (ml/hd/day) of Control vs IF treatments over time. Shaded box

represents treatment period. Error bars represent standard errors. ................................... 171

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Figure 11.3 Change in eye grade over time - Control vs IF treatments. Shaded box represents

treatment period. Error bars represent standard errors. ..................................................... 172

Figure 12.1 Adjusted IOK mean scores for each treatment and day. .................................... 179

Figure 12.2 Average eye scores on each day, grouped according to Score on Day 0. .......... 181

Table of Tables Table 4.1 Historical shipping data showing numbers and percentages of sheep rejected on

arrival at the feedlot due to Imfectious Ovine Keratoconjunctivitis. * depicts significant

result. ....................................................................................................................................... 56

Table 4.2 Historical shipping data provided by the exporter outlining, by season, the

number of sheep rejected due to clinical Infectious Ovine Keratoconjunctivitis. *depicts

significant findings. ................................................................................................................. 57

Table 4.3 Data gatherd by questionnaire showing maximum and minimum percentages of

IOK cases per shipment ........................................................................................................... 57

Table 4.4 Data gatherd by questionnaire showing maximum and minimum percentages of

mild IOK cases per shipment in lambs, wethers and ewes. .................................................. 58

Table 5.1 Treatments tested for control of Infectious Ovine Keratoconjunctivitis .............. 66

GNR = Gram-negative rod; GPC = gram-positive cocci; GPR = gram-positive rod

Table 5.2 Microbial flora isolated from clinically healthy eyes, Mycoplasma conjunctivae

positive is a percentage of total Mycoplasma spp. ................................................................. 67

Table 6.1 Linear models and P-values for the analysis of water intake, feed intake, clinical

eye score and plasma OTC concentration after treatments. .................................................. 84

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Table 6.2 Mean feed intake (kg/head/day) for treatments on different days. Within Day,

means with different letters differ significantly (P<0.05). ....................................................... 86

Table 6.3 Mean water intake (L/head/day) for treatments on different days. Within Day,

means with different letters differ significantly (P<0.05). ....................................................... 88

Table 6.4 Mean clinical eye grade (average of both eyes) for treatments on different days.

Within Day, means with different letters differ significantly (P<0.05). ................................... 90

Table 6.5:OTC dose received by the Oral treatment group. ................................................... 90

Table 7.1: Treatments in experiment to optimise dose and palatability .............................. 100

Table 7.2: Significance levels (P-Values) for terms in model with respect to change in feed or

change in water intake........................................................................................................... 101

Table 9.1: Faecal scoring system (Le Jambre et al., 2007) ..................................................... 131

Table 9.2: Significance table for change in measurements between pre- and post- treatment

parameters. ............................................................................................................................ 139

Table 9.3 One-way ANOSIM of rumen microbial communities associated with OTC

treatment. For each microbial group the influence of oral OTC treatment was investigated.

Where significant differences in rumen microbiota were detected, the pairwise* differences

between treatments were investigated further. ................................................................... 149

Table 10.1 Feed intake between the groups throughout the experiment ............................ 163

Table 11.1 Significance levels (P values) for terms used in models to analyse data. Cov

=covariate .............................................................................................................................. 170

Table 12.1: Significance levels (P Values) for analysis of pink eye scores on each day post-

treatment (control vs in-feed vs injection) ............................................................................ 178

Table 12.2 Significance levels (P values) for analysis of change in pink eye score over time

................................................................................................................................................ 179

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Table 12.3 Change in eye score from day 0 to day 10 ........................................................... 180

Table 12.5: Treatment recommendations (Treat vs Not effective) of different routes of

administration (In-feed vs Injection) at different degrees of severity of disease (Score) on

day 8 of experiment. .............................................................................................................. 182

Table of equations Equation 1: Calculation of bioavailability .............................................................................. 111

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Acknowledgments Throughout the course of this PhD I have been privileged to have the unending support and

guidance of a host of dedicated and encouraging people. Firstly, sincere thanks must go to

the Australian Postgraduate Authority for providing me with financial support over the

course of the PhD and also to Meat and Livestock Australia for funding the project in its

entirety.

Many people ask why I decided to undertake a PhD and in truth that is a difficult question

with no simple answer. It was under the guidance and tutelage of Helen Chapman that I first

became involved in this project and her infectious enthusiasm for sheep medicine inspired

me both to continue with the project and to pursue a career in small ruminant medicine and

production. In a similar vein, I had many friends who were either presently undertaking or

had recently completed PhDs and their positive experiences encouraged me to undertake

one myself.

Of course this thesis wouldn’t have come together without the support of my supervisors.

Firstly, Mike Laurence, my principal supervisor, has been a remarkable help throughout. It is

hard to express my gratitude to him for his continued support and guidance, and his

commitment to the project has kept me going despite falling behind targets at times. Never

again shall I dare to put two spaces after a full stop. I feel honored to consider Mike a

supervisor, colleague and friend.

Ian Robertson has been an equally supportive supervisor. A man who always has time to

help no matter how many other things he has to deal with, Ian has always showed genuine

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interest in my career. He has already given me great advice which I will continue to benefit

from.

As with all projects, this one has very much been a team effort. Huge thanks must go to

Stacey McFarlane for all her technical assistance; no one knows how to keep track of labels

better! Equally, thanks must go to John Abbott who always brought a smile to those days in

the sheep pens. Without the support and cooperation of the staff at the feedlots we would

not have been able to undertake this project. Sincere thanks to our partners at DAFWA,

SARDI, and CSIRO, and to Garth Maker from the Metabolomics department, for all their

invaluable assistance and contributions to the analysis of samples generated from the

project.

Finally huge thanks to my friends and family. Repeatedly hearing the question “Is your thesis

finished yet?” certainly does grate but it provided constant motivation and I know that

everybody asked only because they genuinely cared.

Lastly thanks must go to Jill: I couldn’t have done this without you. Thank you for enabling

me to go on this journey, and for believing in me. I hope you feel it has all been worth it!

I would like to dedicate this thesis to my close friend Serena Finlayson, who taught me that

no matter what life throws at us we must strive to make the most of each and every day.

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Abbreviations

bwt – bodyweight

CSIRO – Commonwealth Scientific and Industrial Research Organisation

CTC – Chlortetracycline

DAFWA – Department of Agriculture and Food, Western Australia

GI – gastro-intestinal

HPLC-MS – High performance liquid chromatography – Mass-Spectrometry

IBK – Infectious bovine keratoconjunctivitis

ID – Identification

IOK – Infectious ovine keratoconjunctivitis

LA – Long acting

LSD – least significant difference

MIC – Minimum inhibitory concentration

nMDS – non-metric multidimensional scaling

OTC – Oxytetracycline

OTU – operational taxonomic units

PCR – polymerase chain reaction

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RDP – ribosomal database project

SARDI – South Australian Research and Development Institute

TRF – terminal restriction fragment

T-RFLP – terminal restriction fragment length polymorphism

UK – United Kingdom

VFAs – Volatile Fatty Acids

WA – Western Australia

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1 Introduction

Western Australia started exporting live sheep in 1845 (Georges et al., 1985). Today

Australia remains overall the world’s largest exporter of livestock and the industry’s welfare

standards are a global benchmark (Australian Government Department of Agriculture,

2011). Sheep are a major part of this export trade. Although numbers have declined since

their peak of around seven million per annum in 2001, there were almost 2.3 million head of

sheep exported in 2012. Of these sheep exported, 80% left from Western Australia and the

remainder from South Australia and Victoria. The Middle East remains the strongest market

for exports with 98% of exports going to this region. Other markets include southern and

northern Asia. As wealth and development increase in these countries so too does the

demand for protein, and consequently the demand for meat increases.

The live export trade remains a heavily scrutinised and controversial industry. Many

countries that import animals are reliant on these imports for food. Supply of processed

meat has increased, but trade in live animals from Australia remains dominant because the

market for processed meat is curtailed by limited availability of refrigeration and freezers

among large proportions of the population in countries of destination. Furthermore, cultural

and religious beliefs are such that the fresh meat market continues to dominate. Halal and

Kosher meat have strict requirements for the treatment of animals pre, during and post-

slaughter. Although these requirements can be addressed in Australia allowing suitably

prepared meat to be exported chilled or frozen, many countries still prefer to slaughter

under the supervision of local religious officials to ensure strict adherence to requirements.

All exported animals are subject to the Australian Standards for the Export of Livestock

(ASEL) legislation (Australian Government Department of Agriculture, 2011). These

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standards are in place to ensure animal health and welfare remain a priority throughout the

export chain and that livestock sourced for export are fit and healthy to travel. Any animals

failing to meet these standards should be removed from the export chain. These standards

help to maintain the health and welfare of the animals and market and public confidence in

the industry. ASEL stipulates a number of reasons why an animal would be unfit for travel,

ranging from body condition to disease status (Australian Government Department of

Agriculture, 2011). Infectious ovine keratoconjunctivitis (IOK) is identified as one reason for

rejection. There is also the requirement that animals have vision in both eyes.

Anecdotal figures from exporters indicate that approximately 0.5-1% of sheep are rejected

annually because of the presence of IOK (G.Robinson Per. comms). This figure relates to only

those animals permanently removed from the export chain and covers only those animals

deemed untreatable or those that fail to respond to treatment. These sheep are deemed to

have no commercial value and are typically sold to the pet meat trade. However, this figure

excludes those sheep held back for treatment and carried over to subsequent shipments. An

independent review of the industry reported that IOK is the most important reason for

rejection (Farmer, 2011).

Although sheep will typically recover from IOK with appropriate treatment, there is a cost

associated with this treatment in medication, labour hours and additional feeding. In

addition to the financial costs associated with the disease, IOK has been recognised as

having a detrimental effect on animal welfare. Conjunctivitis is considered to be an irritating

condition in humans (Leibowitz, 2000) and corneal ulceration is considered a very painful

condition in humans (Wirbelauer, 2006). Assessment of pain and discomfort in a prey

animal such as a sheep is notoriously difficult (Murdoch et al., 2013). Considering the

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findings of Leibowitz (200) and Wirbelauer (2006) it is reasonable to believe that sheep

suffering from these conditions are also feeling pain, even though they show little or no

signs of discomfort. However, it is essential to act on the assumption that the sheep are in

pain, so that animals suffering from IOK receive effective treatment to minimise any

possible impact on their welfare and long-term health. This is not only in the obvious

interest of the animals but in the interest of the live export industry, whose animal welfare

standards must bear rigorous scrutiny.

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2 Literature Review

Infectious Ovine Keratoconjunctivitis (IOK), an infectious eye disease of sheep, has been

reported throughout the world. Early reports document its presence in Australia (Symons,

1912; Beveridge, 1942). Outbreaks have also been reported throughout the world: United

Kingdom (Hosie, 1988); Pakistan (Shahzad, 2013); Norway (Akerstedt, 2004); Netherlands

(Ter, 1988); South Africa (Van Halderen et al., 1994); Ivory Coast (Formenty and Domenech,

1992); New Zealand (Cooper, 1967); Switzerland (Janovsky et al., 2001); Spain (Fernandez-

Aguilar et al., 2013); Slovakia (Kovac et al., 2003) and Israel (Lysnyansky et al., 2007; Hadani

et al., 2013).

Accurate figures relating to the numbers of outbreaks per year are sparse. In the UK up to

25 outbreaks per annum have been reported (Greig, 1989) whereas in Norway over 4000

individual cases were reported in one year (Akerstedt, 2004). Severe outbreaks can occur up

to 10 times per year in the Netherlands with morbidity ranging from a low percentage up to

100% (Konig, 1983a). In all of these instances it is likely that these figures do not reflect the

true incidence as many cases will either be treated by farm staff or will resolve

spontaneously and go unreported.

Although IOK is sometimes regarded as a relatively mild condition, farmers in New Zealand

still considered it the seventh most important disease affecting sheep (Simpson and Wright,

1988). Those farmers considered parasites, lambing related problems, footrot and lameness

more important than IOK.

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2.1 Infectious keratoconjunctivitis in other species

Infectious keratoconjunctivitis is seen in species other than sheep, although the aetiological

agents vary. Infectious bovine keratoconjunctivitis (IBK) presents with similar clinical signs in

cattle (McConnel et al., 2007). Although Moraxella bovis is the most common agent

associated with IBK, Moraxella bovoculi (Angelos et al., 2007) and Moraxella ovis have both

been implicated (O'Connor et al., 2012). Disease has also been reported in yaks and yak

crosses (Bandyopadhyay et al., 2010).

Domestic goats have been reported to be affected by infectious keratoconjunctivitis (Baas et

al., 1977; Busch, 1988) and the agents responsible for IOK are also associated with disease

in goats (Walridge B.M. , 2002). Wild caprinae: Chamois; Ibex; Mouflon and Tar are also

susceptible to infectious keratoconjunctivitis (Tschopp et al., 2005). Although the disease is

typically relatively mild in domestic animals, disease in wild populations is more severe and

often fatal (Nicolet, 1975; Degiorgis et al., 2000; Belloy et al., 2003). Fatalities were

attributed to mis-adventure and increased predation risk associated with impaired vision.

Infectious keratoconjunctivitis has also been found in populations of wild deer (Gortazar et

al., 1998). Although the agent responsible for infection wasn’t identified it is postulated that

a Chlamydia sp. may have been involved. Several studies highlight the occurrence of

infectious keratoconjunctivitis in reindeer (Rehbinder and Nilsson, 1995; Evans et al., 2008).

It is thought that a herpes virus is the causative agent of disease in this species (Tryland et

al., 2009).

Keratoconjunctivitis has also been reported in camelids, both old and new world. A case in a

llama of keratoconjunctivitis attributed to Moraxella liquefaciens has been described

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(Brightman et al., 1981). Outbreaks due to Moraxella canis (Tejedor-Junco et al., 2010) have

been reported in herds of dromedary camels in Lanzarote.

2.2 Nomenclature

Infectious ovine keratoconjunctivitis is sometimes referred to by other names. Pink Eye is

the most commonly used alternative (Cooper, 1974; Konig, 1983b; Moore and Whitley,

1984; Baker et al., 2001; Motha, 2003; Dun, 2009), but the terms contagious ovine

ophthalmia (Beveridge, 1942), contagious conjunctivo-keratitis (Cooper, 1967), heather

blindness (Sinclair, 1955; Cooper, 1974; Townsend, 2007), snow blindness (Hosie, 1989) and

inclusion body keratoconjunctivitis (Kovac et al., 2003) have all been used to describe the

same disease.

2.3 Clinical presentation

Infectious ovine keratoconjunctivitis is considered to be an irritating (Egwu et al., 1989) and

painful condition (Greig, 1989; Akerstedt, 2004) and can occur in one or both eyes (Greig,

1989).

The clinical disease progression has been differentiated into four phases (Egwu, 1991). The

initial phase is characterised by inflammation of the conjunctiva with sheep showing signs of

blepharospasm (blinking) and increased lachrymation (Egwu et al., 1989). These clinical

signs will often be the first that the producer or stock person notices. Increased

lachrymation is evidenced by dirt on the fleece of the cheek.

The next phase is an early keratitis. Keratitis is inflammation of the cornea and initially

presents as corneal oedema (Grahn B.H. and Peiffer, 2007). Increased lachrymation and

28

blepharospasm due to the increased discomfort associated with keratitis can also be seen at

this stage.

Following the initial keratitis a mucopurulent discharge is described, often associated with a

superficial corneal ulcer. A corneal ulcer occurs if parts of the epithelial and stromal layer of

the cornea are lost, thus exposing the descemet’s membrane layer (Slatter, 2001b). At this

stage blindness may be a feature, which is relatively easy to detect by the producer as

affected sheep are difficult to move and often walk into objects. Cooper (1967) found that

up to 25% of sheep in an outbreak can become blind and the blindness can last for 1-2

weeks.

The final stage involves diffuse ulceration of the cornea and occasionally hypopyon.

Hypopyon is the accumulation of pus in the anterior chamber of the eye (Slatter, 2001a).

Corneal neovascularisation is a feature of more severe cases. When this occurs it typically

starts at the limbus and will move towards the centre of the cornea (Walridge B.M. , 2002).

The pain associated with IOK results through damage to the corneal epithelium exposing the

trigeminal nerve endings (Maggs et al., 2008).

2.4 Impacts on production

Some consider IOK to be of minor importance to the sheep industry (Axelsen, 1961) but it is

associated with a number of issues that can be of economic significance. Studies in Australia

have found that compared to healthy sheep, IOK-affected sheep will have reduced weight

gain for the duration of the disease (Axelsen, 1961). The same study found that cross-bred

ewes lost weight over the period of infection, whereas merino ewes showed weight gain,

but that this gain was significantly lower than in uninfected sheep. Weight loss and poor

appetite have also been described in an outbreak in Nigeria (Osuagwuh and Akpokodje,

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1979). In this outbreak two lambs died and this was attributed to starvation as a result of

blindness associated with corneal oedema. Weight loss has also been attributed to

blindness and anorexia (Akerstedt, 2004) in infected sheep.

Carcass quality can be affected by bruising following mis-adventure. Slaughter weights can

be reduced as a result of secondary anorexia (Akerstedt, 2004). Outbreaks in lamb finishing

feedlots in North America have resulted in weight loss (Hopkins et al., 1973); this outbreak,

however, involved both IOK and polyarthritis. Weight losses at pasture can be attributed to

reduced feed intakes associated with reduced vision (Radostits, 2007a).

Outbreaks of IOK around joining and lambing can have an economic impact through

reduced twinning rates. These have been reported following an outbreak around joining and

ewes could be at a higher risk of developing pregnancy toxaemia (Axelsen, 1961). Pregnancy

toxaemia is associated with a reducing plane of nutrition in late pregnancy (Radostits,

2007b). This can be a direct sequela of IOK infection. In addition to reduced conception

rates, losses can occur around lambing in IOK infected ewes that are associated with the

visual deficits suffered by the sheep. These could include squashing lambs or suffocation of

lambs with placenta (Hofland et al., 1969). It is not a common occurrence, but blind ewes

have been shown sometimes to be capable of rearing lambs (Sinclair, 1955) although

success depends on careful monitoring.

Outbreaks of IOK appear to have no impact on wool production (Axelsen, 1961). This is

interesting as it is generally considered that any disease leading to periods of inappetance

can have an impact on wool production. If prolonged enough, a break in the wool fibre can

be seen 2-6 weeks after the insult. However, if the period of inappetance is not prolonged

fibres may be reduced in diameter and length but without a break (Groverman, 1992).

30

While some authors report little or no negative effect on production (Hosie, 1988), most

suggest that the principal economic impact of an outbreak of IOK relates to the costs

associated with treatment (Hosie, 1988). These costs comprise both drug costs and the costs

of time to treat the animals.

2.5 Infectious ovine keratoconjunctivitis and the export of live animals

Australia is the second largest exporter of live sheep worldwide. In 2012, 2.3 million sheep

were exported live from Australia with 80% of these leaving from Western Australia. The

Middle East remains the major export destination accounting for 98% of sheep exports. In

2012 the value of sheep exported was $201 million (MLA, 2013). Exports continued in 2013,

although total numbers had dropped to just below 2 million sheep worth $172 million.

Western Australia continued to export 82% of these sheep and Kuwait remained the biggest

importer, taking 45% of all sheep exported from Australia (MLA and Livecorp, 2014).

Within the export industry the most significant impact on production associated with IOK is

weight loss, but the costs associated with treatment and rejection of animals from the

export supply chain must also be taken into consideration. The literature attributes weight

loss to decreased feed intake resulting from impaired vision and consequent difficulty in

locating feed. Given that IOK is considered a painful condition (Greig, 1989) it could be

argued that this pain may also contribute to weight loss. Although difficult to compare

directly, studies looking at the pain associated with mulesing have reported weight loss in

the week following mulesing (Paull et al., 2008). Cattle with infectious bovine

keratoconjunctivitis have poor weaning weights compared to those without IBK (Killinger et

al., 1977). The reduced weight gain was attributed to pain and impairment of vision. Across

species feed and water intake is consistently modified by painful stimuli, and bodyweight

31

analysis is a useful tool for monitoring effectiveness of analgesia (Stasiak et al., 2003). It is

likely that any weight loss associated with IOK is multi-factorial.

Standard policy in the export industry states that any animal showing clinical signs of

infectious disease is not fit for export and therefore will be held back (Surman, 1968;

Australian Government Department of Agriculture, 2011), either to be treated before the

next shipment or culled (Surman, 1968). Culled animals are deemed to have no commercial

value and are humanely euthanased and used in the pet food market. The cost associated

with loss of sale in addition to costs associated with treatment, labour and additional feed

to hold the animal over for the next shipment all contribute to the exporter’s financial loss

attributable to IOK.

2.6 Risk Factors

Infectious ovine keratoconjunctivitis is a multi-factorial disease and the aetiology of IOK

outbreaks is often complex. A number of microorganisms have been implicated in outbreaks

of IOK. Aside from the infectious agents, several environmental factors are also considered

important in the aetiology of IOK.

Season

Outbreaks have been reported throughout the year in different parts of the world.

In the United Kingdom outbreaks tend to occur in autumn and winter (Arbuckle and Bonson,

1980; Andrews et al., 1987; Egwu, 1992b). Increased cases in the autumn in Norway are

associated with close contact and increased animal supervision. Sheep are grazed at very

low stocking densities over the summer months (Akerstedt, 2004) thus reducing close

contact and minimizing spread of IOK. However, some cases have been reported in Wales in

spring and summer (Sinclair, 1955).

32

In North America cases are seen in winter and spring (Riley Jr and Barner, 1953). Cases in

Switzerland occur throughout the grazing season (Nicolet, 1975) in sheep and this coincides

with cases seen in wild caprinae in summer and autumn owing to the proximity of domestic

animals to the wild population (Belloy et al., 2003).

Outbreaks in Australia are generally reported in the warmer months (Axelsen, 1961;

Surman, 1968). Outbreaks in South Africa, parts of which have a similar climate to Western

Australia, are also seen in the dry and warm months (Van Halderen et al., 1994). Those in

New Zealand typically occur in summer and autumn (Cooper, 1967; Motha et al., 2003),

however some atypical outbreaks have been reported in winter (Motha et al., 2003). The

reasons for these unseasonal outbreaks were unclear. It is postulated that a novel agent was

involved or an outbreak in a naïve flock. The typical outbreaks in New Zealand have been

attributed to increased droving and yarding of sheep during these times for routine

husbandry coupled with dry and dusty conditions (Cooper, 1967). Like Australia, outbreaks

in Croatia are reported in the warmer spring and summer months (Naglic et al., 1999).

Environmental conditions in Israel are similar to some parts of Australia but cases there are

reported in the colder months (January – March) (Lysnyansky et al., 2007). In Nigeria, which

has a hot a humid climate, cases are reported in October at the end of the rainy season

(Osuagwuh and Akpokodje, 1979). Inclement weather, whether that be cold, wintery

weather or hot, dry and dusty summer weather, has been described as a stressor which can

predispose to outbreaks (Michael, 1953).

Seasonal patterns of IOK outbreaks appear variable throughout the world. Although

outbreaks are generally expected during the warmer months in Australia there is a paucity

of literature relating to IOK outbreaks at other times of year in Australia.

33

Ultra-violet light can be a contributing factor to infection (Hosie, 1988) which is consistent

with outbreaks occurring in the warmer months in Australia. Equally, extreme winter

weather is a contributing factor to development of disease (Hosie, 1988).

Housing

The housing of sheep is considered to be a pre-disposing factor to the spread of disease.

Disease is spread by direct contact between sheep (Hopkins et al., 1973; Hosie, 1989) and by

transmission of infective agents in the lacrimal fluid (Blakemore, 1947). Experimentally,

infection can be confined to a group by the use of solid, high partitions between infected

sheep and non-infected sheep (Blakemore, 1947). The spread of disease through close

transmission goes some way to explaining the seasonal variation of outbreaks in different

countries. In countries where sheep are housed in winter to lamb, forced close contact can

occur (Akerstedt, 2004). Equally over the autumn and winter where supplementary feeding

is required, the use of troughs encourages increased sheep to sheep contact (Cooper, 1967;

Egwu, 1992b; Dun, 2009). High stocking densities will increase sheep to sheep contact and

can predispose to an outbreak of IOK (Osuagwuh and Akpokodje, 1979).

Grouping of sheep in feedlots is considered an important factor in aiding rapid spread of

infection in an outbreak (Hopkins et al., 1973). Outbreaks in the warmer months are often

attributed to dry, hot and dusty conditions (Cooper, 1967). Further to these conditions,

grazing tall, seeding grass is considered to be another risk factor (Osuagwuh and Akpokodje,

1979). It is postulated that the grass, contaminated with infected tear secretions, causes

trauma to the eyes and an infection is then established (Dun, 2009).

Vectors

Vectors can play a significant role in the spread of IOK. Under Australian conditions it has

been suggested that flies (Musca domestica) are involved in transmission (Beveridge, 1942).

34

This conclusion was reached when the increased occurrence of disease in the summer

months was noted. Flies are more active at this time than in the winter months when there

is negligible fly activity. In other countries flies have also been implicated in the spread of

disease: in Europe (Konig, 1983a); Nigeria (Osuagwuh and Akpokodje, 1979) and India

(Nanda and Abdussalam, 1943). Strong evidence for their involvement in the Nigerian case

was suggested given the disappearance of disease after flies were controlled.

It is thought that flies can directly spread infection between sheep by carrying infected

lacrimal fluid (Dun, 2009). Flies are also thought to cause small puncture wounds on the

conjunctiva during feeding and the subsequent irritation could allow colonisation by

bacteria (Graham-Smith, 1930).

In the UK, on the other hand, where most outbreaks occur in the colder months, it is

thought that flies are not always significant vectors, (Egwu, 1992b).

Breed

Although IOK can occur in any breed (Radostits, 2007a), some reports exist of variations in

infection rates between breeds. In Australia the condition was originally considered to be

unique to pure-bred merinos. This was based on an experiment where only pure-bred sheep

were experimentally infected, while in cross-bred sheep an infection could not be

established (Edgar, 1931). The author of this study postulated that the cross-bred sheep

may have an immunity of some form to the infection. Subsequently, the theory that IOK was

unique to merinos was disproved following successful experimental infection of cross-bred

sheep (Beveridge, 1942).

In the United Kingdom some experimental work has been done to determine if there was

any breed predisposition to IOK. These studies looked at Cheviots and Scottish Blackface.

35

Cheviots were initially thought to be more susceptible (Wall, 1982), but a similar study

found there to be no difference (Hosie, 1988).

As there is no conclusive evidence of a breed pre-disposition, the variety of breeds that are

involved in the live export supply chain are not likely to have an impact on the occurrence of

IOK outbreaks.

Age

Sheep of all ages are susceptible to IOK (Beveridge, 1942; Michael, 1953; Jones, 1983), even

lambs as young as 5 to 10 days old (Jones et al., 1976). A number of studies have looked at

the severity of disease in different age groups. Disease is thought by some to be more

severe in adult animals (Greig, 1989; Radostits, 2007a). In contrast some consider IOK to be

more severe and outbreaks more frequent in younger sheep (Sinclair, 1955). Beveridge

(1942) found that outbreaks were most common and severe in weaners in Australia. Dun

(2009) found that outbreaks can occur in weaners where frequent handling was a factor;

IOK can be spread through handling of the face and head while animals are being drenched.

Hosie (1988) concurred when frequent outbreaks were described in weaners and this was

related to frequent handling. Radostits (2007) reports weaners as being the most severely

affected age group.

Hosie (1988) found a seasonal variation in rate of infection depending on age group. Ewes

were found most likely to be affected in winter because at that time of year they had been

gathered for mating. Increased exposure to ultra-violet light meant that both lambs and

ewes could be affected in the summer. Weaned lambs were most likely to be affected in

autumn because of increased handling at that time of year for husbandry procedures.

Just as sheep of all breeds enter the live export supply chain, so do sheep of all age

36

Environmental factors

Environmental, mechanical and climatic factors act together with the causative

microorganisms to establish an infection (Lysnyansky et al., 2007). Numerous such factors

have been implicated in the establishment of IOK.

Routine husbandry procedures such as lamb marking, worming, weaning and pre-mating

checks necessitate forced close contact through yarding. Yarding and high stock densities

are recognised risk factors (Edgar, 1931; Beveridge, 1942; Cooper, 1967; Hosie, 1988). In

addition, many irritants have been considered to predispose animals to IOK. Dust (Edgar,

1931; Cooper, 1967) and long grasses (Edgar, 1931; Michael, 1953; Cooper, 1967; Dun,

2009) are the most commonly cited irritants.

Exposure to ultra-violet light and heat (Cooper, 1967) are also considered risk factors

(Palgrave, 1910; Hosie, 1988) and exposure to cold and wet weather is likely to predispose

to IOK (Palgrave, 1910; Edgar, 1931; Blakemore, 1947).

Concurrent disease

Blakemore (1947) considered the presence of another pre-existing or concurrent disease to

be a risk factor for IOK. Both joint-ill and parasitism were thought to be of significance.

Michael (1953) reported that dosing infected sheep with an anthelmintic improved recovery

from IOK. Deficiencies in Vitamin A have been considered a risk factor in sheep (Hofland et

al., 1969). This is similar to a finding with IBK in cattle (el-Sanousi et al., 1978). This theory

was, however, disproven in sheep in India where sheep were managed in such a way that

Vitamin A deficiency was unlikely nevertheless became infected with IOK and no effect was

seen when infected sheep were treated with Vitamin A supplementation (Patwardhan et al.,

1975).

37

Carrier animals

Outbreaks of IOK are often associated with the introduction of new animals to a flock. These

animals are generally free of clinical disease but act as carriers of the organisms. This was

well illustrated during an outbreak of IOK in Croatia (Naglic et al., 2000). No cases of IOK had

been reported in Croatia before the importation of sheep from Australia. The imported

sheep were quarantined prior to being mixed with native sheep and showed no signs of

ocular disease, suggesting that they were asymptomatic carriers of IOK. Outbreaks of IOK

occurred in native sheep following introduction of the Australian sheep to the native flock.

Palgrave (1910) identified that introducing new animals with clinical IOK enabled spread of

disease. Currently in Western Australia, introducing purchased stock is recognised as a risk

factor for IOK (Suijdendorp, 2005). Introduction of carrier rams (Andrews et al., 1987; Hosie,

1988) can partly explain the outbreaks described around joining time. Cooper (1967) found

in farmer surveys that mixing of bought-in and home-bred animals was a risk factor in an

outbreak of IOK. Radostits (2007) also states that animals bought in, be they clinically

infected or apparently healthy carriers, are the source of infection for a flock. Dun (2009)

agrees that infection is introduced by carrier animals.

It has been shown that immunity to the organisms considered to be two of the primary

pathogens, Mycoplasma conjunctivae and Chlamydophila pecorum, is usually poorly

developed and can lead to recurrence of infection in individuals (Baas et al., 1977; Hosie,

1989). Mycoplasma conjunctivae can persist in the eyes and nares for months (Baker et al.,

1965; Hosie, 1989).

It is difficult to avoid many of the risk factors that pre-dispose to outbreaks of IOK when

sheep are being exported for most of the year from Australia. The requirement to hold

38

animals in a pre-embarkation feedlot for a quarantine period before shipping serves to

exacerbate the risk factors associated with close contact, irritants and forced handling. Pre-

embarkation feedlots are arguably a perfect environment for the combination of risk factors

to maximise the chances of IOK spread and animal morbidity.

2.7 Microbiological causative agents

Numerous studies have looked with varying success at the microbiological aetiology of IOK.

While the aetiology of IBK is more clearly defined, there is still considerable debate

regarding the aetiology of IOK (Lindqvist, 1960; Jones et al., 1976; Egwu, 1991).

Rickettsiae

Rickettsiae were first isolated from sheep and considered to be the causative agent of IOK

from early reports in South Africa (Coles, 1931). Coles named this organism Rickettsia

conjunctivae. An organism that was considered the same was also found in clinical cases of

IOK in Tunisia (Cordier and Menager, 1937), Algeria (Donatien and Lestoquard, 1937),

French Congo (Malbrant, 1939), central France (Lafenetre, 1938) and the French island of

Levant (Pigoury, 1937). Beveridge (1942) described isolating an organism in sheep with IOK

in Australia that matched the description given by Coles (1931). However, further studies

have concluded that this organism is not a Rickettsia sp.

Blakemore (1947) found similar evidence to suggest that Rickettsia conjunctivae was a

causative agent of IOK; however, he considered that the organism could not be classified as

a Rickettsia because of the absence of an arthropod vector. In 1948 it was suggested that

this organism be renamed Colesiota conjunctivae (Rake, 1948). Although the presence of

inclusion bodies in these early studies were considered sufficient to identify the organism,

further work has suggested this may not be the case. Inclusion bodies seen by Coles (1931)

39

have since been considered to be goblet cells (Surman, 1973). Surman (1973) puts forward a

strong argument to suggest that Mycoplasma sp are in fact the primary organisms involved.

Akerstedt (2004) postulates that the organism found in Coles’ early (1931) study thought to

be Rickettsia was in fact a Mycoplasma sp.

Mycoplasma sp.

Mycoplasma sp. are considered a major cause of IOK (Baas et al., 1977; Radostits, 2007a).

Early studies have suggested that the Rickettsia organism found by Coles (1931) was in fact

a Mycoplasma sp. Surman (1968) isolated Mycoplasma sp. from clinical cases of IOK and

was able experimentally to induce clinical infection with the isolates. On the basis of this

result it was proposed to classify the organism as Mycoplasma conjunctivae var ovis.

Langford (1971) was also able to isolate a Mycoplasma sp. from clinically infected IOK sheep

eyes. Langford (1971) postulated that Mycoplasma sp. may be present in healthy ocular

tissue but may become pathogenic following a trauma or stress. This theory was based

around an outbreak that occurred in sheep grazing stubble fields where frequent high winds

created dusty conditions. It is considered that there were a number of organisms involved

and that the primary pathogen was Mycoplasma conjunctivae (Baker et al., 2001).

Cases in Australia, Europe and North America have been attributed to Mycoplasma

conjunctivae (Hosie, 1989). It is thought that other bacteria can be involved as they will

colonise the primary lesion created by Mycoplasma conjunctivae resulting in more severe

clinical signs (Kovac et al., 2003). Hosie (1989) agreed with this finding but stated that this

secondary colonization has not been proven experimentally. Infectious keratoconjunctivitis

has been successfully induced experimentally by the inoculation of Mycoplasma

conjunctivae into the ocular tissue in sheep (Jones et al., 1976) and goats(Baas et al., 1977).

40

In both instances the disease replicated the clinical signs expected in a naturally occurring

IOK infection.

Moraxella sp.

Moraxella species have been referred to in the literature as a possible causative agent of

IOK outbreaks. Moraxella sp. have been re-classified from Neisseria to Branhamella and

finally to Moraxella (Akerstedt, 2004).

Moraxella sp. have been consistently isolated from clinically affected sheep (Baker et al.,

1965); however, it is thought that Moraxella ovis is not pathogenic even in the presence of

corneal damage (Jones et al., 1976). Langford (1971) found that Neiserria ovis (Moraxella

ovis) was part of the normal ocular flora and that this may explain its consistent finding by

Baker (1965). Hosie (1989) also found that Branhamella ovis (Moraxella ovis) was frequently

isolated from normal and abnormal eyes. In a study of infected sheep, 27% were found to

have Mycoplasma conjunctivae and 28% were found to have Moraxella ovis (Akerstedt,

2004) which does highlight its presence in infected eyes.

Chlamydia sp.

Chlamydia sp. have been implicated in outbreaks of IOK (Hopkins et al., 1973; Cooper, 1974;

Andrews et al., 1987; Hosie, 1989; Lysnyansky et al., 2007). Hosie (1989) and Lysnyansky

(2007) consider Chlamydia psittaci ovis to be one of the primary pathogens in IOK.

Chlamydial conjunctivitis is thought to be an acute infectious disease of young lambs and

goats (Wyman, 1983) and it can be associated with polyarthiritis (Hopkins et al., 1973;

Wyman, 1983). Hopkins (1973) found that clinical signs of chlamydial conjunctivitis can be

an early warning sign of subsequent polyarthritis in feedlot lambs. Chlamydia psittaci has

been experimentally shown to induce conjunctivitis, although not severe IOK (Wilsmore et

41

al., 1990). Wilsmore (1990) goes on to conclude that C. psittaci reduces the corneal

immunity so that secondary organisms, like Moraxella ovis, are able to colonise.

2.8 Other causes of keratoconjunctivitis

Aside from the bacterial causes of keratoconjunctivitis discussed, some less common causes

have been described.

Fungal keratoconjunctivitis is considered rare in small ruminants (Moore and Whitley,

1984). Hopkins (1973) isolated Aspergillus spp and Mucor spp from eyes showing clinical

signs of keratoconjunctivitis, although these were not considered to be primary pathogens.

Parasites have been cited as a cause of keratoconjunctivitis in sheep. Oestrus ovis, a nasal

bot fly, can cause similar clinical signs to IOK (Townsend, 2007). The adult fly lays eggs on

the mucus membranes of the face. This typically occurs around the external nares, but it is

also possible for the eggs to be directly laid around the eyes. Following hatching, the larvae

migrate. This migration can be through the nasolachyrimal system to the eye or directly

from the muco-cutaneous junction around the eye to the conjunctiva. In either instance

keratoconjunctivitis can develop (Moore and Whitley, 1984). Oestrus ovis is found in

Western Australia. In some areas of South Africa another nasal bot fly, Gedolstia hasleri, can

cause similar clinical signs (Moore and Whitley, 1984).

Thelazia spp. can also cause similar clinical signs to IOK. The Musca spp of fly is the

intermediate host and they can deposit Thelazia spp. larvae on the conjunctiva while they

feed on ocular secretions (Townsend, 2007).

42

2.9 Treatment

Infectious ovine keratoconjunctivitis is a condition that ranges in severity. Cooper (1967)

found in a survey of farmers that only 23% sought veterinary treatment for outbreaks of

IOK, and those that did tended to do so when they first encountered the disease. Some

consider the disease to be self-resolving (Nanda and Abdussalam, 1943; Toop, 1964). Toop

(1964) found that recovery started on the third or fourth day after clinical signs developed

and that it took a further seven days for clinical signs to resolve altogether. Akerstedt (2004)

found the duration of clinical disease to be longer, with recovery not beginning until one

week after clinical signs were first seen and complete recovery not occurring until 3-4 weeks

after initial signs.

Under experimental conditions, Baas (1977) found that severe cases could take up to 12

weeks to resolve. Nanda (1943) describes cases recovering without any treatment.

Blakemore (1947) observed that only mild cases would resolve within a week of clinical signs

developing, whereas those with more severe clinical signs, such as corneal involvement, had

a very prolonged recovery time. In contrast Moore and Whitley (1984) reported that

spontaneous recovery occurs even in severe cases within three weeks. Cooper (1967) found

little difference in clinical cases between those receiving treatment and those not.

Beveridge (1942) found that in sheep with bilateral IOK where one eye was treated and the

other left untreated there was often no difference in recovery rates between the two eyes,

although this was not always the case. In contrast to this, Edgar (1931) found a significant

difference between treated and non-treated groups. Those treated showed return to

normal vision whereas those left untreated showed no progress and continued to have

visual deficits. The first reported outbreak in Michigan was found to take almost two

43

months for clinical signs to resolve in severe cases despite various treatments being used

(Riley Jr and Barner, 1953).

Early descriptions of IOK recognized the importance of treating cases early to limit spread

throughout a group (Symons, 1912). Symons (1912) goes on to describe the difficulties in

treating large groups but highlights the importance of persevering with treatment as success

in treating diminishes the more severe the clinical signs. Isolating clinical cases will not only

protect clinically healthy sheep from exposure but will allow for easier treatment (Michael,

1953) of the sick animals. Ideally, sheep should be isolated indoors to provide protection

from UV light (Nanda and Abdussalam, 1943; Riley Jr and Barner, 1953; Toop, 1964).

Avoiding bright sunlight is thought to reduce the risk of flies and therefore decrease

potential spread of disease (Nanda and Abdussalam, 1943). Toop (1964) describes the ease

of providing feed and water to sheep with reduced visibility through isolation. Combining

isolation of affected sheep and treatment will reduce the length of an outbreak (Cooper,

1967).

Treatments have varied over time. In the early 1900s sheep were treated both systemically

and topically. Oral administration of a purgative of epsom salts was recommended (Symons,

1912) to be followed up with hyposulphite of soda and mitre which could be added to the

drinking water. In combination with these systemic treatments it was suggested that the

eyes should be bathed with a warm strong solution of boracic acid ideally for half an hour at

a time at least twice daily. Following bathing a dilute solution of mercuric chloride or

salicylic acid should be applied (Symons, 1912). Symons (1912) recommended that in severe

cases where ulceration was present a combination of yellow oxide of mercury, boracic acid

and Vaseline be applied to the eye.

44

Michael (1953) describes a number of home remedies including the application of tobacco

juice to the eye. Cutting the lower palpebral vein and angling the head so as to allow the

blood to flow across the surface of the eye (Michael, 1953) was also suggested. Shepherds

traditionally considered blowing sugar on to the eye to be effective (Sinclair, 1955). Michael

(1953) also suggested sugar as a treatment. More primitive treatments included applying

ground up slate to the conjunctiva (Michael, 1953), the theory being that that the fine

particles would scrape away the lesions. Topical kerosene has been considered as a

treatment. Beveridge (1942), however, found kerosene to have no beneficial effect on

clinical signs and in some cases it made the condition worse. It is not surprising that

kerosene worsened the clinical signs given that it is considered to be an irritant to eyes

(Chilcott, 2006).

Topical Zinc Sulphate has been used in various concentrations. Beveridge (1942) found that

using either 10% or 60% concentration solutions of Zinc Sulphate topically made no

difference between treatment and no treatment, however, using Zinc Sulphate in 30%

solution gave mixed results: some eyes improved and others did not. In contrast to this

Edgar (1931) found a 2.5% solution applied topically to be effective. A combination of 1%

Zinc Sulphate and 2% Boric acid has also been found to be an effective topical treatment

(Nanda and Abdussalam, 1943). In human cases of conjunctivitis Zinc Sulphate is considered

to reduce clinical signs but has no effect on the underlying cause (Wood, 1999).

Topical ethidium bromide has also been used topically to good effect (Cooper, 1961;

Arbuckle and Bonson, 1980). Arbuckle (1980 found a 1% solution to be effective whereas

Copper (1961) found a 0.5% solution to be effective within 48 hours.

45

Michaels (1953) studied the effect of oral doses of Riboflavin (Vitamin B2) in the treatment

of IOK. A dose of 15mg was given daily for three-five days. Those treated with riboflavin

were found to have reduced lacrimal discharge within 48-72 hours compared to those

receiving no treatment. Sheep showing more severe signs had a variable response.

2.10 Antibiotics

A number of antibiotics have been used in the treatment of IOK. The choice of antimicrobial

should ideally be based on known sensitivity to the organism involved. Given the potential

involvement of a variety of organisms, antibiotics that are chosen should have a broad

spectrum of activity, including activity against Mycoplasma sp., and should reach sufficient

concentrations to exceed the MIC in lacrimal fluid (Regnier et al., 2013).

Topical agents

A number of studies have been conducted looking at the efficacy of various topical

treatments. Chloramphenicol, Chlortetracycline (Aureomycin Powder), Cloxacillin and OTC

are the most commonly used. In general, for topical treatments to be effective they require

frequent applications on a daily basis which can make their use in large groups impracticable

(Moore and Whitley, 1984). Normal corneal anatomy plus the constant washing from the

tear film prevent high levels of drug penetration of the cornea (Davidson, 2009). As a result

the contact time for ophthalmic suspensions is short and only about 1-10% of the drug is

absorbed by the corneal stroma therefore frequent applications are required to maintain

minimum inhibitory concentrations in the ocular tissue (Davidson, 2009).

46

2.10.1.1 Chloramphenicol

Riley (1953) found topical chloramphenicol to be effective in the treatment of IOK. Severe

cases required twice daily treatment for 5-10 days whereas milder cases only required once

daily treatment for 2-3 days. Sinclair (1955) also found twice daily topical chloramphenicol

to be effective, but a 14-day treatment course was required. In contrast to these studies a

number of other authors have found Chloramphenicol to be ineffective. When topical

chloramphenicol was compared to topical chlortetracycline (CTC), chloramphenicol was

found to be no better than giving no treatment and in some cases sheep were clinically

worse following treatment (Dickinson and Cooper, 1959). Cooper (1961) also found

chloramphenicol to be ineffective. Resistance has been identified to chloramphenicol in

Mycoplasma conjunctivae (Naglic et al., 1999).

Chloramphenicol has been found to cause a severe aplastic anaemia in humans (Page,

1991). This condition can occur indirectly through ingestion of chloramphenicol residues in

meat. Given that a minimum residue could not be established and that there was a risk of

exposure to chloramphenicol residues, the drug has now been withdrawn from use in food

producing animals in Australia (Page, 1991; Australian Government and Australian Pesticides

And Veterinary Medicine Authority, 2014).

2.10.1.2 Chlortetracycline

Topical Chlortetracycline (CTC) commonly comes in a powder form under the trade-name

AureomycinTM. A number of authors have studied the efficacy of CTC in the treatment of

IOK. Dickinson (1959) treated IOK cases twice daily until clinical signs disappeared. A good

clinical response was typically seen within 24-48 hours. Relapses following cessation of

47

treatment were common. The same author compared the efficacy of CTC against

chloramphenicol and against no treatment, and found that CTC was more effective than

both chloramphenicol and no treatment (Dickinson and Cooper, 1959). Cooper (1961) and

Toop (1964) found topical CTC to be an effective treatment. Mycoplasma conjunctivae can

be eliminated following a three day course of CTC applied topically twice daily (Egwu et al.,

1989). Mild cases can be treated with a single application of topical CTC (Hosie, 1988),

although this is likely to be effective only in extensively managed flocks. Intensively

managed flocks would require frequently repeated applications of CTC (Hosie, 1989). Hosie

(1988) found that a single application of CTC was not effective prophylactically. Dun (2009)

also considered a single topical CTC application effective in mild cases.

2.10.1.3 Cloxacillin

Cloxacillin is reported to be effective in treating ocular disease in cattle and sheep. It’s

efficacy in the treatment of IBK has been well documented (Buswell and Hewett, 1983;

Daigneault and George, 1990). Cloxacillin is used in an ointment formulation which provides

an increased contact time with the ocular tissue because of the greater viscosity of the

preparation and the slow release of the drug from droplets which have been found to settle

in the inferior cul-de-sac (McConnel et al., 2007). This allows for less frequent applications

while still maintaining adequate drug concentrations. Although Cloxacillin is an effective

treatment of IBK, where only Moraxalla sp. are thought to be involved, the involvement of

Mycoplasma sp. in IOK will potentially limit the use of cloxacillin due to its mechanism of

action.

A single application of cloxacillin was found to be more effective than no treatment or

treating with a powder containing neomycin/sulphacetamide (Webber et al., 1988).

48

Cloxacillin was found to reduce clinical signs but signs reappeared following cessation of

treatment (Lysnyansky et al., 2007). However, when compared to either CTC (Hosie, 1988)

or OTC (Greig, 1989) both were found to be more effective than cloxacillin.

2.10.1.4 Oxytetracycline

Topical oxytetracycline (OTC) preparations are available in powder and aerosol forms in

Australia and are registered for the treatment of IOK.

Topical OTC given 3-4 times daily has been found to be effective in shortening the course of

disease (Wyman, 1983; Moore and Whitley, 1984; Townsend, 2010). If uveitis or ulceration

is present then the addition of 1% atropine topically three times a day is beneficial (Wyman,

1983). Greig (1989) considers topical treatment with OTC applied at least once daily for 5-6

days to be effective if treatment is initiated in early/mild cases. Naglic (2000) found that

topical OTC was only effective in mild cases when it was applied 2-3 times per day for one

week, however clinical signs usually returned following cessation of treatment.

Injectable

2.10.1.5 Systemic injection

Konig (1983b) conducted a study of various injectable drugs used in the treatment of IOK.

Spiramycin was found to be an effective treatment, but following cessation of treatment

there were a number of relapses. A high number of relapses followed treatment with

tiamullin, which was found to be less effective than spiramycin or OTC even though it

achieved high concentrations in the lacrimal fluid.

Injectable OTC was found to be an effective treatment. Naglic (1999) found that

tetracyclines, tiamulin, tylosin and enrofloxacin were effective systemic treatments for IOK.

49

In order to limit the emergence of resistance in the fluroquinolone (e.g. enrofloxacin) group

of antibiotics, their use in food producing animals has been prohibited in Australia (Cheng et

al., 2012).

Hosie (1995) found OTC injection effective in rapidly reducing the clinical signs of IOK in all

stages of infection. This was also found by Kovac (2003), although this study found that in

severe cases it took four days for clinical signs to resolve. Despite reduction in clinical signs

of IOK, Mycoplasma conjunctivae is not eliminated and the sheep therefore remain carriers

(Kovac et al., 2003). Andrews (1987) and Townsend (2010) also found injectable OTC to be

effective in severely affected sheep. Systemic long acting OTC can be useful in controlling an

outbreak of IOK (Greig, 1989). Dun (2009) considers that systemic long acting OTC can be

used to halt the progress of an outbreak.

Injecting Florfenicol at a dose of 40 mg/kg intra-muscular results in adequate concentrations

in the lacrimal fluid to exceed the minimum inhibitory concentration (MIC) of Mycoplamsa

conjunctivae and therefore makes it a suitable choice for the treatment of IOK (Regnier et

al., 2013).

Injectable cephalosporin was found to be less effective than injectable OTC in the treatment

of IOK (Greig, 1989).

2.10.1.6 Sub-conjunctival injections

Sub-conjunctival injections have been used to treat infections of the cornea and anterior

tissues of the orbit (Regnier, 2007). Injections should be placed in the bulbar conjunctiva

(Whitley and Moore, 1984; Regnier, 2007) with a maximum volume of 0.5 mL per eye in

50

sheep (Whitley and Moore, 1984). The benefit of this method lies in providing sustained

drug concentrations at the site of infection and can be used in place of frequent topical drug

application. Injections of water soluble substances like short acting OTC are likely to provide

therapeutic levels for 8-12 hours (Regnier, 2007). Dun (2009) found sub-conjunctival

injections of OTC to be effective. Sub-conjunctival injection of a penicillin-streptomycin

combination drug was found to be less effective than a systemic injection of OTC (Greig,

1989).

Aside from the risk of intraocular injections (Whitley and Moore, 1984), subconjunctival

injections are difficult to do and time consuming, so that this method of treatment is less

practicable in large groups of sheep (Dun, 2009).

2.10.1.7 Combinations: topical and systemic

Hosie (1988) found that for severe cases of IOK a combination of a single CTC topical

treatment with a systemic OTC injection was the most effective treatment. Konig (1983) also

found combinations to be effective for severe cases. Naglic (2000) considers the optimum

treatment for IOK to be once daily topical OTC in combination with LA OTC injections given

every three days for three consecutive treatments.

Oral treatments

There is limited information in the literature about oral medications for the treatment of

ocular infections. Because of the difficulty of administering frequent topical treatments to

young children oral antibiotics have been shown to be an effective alternative in the

treatment of conjunctivitis in humans (Wald, 1997). Oral erythromycin was found to be

more effective than topical erythromycin at eliminating Chlamydia sp. in ocular infections of

51

children (Patamasucon et al., 1982). There is some evidence that oral doxycycline can be of

use in the treatment of ocular infections in horses (Matthews, 2009). A combination of

injectable OTC and in feed OTC in the treatment of IBK in cattle suggested that this

combination was more effective than procaine penicillin administered sub-conjunctivally

(Eastman et al., 1998). This experiment wasn’t able to conclude whether the oral OTC

medication was effective or not.

Although numerous studies have been done on both treatment and pre-disposing factors of

IOK, there is a paucity of information regarding the unique environment of a pre-export

feedlot. This thesis aims to establish a suitable treatment protocol that is both effective and

practical to use in the feedlot environment where up to 80,000 sheep are held prior to being

exported.

52

3 General Aims

The review of the current literature reveals a paucity of information relating to the

epidemiology and control of IOK in pre-export feedlots. For this reason the following were

considered as general aims for this thesis:

to record data on IOK cases entering a feedlot;

to document whether any of the risk factors identified in the literature are present

in live-export pre-export feedlots and to determine whether anything can be done

to mitigate these factors

considering that much research has been published on microbiological causative

agents of IOK, to determine if these agents are present in feedlot conditions and

associated with clinical disease

to test the efficacy and impact on the animals of various common treatments for

these organisms

to measure bioavailability, clinical efficacy and the impact on the rumen

microbiome of either in-feed or in-water oxytetracycline

to identify practical and effective treatments for IOK in pre-export feedlots that

house up to 80,000 sheep

given that IOK is a progressive disease, to determine if a severity of disease

treatment cut-off can be established so that a decision can be made on which

treatment, if any, would be most appropriate at different stages of the disease

53

4 Epidemiology of infectious ovine keratoconjunctivitis at a pre-

export feedlot

4.1 Introduction

With increasing scrutiny of the live export trade in Australia, it is essential that the industry

address areas of concern throughout the chain. Infectious ovine keratoconjunctivitis (IOK)

has been cited as a problem throughout the export chain and to date only anecdotal

evidence exists as to the gravity of the issue. It has been estimated by exporters that 0.5-1%

of sheep are rejected from a shipment because of IOK (G. Robinson and M. Curnick pers

comms) at departure from the pre-export feedlot. In real terms this equates to

approximately 10,000-20,000 sheep per year, based on 2013 totals. However, this figure

does not include those animals that are held over for treatment until the following

shipment.

The aims of the experiment described in this chapter were to:

1. Establish whether accurate data could be obtained of cases of IOK on arrival and

departure from a pre-export feedlot

2. Determine the prevalence of IOK on arrival and on departure from a pre-export

feedlot

3. Determine seasonal patterns in the prevalence of IOK at a pre-export feedlot

4.2 Materials and Methods

Data were received from an exporter of live sheep who recorded cases of IOK on arrival at

the feedlot. The feedlot is located approximately 50km south of metropolitan Perth in

54

Western Australia and is a designated pre-export quarantine facility with capacity to

accommodate more than 80,000 sheep. All sheep were inspected on arrival at the feedlot. It

is at this point that the exporter decided which animals were not fit for entry to the feedlot.

Rejected animals were deemed to have no commercial value and the producer received no

payment for them.

Sheep were rejected for a number of reasons. These included severe lameness, IOK,

emaciation/weakness and wounds. Stock inspectors were employed at the feedlot to carry

out these inspections. Only sheep with advanced IOK, whereby vision was impaired and/or,

in the opinion of the inspector, treatment was unlikely to be successful, were rejected.

Sheep showing more mild clinical signs of IOK were not rejected but were treated.

Sheep arriving at the feedlot will have come from a number of different properties from

around the state of Western Australia. Esperance was the furthest recorded origin at 690km

from the feedlot. Most sheep originated from 150-300km from the feedlot.

On arrival at the feedlot, sheep were grouped into types based on the commercial market

they were destined for. Each batch of sheep are classified as a line. This could involve mixing

of different lines of sheep. Once grouped, sheep were moved into the sheds where they

were housed for the duration of the quarantine period pertaining to their countries of

ultimate destination.

Odds ratios and their 95% confidence intervals (95% CI) were calculated to assess the risk of

IOK in different months and to identify a seasonal pattern.

Feedlot inspectors were invited to complete a questionnaire (Appendix 1). The

questionnaire was designed to get an overall impression of the percentage of animals

55

presenting at the feedlot with IOK of varying severity and was approved for use by the

Murdoch University Human Ethics Committee. Data from the exporter represented those

animals with severe IOK only; the questionnaire was designed to capture information about

the animals that had milder IOK.

4.3 Results

Historical data were obtained for a twelve-month period from an exporter (Table 4.1).

Within that period 1,168,372 sheep were exported from that pre-export feedlot. Of those

sheep exported, 681 (0.06%) were rejected on account of IOK.

56

Shipment Date

Total number sheep in shipment

Number rejected with IOK

Percentage of shipment

rejected with IOK

Odds Ratios

Lower 95% Confidence

interval

Upper 95% confidence

interval

7/08/2012 74272 7 0.01 1.03 0.36 2.94

12/08/2012 19421 5 0.03 2.82 0.90 8.89

1/10/2012 73541 13 0.02 1.94 0.77 4.85

10/11/2012 69641 100 0.14

15.75* 7.32 33.90

10/12/2012 67303 62 0.09

10.10* 4.62 22.07

7/01/2013 73456 47 0.06

7.01* 3.17 15.52

1/02/2013 29450 40 0.14

14.90* 6.67 33.26

26/02/2013 28224 25 0.09

9.71* 4.20 22.46

8/03/2013 75170 60 0.08

8.75* 4.00 19.14

6/04/2013 70510 76 0.11

11.82* 5.45 25.64

20/04/2013 76602 18 0.02

2.57* 1.08 6.16

15/05/2013 76164 46 0.06

6.62* 2.99 14.66

1/06/2013 23034 3 0.01 1.43 0.37 5.52

23/06/2013 77627 23 0.03

3.25* 1.39 7.57

14/07/2013 70548 9 0.01 1.40 0.52 3.75

21/08/2013 68319 22 0.03

3.53* 1.51 8.26

22/09/2013 76685 7 0.01 1.00

4/10/2013 72029 26 0.04

3.96* 1.72 9.11

7/11/2013 46376 92 0.20

21.77* 10.10 46.96

Table 4.1 Historical shipping data showing numbers and percentages of sheep rejected on arrival at the feedlot due to Imfectious Ovine Keratoconjunctivitis. * depicts significant result.

57

Sheep being shipped in Spring, Summer or Autumn were more likely to have IOK than those

shipped in Winter (Table 4.2). Sheep being shipped in either Spring and Summer were 1.8

(1.55-2.10) times more likely to have IOK than those shipped in Autumn and Winter.

Season

Total number sheep

Number rejected with IOK

Percentage rejected with

IOK Odds Ratio

Lower 95% Confidence

interval

Upper 95% confidence

interval

Spring 338272 238 0.07 3.40* 2.60 4.40

Summer 198433 174 0.88 4.24* 3.21 5.60

Autumn 298446 200 0.67 3.24* 2.46 4.26

Winter 333221 69 0.02 1.00

Spring and Summer 536705 412 0.08 1.80* 1.55 2.10

Autumn and Winter 631667 289 0.04 1.00

Table 4.2 Historical shipping data provided by the exporter outlining, by season, the number of sheep

rejected due to clinical Infectious Ovine Keratoconjunctivitis. *depicts significant findings.

Questionnaires were sent to five inspectors and three responded (60% response rate). The

three responses were combined and are shown in Table 4.3.

Mild IOK (%) Severe IOK (%)

Minimum Maximum Minimum Maximum

Spring October 15 25 1 5

November 20 35 5 10

Summer January 35 50 10 20

February 30 40 10 20

Autumn April 25 35 5 15

May 15 25 2 15

Winter July 5 15 2 5

August 5 15 2 5 Table 4.3 Data gatherd by questionnaire showing maximum and minimum percentages of IOK cases per

shipment

58

In Table 4.4 the breakdown of mild cases in lambs, wethers and ewes respectively are

summarised. The respondents failed to give data for severe cases in different ages of

animal. The seasonal pattern highlighted by the rejection data from the exporter was

confirmed by the exporters with higher percentages of both mild and severe cases seen

during the warmer months.

Lambs Wethers Ewes

Minimum (%)

Maximum (%)

Minimum (%)

Maximum (%)

Minimum (%)

Maximum (%)

Spring October 15 30 5 10 1 15

November 20 35 10 15 5 10

Summer January 35 50 15 20 5 10

February 30 50 15 20 5 10

Autumn April 30 50 15 20 5 10

May 25 35 10 15 1 5

Winter July 20 20 5 10 1 5

August 15 20 5 10 1 5 Table 4.4 Data gatherd by questionnaire showing maximum and minimum percentages of mild IOK cases per

shipment in lambs, wethers and ewes.

The inspectors reported that, for entire male animals, only ram lambs were exported so no

figure could be given for rams individually. 95% of sheep inspected were Merinos. The

inspectors commented that the proportion of cross-bred sheep with IOK was much lower

than in merinos, although they were unable to enumerate this.

4.4 Discussion

The Australian Standards for the Export of Livestock (ASEL) lists the reasons why sheep

should be rejected from a consignment for shipment. These standards state that animals

with IOK must be rejected (Australian Government Department of Agriculture, 2011). Sheep

59

must also have vision in both eyes. These data received from the exporter were limited in

that only those animals deemed too severely affected with IOK for treatment were

recorded.

The figure of 0.06% rejections represented the point prevalence of severe IOK at entry to

the pre-export feedlot. This data did not capture the mild cases that would be treated

during the quarantine period so that they were fit to be shipped. To conform to current

legislation, no severe cases of IOK should be identified on arrival at the feedlot as they

would not be deemed fit to have travelled from the farm (Moir, 2010). These cases are

deemed to be of no commercial value to the exporter and the producer is not paid for them.

It is likely that this discourages producers from sending inappropriate sheep to the pre-

export feedlots.

Anecdotally IOK is seen more commonly in lambs and in the spring and summer months on

arrival at the pre-export feedlot. This pattern was confirmed by the inspectors’ responses to

the questionnaire. They highlighted that most cases were seen in lambs, followed by

wethers with the fewest cases in ewes. Although IOK has been reported in animals of any

age (Hosie, 1989), infection in lambs is considered to be mild and more transient (Egwu et

al., 1989) compared to more severe infections in adults (Hosie, 1989). Although there is a

paucity of data relating to the situation in Australia, one contradicting report highlights that

outbreaks in weaners are most common and when they do occur are severe (Beveridge,

1942). Although the responses from the inspectors did not give definitive figures, they did

suggest that disease was seen most commonly in younger animals. The responses did not

allow differentiation of severity of disease between the different age groups.

60

The data confirmed a significant seasonal pattern to the prevalence of IOK on entry to the

pre-export feedlot. Axelsen (1961) and Surman (1968) reported that cases of IOK in

Australia were most commonly seen in the spring and summer months. As spring and

summer in Western Australia are typically hot and dry months, sheep are likely to be

exposed to a number of the recognised risk factors for IOK. Hosie (1988) considered

exposure to ultra-violet light a risk factor for developing IOK. Cooper (1967) also identified

heat as a risk factor. Yarding is recognised as a time for the spread of disease (Edgar, 1931;

Michael, 1953; Cooper, 1967; Dun, 2009) and in Western Australia yarding in the dry

months will lead to increased exposure to dust, which Edgar (1931) and Cooper (1967)

identified as another risk factor for IOK. Given the high degree of exposure to a number of

significant risk factors it is not surprising that cases of IOK were seen in lines of sheep on

arrival at the feedlot.

In addition to the sheep identified as suffering from severe IOK on arrival at the pre-export

feedlot, a number of sheep arrived with milder IOK. These sheep typically presented with

tear staining of the wool on the cheek and conjunctivitis. Sheep with milder IOK are

considered treatable and would usually be separated into a batch for treatment. The feedlot

management considered that many of these milder IOK cases had resulted from irritation to

the eyes during the truck journey. Although this hypothesis could not be confirmed it is

possible that sheep would have been exposed to irritants in the preparation for loading and

during the transport itself. The questionnaire to the inspectors aimed to capture an

impression of the percentage of mild cases presenting at the pre-export feedlot. Their

responses highlighted the high numbers of sheep arriving at the feedlot with clinical signs of

61

mild IOK. Although these animals were not rejected outright they did require treatment to

enable them to be fit for export.

Attempts were made to validate this data by independently analysing a shipment. At the

pre-export feedlot sheep typically arrive over a 2-day period for a shipment which can

contain up to 80000 sheep. There are six loading banks for trucks, so 12 people would be

required, two at each loading bank, to carry out the inspection. Two people would be

required at each loading bank so that both eyes of the sheep could be inspected. Feedlot

management were concerned that the additional staff would slow down the procedure and,

despite lengthy discussions, the experiment could not proceed. The experiment was

designed to be two-pronged: the attempt to analyse a shipment on arrival, and an attempt

to track cohorts of sheep through the quarantine period to identify if there were certain

groups that were more likely to develop IOK. After unloading, however, groups of sheep

were sorted into batches according to sheep type, and with this mixing it was not possible

to follow farm cohorts through the quarantine period.

Mixing sheep poses two potential risks with reference to IOK. Firstly, introducing animals

from different farms will create the risk that naïve sheep will be exposed to carriers. This

phenomenon was highlighted by Naglic (2000) and cited as the explanation for how IOK was

introduced to native sheep in Croatia in 1995 following contact with imported Australian

sheep. Secondly, sheep are held in pens during the sorting process which allows for close

contact. Hopkins (1973), Hosie (1989) and Akerstedt (2004) described IOK being spread by

close contact between animals. Once housed, sheep are fed an ad-lib pellet ration. Trough

feeding allows for further close contact and this has also been linked to the spread of IOK

(Cooper, 1967; Egwu, 1992b; Dun, 2009). Once infection is identified, Hopkins (1973) found

62

that grouping of sheep in a feedlot was an important factor in aiding the spread of an

outbreak of IOK.

Data were not available from the exporter for rejections on leaving the pre-export feedlot.

Currently at Fremantle Port, final inspections are carried out at the wharf by official AQIS

inspectors rather than during loading for outward transport from the feedlot. This process is

currently under review as concerns have been raised about unsuitable sheep being

presented at the wharf (Farmer, 2011). Any cases of IOK that have been unresponsive to

treatment during the quarantine period will be re-assessed at the feedlot. These cases will

either be held back for the next shipment or culled depending on the assessment of the

feedlot management regarding their likely response to treatment. Guidelines have been

generated as part of this project to aid their decision making with these cases.

4.5 Conclusions

It was not possible to validate the data obtained from the exporter for rejection rates of IOK

on arrival at the feedlot or data for rejection rates on leaving the feedlot. The historical data

obtained shows a prevalence of severe IOK on arrival at the feedlot of 0.06% over the year.

Sheep were more likely to have IOK in spring and summer than in autumn and winter. Mild

cases of IOK are frequently identified in sheep arriving at the pre-export feedlot; this can

affect as many as 50% of sheep during summer. Mild cases are more commonly seen in

lambs; there are very few cases among ewes.

With high numbers of sheep arriving at the feedlot with clinical signs of mild IOK it is

essential that an appropriate treatment strategy is identified to minimize the impact of an

outbreak in the pre-export feedlot and to enable these animals to be exported following the

quarantine period.

63

5 Identification of the microbiological causes and possible

treatment of infectious ovine keratoconjunctivitis in a pre-export

quarantine feedlot.

Infectious ovine keratoconjunctivitis continues to be a concern within the sheep live-export

chain and in particular at the pre-export feedlot. The work described in Chapter 4 has

highlighted that although outright rejections represent a small percentage of sheep arriving

at the feedlot, there is a high percentage of animals presenting with mild IOK that require

treatment. The cost of both the rejections and the treatment of the disease represents a

significant loss to the industry.

A number of treatments are used throughout the world to try and alleviate the clinical signs

of IOK. Although some work has been done to assess treatments in other countries (Konig,

1983b; Hosie, 1995) there is a paucity of literature evaluating treatments used in Australia,

and in particular in the unique environment of a pre-export feedlot.

The aims of this experiment were to:

1. describe normal ocular flora in sheep in a pre-export feedlot

2. assess the efficacy of a number of commonly used treatments at reducing the clinical

appearance of IOK in naturally infected sheep

3. assess the impact of those treatments on the bacterial population in the ocular

environment.

64

5.1 Materials and methods

Fifty-one merino and merino cross sheep with no clinical signs of ocular disease were

selected from the pre-export feedlot. These sheep had been at the feedlot for five days and

were randomly selected from a group with no clinical signs of IOK. Both eyes were swabbed

using a cotton-tipped, wooden-shafted swab which was inserted into the fornix, between

the conjunctiva and the globe. The swab was gently rotated for ten seconds and then

removed. Swabs were plated onto horse blood agar plates in a manner designed to obtain

isolated colonies (Harrigan and McCance, 2014) and the shaft was then cut and the swab tip

placed in a Mycoplasma broth. New gloves were worn for each sheep to minimize the risk

of contamination between animals.

Eighty merino and merino cross sheep with naturally occurring IOK infections were selected

from those sheep held back from a shipment owing to the presence of clinical eye disease.

Sheep were selected on the basis of the clinical grade of infection. The grading system

(Appendix 2) was adapted from existing systems (Konig, 1983b) making the grades

numerical. Only sheep with grade 2 (conjunctivitis) or grade 3 (corneal oedema) in both eyes

were selected for this experiment. Affected sheep showed no other clinical signs of systemic

disease. All sheep were transported to the Murdoch University on-campus farm (Perth,

Western Australia) where they were randomly assigned on passing through the race into 8

groups (n=10) and housed in group pens in an open sided shearing shed. This allowed for

ease of monitoring and for treatments to be easily administered.

Both eyes were photographed and graded on day zero and a swab was taken for bacterial

culture in the same manner described for those without clinical disease. All eyes were

graded by the same two people on each occasion using the grading system in Appendix 2.

65

All eyes were graded and photographed on days 0, 4, 6, 8 and 10. The left and right eye

scores were added to give group scores for both eyes and the change from day 0 to the end

of the trial. Swabs were repeated on day 10 as previously described.

The 8 groups were subdivided, as shown in Table 5.1, into a control group and 7 treatment

groups. The control group was monitored until day 20 and eyes were graded and

photographed on days 0, 4, 6, 8, 10, 12, 18 and 20.

Group Treatment given

1 Oxytetracycline injectable (Alamycin LA 300, Norbrook Lab. Aust. Pty Limited) 20mg/kg given as a single intramuscular injection into neck muscle. Dose calculated based on accurate sheep weights.

2 Cloxacillin 500mg/3g (Orbenin Eye Ointment, Zoetis) administered at a dose of 125mg per eye (one quarter of a tube applied as a streak to each eye, repated after 2 days if still clinical signs).

3 No treatment, control group

4

Oxytetracycline powder 200mg/g administered orally in the drinking water for 5 days.

2g of powder (400mg drug)/head/day based on each sheep drinking 4L water/day.

5 Oxytetracycline 20mg/g, (Terramycin Pink Eye Powder, Pfizer Animal Health),

applied topically twice daily to eyes for 5 days.

6

Oxytetracycline 20mg/g, (Terramycin Pink Eye Powder, Pfizer Animal Health). Applied topically to eyes twice daily for 5 days PLUS

Oxytetracycline injectable 20mg/kg (Alamycin LA 300, Norbrook Lab. Aust. Pty Limited) given as single intramuscular injection into the neck muscle. Dose calculated based on accurate sheep weights.

7 Oxytetracycline 2.0mg/g, (Terramycin Pink Eye Aerosol, Pfizer Animal Health). Applied topically to the eyes twice daily for 5 days.

8 Oxytetracycline (Alamycin 300 LA Norbrook Lab. Aust. Pty) at a dose of 20mg/kg on day 0 and day 4 by intramuscular injection into the neck muscle. Dose

66

Table 5.1 Treatments tested for control of Infectious Ovine Keratoconjunctivitis

Statistical analysis

Statistical analyses were performed using statistical package SPSS version 17. An ANOVA

with Bonferroni corrections were used for normally distributed data. The Kruskal-Wallis

ANOVA was used for continuous data that were not normally distributed. Paired tests were

used to measure the changes in individual sheep between the start and the end of the trial.

5.2 Results

A total of 20 different organisms were cultured from the 102 control eyes. Of these

organisms, Bacillus species were the most commonly isolated with 87.3% positive.

Moraxella ovis was the second most commonly isolated organism with 38.2% followed by

Staphylococcus chromogens with 33.3%. Only 4.9% of control animals were positive for

Mycoplasma species, only one of which was positively identified as Mycoplasma

conjunctivae (Table 5.2).

calculated based on accurate sheep weights.

67

Organism Positive N (%)

Moraxella ovis 39 (38.2)

Mycoplasma species 5 (4.9)

M.conjunctivae positive 1 (25.0)

M.ovis + Mycoplasma sp. 3 (15)

M.ovis + M.conjunctivae 1 (0.9)

Staphylococcus epidermis 20 (19.6)

Bacillus species 89 (87.3)

Bacillus sp. Dry yellow 12 (11.8)

Staphylococcus chromogens 34 (33.3)

Streptococcus pyogenes 0 (0)

Arcanobacterium pyogenes 0 (0)

Mannheima haemolytica 2 (2)

Non-haemolyticMoraxella sp. 2 (2)

Gram-negative cocco-bacilli 0 (0)

Acinetobacter sp. 8 (7.8)

Unidentified GPR 0 (0)

Shewanella sp. 0 (0)

Pasteurella multocida 0 (0)

Unidentified 0 (0)

Corynebacterium sp. 0 (0)

Staphylococcus haemolyticus 38 (37.3)

Gram-negative diplococcic 0 (0)

Unidentified GNR 0 (0)

Staphylococcus aureus 3 (2.9)

Neisseria sp. 2 (2.0)

Proteus sp. 0 (0)

Unidentified GPC 0 (0)

Staphylococcus intermedius 0 (0)

Coliform 2 (2)

Pseudomonas aeruginosa 0 (0)

E.coli 0 (0)

Staphylococcus hyicus 0 (0)

Streptococcus Group G 1 (1)

Streptococcus species 4 (3.9)

Pseudomonas sp. 0 (0)

Aeorcoccus viridians 5 (4.9)

Micrococcus sp. 2 (2)

Kocuria varians 1 (1)

Enterococcus faecalis 0 (0)

Brevundimonas dimuta 13 (12.7)

GNR = Gram-negative rod; GPC = gram-positive cocci; GPR = gram-positive rod Table 5.2 Microbial flora isolated from clinically healthy eyes, Mycoplasma conjunctivae positive is a percentage of total Mycoplasma spp.

68

All sheep continued to eat and drink normally throughout the course of the experiment. An

estimation of water intake was done for animals in group 4 based on residual water

volumes.

The group scores for eyes and the change from day 0 to the end of the experiment is shown

in Figure 5.1. A one-way ANOVA applied to the difference between the total score at day 0

and the total scores at day 10 for the different groups showed a significant difference

overall between the groups (p < 0.001). The order of improvement, ranging from the

greatest reduction in clinical score to the least reduction, was 8, 6, 1, 4, 2, 3, 7 and 5.

It was found that two injections of long acting Oxytetracycline (OTC) treatment (Group 8)

and Oxytetracycline single injection combined with topical oxytetracycline powder (Group 6)

were the most effective treatments. Both topical OTC preparations, powder (Group 5) and

aerosol (Group 7), were less effective than giving no treatment at all. Group 8 was found to

be significantly better than Groups 2, 3, 4, 5 and 7, and Group 6 was significantly better than

Groups 3, 5, and 7. Therefore the study showed that Group 3, the control group, was

significantly worse than Groups 6 and 8. No significant difference was noted between

Groups 8 and 6.

69

Figure 5.1 Eye grades over time

Group 1: Single Alamycin LA 300™ injection 20mg/kg. Group 2: Orbenin Eye Ointment™. Group 3: No treatment. Group 4: Oxytetracycline powder in the water. Group 5: Terramycin Pink Eye Powder™. Group 6: Terramycin Pink Eye Powder™ plus Alamycin LA 300™ 20mg/kg single injection. Group 7: Terramycin Pink Eye Spray™. Group 8: Two injections Alamycin LA 300™ 20mg/kg 4 days apart.

0

10

20

30

40

50

60

0 1 2 3 4 5 6 7 8 9 10 11 12

Tota

l Eye

Gra

de

s

Days post-treatment

Eye grades over time Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8

70

All groups showed a significant reduction in Moraxella ovis autoagglutinating (p<0.05) and

Mycoplasma spp. (p<0.05) indicating that regardless of treatment chosen there was a

reduction in the growth of M. ovis and Mycoplasma spp. over the course of the experiment.

For Mycoplasma spp the change is less in the control group than in group 7 (topical OTC

aerosol) (p<0.033). This indicates that giving no treatment is more effective at reducing

Mycoplasma spp. growth than treating with topical OTC aerosol.

Moraxella ovis and Mycoplasma spp. were reduced in all treatment groups and in the control group.

The use of the topical powder treatment gave a better result than no treatment for Moraxella ovis.

The use of the topical powder treatment gave a poorer result than no treatment for Mycoplasma

spp.

5.3 Discussion

Despite the fact that the sheep were showing no clinical signs of IOK, a number of organisms

were cultured in this current experiment. There is limited literature describing the normal

71

ocular flora of sheep eyes. One study found that Micrococcus spp., Streptococcus spp.,

Achromobacter spp., Corynebacterium spp., Bacillus spp., and Moraxella spp. were found in

the ovine conjunctival sac of a healthy sheep showing no clinical signs of ocular disease

(Spradbrow, 1968). Bacillus spp. were the bacteria most frequently isolated from healthy

eyes in this experiment. It is likely that this frequent occurrence was due to the presence of

Bacillus spp. in dust (Lee et al., 2009). Pre-embarkation feedlots are particularly dusty

environments. Dust is stirred up by the constant movement of sheep through the yards at

the feedlot.

Staphylococcus chromogenes was also frequently isolated from healthy eyes. S.

chromogenes has been associated with sub-clinical mastitis in sheep (Fthenakis, 1994) and

cattle (Devriese et al., 2002), but it has not been reported as an ocular pathogen. It is

postulated that this bacteria is another environmental organism that contaminated eyes in

dusty conditions.

The presence of both Moraxella ovis and, to a lesser extent, Mycoplasma spp. in ocular

tissue of healthy sheep highlights the potential for sheep to remain carriers of the organisms

responsible for IOK. Infectious ovine keratoconjunctivitis is thought to have been introduced

into the native sheep population of Croatia through the introduction of asymptomatic

carrier sheep from Australia (Naglic et al., 2000). Naglic (2000) found that Mycoplasma

conjunctivae was always identified in sheep flocks where IOK was present even in animals

without clinical disease, and this matches the findings of the current experiment.

The presence of IOK-causing organisms in ocular tissue alone is not always enough to result

in clinical disease. Spradbrow (1968) considered that the organisms worked synergistically

with environmental factors to cause clinical disease.

72

Numerous treatments have been recommended for IOK, many of which are no longer used.

Treatments suggested by stockmen to the researchers as being effective for IOK included

kerosene and cod liver oil. Kerosene is frequently used in the developing world as an

emergency treatment for ocular injuries prior to attending hospitals (Ajite and Fadamiro,

2013) despite its being irritant to the eye (Pe'er and BenEzra, 1988). Cod liver oil has also

been reported as an effective treatment for external infections of the eye in humans

(Stevenson, 1936; Ajite and Fadamiro, 2013). Neither treatment was considered suitable for

testing in this experiment.

The results of this experiment highlighted the variation in clinical response seen following

treatment of IOK with different compounds. Previous work in the United Kingdom

demonstrated that a combination of injectable OTC and topical OTC was an effective

treatment for IOK (Hosie, 1988). This combination was ranked as one of the most effective

treatment in this experiment and it was found that utilising topical OTC in conjunction with

injectable OTC resulted in a more effective treatment than a single injection of OTC alone.

However, due to the increased labour requirements required for topical treatment it was

considered practically to be less effective than 2 injections of injectable OTC.

Considering the pharmokinetics of topical OTC, this finding was unexpected. Aqueous

topical treatments have been shown to have a half-life of a few minutes in lacrimal fluid

(Ward, 1991), therefore any additional benefit of the topical preparation would be minimal.

When used alone, in Group 5, OTC topical powder was found to be the least effective

treatment, with the sheep showing a worsening of clinical signs following cessation of

treatment.

73

Animals in Group 7 and Group 5 received topical aerosol OTC spray and OTC powder

respectively, and neither group demonstrated a significant clinical improvement following

treatment. On the contrary, topical OTC treatments (both powder and aerosol) were found

to reduce clinical signs during application of the treatment, but clinical signs worsened

following cessation of treatment and in many individuals, the clinical signs became worse

than on initial presentation. Given the limited contact time of aqueous topical treatments it

is likely that therapeutic concentrations of drugs would not be maintained in the ocular

tissues.

The tear half-life of aqueous topical preparations is short (McConnel et al., 2007). McConnel

et al (2007) also found that any antimicrobial preparations sprayed onto the eye were likely

to be washed away by the tear film within a few minutes. The topical application of powders

and solutions containing dyes have been found to be irritant, resulting in increased tear

production (Brown et al., 1998) and therefore decreased contact times. Only about 1-10% of

drug solutions and suspension applied topically will be absorbed by the corneal stroma

(Davidson, 2009). As a result of these pharmacokinetic principles, frequent applications are

required to maintain minimum inhibitory concentrations in the ocular tissue.

It was found that sheep showed aversion to topical treatments, in particular the topical OTC

spray. Sheep were reluctant to move into the race for treatments or examination following

commencement of treatment with the aerosol. The results of the experiment showed that

those animals that received no treatment showed a significantly greater clinical

improvement than those treated with topical oxytetracyclines.

As a treatment protocol, both powder and aerosol topical treatments are labour intensive

and therefore impractical for use on a large number of animals, aside from the issues of

74

efficacy and aversion to application seen in this trial. This agreed with Davidson’s (2009)

findings that drugs manufactured in powder form and those containing dyes, such as the

topical OTC spray used, are irritating and have low drug bioavailability and are not

recommended for application to eyes.

Cloxacillin was also investigated as a topical agent (Treatment group 2). Unlike the aqueous

OTC topical medications, Cloxacillin is in an ointment base and this formulation allows for

increased contact time with the ocular tissue because of the increased viscosity of the

preparation and the slow release of drug from droplets which have been found to settle in

the inferior cul-de-sac (McConnel et al., 2007). Cloxacillin has been advocated as a

treatment for infectious keratoconjunctivitis in both cattle and sheep. In cattle the clinical

effect of cloxacillin treatment has been well documented (Buswell and Hewett, 1983;

Daigneault and George, 1990) and the drug has been shown to be effective. However,

Infectious Bovine Keratoconjunctivitis (IBK) is known to be caused by Moraxella spp. only,

organisms which have good sensitivity to cloxacillin. In IOK the involvement of Mycoplasma

spp. will limit the efficacy of cloxacillin. Cloxacillin has limited to no effect against

Mycoplasma spp. owing to the absence of a cell wall. Penicillins like cloxacillin act on

receptors in the cell wall of bacteria to cause bacterial death (Rang et al., 2000).

Hosie (1988) found that a single dose of Aureomycin Powder ™ (Chlortetracycline)

administered topically was more effective than cloxacillin ointment at treating clinical IOK in

hill sheep in the United Kingdom. Sheep in Group 2 received treatment with cloxacillin

ointment. Of those animals treated with topical preparations, these sheep showed the

greatest clinical improvement. Overall, however, animals in Group 2 showed only the fifth

greatest clinical improvement. Given the increased labour effort required in applying the

75

treatment and lower levels of drug bioavailability weighed against that of more effective

treatments, cloxacillin ointment could not be recommended as a treatment of choice.

In-water medications would appear to be the least labour-intensive and therefore the most

suitable treatment protocol for use on large numbers of animals. The use of in-water or in-

feed medications makes control of dosing difficult; some animals are likely to be under-

dosed and potentially some will be over-dosed. It was found during this treatment

experiment that, on average, the sheep were drinking two litres of medicated water per

head per day, which would deliver a sub-therapeutic dose, according to the manufacturer’s

guidelines.

Davidson (2009) recommended a dose of 22mg/kg once daily for five days for oral

administration of tetracycline – this is more than double the dose rate used in the current

experiment (880mg for a 40kg sheep versus the 400mg active ingredient used in the current

experiment). Normal levels of water intake for sheep are known to be around four litres per

day. This can vary widely as a result of a number of factors including weather, diet, water

quality and access to water. Although a clinical improvement was seen in the sheep in

Group 4 (OTC in the water), this improvement was less than seen with other treatment

protocols. Animals in Group 4 (OTC in the water) showed the 4th greatest clinical

improvement. These animals improved more than those in the control group and those

receiving topical medications. Although the use of in-water antibiotic medication shows

promise, further work is required to establish individual intakes and to assess any impact on

rumen health.

The use of systemic antibiotic therapy for IOK has been studied throughout the world.

Systemic antibiotic therapy is usually given by way of an intramuscular injection of a long-

76

acting antibiotic preparation. This limits the labour requirements for treatment and has

been found to be effective (Hosie, 1995). Hosie et al (1995) found that a single injection of

long-acting oxytetracycline at a dose of 20mg/kg body weight halted signs and produced a

rapid clinical cure of severe conjunctivitis. Konig (1983) also found a single injection of long-

acting oxytetracycline to be effective in treating clinical cases. The findings of this

experiment align with the work by Hosie (1995) and Konig (1983), and demonstrate that

general principles of treating IOK can be applied to treatment of the disease in a feedlot.

Axelson (1961) reported that feed intakes can be reduced and animals will lose weight as a

result of infection with IOK. It would be expected that sheep with severe clinical signs would

have limited vision and would be likely to be disorientated and might struggle to locate feed

and water troughs, and that this might be a limiting factor with the use of in-feed or in-

water medications.

However, the sheep in the trial showed only mild signs of IOK, and it was noted that all

animals in the experiment continued to eat and drink as normal.

77

5.4 Conclusions

Up to 20 different species/genera of organisms were isolated from the eyes of healthy

sheep including Moraxella ovis and Mycoplasma conjunctivae. This result highlights the

potential carrier status of healthy sheep that can act as a source of infection to naïve

animals.

This experiment has highlighted the limited value of using topical treatments for IOK in

sheep. The use of topical OTC products has been found to worsen the clinical signs of IOK

and giving no treatment is more effective that using either of these products. Injectable OTC

was shown to be the most effective treatment, and this result is similar to studies carried

out under more extensive situations in other countries. Although not the most effective

treatment tested, in-water OTC was more effective than giving no treatment. Given the

relative ease of administration of medication in water to large numbers of animals, this

treatment warrants further investigation. Establishing that injectable OTC is the most

effective enables this treatment to be used as a useful standard with which to compare any

treatments used in subsequent experiments.

78

6 Determination of the efficacy of in-water medication in the

treatment of ovine infectious keratoconjunctivitis

6.1 Introduction

The experimental work of the pilot study described in Chapter 5 highlighted the potential

for the use of in-water OTC as a treatment for IOK. Although this treatment wasn’t the most

effective of those tested, its ease of use in a large feedlot scenario suggested that it

warranted further investigation. It is likely that high numbers of sheep will require

treatment for IOK in pre-export feedlots. Logistically, injecting thousands of sheep requires

a high number of work hours and could divert labour away from other jobs. High standards

of hygiene and good injection technique, which can be difficult to maintain when treating

large numbers of animals, are essential in order to minimize the risk of carcass damage

resulting from intra-muscular injections.

As described in Chapter 5, a dose of 400 mg Oxytetracycline powder was administered per

head in the water as per the manufacturer’s recommendations. This rate of administration

resulted in a 50kg sheep drinking 4 L of water per day consuming a dose of 8 mg/kg OTC.

There is no evidence in the literature of successfully treating ocular infections in sheep with

oral medication. In cattle, IBK has been treated with orally administered OTC at a dose of 22

mg/kg bodyweight for five days (Davidson, 2009). Research undertaken to establish an

effective oral dose of tetracycline in calves demonstrated that a dose of 25 mg/kg was

effective in achieving plasma concentrations of OTC greater than 1µg/mL (Luthman et al.,

1989). The effect of adding OTC to water on palatability and intakes in ruminants hasn’t

been quantified. Luthman (1989) reported that the addition of OTC at doses of 400 mg/L or

79

800 mg/L water had no negative effect on water intake in pigs, however ruminants may

respond differently.

Given that the clinical improvement seen in the oral OTC group in the pilot study was

greater than in those receiving no treatment, it is hypothesised that adequate plasma

concentrations of OTC are achieved to result in a clinical effect when the OTC is given as a

solution. Tetracyclines are known to be absorbed following oral administration in many

species (Pijpers et al., 1991; Levison and Levison, 2009). However, there is a paucity of

literature relating to the absorption of OTC following oral administration in sheep and its

use in the treatment of disease. The pharmacokinetics of doxycycline (Castro et al., 2009)

and enrofloxacin following oral administration (Bermingham, 2002) have been documented

in sheep. Existing literature on the oral administration of OTC in water to sheep is limited to

a study that considered the impact on rumen microbial population. (Munch-Petersen and

Armstrong, 1958). In that study no effect of oral OTC administration on the appetite of the

sheep was found, but this was not quantified and no comment was made on the effect, if

any, on water intake.

Given the subjective nature of assessment of feed and water intake in the pilot study and a

lack of published evidence of any impact of in-water OTC on feed and water intake, it is

important to undertake further work to quantify any change following OTC administration.

This forms the basis for the study outlined in this chapter.

80

6.2 Hypothesis

1. Addition of OTC in the drinking water such that a 50kg sheep drinking 4L of water will get

a 22 mg/kg dose for 5 days will have no adverse effect on feed and water intake.

2. Inclusion of OTC in the drinking water such that a 50kg sheep drinking 4L of water will get

a dose of 22 mg/kg for 5 days is an effective treatment for IOK.

3. OTC is absorbed into the blood stream at detectable levels following administration by

both intra-muscular and in-water routes.


Twenty seven Merino and Merino cross, mixed age and mixed sex sheep were selected from

a pre-export feedlot in the South West of Western Australia. All sheep selected had

naturally occurring clinical signs of IOK and were recent arrivals at the feedlot. None of the

sheep had undergone any antibiotic treatment for IOK or any other bacterial disease.

Selected sheep were in apparent good health aside from the presence of IOK.

At selection all sheep’s eyes were examined and graded. Only those with a grade 2 or grade

3 in both eyes were selected for the experiment. The grading scale was as used in Chapter 5:

Grade 2 being conjunctivitis and epiphora and Grade 3 additionally having corneal oedema.

All identification (ID) tags were recorded and the sheep were transported to Murdoch

University Animal House.

Sheep were housed in purpose built, individual raised pens with visual contact with other

sheep on three sides. Individual housing was chosen to allow close monitoring and to enable

individual measurements to be taken daily. The housing was a new environment for the

81

sheep so an acclimatisation period of two days was allowed before the start of the

experiment. All sheep were weighed on entering the animal house and again at the end of

the experiment. Throughout the experiment sheep were fed the standard export pellet

ration that is fed in the feedlot of origin. Feed was delivered in a trough and water was

provided in a bucket. Sheep were randomly assigned to one of three Treatment groups:

Group 1: control animals, which received no treatment. (Control)

Group 2: treated with OTC (Alamycin LA 300, Norbrook laboratories, Australia) given

by intra-muscular injection at a dose of 20 mg/kg on day 0 and day 4. (IM)

Group 3: treated with water soluble OTC (CCD OTC, CCD Animal Health, Australia)

administered in the drinking water for 5 days. Water concentration was calculated so

that a sheep that drank 10 % of its live-weight per day received a dose of 22 mg/kg

OTC.

Ocular swabs were taken from all sheep on days 0, 5 and 10. A sterile, cotton-tipped

wooden-shafted swab was placed into the fornix, the area between the eyeball and the

eyelid, and held there for 15 seconds during which time the swab was gently rotated. Swabs

were plated onto horse blood agar plates in a manner for achieving a single culture (Bonner

and Hargreaves, 2011). The swab tip was then placed in Mycoplasma broth. Samples were

submitted to the Animal Health Laboratory at the Department of Agriculture and Food WA,

South Perth. Here they were incubated at 37oC for 24 hours before the plates were read and

colonies identified.

Individual feed and water intakes were calculated daily by measuring feed and water

provided and quantity of residuals. Faecal scoring was performed daily to crudely assess any

82

impact on rumen function. Faecal scoring was done using a 1-5 scale with 1 being firm and 5

being diarrhoea (Greeff and Karlsson, 1997).

All eyes were graded on every second day during the experiment to assess the clinical eye

grade. The grading system used is described in the pilot study (Chapter 4).

To determine some of the pharmacological properties of OTC following administration by

the two different routes, IM and Oral, samples of blood and lacrimal fluid were taken from

all sheep in Groups 2 and 3. To collect lacrimal fluid an un-stained schirmer tear test strip

(Haag-Streit, UK) (STT) was placed in the fornix of the eye and held in place for 15 seconds.

Strips were placed individually in a plain 5 mL tube and then frozen at -80oC prior to

analysis. Lacrimal fluid samples were taken on days 0, 1, 3, 5, 7 and 10.

Blood samples were taken at the same time points as lacrimal fluid samples from all sheep

in Groups 2 and 3. A 3 mL sample was taken from the jugular vein using a 20 gauge, 1 inch

needle collecting into a sterile EDTA vacutainer tube. Following collection, samples were

centrifuged for 3 minutes at 4000 rpm and the plasma was transferred into a sterile cryovial

tube and stored at -80 oC until analysis.

6.3.1.1 Sample Analysis

Tear strips were extracted in methanol, while plasma samples were cleaned using solid

phase extraction (SPE) prior to analysis. Samples were analysed using a specially developed

liquid chromatography-tandem mass spectrometry method (LC-MS/MS). High-performance

liquid chromatography-mass spectrometry (HPLC-MS) is well suited to analysis of OTC

concentrations (Oka et al., 1997). This method was developed using OTC solution to

83

determine the appropriate LC conditions and MS/MS transitions. In total five transitions

were monitored to reduce the chance of matrix interference.

6.3.1.2 Statistical Analysis

Linear mixed models (LMM), which included treatment effects, covariates and appropriate

random effects, were used to analyse the data. Covariance structures were defined for

random terms as required and simplified where likelihood ratio tests indicated that this was

possible. Hierarchical tests (Type I sums of squares) and a 5% level of significance were used

to assess whether treatment and covariate effects were significant. When covariates were

fitted after treatment effects they explained within treatment variance; when they were

fitted before treatment effects, treatment effects were adjusted for covariance.

Predicted treatment means and standard errors (SE) were corrected to mean and covariate

values where appropriate.

6.4 Results

In Table 6.1 the structure of the models used for the analysis of water and feed intake, and

the clinical eye score, are summarised.

84

Linear models and P-values

Water Intake Feed Intake Pink eye score

Plasma OTC

Fixed term P-Value P-Value P-Value P-Value

Liveweight <0.001 <0.001 0.095 <0.001

Treatment 0.008 0.162 0.136 <0.001

OTC Dose

0.001 <0.001 0.062

Day <0.001 <0.001 0.006 0.077

Day.Treatment 0.002 <0.001 <0.001 0.275

Table 6.1 Linear models and P-values for the analysis of water intake, feed intake, clinical eye score and plasma OTC concentration after treatments.

The liveweight range was 30.5 kg to 57.5 kg (mean 44.15 kg) at the beginning of the

experiment and 33.5 kg to 67.5 kg (mean 46.25 kg) at the end of the experiment.

Feed intake was significantly affected by liveweight (p value <0.001). Data were corrected

for liveweight and a significant difference in feed intake was seen between the treatments

on different days (Table 6.1 and Figure 6.1).

85

Figure 6.1 Mean feed intake (kg/head/day) over time. Shaded area represents the duration of Oral treatment. Error bars represent standard errors

86

Feed intake – kg/head/day

Day Control IM Oral

0 1.442 1.37 1.429

1 1.458a 1.285 1.185b

2 1.361a 1.241a 0.579b

3 1.572a 1.882a 0.903b

4 1.295a 1.25a 0.746b

5 1.896a 1.659a 1.268b

6 1.632a 1.414 1.033b

7 1.812a 1.697 1.387b

8 1.48 1.33 1.081

9 1.815 1.695 1.627

10 1.728a 1.49 1.12b

Table 6.2 Mean feed intake (kg/head/day) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05).

Feed intake decreased in the Oral treatment group during the treatment period but

returned to maintenance levels following cessation of treatment.

Feed intake was significantly lower in the Oral treatment compared to the Control

treatment on days 1, 2, 3, 4, 5, 6, 7 and 10 (Table 6.2). Feed intake was significantly lower in

the Oral treatment compared to the IM treatment on days 2, 3, 4 and 5 (Table 6.2). There

was no significant difference in feed intakes between the Control and the IM treatments on

any of the days (Table 6.2).

87

Water intake was significantly affected by liveweight (P < 0.001). There was a significant

difference in water intake between the treatments and on different days (p =0.002) (Figure

6.2).

Figure 6.2 Mean water intake (L/head/day) over time. Shaded area represents the duration of oral treatment. Error bars represent standard errors.

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9 10

Me

an w

ate

r in

take

- L

/he

ad/d

ay

Day

Control

IM

Oral

88

Water intake – L/head/day

Day Control IM Oral

0

1 3.273 3.623 3.981

2 2.586a 4.289b 2.203a

3 3.336a 3.845a 2.092b

4 2.577 3.158a 1.481b

5 4.053a 4.567a 3.537b

6 3.836 3.475 2.814

7 4.046 3.678 3.204

8 3.461a 3.123 2.37b

9 4.324 4.126 3.645

10 3.308 2.845 2.703

Table 6.3 Mean water intake (L/head/day) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05).

Water intake in the Oral treatment group was significantly lower than in the Control or IM

treatment groups during the in-water treatment period. Following cessation of in-water

treatment, water intake in the Oral treatment group returned to pre-treatment levels

following cessation of treatment. Day 8 was the exception, where intake of the Oral

treatment group reduced for one day before returning to similar levels to the other groups

for the remainder of the monitoring period.

Sheep in the Oral treatment group drank significantly less than sheep in the IM treatment

on days 2, 3, 4 and 5 and drank significantly more than the Control treatment on day 2

(Table 6.3). The Oral treatment group drank significantly less water than the Control

treatment group on days 3, 5 and 8 (Table 6.3).

89

Clinical eye grades changed over the course of the experiment (Figure 6.3). A significant

difference was seen between the treatment groups on different days throughout the

experiment (p<0.001) (Table 6.4).

Figure 6.3 Mean clinical eye grade (average both eyes) over time. Shaded area represents the duration of Oral treatment. IM OTC treatment given on day 0 and 4. Error bars represent standard errors.

Clinical eye grades did not differ significantly at the end compared to the start for both the

Control and the Oral treatment groups. After initially worsening, eye grades of the Oral

treatment group made a marked improvement on day 5. This improvement, however, was

not sustained and clinical eye grades in this group returned to levels similar to those at the

start of treatment (Figure 6.3).

0

0.5

1

1.5

2

2.5

3

0 1 2 3 4 5 6 7 8 9 10

Clin

ical

eye

gra

de

s

Day

Control

IM

Oral

90

Clinical Eye Grades

Day Control IM Oral

0 2.303 2.238 2.104

3 1.865a 1.849a 2.382b

5 1.99a 1.46 1.271b

7 1.803 1.626a 2.106b

10 1.928 1.46a 2.104b

Table 6.4 Mean clinical eye grade (average of both eyes) for treatments on different days. Within Day, means with different letters differ significantly (P<0.05).

Animals in the IM treatment group received a dose of 20 mg/kg of OTC on days 0 and 4.

Those in the Oral treatment group received a variable dose over time as a result of variable

water intake (Table 6.5).

Day 1 Day 2 Day 3 Day 4

Mean mg OTC consumed

662.4 372.0 353.9 254.1

Average dose mg/kg

14.7 8.2 7.8 5.6

Table 6.5:OTC dose received by the Oral treatment group.

91

Plasma OTC concentrations measured on day 1 and day 5 of treatment differed between the

Oral and IM treatment groups. Serial measurements taken between these two points over

the treatment period were not significantly different between the groups (Figure 6.4).

6.4 Mean plasma OTC concentrations at different time points during the experiment.

For mean plasma OTC concentrations over the treatment period, the IM treatment group

was significantly higher than the Oral treatment group (Figure 6.5).

Figure 6.5 Mean plasma OTC concentration during treatment period for the Oral and the IM treatment groups

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 1 3 5 7 10

Oxy

tetr

acyc

line

Pla

sma

con

cen

trat

ion

(µ

g/m

L)

Days

oral

IM

0

0.2

0.4

0.6

0.8

1

1.2

IM Oral

Me

an p

lasm

a O

TC c

on

cen

trat

ion

(µ

g/m

L)

92

Analysis of the lacrimal fluid proved to be unsuccesful. Following extraction of the samples

from the STTs they were run through the liquid chromatography-tandem mass

spectrometer. Unfortunately the results proved difficult to interpret. All samples, including

those in the control group, showed a spike reaction at the site where an OTC spike was

anticipated.

6.5 Discussion

Feed intake decreased during the in-water treatment period in the Oral treatment group.

This coincided with a decrease in water intake in the same animals during this time. It is

hypothesised that the decrease in feed intake was a consequence of the reduced water

intake. All animals were fed a dry, pellet-based ration. As the water intake decreased in

these animals the hydration state would also decrease resulting in a decreased palatability

of a dry feed and a decreased appetite. Despite the decreased water intake, no animals

appeared clinically dehydrated. There were no signs of skin tenting or sunken eyes on clincal

examination. Water intake can be linked to dry matter intake in sheep. Within two days of

reduced water intake, feed intake will be noticeably reduced (Forbes, 2007b). Forbes

(2007b) attributed this to the requirement of water to enable passage of feed through the

digestive tract. Frequency of drinking has also been shown to decrease feed intake (Squires

and Wilson, 1971) whereby decreasing the number of drinks per day will also decrease feed

intake. Animals will typically drink twice the weight of dry matter intake (Forbes, 2007a).

Despite alterations in feed and water intake this is a pattern that can be seen in the results

of this experiment.

93

It is also hypothesised that the decrease in water intake during the in-water treatment

period in this oral treatment was an effect of the palatability of the OTC dissolved in water.

Both feed and water intakes were decreased following the commencement of the Oral

treatment. Palatability effects are typically seen immediately after a change occurs (Arnold

et al., 1980). This is possibly associated with an unpleasant taste in the water caused by the

addition of OTC powder, and following cessation of treatment, feed and water intakes did

increase. Although post-treatment measurements fluctuated over the monitoring period,

the trend was towards pre-treatment levels. The return to normal intake levels following

cessation of treatment coupled with the dramatic drop in intake on day one of treatment is

sufficient to conclude that OTC powder dissolved in the water was unpalatable to the

sheep.

During the treatment period intakes appeared to remain constant. It is not known if

extending the treatment period would allow the sheep to adapt to the new taste, possibly

leading to improved intake over time.

Although sheep are not expected to gain weight during the live export process, it is

important that they maintain their weight. A number of studies have looked at risk factors

associated with deaths within the export process (Norris et al., 1989a; Norris et al., 1989b;

Richards et al., 1989; Richards et al., 1991). Inanition and salmonellosis have been

highlighted as major causes of death during the export process (Richards et al., 1989).

Richards et al. (1989) highlighted the importance of maintaining regular feed intake to

minimise the risk of either of the major causes of death. Although feed intake decreased

during the Oral treatment it did not drop to zero intake. Despite maintaining some intake it

94

would be preferable to avoid a decrease in feed intake, and therefore methods to reduce

the impact of the Oral treatment on feed intake should be explored.

An improvement in the clinical eye score was seen only in the IM treatment group. In

contrast to Chapman et al. (2010), who found a clinical improvement in the oral treatment

group, there was no overall improvement despite some initial improvement. It is suggested

that the unpalatable taste of the OTC dissolved in water at a concentration that would

deliver a dose of 22mg/kg if an animal drank 10% of its liveweight, caused the animals to

drink too little water to deliver an effective dose of OTC. Intake of such a low dose of OTC is

unlikely to allow concentrations in the plasma and ocular tissues to be sufficiently high to

exceed the MIC of the pathogenic organisms. Further work needs to be done to determine

the concentration of OTC available following oral administration.The benefits of giving IM

medication over medication in the water is that a known concentration is given. In-water

doses are very variable depending on intake. Intake itself is also dependant on a number of

factors: climatic, availability of clean water, and adequate trough space. However, if it were

possible to optimise the in-water concentration such that animals received an effective

dose, this treatment would be suitable for a feedlot and would be less labour intensive than

giving multiple IM injections. Additionally, blanket treatment of a group with a high number

of clincal cases would allow treatment of those animals with early clincal disease, whose

clinical signs might not have been seen by feedlot staff.

Changes in clinical appearance between the Oral and IM treatment groups can be attributed

to the difference in the OTC dose. In the Oral treatment group the highest average dose was

14 mg/kg OTC on a single day. It is difficult to compare this directly with the intramuscular

dose of 20 mg/kg OTC (Alamycin LA 300, Norbrook laboratories, Australia) which is a long

95

acting preparation. Shorter acting preparations, those designed to last 24 hours, are given

daily at a dose of 8 mg/kg (Engemycin Intervet/Schering-Plough Animal Health Australia).

Comparing daily mg/kg doses alone, sheep in the Oral treatment group had a potentially

effective oral dose on days 1 and 2 only, and the remainder of the course was therefore at

sub-therapeutic levels. Comparing given doses alone does not take into account the

concentration of drug available. This highlights the requirement for further work to establish

the bioavailability of OTC through various methods of administration.

This experiment indicates that OTC can be detected in plasma following both IM and oral

routes of administration. Although plasma concentrations are higher following IM injection,

the experiment shows that this difference is not dose related as dose was not a significant

covariate in the model. Therefore, the hypothesis is that the difference was due to a

different bioavailability of the drug when given by different routes of administration.

Failure of lacrimal fluid analysis has been attributed to a substance that shows a similar

structure to OTC through the liquid chromatography tandom mass-spectrometer, a

phenomenon known as matrix effects (Trufelli et al., 2011). Although HPLC-MS is considered

to be highly selective it is still possible for endogenous impurities to exist which intereferes

with the reliabilty of the results (Trufelli et al., 2011). The presence of these matrix effects

makes interpretation of the data impossible. To confirm that the substance could not be

attributed to the STT strips and not to the lacrimal fluid collected plain STT strips were

prepared as described and the fluid generated run through the liquid chromatography

tandem mass-spectrometer. The plain STT strip showed no reponse at the OTC site. It was

considered that STT strips may not be suitable for such analysis, however they have been

96

used to harvest tears for analysis and been successfully used in elephant seals for the

analysis of doxycycline (Freeman et al., 2013).

Ocular tissue is protected by a tear film which has many functions including the provision of

white blood cells to the cornea and conjunctiva and protection via specific and non-specific

antibacterial substances (Davidson and Kuonen, 2004). These substances can act directly on

the bacteria themselves or can act to modulate the host’s immune system. It is possible that

such substances could themselves provide matrix effects.

Alternative methods of sample collection were considered including cathetirisation of the

naso-lacrimal gland. The use of this technique in studies in calves has shown that OTC

concentrates in lacrimal fluid and ocular tissue (George, 1985). Studies in sheep where a

bioassay technique was used have also shown that OTC will concentrate in the ocular tissues

(Nouws and Konig, 1983), albeit at low concentrations. Both these studies were conducted

using intra-muscular injection only. Given the higher bioavailability of OTC following intra-

muscular injection compared to oral dosing, it is possible that drug concentrations in the

lacrimal fluid are below detectable levels following oral administration of the drug. Further

exploration of lacrimal fluid analysis was ruled out. Nouws and Konig (1983) discuss the

passive diffusion of the non-protein bound fraction of drugs into lacrimal fluid. Given the

low proportion of protein binding of OTC (Ziv and Sulman, 1972) it is feasible for the drug to

concentrate in lacrimal fluid. Although concentration of OTC in the lacrimal fluid has not

been confirmed in this study, previous written reports of this and the clincal improvement

seen in the groups treated with OTC compared to the control group in this experiment

indicate that OTC must reach sufficient concentrations in the ocular tissue/fluid to result in a

clinical response. Following systemic administration of OTC in cattle, OTC has been shown to

97

concentrate in the corneal tissues (Davidson and Pickett, 2009). If this is true in sheep this

may go some way to explaining any clinical effect.

6.6 Conclusion

This experiment showed that OTC can be detected in plasma following both IM injection

and oral routes of administration. Although significant clinical response to treatment in the

Oral treatment group was not seen, this lack of response can potentially be attributed to the

low intakes seen following commencment of medication in the water. This observation,

combined with the promising findings in the pilot study (Chapter 5), led the researchers to

believe that it may be possible to alter the dose such that optimal intakes are achieved. The

goal would be to alter the concentration of OTC in the water in such a way and to such an

extent that water intake was not adversely affected, resulting in greater concentrations of

OTC available for absorption from the GI tract. This aim formed the basis for the experiment

summarised in the following chapter.

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7 Maximising intake of in-water oxytetracycline

7.1 Introduction

The previous experiment highlighted the negative impact on water and feed intake

associated with a high concentration of OTC in the drinking water. In order to maximise

water intake and mitigate some of the palatability issues it was hypothesised that reducing

the dose to the point where intake wasn’t affected might enable therapeutic concentrations

of OTC to be achieved in plasma. It was also hypothesised that mitigating the adverse taste

by adding dextrose to the water might be effective in reducing the negative impact on water

intake.

It is generally considered that most domestic animals are attracted to sweet tastes

(Goatcher and Church, 1970b). Sugars are often added to medicated feedstuffs and water to

increase palatability (Pers. comm. K. Nairn). Fowl demonstrate a preference for sucrose-

flavoured water (Kare et al., 1957). However, studies have found that sheep can remain

indifferent to sweet flavours (Goatcher and Church, 1970b). Goatcher (1970) found that

sheep showed minimal preference for any sugar added to water, but once concentrations

exceeded 20% sheep began to show a dislike for additional sugars in their water.

Concentrations of glucose above 5% in water have been shown to decrease the sheep’s

intake of water; concentrations lower than 5% have a minimal impact on water intake

(Arnold et al., 1980). In contrast, it is thought that increased dry matter intake of pasture

species with high water soluble carbohydrate (sucrose) concentrations result because of

increased palatability (O'Donovan et al., 2011).

99

Although there is evidence in other species of a predilection for sweet tastes, the existing

evidence surrounding taste preferences in sheep is inconclusive. The trend is such that

sheep are not averse to sweet tastes at low concentrations in water. Increasing the

concentration of OTC in-water between the experiments in Chapters 5 and 6 resulted in a

reduction in water intakes and consequently in OTC intakes. If the concentration of OTC in

the water were reduced, but this reduction were more than off-set by the consequent

increase in water intake, it could mean that higher concentrations of OTC were available for

absorption.

7.2 Hypotheses

1. A lower concentration of OTC in the water will increase palatability and lead to a

subsequent increase in water intake. The result would be an effective increase in the dose

of OTC.

2. The addition of dextrose to water medicated with OTC will not improve the palatability of

water medicated with OTC.


Thirty adult Merino cross ewes were randomly selected from the Murdoch University

teaching flock. Animals were housed in a purpose built animal house in individual raised

pens with sheep contact on at least one side.

As in previous experiments, sheep were randomly assigned to five treatments (n=6, see

Table 7.1). All sheep were weighed on entry to the animal house and again on leaving the

animal house. A two-day acclimatisation period was allowed prior to commencing

100

measurements to allow the sheep to adapt to the pellet ration and the individual pens. All

sheep were fed a maintenance pellet ration during this period from a trough and water was

given in a bucket.

The concentrations of OTC in the water were calculated such that a 50kg sheep drinking

four litres per day would consume the treatment dose. Treatments started after two days of

baseline measurements and lasted for five days. Following this there was a further five days

of monitoring. Measurements of feed and water intake were taken daily.

Treatment Dose rate and water additive

1 2% dextrose (Control)

2 2% dextrose and 11 mg/kg OTC

3 2% dextrose and 16.5 mg/kg OTC

4 11mg/kg OTC

5 16.5 mg/kg OTC

Table 7.1: Treatments in experiment to optimise dose and palatability


For each animal all pre-treatment feed and water intakes were averaged over the

acclimation period and change from the pre-treatment average was calculated for each

post-treatment date. A linear mixed model was fitted to the calculated changes on all post-

treatment dates. The model included fixed effects for pre-treatment liveweight, treatment,

post-treatment date and treatment by date interaction; and random effects for animal and

the animal by date interaction. An autoregressive model allowed for correlations between

measurements made on the same animal on different dates and different residual variance

on each date. 5% LSD’s were calculated to compare treatment means to zero, i.e. whether

treatment caused a change from the pre-treatment mean, and to compare treatments.

101

7.4 Results

The addition of dextrose made no significant difference to either feed or water intake, with

or without the addition of OTC (feed intake P = 0.099, water intake P = 0.154, see Table 7.2).

Adding OTC to water at a dose rate of 16 mg/kg and 11mg/kg caused both feed and water

intake to fall significantly after treatment began (feed intake P = 0.003, water intake P =

0.007, see Table 7.2 and Figure 7.1). Water intake fell more at the 16 mg/kg dose rate

compared to the 11mg/kg dose rate (P = 0.051, see Table 7.2 and Figure 7.2).

Over time, particularly after the OTC treatment ended, feed and water intakes recovered to

near the levels of the no OTC treatment. Recovery of intakes commenced from day 2 of

treatment.

Total OTC quantities consumed per day were no different for the 11mg/kg treatment

compared to the 16 mg/kg dose rates (see Figure 7.3).

Term Change in feed intake

Change in water intake

Weight 0.846 0.323

OTC (with or without OTC) 0.003 0.007

OTC.dextrose (OTC with or without dextrose)

0.099 0.154

OTC dose (11 or 16mg/kg) 0.951 0.051

OTC.dextrose.dose (11 or 16mg/kg dextrose)

0.626 0.437

Time <0.001 <0.001

time.OTC 0.004 0.005

Table 7.2: Significance levels (P-Values) for terms in model with respect to change in feed or change in water intake.

102

Figure 7.1 Change in feed intake (g/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors

Figure 7.2 Change in water intake (ml/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors.

103

Figure 7.3 OTC intakes (based on Experiment 1) for different doses of OTC during treatment period.

mg

OTC

co

nsu

me

d

104

7.5 Discussion

Following results from the experiment described in Chapter 6, the current experiment was

designed to try and mitigate the assumed negative palatability effects of including OTC in

water. This pilot trial was designed using knowledge from previous experiments where feed

and water intakes were reduced following the addition of OTC to plain water. As such no

control group was included of just plain water. This limited our ability to draw conlusions on

the effect of adding dextrose to plain water alone. Dextrose was chosen as an additive

because anecdotal evidence shows that it has been used in the poultry industry as a

supplement in medicated water to improve water intake. Additions of 1 and 2% glucose to

drinking water for dairy cows has shown to have no negative impact on intakes (Osborne et

al., 2002). However, sheep appear to be less attracted to sweet tastes compared to other

domestic species (Goatcher and Church, 1970b). Despite this there are no reports in the

literature of a negative impact on water or feed intake by sheep associated with the

inclusion of low concentrations of sugars. Increasing concentrations of sucrose in water had

no impact on intake for sheep until an inclusion rate of 20% was reached which resulted in a

slight decrease in intake (Goatcher and Church, 1970a). Although a preference for sweet

tastes is typical of some animals like cattle and goats, the same is not consistently true with

sheep where significant variation exists between animals (Goatcher and Church, 1970b).

Dextrose is readily soluble in water and has a sweet taste. Given the lack of strong evidence

that this taste would be attractive to sheep it was unknown whether adding dextrose to the

water would counteract any negative effects of OTC on palatability. The previous

experiment looked at the effects of using 22mg/kg OTC dissolved in water to treat IOK.

105

Lower OTC doses were selected in this experiment with the hypothesis that if sheep

tolerated the taste they would consume more water and increase the therapeutic dose.

The lack of effect of adding dextrose to water in order to mitigate taste mirrors the results

of the previous experiment with regard to decreased intakes. This correlates well with work

done in pigs where sweetening the water was also found to be ineffective in increasing

water intakes (Maenz et al., 1993). The addition of tetracycline to the drinking water of pigs,

however, had no impact on water intake (Luthman et al., 1989) which may further highlight

the peculiar palate of the sheep compared to other domestic animals.

The addition of in-water OTC reduced feed and water intake to different levels when added

at different concentrations. Total consumption of OTC data from the previous experiment

was used to extrapolate daily consumption of OTC in this experiment. This showed that

there was no difference in OTC intakes at differing dose rates of OTC (Figure 7.3). Despite

lowering the concentration of OTC in the water, intake was still reduced. It would appear

that the higher the concentration in water, the less the sheep will drink and consequently

the lower the amount of OTC consumed.

Although growth is not required in sheep within the live export chain, it is important that

feed and water intakes are maintained. Any factors which result in a reduction of feed or

water intake should be minimized to reduce the risk of deaths associated with inanition and

salmonellosis (Richards et al., 1989). Given that the administration of in-water OTC is

associated with decreased feed and water intakes it is prudent to address the significance of

this before contemplating this as a suitable treatment for IOK.

106

7.6 Conclusions

The results of the experiment have disproved the hypothesis that lowering the

concentration of OTC in the water will result in increased water intakes. Adding dextrose in

an attempt to improve the palatability of the water had no effect in mitigating the negative

effect of OTC on daily water consumption.

107

8 Determination of oral bioavailability of oxytetracycline in sheep.

8.1 Introduction

Although it has been demonstrated that OTC is absorbed at detectable concentrations

following oral administration, it is important to determine how bioavailable OTC is following

oral administration in sheep. Once this has been established the dose of OTC could be

modified with a view to maximizing water intake without decreasing available

concentrations of OTC in the blood.

Only when a drug is administered intravenously can it be assumed that all of the drug will be

active; administration by any other route is subject to the bioavailability of that drug

(Baggot, 2004). The bioavailability of any drug can be defined as the concentration at which

a drug enters the circulation without having undergone any change (Baggot and Giguere,

2013). It is not the dose of the drug given that determines the therapeutic effect, rather the

concentration of the drug in the bloodstream following absorption (Neuschl, 2000), and

ultimately the concentration at the target site. It is important when a drug is used

therapeutically to treat or prevent disease that it is absorbed sufficiently to achieve

adequate concentrations within the body fluids and tissues (Luthman et al., 1989).

The addition of OTC to water results in a solution that is typically readily absorbed from the

gastro-intestinal tract. While the rumen is considered to have good absorptive ability,

dilution of drug in the large volume of rumen fluid can slow absorption (Droumev et al.,

1992). In addition to the large volumes of fluid the presence of microorganisms in the

rumen and a pH of 5.5-6.6 can result in denaturing of some antibiotics (Baggot and Giguere,

2013). Rumen microflora can inactivate drugs through metabolic or chemical reactions

(Toutain et al., 2010). An example of this is Chloramphenicol which is known to be

108

deactivated by reduction of the nitro group in the rumen of goats by rumen microbes

(Theodorides et al., 1968).

Absorption across the mucosal wall relies on the drug being soluble in lipids. Oxytetracycline

is lipid soluble but newer generation tetracyclines, like doxycycline, are more lipid soluble

and therefore more readily absorbed (Castro et al., 2009). Doxycycline is 5-10 times more

lipid soluble than OTC (Abd El-Aty et al., 2004). Absorption across cell membranes is optimal

between pH 4-7 (del Castillo, 2013) which suits the environment within the rumen.

Tetracyclines have extended half-lives of 6-10 hours because of entero-hepatic circulation

whereby drug excreted in the bile is reabsorbed in the intestine (del Castillo, 2013). If

adequate drug concentrations are maintained, entero-hepatic circulation will prolong the

pharmacological effects of the drug (Gibson and Skett, 2001b).

In order to achieve as high a circulating concentration of OTC as possible, it is important to

understand the oral bioavailability of the drug. If a concentration of OTC can be delivered

through the drinking water or through medicated feed such that an effective plasma

concentration is achieved then the use of oral medications in the feedlots would be a viable

option. An effective drug concentration is one that exceeds the Minimum Inhibitory

Concentration (MIC) of the target organism where the MIC is the minimum concentration of

a drug that will inhibit growth of an organism in vitro (Boothe, 2001b).

This experiment was modelled on a similar study investigating the pharmacokinetics of

enrofloxacin given to sheep orally and by IV injection (Bermingham, 2002) and was designed

to determine the bioavailability of OTC following oral administration in both water and feed.

109


Six adult Merino cross ewes were selected at random for the experiment. A three-way cross

over study design was used with a 10-day rest period between study periods to ensure

adequate clearance of drug. The sheep were randomly assigned into three groups of two

animals per group. They remained in that assigned group for all three study periods.

Sheep were housed for 36 hours prior to the start of the experiment to allow them to

become accustomed to the housing and feed. All animals were offered a diet of oaten chaff

and commercial maintenance pellets.

The three treatments were:

IV: 8mg/kg OTC (Engemycin, MSD Animal Health, Bendigo, VIC, Australia) given by

intravenous injection once

IW: 22mg/kg OTC (CCD OTC, CCD Animal Health, Geelong VIC, Australia) given orally in-

water once.

IF: 20 mg/kg OTC (Terramycin 200, Phibro Animal Health, Girraween, NSW, Australia) given

orally in-feed once.

The two sheep in each group were rotated for each experiment period meaning that all

sheep were used in each treatment group once. All animals were weighed and housed in

individual, raised pens with sheep contact on at least one side. Fleece was clipped over the

neck to facilitate catheter placement. Indwelling catheters (16 gauge, 2 ¾”) were placed in

the jugular vein of each sheep and an extension set was attached to facilitate easier

sampling. Catheters were sutured in place using nylon sutures and a net bandage was used

to provide additional protection over the neck area.

110

The two sheep in the IF treatment had feed withheld for 12 hours prior to commencement

of the experiment to improve feed intake over a short period of time. Sheep in the IW

treatment were given their dose by a drench using a Magrath feeder tube (Springer Magrath

Co. MN, USA). Using a Magrath feeder ensured the medicated fluid was placed in the

oesophagus to gaurantee that the required dose was given.

Blood samples were taken via the catheter. On each occasion the first 2 mL of blood was

discarded as this removed any blood or flush fluid that remained in the extension set. A

blood sample was taken from all the sheep five minutes prior to administration of any

treatment. This sample provided a base-line value. Serial blood samples were then taken at

1, 2, 3, 5, 10, 15, 20, 30, 40, 60 minutes and 2, 4, 6, 8, 12 and 24 hours following treatment.

The catheter and extension set were flushed using heparinised saline following each sample.

Blood was transferred to a plain vacutainer and then centrifuged at 2000rpm for 15

minutes. Serum was decanted off and stored in cryovials at -80 C until analysis.

Sample analysis

All serum samples were analysed using High Performance liquid Chromatography with

Tandem Mass Spectrometry as described in Chapter 6.


Plasma OTC concentrations over a 24 hour period for animals in each treatment were

examined graphically. Average OTC values for each treatment at each time of measurement

were estimated using split plot analysis of variance and these values were used to calculate

the total plasma OTC over a 24-hour period (the area under the concentration vs. time

curve). OTC bioavailability for IF and IW treatments were calculated by comparing their total

111

plasma OTC over a 24 hour period to the same value for the IV treatment, Equation 1

describes this calculation.

Equation 1: Calculation of bioavailability

112

8.3 Results

Oxytetracycline was detected in the plasma of sheep in all three treatment groups. Plasma

concentrations peaked at a mean of 415.57 (+/- 51.3) µg/mL at 1 minute post-injection

following IV administration. Following this peak, plasma concentrations steadily declined

(Figure 8.1). In both the IF and IW treatments, plasma concentrations were not detected

within the first 30 minutes of monitoring. In the IF group OTC was first detected in the

plasma at 120 minutes, followed by peak concentration of 4.12 (+/- 0.03) µg/mL at 6 hours

(Figure 8.2). In the IW group OTC was first detected in plasma at 30 minutes post-treatment.

Peak plasma concentrations of 5.34 (+/-0.04) µg/mL OTC were seen in the IW group at 12

hours (Figure 8.3).

Figure 8.1 Plasma OTC concentrations over time following IV treatment, error bars show standard error

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

-5 1 2 3 5 10 15 20 30 40 60 120 240 360 480 720 1440

Pla

sma

OTC

co

nce

ntr

atio

ns

(µg/

mL)

Time (mins)

113

Figure 8.2 Plasma OTC concentrations over time following medication with in-feed (IF) OTC with error bars showing standard error.

Figure 8.3 Plasma OTC concentrations over time following medication with in-water (IW) OTC. Error bars show standard error.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

-5 1 2 3 5 10 15 20 30 40 60 120 240 360 480 720 1440

Pla

sma

OTC

co

nce

ntr

atio

n (

µg/

mL)

Time (mins)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

-5 1 2 3 5 10 15 20 30 40 60 120 240 360 480 720 1440

Pla

sma

OTC

co

nce

ntr

atio

n (

µg/

mL)

Time (mins)

114

The half-life of OTC for the IV group was 3.96 hours, IF group 279.9 hours and IW group 51.5

hours.

The oral bioavailability of OTC in the IF treatment was 18% and the bioavailability of OTC in

the IW treatment 27%.

8.4 Discussion

Oral bioavailability is defined as the fraction of the drug that reaches the systemic system

unchanged following oral administration (Rang et al., 2008). This measurement is

considered to be of greater clinical importance than the rate of drug absorption (Baggot and

Giguere, 2013). Oxytetracycline was shown to be absorbed and detectable in plasma

following administration IV and both IF and IW. IW medication was more bioavailable than

IF (27% vs 18%).

Boluses of drug given IV are instantly available in the body and require no absorptive

process. This results in the rapid achievement of maximum plasma concentrations. Al-

Nazawi (2003) found that a peak plasma concentration of OTC of 850 µg/mL was reached at

two minutes post-injection of 5 mg/kg OTC. Similar results were found in this experiment

where a dose of 8 mg/kg was given IV. A dose of 20 mg/kg tetracycline given IV has been

shown to achieve a peak plasma concentration of 200 µg/mL (Rajaian, 2007). Despite being

a comparison between OTC and tetracycline, there is a considerable difference in the peak

plasma concentrations reported by Rajaian (2007) and Al-Nazawi (2003). Rajaian (2007)

analysed samples by spectrofluorimetric analysis whereas Al-Nazawi (2003) analysed

samples by fluorescence. Spectrofluorimetric analysis is considered to be less sensitive in

detection of OTC or tetracycline than fluorescence (Hayes Jr and DuBuy, 1964). This

115

difference in sensitivity would, most likely, explain the difference in peak plasma

concentrations.

A number of factors affect the amount of active drug in the plasma following oral

administration. In humans approximately 60-80% of OTC is absorbed following oral

administration (Chambers, 2006; Rang et al., 2007a). In humans OTC is absorbed primarily

from the stomach and the proximal small intestine (Chambers, 2006). Any alteration in gut

motility will impair absorption of the drug (Rang et al., 2007a). Oxytetracycline is known to

be chelated by a number of ions that can be found in the feed. Calcium ions are the most

common chelating agents (Rang et al., 2007a), but magnesium, aluminium, iron or zinc can

also bind OTC (Chambers, 2006). Once chelated, OTC is no longer available for immediate

absorption and therefore the rate of absorption will be prolonged. It is recommended that

tetracyclines are given orally to calves or lambs in milk. Given the high levels of calcium in

milk it is not surprising that this leads to a poorer absorption than if they were given in

water (Neuschl, 2000). Neuschl (2000) conducted a study comparing chlortetracycline (CTC)

administered in milk and in water. The oral bioavailability of CTC was 33% lower in those

sheep dosed with milk than with water, although the author did not state the exact

bioavailability. In the same study, the maximum concentrations of CTC in plasma were lower

in medicated milk-fed animals compared to those that received medicated water, 1.72

µg/mL and 2.2 µg/mL respectively. The half-life of CTC was also longer in the medicated milk

group compared to the medicated water group, 8.8 hours and 8.6 hours respectively. This

longer half-life implies that there is a delay in absorption from the gut (Neuschl, 2000)

caused by the chelation of CTC with milk calcium.

116

Chlortetracycline is considered to be the most poorly and inconsistently absorbed of the

tetracycline group (Neuschl, 2000). By comparing the maximum plasma concentrations

obtained in this experiment, it is concluded that a larger concentration of OTC is absorbed

compared to CTC. Additionally the concentrations of OTC in plasma after IF and IW

administration remained above the MIC for sensitive organisms, reported as 0.5 µg/mL

(Neuschl, 2000). Oral dosing with tetracycline in milk can achieve effective plasma

concentrations (Luthman et al., 1989). Luthman et al. (1989) found that the maximum

concentration was reached in plasma four hours after administration and that levels were

maintained above 1µg/mL with a dose of 25 mg/kg.

Drugs are absorbed from the gastro-intestinal tract through the mucosa. Following

absorption they are taken via the circulation to the liver and then distributed throughout

the body. Even after absorption there is the risk that the drug can be inactivated by

enzymatic activity in the wall of the gastrointestinal mucosa or liver (Rowland and Tozer,

1995a; Rang et al., 2007a) making less active drug available. In mono-gastrics, splanchnic

blood flow increases after eating, and this improves circulation between the gut and the

liver, which in turn can increase drug availability (Rang et al., 2007a). In order to achieve

maximal absorption, it is best for the drug to be in an area with a large area of permeable

mucosal surface (Rowland and Tozer, 1995a). This is found in the small intestine. To achieve

maximal absorption it is desirable for the drug to reach this area as quickly as possible. In

humans this occurs within 2-4 hours. If maximal absorption does not occur within this time

then there is a greater chance of drug de-activation and therefore less active drug available

for absorption (Rowland and Tozer, 1995a).

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In ruminants, any oral dose must pass through the reticulum, rumen, omasum and finally

abomasum before reaching the small intestine. Transit times in the ruminant are far greater

than in the mono-gastric owing to the retention of material in the rumen. For feeds to pass

through the entire gastro-intestinal tract in a ruminant the time taken can vary from 30-50

hours for highly digestible feeds, such as concentrates, and up to 50-80 hours for more

fibrous feeds (Fuller, 2004). Fluid, however, will pass through quicker than any particulate

matter. The reported liquid half-life in a ruminant varies in the literature ranging from 15-20

hours (Herdt, 2002; Toutain et al., 2010). Water will only leave the rumen as it is replaced.

Replacement fluid comes only via the oesophagus from either saliva, drinking water or

succulent feeds (Herdt, 2002). As concentrate feeds are not succulent and do little to

stimulate the rumen, leading to a reduction in saliva production, there is potential for

prolonged liquid transit times in the rumen as fluid is not being replaced (Herdt, 2002). The

same is true with animals that have reduced feed intakes, whether that be due to shy

feeding, concurrent disease or other reasons. If the drug becomes bound to fibrous material

with high cellulose contents, the time taken to pass through the gastro-intestinal tract can

be 50-60 hours, this being adequate time to break down the cellulose (Toutain et al., 2010).

Although absorption is considered to occur only within the abomasum and the small

intestine, the results of this experiment indicate some absorption must occur across the

rumen mucosa. Oxytetracycline was first detected in plasma at 30 minutes for the IW group

and two hours for the IF group. Given that the transit time for liquid through the rumen is

considered to be from 6-20 hours it is unlikely that these plasma concentrations would be

the result of absorption from more caudal parts of the gastro-intestinal tract. The rumen is

lined by a keratinised stratified squamous epithelium providing a small surface area for

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absorption (McKellar, 1994). Absorption of OTC occurs by passive transfer across the

mucosal membrane (Rang et al., 2007a) until an equilibrium is reached.

The reticulo-rumen volume can be estimated by the following equation (National Research

Council, 2007)

Volume of reticulo-rumen (L) = 0.77 x bwt (kg)0.57-3.49

As an example, for a 50kg sheep the reticulo-rumen is likely to have a volume of 4.5 L. The

volume of the reticulo-rumen in sheep is generally within the range of 3-15 L (Dehority,

2004). Given this large volume it is likely that a low concentration gradient will be present.

This results in slow absorption of OTC across the rumen mucosa (McKellar, 1994).

Lipid soluble drugs will easily cross the rumen mucosa (McKellar, 1994). Tetracyclines have

varying lipophicity with Doxycycline being the most lipophillic, and OTC slightly less lipophilic

(Ziv and Sulman, 1974; Boothe, 2001a). Ionisation of a drug also affects its ability to cross

the gastrointestinal mucosa. Tetracyclines are ionised at all pHs so they cannot easily cross

this membrane (Ziv and Sulman, 1974). It has been suggested that ionised drugs will form

bonds with a material in the gastro-intestinal tract to reduce their polarity (Ziv and Sulman,

1974). Ziv (1974) also postulated that ionised lipophillic drugs could be absorbed by passing

through a small number of large pores within the mucosal wall.

This experiment supports the hypothesis that OTC is absorbed through the rumen mucosa,

despite being of relatively low lipophillicity and being ionised.

The bioavailability of a drug depends on the characteristics of the preparation. For example

oxytetracycline hydrochloride is more readily absorbed than oxytetracycline dihydrate

(Baggot, 2008). Bioavailability also depends on the individual animal. Between animals there

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will be a variation in enzyme activity, intestinal motility and pH of the gastrointestinal tract

(Rang et al., 2007a) all of which can have an influence on bioavailability. Therefore, oral

bioavailability can vary between animals. This is particularly important in animals that have

gastrointestinal disease or conditions affecting the circulatory system (Rang et al., 2007a).

Disease status of the animal can also impact on the oral bioavailability. In pigs, ill animals

were found to have a higher oral bioavailability of tetracyclines (Pijpers et al., 1991). It is

thought that this is due to a decreased gastric function which increased the overall time

available for absorption of the drug (Pijpers et al., 1991). The decreased gastric function

resulted in the time to reach maximum plasma concentrations to be increased from 1.7

hours in healthy pigs to 7 hours in ill pigs.

Droumev (1992) studied the oral bioavailability of tetracycline in sheep using a slow release

intra-ruminal bolus. The oral bioavailability was found to be 12-13%. This value is lower than

the values calculated for either IF or IW in this experiment. Droumev (1992) attributed the

low bioavailability to the design of the bolus, which ensured that it remained in the reticulo-

rumen, and this caused the released drug to be greatly diluted. In addition to the anatomical

location it was considered that additional gastro-intestinal factors had an influence:

attachment of the drug to food particles; chelation; and disintegration of the drug in the

rumen microflora (Droumev et al., 1992).

In this experiment, incorporating OTC in water or in rapidly digestible pellets allowed the

drug to be available for absorption more quickly than through a slow release mechanism.

This minimized the risk of denaturing the OTC and increased its bioavailablity.

The time of peak plasma concentration varied between the IV group and the IF and IW

groups. When a drug is given by an extra-vascular route it has to rely on absorption into the

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vascular system which accounts for the delay in achieving plasma concentrations (Rowland

and Tozer, 1995b). The peak plasma concentration represents the point at which

elimination of the drug exceeds absorption resulting in decreasing concentrations of the

drug (Rowland and Tozer, 1995b). Given the number of factors which can impact absorption

of OTC following oral administration it is expected that the peak plasma concentration will

be lower than following IV administration. In contrast, the plasma concentration of oral OTC

was found to remain higher than that of IV OTC over time. This relates to the slow

absorption of drug from the gastrointestinal tract over time which offsets the rate of

elimination to a degree (Rowland and Tozer, 1995b). The same is true for any dosage regime

where the drug is given extra-vascularly, e.g. intra-muscular injection. Despite a delay in

reaching peak plasma concentrations, both the IF and IW groups maintained plasma

concentrations above the MIC for 22 hours.

With some drugs the peak plasma concentration can be increased by increasing the dose of

the drug, but this would be unlikely to affect the time to peak concentration. However,

tetracyclines are incompletely absorbed from the gastrointestinal tract and the remaining

drug is eliminated unchanged (Chambers, 2006). This remaining active drug has the

potential to be active against gastrointestinal microflora. In humans this can result in

overgrowths of yeasts, Pseudomonas spp. and Enterococci spp. This is typically only seen

following long-term use of the drugs (Chambers, 2006). By increasing the dose, it is likely

that more active drug will be left in the gastrointestinal tract resulting in a higher risk of

negative effects on the microflora. Droumev (1992) found that giving 60-70 mg/kg of

tetracycline orally resulted in a maximum plasma concentration of 6.6 µg/mL. This dose is

three times higher than what was used in this experiment, yet the maximum concentration

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is only marginally higher than the IF and IW plasma concentration. It would appear that

significantly increasing the dose administered of oral OTC has little effect on the maximum

plasma concentration and given the risk to the gastro-intestinal microflora it would not be

advisable.

The degree of distribution throughout the body that follows absorption impacts on the

efficacy of an antimicrobial (Al-Nazawi, 2003). The tetracyclines are generally widely

distributed (Chambers, 2006; Papich and Riviere, 2009). The volume of distribution of a drug

is defined as the volume of fluid required to hold the amount of drug within the body at the

plasma concentration (Rang et al., 2008) Al-Nazawi (2003) found that the volume of

distribution in sheep was 13.4 L/kg. This is greater than the volume of the sheep’s body

water which is an indication of excellent penetration of the drug throughout all the body

tissues (Al-Nazawi, 2003). Volumes of distribution greater than 1 L/kg show that the drug is

distributed outside of the extracellular fluid. This indicates penetration into intracellular

spaces or binding to tissue sites (Papich and Riviere, 2009). A wide volume of distribution

will maximise the potential of OTC reaching the ocular tissue where it is required in IOK

cases. There is reported to be an age-related change in the volume of distribution. The

volume of distribution is higher in young animals and decreases with age because of

decreasing extracellular and total body fluid and the increased volume of the

gastrointestinal tract (Nouws et al., 1983). Given the wide age range amongst sheep at the

feedlot, there may be a varying efficacy of OTC treatment.

All tetracyclines are protein bound within plasma. Protein binding of drugs limits the

availability of active forms in the plasma (Papich and Riviere, 2009) as it is the unbound form

which is active (Rang et al., 2007a). Oxytetracycline is the least protein bound of the

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tetracyclines (Ziv and Sulman, 1974). In sheep 21-25% of OTC is protein bound (Papich and

Riviere, 2009).

To ensure that a steady state of plasma concentration is maintained it is important to have

an understanding of the elimination characteristics of the drug. The tetracyclines are

eliminated from the body primarily through glomerular filtration in the kidneys but also

through the liver in the bile to the gastrointestinal tract (Short, 1994; Chambers, 2006). In

the liver OTC is conjugated with glucoronides which makes them hydrophilic (Rang et al.,

2007a). This conjugated OTC is excreted back into the intestines via the bile ducts. The

bacteria in the small intestine produce a high concentration of ß-glucuronidase enzymes

which can break down these glucoronide-OTC conjugates, thus releasing OTC (Gibson and

Skett, 2001b). The hydrolysed OTC is then available to be re-absorbed back into the

circulation whereas the OTC which is not hydrolysed in this way is excreted from the body in

the faeces (Gibson and Skett, 2001b). This reabsorption is known as enterohepatic

circulation (Chambers, 2006) and can increase the duration of action of the drug (Gibson

and Skett, 2001b) because more drug becomes available for absorption following the initial

absorption.

The half-life of the drug is the determining factor in the duration of action. The half-life is

defined as the time taken for the concentration of drug in plasma to reduce by 50% (Rang et

al., 2007b). The longer the half life, the longer the duration of action of a drug (Rang et al.,

2007b). In humans the half-life of tetracyclines varies between 6-12 hours (Chambers,

2006). In sheep following intra-venous administration of OTC the half-life is 3.2 hours (Al-

Nazawi, 2003), which is similar to the 3.96 hours found in this experiment. Half-life

determines the dosing frequency of a drug. If the half life is long, then to maintain steady

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plasma concentrations, the frequency of administration can be reduced (Gibson and Skett,

2001a). With antimicrobial therapy it is important to maintain a steady therapeutic level to

minimise the risk of under-dosing, which can lead to development of resistance. To maintain

this the dosage frequency should be equal to or less than the half-life (Gibson and Skett,

2001a). However, other factors can impact on the plasma concentration achieved, mainly

the absorption of the drug and then the subsequent distribution of the drug. As expected

because of the slower absorption of the drug, half-life for OTC following oral administration

is much longer. Available data from this experiment showed that the half-life of the IW

group was 51.5 hours and of the IF group 279.94 hrs. These values cannot be guaranteed

given the limited data available; further samples beyond 24 hours would be required to

confirm these values. Equally the length of half-life is only part of the requirements for the

drug to be effective, time above the Minimum Inhibitary Concentration is a great

importance also.

The oral bioavailability varied between the IF and the IW group. Many of the factors

described can be used to explain this. Medication incorporated into a feed pellet requires

breakdown of that pellet before it can be available for absorption. This is in contrast to the

IW group where the drug is dissolved in the water and immediately available for absorption.

Increasing the time within the gastro-intestinal tract results in less drug being available for

absorption, and therefore a lower oral bioavailablity.

One limitation of the experiment is that in both the IF and IW groups the drug was

administered over a short period of time which would not reflect what would happen in

sheep fed a medicated pellet on an ad lob ration or medicated water where small quantities

would be consumed throughout the day altering the drug pharmacokinetics.

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8.5 Conclusions

Oxytetracycline was detectable in plasma following both intravenous and oral

administration. Plasma concentrations were relatively low following oral administration.

This may limit their use in a clinical context if the concentrations achieved are lower than

the MIC of the target bacteria. Despite this concern, initial impressions from the work

described in Chapters 5 and 6 indicate a degree of clinical effect following dosing with OTC

in the drinking water. Further work is required to establish a concentration that is well

enough tolerated in water for adequate water intake to be maintained during treatment.

Given the percentage of drug that is not absorbed and therefore theoretically remaining

active within the gastrointestinal tract, it is important to determine what, if any, effect this

active drug would have on the rumen microflora. Since palatability of medicated water was

considered a limiting factor in intake, this could be a limiting factor in delivering an effective

dose via the oral route in sheep, given the pharmacokinetic data discussed in this chapter.

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9 The impact of oral oxytetracycline on rumen health

9.1 Introduction

As with all herbivorous animals, ruminants rely on micro-organisms for their general health

(Doetsch and Robinson, 1953) and for the digestion of plant constituents (Bell et al., 1951;

Hobson, 1969; Cunha et al., 2011). The rumen houses a complex microbial environment.

The ruminant and the microbial population within the rumen are reciprocally beneficial

(Hungate, 1966a). The rumen continually mixes incoming feed with micro-organisms and

saliva secreted by the host through frequent rumen contractions. In addition to mixing, the

contractions enable movement of organisms and digesta from the rumen into the omasum

and abomasum (Hobson, 1969). The ruminant supplies feed which the microbial population

break down into Volatile Fatty Acids (VFAs) and gases. The VFAs are absorbed and used by

the ruminant to provide energy (Hungate, 1966a; Kamra, 2005). In forage fed ruminants,

saliva serves to maintain a neutral pH within the rumen, thus maintaining a stable microbial

population (Hobson, 1969). Gases produced, mainly carbon dioxide and methane, sit in the

gas layer in the dorsal rumen prior to being excreted (Hobson, 1969).

Early research highlighted the role of bacteria in the fermentation of plant material

(Pasteur, 1863) revealing that this bacterial digestion could generate products that could be

used by the ruminant host (Zuntz, 1879). Given that ruminants typically consume a diet high

in plant material it is important that they can digest the cellulose in the plant matter (Lodge

et al., 1956). Mammals do not produce the cellulase enzyme required for the breakdown of

cellulose (Doetsch and Robinson, 1953). It has long been known that microbes in the rumen

can digest cellulose (Hofmeister, 1881) through the production of cellulase (Doetsch and

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Robinson, 1953). Since these early researchers, many others have looked at the complex

microbial population within the rumen and what effects diet can have.

Most of the bacteria within the rumen microbial population are obligate anaerobes

(Hungate, 1966b; Flint, 1997; Kamra, 2005). The rumen maintains an anaerobic environment

to support these organisms (Hungate, 1966b; Dehority, 2004). For optimal function of the

rumen microbes the pH should be maintained between 6.0-7.0 (Hungate, 1966b; Dehority,

2004; Kamra, 2005) and at a temperature of 39-40 oC (Hungate, 1966b; Dehority, 2004;

Kamra, 2005).

The rumen microbial ecosystem exists in a fairly constant state maintained through the

frequent addition of food material and removal of products of digestion (Hungate, 1966b).

The ecosystem is made up of 1010-1011 cells/mL of bacteria, 104-106 cells/mL ciliate

protozoa, 103-105 zoospores/mL of anaerobic fungi and 108-109 cells/mL bacteriophages

(Kamra, 2005). The different groups work together, both synergistically and antagonistically.

Some microbes depend on others for the supply of nutrients (Hobson, 1969) whereas some

are antagonised by compounds released by others (Kamra, 2005). This complexity of

microbes in the rumen is required to enable digestion of the wide variety of components of

the diet (Hungate, 1966b). A variety of microbes also allows for maximum biocatalytic

capacity within the rumen as multiple bacteria will provide multiple pathways for

breakdown of feedstuffs, thus maximising the amount of energy generated (Hungate,

1966b).

The fungi found in the rumen are obligate anaerobes and are involved in the breakdown of

fibre (Kamra, 2005). It is estimated that fungi make up 10% of the rumen micro-organism

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population (Chaucheyras-Durand and Ossa, 2014). Given the low fibre content, lower pH

and higher transit times seen with pelleted diets, growth of fungi is limited (Kamra, 2005).

Bacteriophages are present in the rumen and are specific to certain bacteria (Kamra, 2005).

They are responsible for turnover of bacteria; lysis of bacteria releases protein which can be

useful to the ruminant (Kamra, 2005).

More than 200 species of bacteria have been identified using culture-based techniques

(Chaucheyras-Durand and Ossa, 2014). With the use of PCR techniques many more

organisms have been identified (Chaucheyras-Durand and Ossa, 2014). Using 16s ribosomal

rRNA sequences approximately 7000 bacterial species have been identified within the

rumen micro-organism population (Chaucheyras-Durand and Ossa, 2014). Most bacteria in

the rumen are cocci and short rods measuring 0.4-1 µ in diameter and 1-3 µ in length

(Hungate, 1966b). The nature of the bacteria varies depending on the diet given. Hungate

(1966b) found that in ruminants on a high fibre diet of hay and forage the bacteria were

mainly gram negative, whereas those in ruminants on a low fibre diet high in concentrates

were mainly gram positive. High concentrate diets lead to an increased proportion of

Lactobacilli sp. which are better suited to the more acidic conditions in the rumen

associated with concentrate feeding (Hungate, 1966b). The diversity of rumen micro-

organisms is greater in animals fed forage based diets than in those on concentrate-based

diets (Fernando et al., 2010). The most common phyla of bacteria are Bacteroidetes,

Firmicutes (Cunha et al., 2011; Castro-Carrera et al., 2014) and Proteobacteria

(Chaucheyras-Durand and Ossa, 2014). The primary functions of bacteria within the rumen

are the breakdown of cellulose, production of fatty acids and the synthesis of proteins and

vitamins (Doetsch and Robinson, 1953).

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The microbial population of the rumen is susceptible to daily fluctuations in the

environment, micro-environment of the rumen and the physiology of the animal (Ziemer et

al., 2000; Chaucheyras-Durand and Ossa, 2014). Diet is a key factor in changing the micro-

environment of the rumen (Hungate, 1966b). Many of the organisms within the rumen are

sensitive to pH, particularly a reduction in pH (Flint, 1997). Concentrate diets are known to

reduce pH (Flint, 1997) and potentially create an unstable microbial population (Lee et al.,

1982). Lee (1982) found that the instability of the microbial environment was due to

increased bacterial activity resulting in an accumulation of acid. Feeding of roughage with

the concentrate can help to stabilise the micro-environment (Lee et al., 1982). Ziemer

(2000) considered that although diet changes will alter the microbial population there is

little evidence to show that the whole microbial community is affected.

Although there is a wide variety in microbial population it is difficult to quantify the exact

role played by any of the groups of microbes (Doetsch and Robinson, 1953; Kamra, 2005).

The earliest recorded attempts at culturing rumen bacteria were in 1884 (Tappeiner, 1884).

No successful attempts to isolate rumen bacteria occurred until the late 1940s (Hobson,

1969). The earliest success at culturing rumen bacteria was in 1947 when some were

distinguished but full characterisation of the bacteria was not completed (Gall et al., 1947).

The difficulty in culturing rumen bacteria lies with provision of suitable conditions mimicking

the rumen environment and the supply of primary nutrients normally provided by other

bacteria within the rumen (Hobson, 1969). Only a small percentage of the rumen microbial

population can be identified by culture (Tajima et al., 1999), estimated to be 20%

(Chaucheyras-Durand and Ossa, 2014). Reliance on culture-based techniques alone will

severely underestimate microbial diversity within the rumen (Fernando et al., 2010). Most

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rumen bacteria are similar in size and shape and therefore indistinguishable under a

microscope (Hungate, 1966b). Prevotella spp. and Butyrivibrio spp. appear to be the most

common rumen bacteria to be cultured (Flint, 1997).

Advances in molecular techniques have allowed a greater understanding of the genetic

diversity of the microbial population (Tajima et al., 1999). Most of the rumen microbes are

found in the fibrous layer of the rumen (Brosh et al., 1983) and are therefore difficult to

identify by culture. Molecular techniques enable identification of organisms attached to

plant material within the fibrous layer as their nucleic acid can still be recovered (Ziemer et

al., 2000). The use of rRNA-based techniques is essential to advance knowledge and

understanding of the rumen microbial population (Flint, 1997). Using such techniques allows

for a direct comparison to be made of the rumen microbial population before and after an

alteration to either the rumen environment or diet (Ziemer et al., 2000).

Given the importance of a stable rumen microbial population to the general health and

productivity of ruminants, it is essential to fully determine the effect of oral antibiotic

medication on the rumen microbial population. Much has been done to determine the

suitability of antibiotics for inclusion in rations for the purposes of growth promotion

(Bartley et al., 1951; Neumann et al., 1951; Munch-Petersen and Armstrong, 1958;

Klopfenstein et al., 1964). Research has also been done to assess the impact of antibiotics

given orally on milk production (Rusoff et al., 1952). Most work to date has been done

looking at the use of aureomycin (chlortetracycline). Klopfenstein (1964) found that

including aureomycin resulted in increased weight gains and faster growth. Neumann

(1951), however, found no difference in growth with the inclusion of aureomycin.

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Aureomycin has been found to alter the rumen microflora when given orally (Bartley et al.,

1951; Neumann et al., 1951; Horn et al., 1955; Munch-Petersen and Armstrong, 1958;

Klopfenstein et al., 1964; Purser et al., 1965). Neumann (1951) and Purser (1965) found that

total bacterial counts were unchanged by inclusion of antibiotics, but the diversity of

bacteria was different. In contrast Bartley (1951) found no difference in direct microscopic

examination of the rumen microflora between those treated and those untreated. The

addition of OTC has been found to change the microbial population, but as no cultures were

obtained the true nature of the change could not be identified (Munch-Petersen and

Armstrong, 1958). Early research focused more on the effects of antibiotics on digestibility

as opposed to directly on the organisms (Bell et al., 1951; Horn et al., 1955).

This experiment was designed to determine the effects of OTC on the rumen microflora.

Given the paucity of reports in the literature relating to the effects of OTC it was an

important experiment to carry out. As ruminants rely heavily on a stable and functional

microbial population for the digestion of feedstuffs and maintenance of general health, it is

important to assess any effects on this associated with OTC before recommending its use.

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Twelve mixed-age Merino-cross wethers with permanent rumen fistulas in place were used

in this experiment. The wethers were of varying ages and sizes, so attempts were made to

put equal numbers of old and young animals in each group to minimise overall differences

in weight. The sheep had previously been fistulated using a standard two-stage process

(Dougherty, 1955; Saeed et al., 2007). The fistula was located in the left paralumbar fossa

and enabled direct access to the rumen. All sheep were housed in a purpose-built animal

house at CSIRO Floreat, WA. Sheep were housed in individual pens and contact between

sheep through pens was allowed. An acclimatisation period of one week was allowed prior

to commencing the experiment for sheep to adapt to the housing and pellet ration. Sheep

were weighed and body conditions were measured (Russel et al., 1969) by the same

operator three times: on entry to the animal house, once during the experiment and again

at the end of the experiment. Sheep were split into two treatment groups and to balance

the treatments each group consisted of three older and three younger wethers. Individual

feed and water intake and faecal scores were measured on a daily basis throughout the

experiment. Faecal scores were measured as a visual assessment of faecal consistency using

a 1-5 scale, with 1 being firm pellets and 5 being diarrhoea (Le Jambre et al., 2007), Table

9.1, and were conducted by the same person each time.

Faecal Score Description

1 Firm, well formed pellet

2 Soft, pellet form

3 Soft faeces, loss of pellet structure

4 Paste like faeces

5 Watery, liquid diarrhoea Table 9.1: Faecal scoring system (Le Jambre et al., 2007)

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Alternative feedstuffs (chaff) were offered to sheep to mitigate the decrease in feed intake

when it occurred during the experiment.

Treatments

Both treatment groups were treated for five days with water soluble Oxytetracycline (OTC)

(CCD OTC, CCD Animal Health, Geelong VIC, Australia). Treatment group 1 was given

22mg/kg liveweight of OTC and Treatment group 2 was given 11mg/kg liveweight OTC. OTC

powder was diluted in water and administered directly into the rumen via the rumen fistula

each day for 5 consecutive days.

Rumen fluid samples

Rumen fluid samples were taken on days -5, -3, -1, 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, 16 and 18.

On day 0 a rumen fluid sample was taken immediately prior to administration of medication

and a follow up sample was taken 1 hour after medication. Rumen fluid samples were taken

by inserting a rigid plastic tube through the fistula and removing fluid by capillary action.

Samples were placed on ice after collection. Samples taken prior to commencement of

treatment were used as controls.

Rumen pH of the samples was measured immediately on collection using an electronic pH

meter, which was calibrated daily. Rumen fluid samples were frozen (-20 oC)and analysed

later for Volatile Fatty Acid (VFA) and Ammonia concentrations. Separate samples were

taken immediately prior to drug administration on day 0 and on day 5. These were frozen at

-80oC and sent for microbial profiling.

Rumen microbial profiling

The microbial profiling methodology used to investigate rumen bacterial, archaeal, fungal

and protozoan communities was based on the terminal restriction fragment length

polymorphism (T-RFLP) technique. T-RFLP is a culture-independent technique for profiling

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microbial communities based on differences at the nucleic acid or genome level (Torok et

al., 2008). This tool has been used to investigate changes in gut bacterial communities

associated with dietary modification, such as addition of feed enzymes, prebiotics, organic

acids and antimicrobials in the poultry industry (Geier et al., 2009; Geier et al., 2010; Torok

et al., 2011). The advantage of the technique is that it is high-throughput, high resolution

and capable of providing a “snap shot” of the entire microbial community at any particular

time. Hence, it is an ideal initial screening tool that requires no prior knowledge of the

actual microorganisms present within the community. This method was deemed

appropriate for the purposes of this experiment, namely to assess the impact of oral OTC on

rumen microflora.

Where significant treatment differences are detected other molecular techniques can be

used to identify organisms involved. In this experiment, the organisms identified as being

affected by OTC treatment were further characterised by pyrosequencing technology.

Pyrosequencing is a method of DNA sequencing based on the sequencing by synthesis

principle. Pyrosequencing can be used to identify the microbial genome sequence and

assign phylogenetic information to the population. In this experiment taxonomic assignment

was done using the ribosomal database project (RDP), which assigned taxonomy to family

level.

Methodolgy for T-RFLP

Total nucleic acid was extracted from the 24 freeze dried samples, representing the pre and

post treatment sample from both Treatment groups, using a South Australian Research and

Development Institute (SARDI) proprietary method (Torok et al., 2008). The bacterial

ribosomal DNA from the extracted material was amplified with universal 16S bacterial

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primers, one of which was labelled with fluorescent dye. Archael, fungal and protozoan

communities were also amplified using universal group specific primers. The resulting

amplicons were restricted with a specific recognition sequence restriction enzymes and

electrophoretically separated on a capillary DNA sequencer (ABI 3730, Applied Biosystems).

Data were analysed using GeneMapper (Applied Biosystems) to determine positions of

terminal restriction fragments (TRF).

Methodolgy for Pyrosequencing

Bacterial 454 pyrosequencing was done with primers 27F and 534R (Nossa et al., 2010). This

primer pair amplified a 598 bp product from the most variable region of the 16S rRNA (V2-

V3). Bacterial DNA was amplified from total nucleic acid using the FastStart HiFi PCR system

(Roche Diagnostics). Each primer included sequences to facilitate the sequencing of

products in the Roche/454 system. The forward PCR primer consisted of a related set of

primers with different “barcode” sequences, which enabled the identification of individual

samples. PCR amplicons were analysed for specificity by electrophoresis on a 2% agarose

gel. PCR amplicons were purified using Agencourt® AMPure® XP (Beckman Coulter) and

quantified according to the “Amplicon Library Preparation Methods Manual GS Junior

Titanium Series” protocol (Roche Diagnostics). Pooled samples (n=12 (controls)) were

sequenced using the Roche/454 GS Junior Genome Sequencer and Titanium chemistry

according to the manufacturer’s instructions (Roche Diagnostics).

The rumen bacterial communities from the control (n=12), Treatment group 1 (22 mg/kg)

(n=6) and Treatment group 2 (11 mg/kg) (n=6) were analysed. 16S rRNA sequences were

assigned to operational taxonomic units (OTU) with 97 % within group similarity, a level

approximately equivalent to a species designation. OTU tables were produced in Qiime v

1.4.0 (Caporaso et al., 2010) with standard defaults and taxonomic assignment determined

135

using the greengenes RDP classifier database with a cut-off threshold of 80%. OTU tables

were filtered to remove the possibility of sequencing errors and chimeras, by eliminating

OTU with less than five sequences and present in less than two samples BLASTn with

nucleotide collection (nr/nt) databases and the Megablast algorithim (National Center for

Biotechnology Information [NCBI]) were also used to identify similarity of OTU of interest to

sequences available in public genome sequence databases (Altschul et al., 1990).

Blood samples

Blood samples were collected from the jugular vein using a 20 gauge, 1” needle and a

vacutainer tube, plain tube for OTC concentrations and lithium heparin for BHB analysis.

Blood samples were centrifuged at 2000 rpm for 15 minutes and the plasma titrated off.

Plasma was frozen and analysed at a later date. Blood samples collected on days -3, 0, 1, 2,

3, 4, 5, 7, 9, 11, 14, 16 and 18 were analysed for BHB concentrations. Blood samples were

also collected on days 0, 1, 2, 3, 4, 5, 7, 9 and 11 for measurement of plasma OTC

concentrations.

Statistical Analysis

For all data except those associated with rumen microbe profiling the following analyses

were carried out. For each animal all pre-treatment values were averaged and change from

the pre-treatment average was calculated for each post-treatment date. A linear mixed

model was fitted to the calculated changes on all post-treatment dates. The model included

fixed effects for Treatment (11 vs 22), post-treatment date and their interaction; and

random effects for animal and the animal by date interaction. An autoregressive model

allowed for correlations between measurements made on the same animal on different

dates. 5% LSD’s were calculated to compare Treatment means to zero, i.e. whether

treatment caused a change from the pre-treatment mean, and to compare the two

136

Treatments. For pellet and total ME intake the analysis has also been performed with water

intake included in the fixed model, i.e. as a covariate.

For the rumen profiling analysis, data points from GeneMapper analysis were validated and

outputs generated for statistical analysis using queries within a custom built database

(Torok et al., 2008). Queries in the database were used to compare duplicate T-RFLP profiles

and identify synonymous fragment sizes (±2 bp). The resulting fragments were treated as

OTU, representing particular microbial species or taxonomically related groups. Operational

taxonomic units (OTU) obtained from the 24 rumen samples were analysed using

multivariate statistical techniques (PRIMER 6, PRIMER-E Ltd., Plymouth, UK). These analyses

were used to examine similarities in sheep rumen microbial communities and to identify

OTU accounting for the differences observed in microbial communities (Torok et al., 2008).

Bray-Curtis measures of similarity (Bray and Curtis, 1957) were calculated to examine

similarities between rumen microbial communities from the T-RFLP generated (OTU) data

matrices, following standardization and fourth root transformation. The Bray-Curtis

similarity co-efficient (Bray and Curtis, 1957) is a reliable measure for biological data on

community structure and is not affected by joint absences that are commonly found in

microbial data (Clarke, 1993). Analysis of similarity (ANOSIM) (Clarke, 1993) was used to test

if rumen microbial communities were significantly different between treatments. This is a

non-parametric method of ranked siddimilarities. The R-statistic value describes the extent

of similarity between each pair in the ANOSIM analysis, with values close to unity indicating

that the two groups are entirely separate and a zero value indicating that there is no

difference between the groups.

137

Similarity percentages (SIMPER) (Clarke, 1993) analyses were done to determine which OTU

contributed most to the dissimilarity between treatments. The overall average dissimilarity

( ) between microbial communities of ruminants on two treatments shown to significantly

differ were calculated and the average contribution of the ith OTU ( i ) to the overall

dissimilarity determined. Average abundance ( y ) of important OTU in each of the groups

was determined. OTU contributing significantly to the dissimilarity between treatments

were calculated ( i/SD(δi)>1). Percent contribution of individual OTU ( i%) and cumulative

percent contribution (Σ i%) to the top 65% of average dissimilarities were also calculated.

Unconstrained ordinations were done to graphically illustrate relationships between

treatments using non-metric multidimensional scaling (nMDS) (Shepard, 1962a; Kruskal,

1964). nMDS ordinations attempt to place all samples in an arbitrary two-dimensional space

such that their relative distances apart match the corresponding pair-wise similarities.

Hence, the closer two samples are in the ordination the more similar are their overall

microbial communities. “Stress” values (Kruskal’s formula 1) reflect difficulty involved in

compressing the sample relationship into the 2-D ordination.

For pyrosequencing analysis OTU obtained from the rumen of 24 samples were analysed

using multivariate statistical techniques (PRIMER 6, PRIMER-E Ltd, Plymouth, UK). These

analyses were used to examine similarities in sheep rumen bacterial communities and to

identify OTU accounting for differences observed in bacterial community structure. Bray-

Curtis measures of similarity (Bray and Curtis, 1957) were calculated to examine similarities

between ruminal bacterial communities from the pyrosequencing generated (OTU) data

matrices, following standardization and fourth root transformation. Similarity percentages

(SIMPER) (Clarke, 1993) analyses were done to determine which OTU contributed most to

138

the dissimilarity between treatments. Bacteria OTU contributing significantly to the

dissimilarity between treatments were calculated (Diss/SD>1). Unconstrained ordinations

were done to graphically illustrate relationships between treatments using non-metric

multi-dimensional scaling (nMDS) (Shepard, 1962b, a; Kruskal, 1964). nMDS ordinations

were done to place all samples in an arbitrary two-dimensional space such that their relative

distances apart match the corresponding pair-wise similarities. Hence, the closer two

samples are in the ordination the more similar are their overall bacterial communities.

“Stress” values (Kruskal’s formula 1) reflect difficulty involved in compressing the sample

relationship into the 2-D ordination.

9.3 Results

In all cases for each animal all pre-treatment values were averaged and change from the

pre-treatment average was calculated for each post-treatment date. In Table 9.2 the level of

significance for all the measurements taken is summarised.

Intake - kilo’s of feed

Intake - MJ ME

Water intake

Faecal score

rumen pH

Plasma BHB

Plasma OTC

Term P-value P-value P-value

P-value P-value

P-value

P-value

Time <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Water <0.001 <0.001

Treatment 0.646 0.745 0.670 0.948 0.184 0.265 0.154

139

Time.Treatment 0.455 0.164 0.019 0.858 0.140 0.122 0.258

Table 9.2: Significance table for change in measurements between pre- and post- treatment parameters.

140

Bodyweight and body condition score

The mean bodyweight of animals in the Treatment 1 group (11mg/kg OTC) was 72.7 kg prior

to commencement of treatment compared to 65.3kg for animals in the Treatment 2 group

(22 mg/kg OTC). In both groups, bodyweight decreased up to day 3 before increasing again.

The mean weight loss in both Treatments was 2.8 kg. There was no significant difference

between the Treatment groups.

Feed Intake

Feed intake reduced over time following commencement of the treatment, although it

returned to pre-treatment levels following cessation of treatment (Figure 9.1). There was no

significant difference between the two Treatment groups. When water was fitted as a co-

variant it was highly significant (p<0.001) suggesting a significant association between feed

and water intake.

141

Figure 9.1 Graph of mean feed intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

-200

0

200

400

600

800

1000

1200

1400

-10 -5 0 5 10 15 20

mean

gra

ms (

g)

of

pellets

co

nsu

med

days

11 mg/kg OTC

22 mg/kg OTC

142

Metabolisable energy

The total mega-joules (MJ) of metabolisable energy (ME) were calculated (Figure 9.2) to

account for the addition of chaff when intake dropped significantly. As with feed intake,

energy intake decreased following the start of treatment and recovered after treatment

finished. It appeared that sheep preferentially ate chaff over pellets during the treatment

period. As with pellet intake, there was no significant difference between the Treatments

over time.

Figure 9.2 Graph of mean ME intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

-10 -5 0 5 10 15 20

Me

an

MJ

ME

co

ns

um

ed

Days

11 mg/kg OTC

22 mg/kg OTC

143

Water Intake

Mean water intake fell significantly following the start of treatment (Figure 9.3). There was a

significant difference in mean water intake over time and also a significant difference

between the Treatments on days 4, 10, 14 and 15.

Figure 9.3 Graph of mean water intake over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

-10 -5 0 5 10 15 20

me

an

lit

res

of

wa

ter

co

nsu

me

d

Days

11 mg/kg OTC

22 mg/kg OTC

144

Plasma Beta-Hydroxybutyrate (BHB) concentrations

Mean BHB concentrations rose significantly following commencement of treatment.

Concentrations peaked at day 8 and returned to pre-treatment concentrations on

approximately day 13 (Figure 9.4). There was no significant difference between Treatments

over time.

Figure 9.4 Graph of mean plasma BHB concentrations over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors. Black line represents BHB concentration at which sheep are at risk of sub-clinical ketosis.

0.000

0.200

0.400

0.600

0.800

1.000

1.200

-5 0 5 10 15 20

mean

pla

sm

a B

HB

co

ncen

trati

on

s (

mm

ol/L

days

11 mg/kg OTC

22 mg/kg OTC

145

Faecal Score

Faecal score changed significantly over time and increased significantly after the start of

OTC treatment (Figure 9.5). There was no significant difference between the Treatments

over time.

Figure 9.5 Graph of mean faecal score over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

1

2

3

4

5

-10 -5 0 5 10 15 20

Faec

al s

core

Days

11 mg/kg OTC

146

Rumen pH

Rumen pH changed significantly over time and increased significantly after the start of OTC

treatment (Figure 9.6). There was no difference between the Treatments over time.

Figure 9.6 Graph of mean rumen pH over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

6.00

6.20

6.40

6.60

6.80

7.00

7.20

7.40

7.60

7.80

-10 -5 0 5 10 15 20

me

an

ru

me

n p

H

days

11 mg/kg OTC 22 mg/kg OTC

147

Plasma OTC concentrations

OTC was detected in the blood of all animals during the OTC treatment period.

Concentrations peaked at day 5 and low levels were still detected on day 9, 4 days after

treatment ceased (Figure 9.7). There was no significant difference in the concentration of

OTC in the plasma in the two treatment groups over time.

Figure 9.7 Graph of mean plasma OTC over time for Treatments (11mg/kg and 22mg/kg oral OTC). Shaded box represents OTC treatment period. Error bars represent standard errors.

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0 2 4 6 8 10

me

an

pla

sm

a O

TC

co

nc

en

tra

tio

n (

µg

/mL

)

days

11mg/kg OTC

22 mg/kg OTC

148

Rumen fluid parameters

There was a significant effect of OTC treatment on rumen bacterial, archael and fungal

populations, but no effect was seen on protozoal populations. Despite this inter-animal

variability, a subjective judgement can be made for the bacterial, archaeal and fungal

communities in the rumen communities of animals pre-treatment compared to after the

start of OTC treatment. This difference relates to both presence/absence of unique OTU, as

well as shifts in abundance of common OTU. The number of OTU shared among the animals

post-treatment was reduced compared with the number observed pre-treatment, indicating

a reduced biodiversity in micro-organisms within the rumen after OTC treatment

commenced.

Multivariate statistical analyses were used to investigate differences in rumen bacterial,

archaeal, fungal and protozoan communities associated with OTC treatment. Significant

differences (P=0.001) associated with OTC treatment were detected in the rumen bacterial,

archaeal and fungal communities (Table 9.3). OTC treatment did not influence the rumen

protozoan communities (Table 9.3). Both Treatments altered the rumen bacterial, archaeal

and fungal communities when compared with pre-treatment populations. However, it was

only within the bacterial communities that there was also a significant difference associated

with the OTC dose (Table 9.3). The influence of OTC dose on rumen bacterial communities is

summarised in Figure 9.8.

149

Bacteria (R=0.703, P=0.001) pre-treatment 11 OTCa 22 OTCb

pre-treatment 0.774 0.816 11 OTC 0.001 0.141

22 OTC 0.001 0.050 Archaea (R=0.448, P=0.001) pre-treatment 11 OTC 22 OTC


22 OTC 0.001 0.366 Fungi (R=0.489, P=0.001) pre-treatment 11 OTC 22 OTC


22 OTC 0.002 0.177 Protozoa (R=0.063, P=0.203)

* For each pairwise comparison the R value (Grubb et al.) and P value (italics) are indicated.

P < 0.05 is significant.a 11 OTC = Daily oral dose of 11 mg OTC per kg liveweightb 22 OTC =

Daily oral dose of 22 mg OTC per kg liveweight

Table 9.3 One-way ANOSIM of rumen microbial communities associated with OTC treatment. For each microbial group the influence of oral OTC treatment was investigated. Where significant differences in rumen microbiota were detected, the pairwise* differences between treatments were investigated further.

150

Figure 9.8 nMDS of rumen bacterial communities associated with oral dose of OTC taken on day 5.

Treatments are: 11mg OTC/kg LW/day () and 22mg OTC/kg LW/day (). Each point in the ordination shows the overall microbial profile of an individual animal. The closer two points are in the ordination the more similar are their profiles.

Pyrosequencing of bacterial communities generated 90396 reads with an average of 3700

reads per sample. 1631 OTU were detected once data had been filtered. Fifteen bacterial

phyla as well as unclassified bacteria were identified in the rumen of the sheep (Figure 9.9).

151

Figure 9.9 Abundance of rumen bacteria classified to level of phyla for individual control and OTC treated sheep. (Cont) untreated control sheep, (Low) treated with 11 mg OTC/kg live weight/day and (High) treated with 22 mg OTC/kg live weight/day.

152

The majority of sequences identified belonged to the Bacteroides (23.1%), Firmicutes

(41.3%), Proteobacteria (14.9%), Fibrobacteres (8.2%) and unclassified bacteria (8.5%).

Rumen bacteria could be further classified into 45 groups based on class, order or family

(Figure 9.10).

153

Figure 9.10 Abundance of rumen bacteria classified, where possible, to level of family for individual control and OTC treated sheep. (Cont) control sheep, (Low) 11 mg OTC/kg live weight/day and (High) 22 mg OTC/kg live weight/day.

154

Control samples had a more diverse rumen microbiota (6-14 bacterial phyla detected per

sheep with an average of 11.1 phyla) compared to the post-treatment samples (3-7 bacterial

phyla per sheep with an average of 4.7 phyla). Bacteroidetes were more abundant in the

rumen of control sheep (46.1%) compared to treated sheep (0.1%). The differences in

Bacteroidetes were due to greater abundance of unclassified Bacteroidales (10.6%) and

Prevotellaceae (32.2%) in the control sheep than in the treated sheep (less than 0.1% of

each). Control sheep also contained Fibrobacteres (16.3%) and Spirochaetes (2.6%), while

these phyla contributed <0.01% to the rumen bacterial population in OTC treated sheep.

Control sheep contained 16.3% Fibrobacteraceae and 2.5% Spirochaetaceae, while the OTC

treated sheep contained less than 0.1% each of these families. There was a greater

abundance in OTC-treated sheep of Firmicutes (65.1%), Proteobacteria (19.4%) and

unknown bacteria (14.9%) than in the control sheep (17.6%, 10.3% and 2.1% respectively).

The difference in Firmicutes was predominantly due to the Lachnospiraceae, which were

more abundant in the treated sheep (55.1%) than in the control sheep (7.2%). The increased

Proteobacteria detected in treated sheep was due to increased abundance of

Enterobacteriaceae (19.0%) which accounted for <0.1% in control sheep. Although

Proteobacteria were less abundant in the controls, Aeromondales (8.6%), were more

abundant in the controls which were absent from the treated sheep. The percentage

contribution of bacterial taxa to overall community structure for individual sheep is shown

in Appendix 3.

155

9.4 Discussion

The two dosage regimes used in this experiment were chosen to assess the recommended

dose (22 mg/kg) and half the recommended dose of OTC. The dose of 11 mg/kg OTC was

used to determine the impact of a lower dose such that if a severe impact was seen with the

higher dose recommendations could potentially be based on the lower dose.

Experimental work in Chapter 6 highlighted a decrease in both water and feed intake

following inclusion of OTC medication in water. The results of this experiment also

demonstrated negative effects of OTC on feed and water intakes. In Chapter 6 the reduced

feed intake was attributed to a concurrent decrease in water consumption. In this

experiment sheep were offered non-medicated water and the OTC was administered

directly into the rumen via the fistula. This was done to ensure each sheep received the

correct dose of OTC to allow a true assessment of the effect of the two doses on intake and

rumen health.

It is postulated that the decrease in feed intake is directly related to the effects of the OTC

on the rumen microflora. Analysis of the data from the rumen fluid showed that the

Treatments significantly changed the populations of rumen microbes. What is evident from

these depictions is that the rumen microbiota were variable among animals on the same

treatment and that there was a need for robust statistical analysis when investigating

treatment differences. The two Treatments caused the populations to change in different

ways such that the rumen microbe profile at the end of the experiment differed between

Treatments. This, however, did not lead to a difference between Treatments in any of the

other variables, namely feed intake, energy intake, faecal score, BHB concentrations or

156

rumen pH. Pyrosequencing was done to speciate the microflora populations that were

affected and the results are included as Appendix 3.

Bacteroidetes spp., Firmicutes spp. and Fibrobacter spp. are the most common genera of

the rumen bacterial population (Fernando et al., 2010; Castro-Carrera et al., 2014) and

these were the main genera impacted by the treatment administered.

Bacteroidetes spp. were reduced following treatment. Prevotellacae are the most common

species of the phylum Bacteroidetes (Stewart et al., 1997). Prevotellacae are gram negative,

anaerobic rods or coccibacilli which are found in the rumen in association with a wide

variety of feedstuffs (Stewart et al., 1997). They have no action against cellulose but will

degrade and utilise starch and are important in the degradation of protein (Stewart et al.,

1997). Fermentation products of Prevotellacae include proprionate, which is an important

energy precursor in the ruminant. Members of the Prevotellacae genera have been shown

to develop plasmids conferring resistance to tetracyclines (Flint and Stewart, 1987);

however, given the reduction in population seen in this experiment the significance of this is

unclear. Longer term monitoring of the rumen microflora population would be required to

assess if this had the potential to be a significant concern.

Fibrobacter spp. were seen to reduce following treatment. Fibrobacter succinogenes is a

common bacteria found in the rumen microflora and its main function is the breakdown of

cellulose (Stewart et al., 1997). Interestingly F. succinogenes is considered to be resistant to

many in-feed antibiotics such as Avoparcin (Stewart et al., 1997). Given that OTC is also

effective against gram positive organisms it would be reasonable to expect no change in the

population following treatment; however, the decrease would indicate that F. succinogenes

is not resistant to OTC. Given the importance of Fibrobacter spp. in the initial breakdown of

157

cellulose, a reduction in their activity will limit further breakdown of cellulose by other

organisms (Mackie et al., 2002) and consequently minimize effective digestion of feed.

Animals in both Treatment groups altered their body weight following the introduction of

OTC. This weight loss is probably attributable to loss of rumen mass as opposed to loss of

muscle mass. This can be confirmed by only a mild increase in BHB concentrations that

indicates a relatively low level of fat catabolism. None of the animals entered a clinically

significant state of negative energy balance which is evident when the BHB level exceeds 1.5

mmol/L. Beta-hydroxy butyrate is produced in a number of ways including from the rumen

microbes and through fat catabolism in the liver. Although BHB concentrations increased

during treatment, they only briefly exceeded 0.8 mmol/L which is considered a safe value

for the estimation of the nutritional status of a flock (O'Doherty and Crosby, 1998).

O’Doherty and Crosby (1998) found that ewes consuming half their ME requirements had

BHB concentrations of 1.18-1.25 mmol/L, which was in excess of values found in this

experiment.

Not all bacterial genera were reduced following treatment. Firmicutes spp. increased.

Lachnospiraecae, of which Lachnospira multiparus is a member, were responsible for this

increase. These bacteria further breakdown cellulose following initial digestion (Mackie et

al., 2002). Proteobacteria populations also increased following treatment. There is some

debate surrounding the importance of Proteobacteria in the rumen microflora (Kang et al.,

2013): originally they were thought to be of limited importance because of their relatively

low numbers, but recently their metabolic importance has been considered more important

than previously thought when judged on their numbers alone. (Kang et al., 2013).

158

Despite the observed changes within the rumen microflora it is important to recognize the

limitations of the pyrosequencing technique in identifying all changes present. The rumen

functions through the interactions of bacteria, fungi and protozoa (Mackie et al., 2002).

Given that there were no significant changes seen in fungal or protozoal populations, and

that the animals returned to normal feed intakes following cessation of treatment it is likely

that any changes to bacterial populations were transient and rumen microbiota returned to

normal in a relatively short time post-OTC treatment.

The decrease in feed intake appeared to be offset to a degree by the introduction of chaff.

Chaff was mixed through the pellet ration and sheep appeared to preferentially eat the

chaff during the treatment period and for a number of days following treatment. Further

work would be required to definitively confirm that feeding chaff during the treatment

period would maintain feed and energy intake. The decrease in feed intake was only

temporary, and intake levels returned to levels similar to those pre-treatment, 1 week post-

treatment.

Rumen pH increased over time but the mean pH did not exceed that of a healthy sheep,

which should be in the range of 6.5-7.5. Prior to introduction of OTC the pH was lower than

6.5. This is to be expected in sheep on a diet consisting of only pelleted feed (Briggs et al.,

1957; Mould and Orskov, 1983).

It is encouraging that OTC was absorbed following administration directly into the rumen.

Plasma concentrations of OTC were comparable to those in the previous experiment, even

though the OTC dose was administered directly into the rumen as opposed to relying on

water intake.

159

There were no significant differences between the two different doses of OTC given in this

experiment and it appears that the responses to soluble OTC were not dose-dependent.

There was a cumulative dose effect whereby peak plasma OTC concentrations were

achieved after four days in the 22 mg/kg Treatment and after five days in the 11 mg/kg

Treatment. Plasma concentrations were lower than those found following intra-muscular

injections of OTC, namely 0.94 µg/mL, (Chapter 6) which is to be expected given the lower

bioavailability seen following oral administration.

9.5 Conclusions

Although some changes in rumen microflora were found in the Treatment groups, these

changes were restricted to the bacterial populations with no significant changes seen in

either the fungal or protozoal populations. As the rumen microflora work symbiotically it is

likely that any change will be transient and the population will modify such that function is

maintained. Despite changes in microflora, all other parameters indicative of rumen health

showed no significant difference.

Feed and water intake were again reduced in this experiment, and this has the potential to

be problematic within the live export chain. To date in-water medication has been assessed.

Work described in Chapter 8 indicated that OTC is absorbed following administration in

feed, however the impact of administering OTC in-feed on feed and water intakes is

unknown.

160

10 The use of medicated feed to treat infectious ovine

keratoconjunctivitis

10.1 Introduction

Previous chapters have described several negative effects associated with in-water

medication on the physiology and behaviour of the sheep. However, the positive clinical

response of IOK to OTC treatment encouraged the researchers to consider alternative

options. It was shown in Chapter 8 that OTC was absorbed when administered in feed.

A number of early studies have considered the impact of medicated feed on rumen function

(Bell et al., 1951; Neumann et al., 1951; Perry et al., 1954; Munch-Petersen and Armstrong,

1958). Both Neumann et al (1951) and Perry et al (1954) found that feed intake was

transiently reduced for 2-4 days following introduction of feed medicated with aureomycin.

Bell et al (1951) reported a marked anorexia and severe diarrhoea associated with

aureomycin medicated feed: it was postulated that this was not a palatability issue but

rather was related to digestive disturbance. In contrast to these reports Munch-Petersen

and Armstrong (1958) found that no change in appetite was seen following the introduction

of medicated feed. Given the conflicting reports in the literature and the paucity of studies

looking at OTC inclusion it was important to determine if there were any negative effects of

in-feed medication with OTC.

With the negative effects on feed and water intakes seen with in-water OTC medication, a

palatability experiment was conducted first to determine the effect of in-feed medication

on feed intake.

161

10.2 Hypothesis

1. The addition of oxytetracycline to feed will have no impact on feed intakes


Thirty adult Merino cross ewes were sourced from the Murdoch University teaching flock.

All sheep selected were in good health and had received no treatments in the month

preceding the experiment. Sheep were housed in individual pens in a purpose built animal

house ensuring they had sheep contact on at least one side. All sheep were weighed on

entry to the animal house and a 2-days acclimatisation period was observed prior to

commencement of the experiment.

The sheep were randomly assigned as they came through the race to two treatment groups

of fifteen sheep.

IF. This treatment received non-medicated pellets for the first 2 days followed by 5 days of

medicated pellets and then 5 days of non-medicated pellets.

Control. This treatment received only non-medicated pellets throughout the study period.

Feed and water measurements were recorded daily at the same time each day for all

animals. All sheep were weighed at the end of the experiment.

Preparation of medicated pellets (in-feed medication).

Medicated pellets were manufactured so that a 50 kg sheep eating 2% of its bodyweight in

pellets per day would consume a dose of 20mg/kg OTC. Pellets were manufactured by

Wellard Rural Exports at their feedmill. The pellets consisted of a mixture of lupins, grain

162

(Triticale) and chopped straw. The OTC powder (Terramycin, Phibro Animal Health Pty Ltd,

Girraween, Australia) was mixed in with the cereal and 15 minutes was allowed in the mixer

to ensure adequate distribution of the powder throughout the mixture. Following this,

chopped straw was added and a further 10 minutes of mixing occurred. The mixture was

then heated to approximately 70oC for 2 minutes. The heat was generated by application of

steam, which moistened the mixture to enable better compaction into the pellet shape.


For each animal all pre-treatment feed and water intakes were averaged. A linear mixed

model was fitted to the feed and water intakes on all post-treatment dates. The model

included fixed effects for pre-treatment liveweight and intakes, treatment, post-treatment

date and treatment by date interaction; and random effects for animal and the animal by

date interaction. An autoregressive model allowed for correlations between measurements

made on the same animal on different dates and different residual variance on each date.

5% Least Significant Differences (LSD) were calculated to compare the two treatments.

10.4 Results

There was no significant difference in the weight of sheep between the groups: sheep in the

IF group had a mean weight of 40.5 (+/- 2.88) kg and those in the control group 42.2 (+/-

4.21) kg pre-treatment and 41.33 (+/- 2.92 kg) and 42.2 (+/-4.54)kg post-treatment.

Both feed and water intakes were significantly impacted by the introduction of medicated

feed to the sheep in the IF treatment when compared to those in the control group

(P<0.001, see Figure 10.1 and 10.2). In Table 10.1 the mean feed intake values are

summarised.

163

Feed intake fell after the introduction of medicated feed and recovered to near the levels of

the control animals by the end of the experiment. Similarly, water intake fell after the

introduction of pellets, recovered and then fell slightly again before the end of the

experiment.

IF group Control group

Mean intake (g) during treatment

820.33 (+/- 330.6) 1173.00 (+/- 45.2)

Mean intake (g) following treatment

1002.57 (+/- 159.7) 1240.29 (+/- 22.1)

Mean intake (g) over whole experiment

993.75 (+/- 256.7) 1308.75 (+/- 38.7)

Table 10.1 Feed intake between the groups throughout the experiment

0

200

400

600

800

1000

1200

1400

1600

1 2 3 4 5 6 7 8 9 10 11

fee

d in

take

g/h

ead

/day

Day

Control

OTC

Figure 10.1 Feed intake (g/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors.

164

0

500

1000

1500

2000

2500

3000

3500

4000

0 1 2 3 4 5 6 7 8 9 10

wat

er

inta

ke m

L/h

ead

/day

Day

Control

OTC

Figure 10.2 Water intake (kg/head/day) over time. Shaded box represents treatment period. Error bars represent standard errors.

165

10.5 Discussion

Feed intake in the IF group dropped sharply in the first two days following the introduction

of the medicated feed. Following this drop, intake slowly increased to levels closer to those

prior to introduction of medicated feed. Water intake also dropped sharply in the early

treatment period before slowly returning to levels comparable with those of the control

group following cessation of treatment.

All sheep were offered a pelleted ration at a standard weight of 1.5 kg daily. The pellet

provides 11 MJ/kgDM of metabolisable energy and was calculated to contain 90% dry

matter (Caporaso et al.). This would equate to a kilogram of feed providing 9.9 MJ of

metabolisable energy as fed. The maintenance energy requirements of sheep vary with age,

type and the environment in which they are kept. As a guide, adult sheep require 0.185 MJ

ME kg -1 (Ball et al., 1998) per day for maintenance, equivalent to 9.25 MJ ME for a 50kg

sheep. The IF group showed a reduction in intake during the treatment period which

resulted in their mean energy intake reducing to 8.1 MJ ME which was above their

maintenance energy requirements according to Ball (1998) of 7.2 MJ ME. Given that

maintenance, and not growth, is the aim for sheep during pre-embarkation, and given the

recovery of intake to previous levels following cessation of treatment, this reduction in

intake may be an acceptable consequence of treatment.

As with feed intake, water intake was seen to drop but still remained within acceptable

volumes for maintenance.

166

11 Clinical efficacy of in-feed OTC

11.1 Introduction

The previous experiment has demonstrated that, although feed intake was reduced, feed

intakes remained sufficient for maintenance despite the addition of medication to the

pellets. In Chapter 5 it was concluded that OTC was absorbed into the blood at detectable

levels following administration in the feed. On the basis of these findings it was judged

important to assess the clinical efficacy of administering OTC by this route to animals with

naturally occurring clinical IOK.

11.2 Hypothesis

1. Administration of pellets medicated with OTC will be effective in the clinical

improvement of IOK.


Thirty mixed age and mixed breed sheep with clinical IOK were selected from a pre-export

feedlot. Sheep were selected with either grade 2 (conjunctivitis) or grade 3 (conjunctivitis

and corneal oedema) infection in both eyes. No treatment had been given to the sheep

prior to selection for this experiment. The sheep were transported to a purpose built animal

house facility at Murdoch University where they were housed in individual pens with sheep

contact on at least one side.

All sheep were weighed on arrival at the animal house and on leaving. Sheep were

accustomed to the pelleted ration prior to selection, therefore a 24-hour period was

allowed for acclimatisation to the change in housing.

The sheep were randomly assigned into 2 treatment groups of fifteen sheep each:

167

Control. These sheep received non-medicated pellets throughout the duration.

IF. These sheep received medicated pellets for 5 days followed by 5 days of un-medicated

pellets.

Medicated pellets were as described in the in-feed palatability trial, section 10.2.

Swabs for bacterial culture were taken from all sheep on arrival at the animal house and

again on leaving. Swabs were taken by placing a sterile cotton-tipped wooden-shafted swab

in the fornix of the eye between the third eyelid and the conjunctiva; these swabs were

then plated on sheep blood agar in the standard manner to obtain a single culture before

the tip was broken off into a Mycoplasma broth. Plates and Mycoplasma broths were

submitted to the Department of Agriculture microbiology laboratory in South Perth for

culture growth and analysis.

Feed and water intake were recorded for individual sheep daily. Eye grades were assessed

and recorded in all sheep on days 0, 1, 3, 5, 7 and 10.


For each animal all pre-treatment feed and water intakes were averaged. A linear mixed

model was fitted to the feed and water intakes on all post-treatment dates. The model

included fixed effects for pre-treatment liveweight and intakes, treatment, post-treatment

date and treatment by date interaction; and random effects for animal and the animal by

date interaction. An autoregressive model allowed for correlations between measurements

made on the same animal on different dates and different residual variance on each date.

168

For each eye on each animal all pre-treatment eye grades were averaged. A linear mixed

model was fitted to the eye grade data on all post-treatment dates (days 0, 2, 4 and 7). The

model included fixed effects for pre-treatment eye grade, treatment, post-treatment date

and treatment by date interaction; and random effects for animal, the animal by date

interaction and eyes within dates and animals. An autoregressive model allowed for

correlations between measurements made on the same animal and eyes on different dates

and different residual variance on each date.

A split plot analysis of covariance was used to analyse bacterial scores from each eye of each

animal made at the end of the experiment. Pre-treatment score for the appropriate bacteria

was used as a covariate measurement and eye within animal was used as the split factor.

5% LSD’s were calculated to compare the two treatments.

Plasma sample analysis

Plasma samples were analysed for concentrations of OTC. The method used for sample

analysis is as described in Chapter 6.

169

11.4 Results

The mean weight at the start of the experiment was 39.3 kg for the control group and 38.5

kg for the IF group. There was no significant difference in weight between the animals in the

groups and no significant change in their weight over the experimental period.

Feed intakes reduced significantly during the treatment period in the IF treatment but

recovered after treatment ended (P=0.049, see Table 11.1 and Figure 11.1)

Water intake was not significantly reduced and there was no significant reduction in water

intake associated with the drop in feed intake in the IF group (Figure 11.2).

Clinically, sheep in the IF treatment showed a significant improvement in eye grade during

treatment compared to those in the control group (P=0.007, see Table 11.1 and Figure

11.3). Following cessation of treatment there was a degree of worsening of eye grades in

the treatment group but they still remained significantly lower than those in the control

group.

Sheep in the treatment group showed a reduction in bacterial load for Moraxella ovis only

following IF treatment compared to the control group (P=0.004, see Table 11.1). There was

no difference seen between the treatments in Mycoplasma conjunctivae load.

170

Term Feed intake Water intake M.ovis Mycoplasma Eye grade

eye grade (cov) 0.035

feed intake (cov) 0.056 0.130

water intake (cov) 0.361 0.034

weight 0.826 0.914

day (time) <0.001 <0.001 <0.001

treatment (cont v IF) 0.049 0.285 0.004 0.231 0.007

day.treatment <0.001 0.054 0.008

Table 11.1 Significance levels (P values) for terms used in models to analyse data. Cov =covariate

171

0

200

400

600

800

1000

1200

1400

1600

0 1 2 3 4 5 6 7 8 9 10

fee

d in

take

g/h

ead

/day

Day

Control

IF

0

1000

2000

3000

4000

5000

6000

0 1 2 3 4 5 6 7 8 9 10

Wat

er

inta

ke m

L/h

ead

/day

Day

Control

IF

Figure 11.1 Feed intake (g/hd/day) of Control vs IF treatments over time. Shaded box represents treatment period. Error bars represent standard errors.

Figure 11.2 Water intake (ml/hd/day) of Control vs IF treatments over time. Shaded box represents treatment period. Error bars represent standard errors.

172

Despite the reduction in intake of medicated pellets, a significant improvement in clinical

eye grade within the IF treatment group was seen in this experiment. In line with the results

in Chapter 10, the sheep in the treatment group exceeded the minimum intake of 7.1 MJ

ME required for maintenance. During the treatment period the mean intake was 9.1 MJ ME

and throughout the whole experiment period the mean intake was 10.8 MJ ME for those in

the IF group.

11.5 Discussion

This experiment was designed to determine if in-feed (IF) medication had any effect on

clinical IOK. It has been shown that there was a reduction in feed intake following

introduction of medicated pellets (Section 10.1). Despite the reduction in intake of

medicated pellets, a significant improvement in clinical eye grade within the IF treatment

group was seen in this experiment. In line with the results in Chapter 10, the sheep in the

treatment group exceeded the minimum intake of 7.1 MJ ME required for maintenance.

0

1

2

3

4

0 2 4 7

Me

an e

ye g

rad

e

Day

control

IF

Figure 11.3 Change in eye grade over time - Control vs IF treatments. Shaded box represents treatment period. Error bars represent standard errors.

173

During the treatment period the mean intake was 9.1 MJ ME and throughout the whole

experiment period the mean intake was 10.8 MJ ME for those in the IF group.

In contrast to the results when OTC was included in water, the results of this experiment

show the degree of feed and water intake reduction was lower when OTC is added to the

pelleted ration. It is likely that the difference relates to the difference in palatability

between in-feed and in-water medications, with OTC in-feed being more palatable than OTC

in-water.

Bacteriology samples taken before and after treatment indicated that effective

concentrations of OTC were reaching the ocular tissue resulting in the reduced bacterial

load seen post-treatment. This is supported by experimental work in Chapter 8 that showed

that following administration of OTC in feed, detectable concentrations were achieved in

the plasma. For an antimicrobial drug to be effective it must exceed the Minimum Inhibitory

Concentration (MIC) of the target bacteria. The MIC of Mycoplasma conjunctivae for OTC is

reported to range from 0.78-6.2 µg/mL depending on the strain (Egwu, 1992a). The MIC for

Moraxella ovis for OTC is 0.5 µg/mL (Catry et al., 2007). Experimental work reported in

Chapter 8 indicated that peak plasma concentrations above the MIC for both bacteria were

achieved.

Oxytetracycline is a bacteriostatic antibiotic (Neu, 1978) and as such only inhibits the growth

of the bacteria through interference with protein synthesis, and does not kill the bacteria.

Most bacteriostatic antimicrobials will also kill a large proportion of bacteria, 90-99%, but

not a sufficient proportion to be classed as bacteriocidal (Pankey and Sabath, 2004). The

inhibition of the bacteria by OTC then relies on the host immune system to remove the

organism (Taylor et al., 2002).

174

Notably, although there was a decrease in Moraxella ovis colonies following IF treatment,

there appeared to be very few other organisms grown from the final sample and in some

cases a pure culture of M. ovis was present following IF treatment. This could potentially

contribute to the slight deterioration in clinical eye grades following cessation of treatment.

The positive results that followed five days of in-feed medication suggest that extending the

duration of treatment might improve the overall result. Sheep are typically in the feedlot for

seven days, therefore an extended course would be feasible. Further studies would be

required to fully assess the effect of altering the treatment course in this way.

11.6 Conclusion

Treating sheep with OTC medicted pellets for 5 days effectively reduces clinical eye grades

and bacterial load. Feed and water intakes are reduced, however adequate levels are

consumed throughout treatment for maintenance.

175

12 The treatment of infectious ovine keratoconjunctivitis with in-

feed medication in a pre-embarkation feedlot.

12.1 Introduction

It was important to test whether the positive responses seen clinically in sheep treated with

OTC medicated feed in controlled animal house environments were replicated in an actual

pre-embarkation feedlot. Up until this point all experiments had focused on small numbers

of animals with frequent and close individual monitoring. The experiment reported here

mimicked the feedlot environment. This experiment was designed to validate the previous

results in an industry context so that the efficacy of in-feed treatment of IOK could be

demonstrated as a practical, affordable and realistic option for exporters. Both productivity

and animal welfare were hypothesised to improve should the results concur with previous

research.

A range of severity of infection was considered in this research, based on selection of sheep

with eye grades between 1 and 6. The experiment aimed to assess the efficacy against

varying grades of IOK of in-feed OTC medication compared with two injections of OTC,

which has been consistently found to be the optimum treatment.

12.2 Hypotheses

Oxytetracycline-medicated feed will be effective in treating mild, up to and including

grade 3, IOK

Two injections of OTC will be effective against all grades of IOK

176


Two hundred and seven Merino cross, mixed age sheep with naturally occurring clinical IOK

were selected from those rejected at a pre-export feedlot. No sheep had received treatment

prior to selection.

Sheep eyes were graded using the grading system previously described (Appendix 1). All

sheep had clinical IOK in both eyes, not necessarily of equal severity. Following grading,

sheep were drafted into two groups, one group with clinical eye grades 2-4 and the other

with clinical eye grades 5-6. From these groups, sheep were randomly drafted into 3

treatment groups:

Group 1 - control group receiving no treatment (n=69 with 40 grade 2-3 and 29 grade 4-5)

Group 2 - intra-muscular injection OTC (Alamycin 300 LA, Nobrook Laboratories Australia

PTY Ltd, Tullamarine, VIC, Australia) (2 doses of 20mg/kg 4 days apart) (n=70 with 45 grade

2-3 and 25 grade 4-5)

Group 3 – in-feed OTC (Terramycin 200, Phibro Animal Health, Girraween, NSW, Australia)

(n= 68 with 36 grade 2-3 and 32 grade 4-5).

Sheep were housed in three separate raised pens in a standard feedlot. Free access to water

was given to all sheep. Those in groups 1 and 2 had ad lib access to pelleted feed as would

be typical in a feedlot. Sheep in group 3 received medicated feed, as described in Chapter

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10, for 5 days. Accurate measurements of feed intake were not taken, but subjective

assessment of residual feed was made; residual feed was removed on a daily basis. Accurate

measurement of residuals was not possible owing to contamination of feed and crumbling

of pellet diet. Ad lib access to non-medicated pellets was given following cessation of

medicated pellets.

Eye grades were recorded every second day from day 0 up to day 10 using the same grading

system as before, Appendix 2.

For each eye on each animal all pre-treatment eye grades were averaged. A linear mixed

model was fitted to the eye grade data on all post-treatment dates. The model included

fixed effects for pre-treatment eye grade, treatment, post-treatment date and treatment by

date interaction; and random effects for animal, the animal by date interaction and eyes

within dates and animals. An autoregressive model allowed for correlations between

measurements made on the same animal and eyes on different dates and different residual

variance on each date.

12.4 Results

Eye score on Day 0 was significantly associated with eye scores post treatment (P <0.001).

When treatment means were corrected for score on Day 0 there was a significant effect of

Treatment (P<0.001) on daily eye score but this effect interacted with day-post-treatment

such that the effect of treatment on eye score was different on different days post

treatment (P<0.001) (Table 12.1). Daily treatment eye score means (corrected for score on

Day 0) are shown in Figure 12.1

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At the end of the experiment animals treated with injections had lower eye scores than

those treated with in-feed medication. Both treatments had lower mean eye scores than

the control animals.

Fixed Term P Value

Day <0.001

Score on Day 0 <0.001

Treatment <0.001

Day.Treatment <0.001

Score on Day 0.Treatment <0.001

Table 12.1: Significance levels (P Values) for analysis of pink eye scores on each day post-treatment (control vs in-feed vs injection)

179

Figure 12.1 Adjusted IOK mean scores for each treatment and day.

The eye score on Day 0 significantly affected the degree of change of eye score over time

(P<0.001) (Table 12.2).

Fixed Term P value

Score on day 0 <0.001

Treatment <0.001

Score on day 0.Treatment 0.048

Table 12.2 Significance levels (P values) for analysis of change in pink eye score over time

180

Treatment Control In feed Injection

-0.898 -1.691 -2.318

Standard errors 0.1316 0.1322 0.1304

Table 12.3 Change in eye score from day 0 to day 10

At an average Score on day 0 value of 3.1, the decrease in eye score from Day 0 to Day 10

for the In-feed group was 0.793 (±0.186) (-0.898 vs -1.691 for the In-Feed and Control

groups respectively). The decrease in eye score from Day 0 to Day 10 for the Injection group

was 1.4206 (±0.186) more than for the Control group (-0.898 vs -2.318). The Injection group

had a significantly higher decrease in eye score than the In-Feed group (Table 12.3).

181

Figure 12.2 Average eye scores on each day, grouped according to Score on Day 0.

Treatment In feed

Treatment Injection

Treatment Control

3

2

5

4

0

9

2

6

4

3 0

0

3

3 6 9

5

1

0

1

Score v Day (1)

Score v Day (4)

Score v Day (3)

Score v Day (2)

Score v Day (5)

182

The eye score means according to the starting eye score are depicted in Figure 12.2. In-feed

OTC for 5 days was found to be an effective treatment for sheep with IOK up to and

including grade 3, whereas injectable OTC was effective in treating IOK up to and including

grade 5 (Figure 12.2, Table 12.5).

Score In-feed Injection

1 Treat Treat

2 Treat Treat

3 Treat Treat

4 Not effective Treat

5 Not effective Treat

Table 12.4: Treatment recommendations (Treat vs Not effective) of different routes of administration (In-feed vs Injection) at different degrees of severity of disease (Score) on day 8 of experiment.

183

12.5 Discussion

The results of the feedlot experiment are consistent with results seen in the previous

experiments. Although, subjectively, feed intake in the medicated feed group did decrease,

the amounts of residual feed remaining were small. In-feed OTC is considered effective in

treating sheep with eye grades up to and including 3. The hypothesis was that in-feed OTC

would be effective up to grade 4; however, the experiment demonstrated that those sheep

with grades 4 and 5 need to be treated with intra-muscular OTC to have the best chance of

recovery to the point where they could be shipped.

The use of in-feed medication is significantly less labour intensive than injecting individual

animals which greatly reduces the cost of the treatment. Within a cohort of sheep at the

pre-export feedlot there is likely to be variation in severity of clinical signs of IOK. Survey

results from inspectors outlined in Chapter 4 indicated that up to 50% of a shipment could

have mild IOK during the high-risk summer period. Given the numbers of animals involved

and the labour required to inject those with mild disease it is encouraging that in-feed

medication is an effective treatment. Given that IOK is considered a painful condition (Greig,

1989; Akerstedt, 2004) having an effective treatment available that requires minimal labour

could contribute to improved welfare of animals in pre-export feedlots.

184

13 General Discussion

Infectious Ovine Keratoconjunctivitis continues to affect the live export industry, both in

pre-export feedlots and onboard voyages. The live export trade attracts some of the most

consistent attention from the public in relation to animal welfare (O'Flynn and O'Dea, 1996).

With ever-increasing scrutiny on the industry it is important to address issues that may

compromise animal welfare and give the public reason for concern. IOK is a multifactorial

disease with several aetiological agents and is associated with numerous risk factors. These,

along with the potential for the development of a carrier state make treating disease and

keeping the sheep free from disease within the export chain a significant challenge.

Infectious ovine keratoconjunctivitis is a common disease of sheep worldwide. Although

numerous authors have investigated causes of the disease (Livingston.C. W et al., 1965;

Cooper, 1967; Surman, 1973; Cooper, 1974; Hosie, 1988; Egwu et al., 1989; Naglic et al.,

2000) there still remains some debate as to the exact agent responsible. To date much of

the published research has investigated outbreaks in the northern hemisphere with few

reports of outbreaks in Australia and no investigations of the disease within pre-export

feedlots.

The unique environment of a pre-export feedlot poses many challenges to the control of

IOK. In the days leading up to a shipment the feedlot will receive up to 80000 sheep from a

number of different sources, some travelling a considerable distance. As discussed in

Chapter 4 this mixing of sheep poses a great risk to the spread of IOK through the exposure

185

of naïve sheep to carriers. Additionally the stress of mixing coupled with a dusty and often

hot environment further adds to the risk of developing clinical IOK.

The feedlots try to address some of the risk factors. Many of the holding pens have concrete

bases and all areas leading up to the unloading bays are concreted. This minimizes dust, but

with that number of sheep moving over a relatively short period of time dust will be

unavoidable. All holding and sorting pens are covered to provide shade, which reduces the

effect of UV light. Upon arrival sheep are sorted into ‘lines’ whereby they are grouped

according to their age and/or type. Keeping age groups together may theoretically be

beneficial in reducing the risk of exposure to carrier animals; however, the literature and the

responses to questionnaires in the current study highlight that there is no age group that is

specifically affected more than any other.

Although IOK is not typically associated with high mortality and therefore, superficially,

could be considered less important than other diseases, e.g. Salmonellosis, it is associated

with high morbidity. As discussed in Chapter 1 IOK is considered a painful condition and

does result in a reduction in feed intake and growth, and this coupled with the rejection

from a shipment leads to significant economic losses to the exporter.

Despite efforts by the feedlot management to mitigate some of the risk factors IOK still

remains an issue, therefore sourcing an effective treatment is of paramount importance for

addressing the welfare concerns raised by the presence of the disease, and for addressing

the economic concerns as well.

The initial pilot work of Chapter 5 concluded that injectable OTC was the most effective

treatment. This is in agreement with many studies from around the world (Andrews et al.,

186

1987; Greig, 1989; Hosie, 1995; Naglic et al., 1999; Kovac et al., 2003; Dun, 2009; Townsend,

2010). This initial pilot work was also useful in concluding that using topical OTC treatments

is no better than giving no treatment. Despite highlighting that injectable OTC was the most

effective treatment, there still remained the serious logistical problem of treating all the

large numbers of affected sheep in this way. If the feedlot were to rely solely on injectable

OTC it is likely that many of the milder cases would be left untreated, therefore controlling

outbreaks would be difficult and welfare issues associated with the disease would not be

addressed across all animals.

In-water medication initially showed promise in the pilot work described in Chapter 5. The

appeal of a mass medication modality is understandable because it means that it is not

necessary to handle animals individually, which is an obvious advantage given the numbers

of animals involved. Further experiments with in-water treatment have, however,

highlighted limitations. The addition of medication appeared to have a deleterious effect on

the palatability of water resulting in low intakes during the treatment period. This leads to

very low concentrations of OTC being consumed resulting in a poor clinical response.

Oxytetracycline appears to be more palatable when mixed in-feed, with a reduction in feed

intake observed but not to levels below those required for maintenance. Consistently, in-

feed medication has resulted in a good clinical response throughout the experiments. The

reduction in feed intakes observed appears to be temporary, with sheep returning to

intakes comparable with those pre-treatment following cessation of treatment. To date

experimental work has focused on a 5-day treatment period and it has been noted that

following initial improvement in clinical signs, clinical signs can begin to worsen in the 5-day

period following treatment. The researchers would recommend testing a longer period of

187

offering medicated feed – possibly seven days. Sheep are typically in the pre-export feedlot

for around 7 days and it may be possible to extend the treatment to this time. In addition to

extending the treatment period it would be beneficial to extend the post-treatment

monitoring period to assess whether treatment improves recovery rates over a 3 weeks

period compared to no treatment. Mimicing the risk factors that the sheep will be subject to

following leaving the feedlot and going to the ship would be difficult in an experimental

environment therefore longer term monitoring is unlikely to reflect true recovery rates

within the live export chain.

Although IOK is a significant cause of rejection from the export chain, there are a variety of

other issues present within the chain that must be considered when implementing a

treatment strategy. Inanition is one of the most important causes of death in sheep within

the live export chain alongside Salmonellosis (Norris et al., 1989a; Barnes et al., 2008).

Failure to eat pellets during the latter part of the quarantine feedlot period has been

recognized as a risk factor for deaths during the subsequent voyage (Norris et al., 1989a).

Non-feeders are at highest risk of dying from inanition and salmonellosis and one of the

keys to reducing this risk is to ensure sheep continue to eat consistently throughout the live

export chain process (Norris et al., 1989a). There are other factors to consider in relation to

onboard deaths, including the season of shipment and fatness of the animals (Higgs et al.,

1999). Links between inanition and the development of Salmonellosis have been well

documented in the literature (Higgs et al., 1993). Stressed sheep that do not eat are at risk

of developing clinical Salmonellosis as a result of environmental contamination (Higgs et al.,

1993). Although the sheep treated with in-feed OTC had a reduced feed intake, intake was

still maintained throughout the treatment period and returned rapidly to levels comparable

188

with pre-treatment levels soon after cessation of treatment. If mild IOK lesions were

allowed to progress it is likely that this would have a more significant impact on feeding

than treating with in-feed OTC.

Advocating the use of antibiotics in-feed, or indeed in-water, could be considered

irresponsible use of antibiotics. The development and presence of antimicrobial resistance

occurs naturally; however, the inappropriate use of antibiotics is known to be a factor in

accelerating the development of antimicrobial resistance (WHO, 2015). The Australian

Government has published a national strategy to minimize antibiotic misuse and

development of resistance (Australian Government, 2015). Although the Government

recognizes that the use of antibiotics in food animals is comparatively low in Australia

compared to worldwide figures, it is still essential to ensure that those that are used are

used appropriately.

Antimicrobial resistance develops through antimicrobial use which selects for resistant

organisms coupled with a genetic change in the organism (Levy and Marshall, 2004). The

genetic change confers resistance by altering the organisms’ ability to defend itself (Witte,

1998). This can be through altering the drug target site, altering metabolism to circumvent

affected pathways or through detoxifying the drug (Witte, 1998). These alterations can

result in resistance to multiple drugs. Tetracyclines work by inhibiting the protein synthesis

of the bacteria. This is the same mode of action as aminoglycosides, macrolides and

lincosamides (Levy and Marshall, 2004), therefore resistance to one can infer resistance to

drugs in the other groups.

Levy and Marshall (2004) refer to a study in chickens which were fed OTC in feed for growth

promotion which led to rapid development of tetracycline resistant E.coli within days but

189

after 2 weeks of use the E.coli was multi-resistant. Antibiotics used in feed for the purposes

of growth promotion are given at sub-therapeutic concentrations for prolonged periods of

time (Wegener, 2003), typically less than 200g per tonne and for greater than 2 weeks

(McEwen and Fedorka-Cray, 2002). Long term use of an antibiotic, for greater than 10 days,

is known to lead to selection of resistant bacteria, not just to that antibiotic but potentially

to others too (Levy and Marshall, 2004). In the 1960s the Swann Committee in the UK

identified the risk of using drugs which are important in human medicine as growth

promoters in animal feed (Witte, 1998). It is recognized that the use of antibiotics in feed at

sub-therapeutic concentrations has led to a reservoir of resistant bacteria, and this

prompted the European Union to ban the use of a number of medically important drugs for

use as growth promoters (Wegener, 2003). Wegener (2003) goes on to discuss the decrease

in prevalence of resistant bacteria found in food, food-producing animals and humans since

this ban was implemented. Although a risk, the contribution of antibiotic use in animal

health to the development of antibiotic resistance affecting humans is considered small and

in general is confined to enteric organisms (Levy and Marshall, 2004).

In contrast to the long-term use of in-feed antibiotics, short term use of antibiotics at

treatment concentrations for treatment of disease is considered responsible use. The use of

antibiotics can always be justified for limited treatment periods in response to disease, or as

a prophylactic against disease (McEwen and Fedorka-Cray, 2002). Tetracyclines are

considered to be of low importance in human medicine (Scott et al., 2012a) making their

choice a responsible one. A study in Canada found no significant link between tetracycline

administration to sheep in food or water and the development of resistant Campylobacter

190

sp. (Scott et al., 2012b), although an association was seen with the development of resistant

faecal E.coli spp.

Advocating the treatment of sheep at a pre-export feedlot with in-feed OTC is defensible on

a number of fronts. Firstly the evidence in the literature points to low risk of development of

resistant bacteria following short courses of in-feed antibiotics when given at therapeutic

doses in feed. The dose advocated is a therapeutic one and the evidence presented in

Chapters 7 and 8 highlight the clinical response to treatment. Secondly, relying on individual

treatment with injectable OTC will result in many sheep not being treated and therefore

compromising their welfare. Aside from the high numbers of animals involved, it can be

difficult to identify the early clinical signs of IOK in individual sheep when they are grouped

together as in a feedlot. The data presented in Chapter 4 highlights the high percentage of

mild cases that arrive at the feedlots; blanket treatment of groups with in-feed medication

is the most effective way of ensuring that all these cases are treated. The work in Chapter

11 has highlighted the limitations of in-feed medication for the treatment of those sheep

with more severe IOK, and given this evidence it would be inappropriate to treat these with

in-feed medication. The numbers affected with severe IOK are much lower and they are

potentially easier to spot making them more suitable candidates for intramuscular

treatment. In-feed OTC medication is not superior to injectable OTC in clinical effect, but it is

cheaper and easier to administer to large groups.

The stability of OTC in in-feed medication is unknown. In all the experiments the feed was

made up within a week of it being used. It is known that OTC is sensitive to degradation by

UV light (Christiano et al., 2010), therefore storage of medicated feed could be problematic.

Although the in-feed medication used in these experiments is registered for use in sheep in

191

Australia, it does not have a label claim for the treatment of IOK. Additionally no export

slaughter interval has been established to the researchers’ knowledge. These issues would

need to be considered prior to the widespread adoption of this treatment strategy.

Through a series of experiments it has been shown that IOK up to grade 5 is treatable. This

information should be useful for feedlot managers to enable them to instigate the most

effective treatment at the earliest possible opportunity. This should result in higher recovery

rates and fewer culls owing to IOK. This improvement in treatment outcomes will improve

animal welfare, which remains a key aim of those involved in the live animal export industry.

Ultimately the aim would be to prevent occurrence of IOK completely. However, as this is a

multi-factorial disease with a number of organisms and risk factors involved that is very

much easier said than done. In cattle, where the causative agent is more clearly understood,

vaccines have been developed to prevent disease. These vaccines are manufactured based

on the pili of Moraxella bovis which is important in the stimulation of immunity. As there

are a number of different pili the current vaccine provides little, if indeed any, protection

against clinical disease (Brown et al., 1998). Given the relative complexity of IOK it is unlikely

that a vaccine could be developed to provide adequate protection against outbreaks

occurring in the feedlots.

This work has identified a sound treatment protocol for use within the pre-export feedlots

in Western Australia. However, anecdotal evidence suggests that outbreaks of IOK continue

to occur on board ships. Tracing sheep through the live export chain beyond the feedlot was

outside the scope of this study. Further work is required to determine what, if any,

difference implementing this treatment protocol in the pre-export feedlot has on

subsequent outbreaks of IOK onboard the ships.

192

14 Conclusions

Infectious ovine keratoconjunctivitis is a disease that continues to challenge not only the live

export industry but also the farming community worldwide. The multitude of factors known

to contribute to the occurrence and severity of an outbreak continue to frustrate veterinary

practitioners and producers alike. Given the presence of a number of key risk factors within

the pre-export feedlots, IOK outbreaks would appear to be unavoidable. It is for this reason

that effective and realistic treatment strategies must be in place to ensure treatment of the

disease, optimization of animal welfare, reduction in economic losses and an assurance to

the general public that the industry is focused on the health and welfare of the animals it

exports.

193

15 Appendix 1

Determination of patterns in numbers of animals presenting with eye disease

at pre-export feedlots.

Controlling eye disease in sheep within the export industry represents an ongoing challenge.

Infectious ovine keratoconjunctivitis (IOK), Pink Eye, is a common cause for animals to be

rejected from shipments due to its infectious nature. Current estimates state that 0.5-1.0%

of all sheep are rejected due to Pink Eye infections. This figure only represents those

animals which are deemed to have severe Pink Eye and therefore have no commercial

value. Accurate figures representing the numbers of mild cases presenting at the feedlots

which will be treated do not exist.

The aim of this study is to get an estimation of numbers of animals seen at feedlot entry

inspections with mild or severe Pink Eye. This project forms part of a bigger project

addressing treatment and control options for Pink Eye within pre-export feedlots. The

project has been funded by Meat and Livestock Australia.

Completion of this study is entirely voluntary. The information gathered will remain

confidential, all data will be analysed and incorporated into a report for Meat and Livestock

Australia in addition to my PhD thesis.

My supervisor and I are happy to discuss with you any concerns you may have about this study.

Sincerely

194

Dr Fraser Murdoch Dr. Michael Laurence

PhD Candidate Senior Lecturer Production Animal Health

[email protected] [email protected]

This study has been approved by the Murdoch University Human Research Ethics Committee (Approval 2014/205). If you have any reservation or complaint about the ethical conduct of this research, and wish to talk with an independent person, you may contact Murdoch University’s Research Ethics Office (Tel. 08 9360 6677 (for overseas studies, +61 8 9360 6677) or e-mail [email protected]). Any issues you raise will be treated in confidence and investigated fully, and you will be informed of the outcome.

mailto:[email protected]



195

Infectious ovine keratoconjunctivitis (IOK), or Pink Eye, is a common problem within the pre-

export feedlots. Data gathered to date quantifies the numbers of rejections at entry to the

feedlot due to IOK. It would be useful to expand on this data and confirm anecdotal

evidence of seasonal and age related patterns. The following survey is designed to

determine an accurate estimate of numbers of sheep presenting with clinical signs of IOK at

the feedlots.

Definitions:

Mild IOK – sheep have reddening of the conjunctiva and discharge from the eyes but no

evidence of decreased vision (not walking into things)

Severe IOK – sheep will likely have clouding of the surface of the eye and do show

decreased vision.

1. For shipments in the following months please state what you consider to be the

minimum and maximum number of mild cases of IOK as a percentage:

Minimum Maximum

Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

196

May _______ _________

Winter: July _______ _________

August _______ _________


minimum and maximum number of cases of severe IOK as a percentage:

Minimum Maximum

Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

3. For shipments in the following months please state what you consider to be minimum and maximum numbers of sheep affected with IOK (separate percentage for mild and severe) in each class as a percentage of the total shipment:

Minimum Lambs Maximum Lambs

Mild/Severe Mild/Severe

197

Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

Minimum Wethers Maximum Wethers


Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

Minimum Ewes Maximum Ewes

198


Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

Minimum Rams Maximum Rams


Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

199


minimum and maximum numbers of sheep affected with IOK (separate percentage

for mild and severe) in each breed as a percentage of the total shipment:

Minimum Merino Maximum Merino


Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

Minimum Cross breed Maximum Cross Breed


200

Spring: October _______ _________

November _______ _________

Summer: January _______ _________

February _______ _________

Autumn: April _______ _________

May _______ _________

Winter: July _______ _________

August _______ _________

We thank you in advance for your time in completing this survey. All data is anonymous and

will be used solely for analysis as part of a PhD thesis. Please return completed surveys to:

Dr. Michael Laurence

School of Veterinary and Life Sciences

College of Veterinary Medicine

Murdoch University

Murdoch, WA 6150

or email to:

[email protected]

201

16 Appendix 2

Grade 0 – normal eye Grade 1 – epiphora (weeping eye)

Grade 2 – conjunctivitis Grade 3 – corneal oedema (clouding of the

cornea)

Grade 4 – corneal ulceration

Grade 5 - corneal neovascularisation

Grade 6 – chronic eye damage

202

17 Appendix 3

Taxon

(phyla, class, order family)

Percentage contribution*

C 1 C 10

C 11

C 12 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 H16 H20 H21 H22 H23 H24 L13 L14 L15 L17 L18 L19

Other; 1.5 2.4 1.9 2.0 2.3 0.6 1.3 3.6 3.6 2.9 1.2 1.5 20.0 9.5

11.6

37.1

15.6

27.8

14.6 0.5

17.8 4.8 1.2

18.1

Actinobacteria; Actinobacteria; Bifidobacteriales; 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.2 0.0 0.4 0.5 0.2 0.4 0.1 0.0 0.1 0.0 0.0 0.5

Bacteroidetes; Bacteroidia; Bacteroidales; Other 0.1 1.7 1.4 0.8 0.7 0.1 1.9 2.1 0.8 2.3 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Bacteroidetes; Bacteroidia; Bacteroidales; 1.9

15.5

15.5

13.7

12.1 5.1

18.1

13.0

12.0

10.7

10.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0

Bacteroidetes; Bacteroidia; Bacteroidales; Porphyromonadaceae 0.5 2.6 9.1 3.0 0.9 1.4 3.2 0.2 1.4 3.3 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.4 0.0

Bacteroidetes; Bacteroidia; Bacteroidales; Prevotellaceae 37.8

30.3

28.0

12.9

24.9

33.8

29.9

23.7

22.5

33.7

25.0

83.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0

203

Taxon



C 1 C 10

C 11


Bacteroidetes; Flavobacteria; Flavobacteriales; Flavobacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0

Cyanobacteria; 4C0d-2; YS2; 0.0 0.1 0.0 0.4 0.0 0.0 0.0 0.0 0.8 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Elusimicrobia; Endomicrobia 0.0 0.6 0.0 0.2 0.1 0.1 0.1 0.0 0.3 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Fibrobacteres; Fibrobacteres; Fibrobacterales; Fibrobacteraceae 8.3

18.5

17.2

10.9

31.1 5.8

11.9

24.9

28.8

16.1

22.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Firmicutes; Bacilli; Lactobacillales; Enterococcaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 0.5 1.1 0.3 5.4 0.5 0.0 0.0 0.4 0.4 1.3 1.3

Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.9 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Firmicutes; Bacilli; Lactobacillales; Streptococcaceae 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.1 0.0

Firmicutes; Clostridia;

0.3 1.8 2.2 2.3 0.4 0.4 4.4 0.7 1.3 2.3 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.8 0.0 0.0 0.0 0.0

204

Taxon



C 1 C 10

C 11


Clostridiales; Other

Firmicutes; Clostridia; Clostridiales; 1.3 4.7 4.0 3.8 3.5 3.8 6.7 3.3 6.3 4.1 6.4 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0

Firmicutes; Clostridia; Clostridiales; Catabacteriaceae 0.1 0.0 0.0 0.2 0.3 0.2 0.2 0.0 0.1 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Firmicutes; Clostridia; Clostridiales; Eubacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.3

Firmicutes; Clostridia; Clostridiales; Lachnospiraceae 7.4 6.9 2.9 7.8 5.4 2.1 7.2

11.8 7.5 5.7

12.5 8.9

41.4

80.8

65.6

55.8 6.5

56.5

68.6

34.1

75.2

79.0

26.3

71.6

Firmicutes; Clostridia; Clostridiales; Ruminococcaceae 1.4 1.6 1.1 3.0 1.2 2.0 4.3 2.6 1.6 4.8 3.8 0.0

28.4 0.0 0.0 0.0 0.1 0.0

13.7

60.5 0.0 0.2 0.1 0.0

Firmicutes; Clostridia; Clostridiales;

0.8 2.1 1.9 2.6 2.3 3.9 1.9 2.9 1.8 3.4 2.1 2.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

205

Taxon



C 1 C 10

C 11


Veillonellaceae

Fusobacteria; Fusobacteria; Fusobacteriales; Fusobacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0

Lentisphaerae; Lentisphaerae; Victivallales; Victivallaceae 0.0 1.5 4.6 0.8 1.0 0.3 1.0 0.3 0.1 4.2 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Lentisphaerae; Lentisphaerae; Z20; 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.2 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Planctomycetes; Planctomycea; Pirellulales; 0.0 0.3 0.0 0.3 0.3 0.0 0.2 0.5 1.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteobacteria; Alphaproteobacteria; Other; Other 0.0 2.4 0.0 0.6 1.6 0.1 0.9 0.9 0.9 0.1 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteobacteria; Alphaproteobacteria 0.0 0.5 0.1 1.7 0.3 0.8 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteobacteria; Betaproteobacteria; Neisseriales;

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0

206

Taxon



C 1 C 10

C 11


Neisseriaceae

Proteobacteria; Gammaproteobacteria; Other; Other 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.4 0.0

Proteobacteria; Gammaproteobacteria; Aeromonadales; 35.2 0.2 2.4

25.7 0.2

37.6 0.1 1.0 0.1 0.0 0.4 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteobacteria; Gammaproteobacteria; Aeromonadales; Succinivibrionaceae 0.9 0.3 0.1 2.4 0.6 0.0 0.1 1.7 0.9 0.1 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteobacteria; Gammaproteobacteria; Enterobacteriales; Other 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.1 0.0 0.1

Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 7.7 9.1

20.7 6.2

71.7

14.6 1.4 3.3 5.9

15.5

63.8 8.1

Proteobacteria; Gammaproteobacteria; Pasteurellales; Pasteurellaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0

207

Taxon



C 1 C 10

C 11


Proteobacteria; Gammaproteobacteria; Pseudomonadales; Moraxellaceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.1 0.0 3.1 0.0

SR1; 0.0 0.0 0.0 0.4 0.0 0.3 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Spirochaetes; Other; Other; Other 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Spirochaetes; Spirochaetes; Other; Other 0.1 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Spirochaetes; Spirochaetes; Spirochaetales; Spirochaetaceae 1.3 2.2 0.6 2.0 8.3 1.1 2.3 4.7 3.4 2.1 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0

Synergistetes; Synergistia; Synergistales; Dethiosulfovibrionaceae 0.0 0.0 0.1 0.4 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

TM7; TM7-3; CW040; F16 0.0 0.1 0.0 0.2 1.3 0.1 0.1 0.1 0.7 0.6 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

208

Taxon



C 1 C 10

C 11


Tenericutes; Other; Other; Other 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.2 0.3 0.0 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Tenericutes; Erysipelotrichi; Erysipelotrichales; Erysipelotrichaceae 0.0 0.1 0.2 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.2 0.0 0.2 0.0 0.0 0.0 0.2 0.0 0.5 0.0 1.2 0.1

Tenericutes; Erysipelotrichi; Erysipelotrichales; vadinHA31 1.2 2.7 6.2 1.4 0.9 0.3 3.2 1.1 2.6 0.5 4.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Tenericutes; Mollicutes; Anaeroplasmatales; Anaeroplasmataceae 0.0 0.3 0.2 0.2 0.0 0.0 0.4 0.1 0.9 0.6 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Tenericutes; Mollicutes; Mycoplasmatales; Mycoplasmataceae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0

Tenericutes; Mollicutes; RF39; 0.0 0.4 0.0 0.2 0.0 0.0 0.2 0.0 0.1 0.2 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

209

18 References

Abd El-Aty, A., A. Goudah, and H.-H. Zhou. 2004. Pharmacokinetics of doxycycline after administration as a single intravenous bolus and intramuscular doses to non-lactating Egyptian goats. Pharmacological research 49: 487-491.

Ajite, K. O., and O. C. Fadamiro. 2013. Prevalence of harmful/traditional medication use in traumatic eye injury. Global journal of health science 5: p55.

Akerstedt, J. 2004. Bacteriological investigation of infectious keratoconjunctivitis in Norwegian sheep. Acta veterinaria scandinavica 45: 19-26.

Al-Nazawi, M. H. 2003. Comparative pharmacokinetic studies on oxytetracycline in camels, sheep and goats. Pakistan Veterinary Journal 23: 187-191.

Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.

Andrews, A., P. Goddard, A. Wilsmore, and G. Dagnell. 1987. A chlamydial keratoconjunctivitis in a British sheep flock. Veterinary Record 120: 238-239.

Angelos, J. A., P. Q. Spinks, L. M. Ball, and L. W. George. 2007. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. International Journal of Systematic and Evolutionary Microbiology 57: 789-795.

Arbuckle, J. B., and M. D. Bonson. 1980. The isolation of Acholeplasma oculi from an outbreak of ovine keratoconjunctivitis. Veterinary Record 106: 15.

Arnold, G., E. Boer, and C. Boundy. 1980. The influence of odour and taste on the food preferences and food intake of sheep. Australian Journal of Agricultural Research 31: 571-587.

Australian Government. 2015. Australia's first national antimicrobial resistance stategy 2015-2019. In: Deaprtment of Health and Department of Agriculture (ed.), Canbera.

Australian Government, and Australian Pesticides And Veterinary Medicine Authority. 2014. Substances Not Permitted for use on Food-Producing Animals in Australia No. 2014, Canberra.

Australian Government Department of Agriculture, F. a. F., ,. 2011. Australian Standards for the Export of Livestock (Version 2.3).

Axelsen, A. 1961. Effect of contagious ophthalmia on multiple lambing and sheep liveweight. Australian Veterinary Journal 37: 60-62.

Baas, E. J., S. L. Trotter, R. M. Franklin, and M. F. Barile. 1977. Epidemic caprine keratoconjunctivitis: recovery of Mycoplasma conjunctivae and its possible role in pathogenesis. Infection and Immunity 18: 806-815.

Baggot, J. D. 2004. The pharmacokinetic basis of species variation in drug disposition. In: J. D. Baggot (ed.) The physiological basis of veterinary clinical pharmacology. p 2-3. Blackwell Science.

Baggot, J. D. 2008. The Concept of Bioavailability and applications to veterinary dosage forms. In: J. D. Baggot (ed.) The Physiological Basis of Veterinary Clinical Pharmacology. p 60. Wiley-Blackwell.

Baggot, J. D., and S. Giguere. 2013. Principles of Antimicrobial Drug Bioavailablity and Disposition. In: J. F. P. a. P. M. D. Steeve Giguere (ed.) Antimicrobial Therapy in Veterinary Medicine. Wiley Blackwell.

Baker, J., W. Faull, and W. Ward. 1965. Conjunctivitis and keratitis in sheep associated with Moraxella (Haemophilus) organisms. The Veterinary Record 77: 402.

Baker, S., J. Bashiruddin, R. Ayling, and R. Nicholas. 2001. Molecular detection of Mycoplasma conjunctivae in English sheep affected by infectious keratoconjunctivitis. Veterinary Record 148: 240-241.

Ball, A., J. Thompson, C. Alston, A. Blakely, and G. Hinch. 1998. Changes in maintenance energy requirements of mature sheep fed at different levels of feed intake at maintenance, during weight loss and realimentation. Livestock production science 53: 191-204.

Bandyopadhyay, H., T. Biswas, D. Sasmal, I. Samanta, and M. Ghosh. 2010. Evaluation of methanolic extract of Allium sativum and Saussurea costus in yaks with infectious keratoconjunctivitis. The Indian Journal of Animal Sciences 80.

Barnes, A., D. Beatty, C. Stockman, and D. Miller. 2008. Inanition of Sheep. Bartley, E., K. Wheatcroft, T. Claydon, F. Fountaine, and D. Parrish. 1951. Effects of feeding aureomycin to

dairy calves. In: Journal of Animal Science. p 1036-1036. Bell, M. C., C. K. Whitehair, and W. D. Gallup. 1951. The Effect of Aureomycin on Digestion in Steers.

Experimental Biology and Medicine 76: 284-286.

210

Belloy, L., M. Janovsky, E. M. Vilei, P. Pilo, M. Giacometti, and J. Frey. 2003. Molecular Epidemiology of Mycoplasma conjunctivae in Caprinae: Transmission across Species in Natural Outbreaks. Applied and Environmental Microbiology 69: 1913-1919.

Bermingham, E. C. 2002. Pharmacokinetics after intravenous and oral administration of enrofloxacin in sheep. American Journal of Veterinary Research 63: 1012-1017.

Beveridge, W. I. B. 1942. Investigations on contagious ophthalmia of sheep, with special attention to the epidemiology of infection by Rickettsia conjunctiva. Australian Veterinary Journal 18: 155-164.

Blakemore, F. 1947. Conjunctivitis and Keratitis of Cattle and Sheep Associated with the Presence of Cell-Inclusion Bodies. Journal of Comparative Pathology and Therapeutics 57: 223-231.

Bonner, P. L. R., and A. J. Hargreaves. 2011. Streak Dilution, Basic Bioscience Laboratory Techniques: A Pocket Guide. John Wiley and Sons Ltd, Hoboken, United States of America.

Boothe, D. M. 2001a. Antimicrobial Drugs In: D. M. Boothe (ed.) Small Animal Clinical Pharmacology and Therapeutics. p 169-170. W.B. Saunders Company.

Boothe, D. M. 2001b. Principles of antimicrobial therapy. In: D. M. Boothe (ed.) Small animal clinical pharmacology and therapeutics. p 131. W.B. Saunders Company.

Bray, J. R., and J. T. Curtis. 1957. An Ordination of the Upland Forest Communities of Southern Wisconsin. Ecological Monographs 27: 325-349.

Briggs, P., J. Hogan, and R. Reid. 1957. Effect of volatile fatty acids, lactic acid and ammonia on rumen pH in sheep. Crop and Pasture Science 8: 674-690.

Brightman, A., S. McLaughlin, and V. Brumley. 1981. Keratoconjunctivitis in a llama. Veterinary medicine, Small Animal Clinician 76: 1776.

Brosh, A., B. Sneh, and A. Shkolnik. 1983. Effect of severe dehydration and rapid rehydration on the activity of the rumen microbial population of black Bedouin goats. The Journal of Agricultural Science 100: 413-421.

Brown, M. H., A. H. Brightman, B. W. Fenwick, and M. A. Rider. 1998. Infectious Bovine Keratoconjunctivitis: A Review. Journal of Veterinary Internal Medicine 12: 259-266.

Busch, T. J. 1988. Infectious keratoconjunctivitis in goats. New Zealand veterinary journal 36: 153-155. Buswell, J., and G. Hewett. 1983. Single topical treatment for bovine keratoconjunctivitis using benzathine

cloxacillin. Veterinary Record 113: 621-622. Caporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. D. Bushman, E. K. Costello, N. Fierer, A. G. Pena, J.

K. Goodrich, J. I. Gordon, G. A. Huttley, S. T. Kelley, D. Knights, J. E. Koenig, R. E. Ley, C. A. Lozupone, D. McDonald, B. D. Muegge, M. Pirrung, J. Reeder, J. R. Sevinsky, P. J. Turnbaugh, W. A. Walters, J. Widmann, T. Yatsunenko, J. Zaneveld, and R. Knight. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat Meth 7: 335-336.

Castro-Carrera, T., P. G. Toral, P. Frutos, N. McEwan, G. Hervas, L. Abecia, E. Pinloche, S. Girdwood, and A. Belenguer. 2014. Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae. Journal of Dairy Science 97: 1661-1669.

Castro, L. J., A. M. Sahagún, M. J. Diez, N. Fernández, M. Sierra, and J. J. García. 2009. Pharmacokinetics of doxycycline in sheep after intravenous and oral administration. The Veterinary Journal 180: 389-395.

Catry, B., F. Boyen, M. Baele, J. Dewulf, A. de Kruif, M. Vaneechoutte, F. Haesebrouck, and A. Decostere. 2007. Recovery of Moraxella ovis from the bovine respiratory tract and differentiation of Moraxella species by tDNA-intergenic spacer PCR, . Veterinary Microbiology 120: 375-380.

Chambers, H. F. 2006. Protein Synthesis Inhibitors and Miscellaneous Antibacterial Agents. In: L. L. Brunton, S. J. Lazo and K. L. Parker (eds.) Goodman & Gilman's The Pharmacological Basis of Therapeutics, . p 1173-1179. McGraw-Hill.

Chaucheyras-Durand, F. d. r., and F. Ossa. 2014. Review: The rumen microbiome: Composition, abundance, diversity, and new investigative tools. The Professional Animal Scientist 30: 1-12.

Cheng, A. C., J. Turnidge, P. Collignon, D. Looke, M. Barton, and T. Gottlieb. 2012. Control of fluoroquinolone resistance through successful regulation, Australia. Emerging Infectious Diseases, 18: 1453.

Chilcott, R. P. 2006. Compendium of Chemical Hazards : Kerosene (Fuel oil). Health Protection Agency, Chemical Hazards and Poisons Division,, Chilton, United Kingdom.

211

Christiano, R. S. C., C. C. Reilly, W. P. Miller, and H. Scherm. 2010. Oxytetracycline Dynamics on Peach Leaves in Relation to Temperature, Sunlight, and Simulated Rain. Plant Disease 94: 1213-1218.

Clarke, K. R. 1993. Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18: 117-143.

Coles, J. D. W. A. 1931. An unknown intracellular organism of the conjunctival epithelium of sheep. Preliminary Reports: 17th Report of the Director of Veterinary Services and Animal Industry, Onderstepoort, Union of South Africa: 187-189.

Cooper. 1961. Treatment of Conjunctivokeratitis of sheep and cattle with ethidium bromide. II Contagious Conjunctivokeratitis of sheep. Veterinary Record 73: 409.

Cooper, B. 1967. Contagious conjunctivo-keratitis (CCK) of sheep in New Zealand. New Zealand veterinary journal 15: 79-84.

Cooper, B. S. 1974. Transmission of a Chlamydia like agent isolated from contagious conjunctivo keratitis of sheep. New Zealand veterinary journal 22: 181-184.

Cordier, G., and J. Menager. 1937. Existence de Rickettsia conjunctivae du Mouton, Coles 1931, en Tunisie. Bulletin de la Societe de Pathologie Exotique 30: 224-227.

Cunha, I. S., C. C. Barreto, O. Y. Costa, M. A. Bomfim, A. P. Castro, R. H. Kruger, and B. F. Quirino. 2011. Bacteria and Archaea community structure in the rumen microbiome of goats ( Capra hircus) from the semiarid region of Brazil. Anaerobe 17: 118-124.

Daigneault, J., and L. George. 1990. Topically applied benzathine cloxacillin for treatment of experimentally induced infectious bovine keratoconjunctivitis. American Journal of Veterinary Research 51: 376-380.

Davidson, H. J. 2009. Opthalmic Therapeutics. In: R. D. Anderson DE (ed.) Current Veterinary Therapy, Food Animal Practice

p445-448. Elsevier, St Louis, Missouri. Davidson, H. J., and V. J. Kuonen. 2004. The tear film and ocular mucins. Veterinary Ophthalmology 7: 71-

77. Davidson, H. J., and J. P. Pickett. 2009. Selected Eye diseases of cattle. In: R. D. Anderson DE (ed.) Current

Veterinary Therapy Food Animal Practice. p 421-427. Saunders Elsevier. Degiorgis, M., J. Frey, J. Nicolet, E.-M. Abdo, R. Fatzer, Y. Schlatter, S. Reist, M. Janovsky, and M. Giacometti.

2000. An outbreak of infectious keratoconjunctivitis in Alpine chamois (Rupicapra r. rupicapra) in Simmental-Gruyeres, Switzerland. SAT, Schweizer Archiv fur Tierheilkunde 142: 520-527.

Dehority, B. A. 2004. Gross Anatomy, physiology and environment of the ruminant stomach. In: B. A. Dehority (ed.) Rumen Microbiology. p 19-41. Nottingham Universtiy Press.

del Castillo, J. R. E. 2013. Tetracyclines. In: J. F. P. a. P. M. D. Steeve Giguere (ed.) Antimicrobial Therapy in Veterinary Medicine. p 257-268. Wiley Blackwell.

Devriese, L. A., M. Baele, M. Vaneechoutte, A. Martel, and F. Haesebrouck. 2002. Identification and antimicrobial susceptibility of Staphylococcus chromogenes isolates from intramammary infections of dairy cows. Veterinary Microbiology 87: 175-182.

Dickinson, L., and B. S. Cooper. 1959. Contagious conjunctivo-keratitis of sheep. The Journal of Pathology and Bacteriology 78: 257-266.

Doetsch, R. N., and R. Q. Robinson. 1953. The bacteriology of the bovine rumen: a review. Journal of Dairy Science 36: 115-142.

Donatien, A., and F. Lestoquard. 1937. Existence de Rickettsia conjunctivae du mouton, Coles 1931, en Algerie. Bull. Soc. path. exot 30: 18-20.

Dougherty, R. 1955. Permanent stomach and intestinal fistulas in ruminants: some modifications and simplifications. The Cornell Veterinarian 45: 331.

Droumev, D., D. Pashov, M. Droumev, S. Vangelov, E. Velikova, I. Kanelov, L. Lashev, and R. Moutafchieva. 1992. Slow release bolus for small ruminants: in vitro release of tetracycline compared with serum concentrations of the antibiotic in sheep. In: Annales de Recherches Veterinaires. p 215-223.

Dun, K. 2009. Ocular Conditions in Sheep. Livestock 14: 41-44. Eastman, T. G., L. George, D. Hird, and M. Thurmond. 1998. Combined parenteral and oral administration of

oxytetracycline for control of infectious bovine keratoconjunctivitis. Journal of the American Veterinary Medical Association 212: 560-563.

Edgar, G. 1931. Studies in infectious ophthalmia of sheep. Veterinary Research Report Department of Agriculture, New South Wales 6: 22-35.

212

Egwu, G. 1991. Ovine infectious keratoconjunctivitis: an update. Veterinary Bulletin. Egwu, G., W. Faull, J. Bradbury, and M. Clarkson. 1989. Ovine infectious keratoconjunctivitis: a

microbiological study of clinically unaffected and affected sheep's eyes with special reference to Mycoplasma conjunctivae. Veterinary Record 125: 253-256.

Egwu, G. O. 1992a. In vitro antibiotic sensitivity of Mycoplasma conjunctivae and some bacterial species causing ovine infectious kerato-conjunctivitis. Small ruminant research 7: 85-92.

Egwu, G. O. 1992b. Outbreak of ovine infectious kerato-conjunctivitis caused by Mycoplasma conjuctivae. Small ruminant research 9: 189-196.

el-Sanousi, S., M. Eisa, and E. el-Badawi. 1978. Infectious bovine keratoconjunctivitis in association with vitamin A deficiency in a feedlot. Bulletin of animal health and production in Africa. Bulletin des sante et production animales en Afrique 26: 293.

Evans, A. L., R. F. Bey, J. V. Schoster, J. E. Gaarder, and G. L. Finstad. 2008. Preliminary Studies on the Etiology of Keratoconjunctivitis in Reindeer (Rangifer tarandus tarandus) Calves in Alaska. Journal of Wildlife Diseases 44: 1051-1055.

Farmer, B. 2011. Independant review of Australia's Livestock export trade, Canberra. Fernandez-Aguilar, X., O. Cabezon, I. Marco, G. Mentaberre, J. Frey, S. Lavin, and J. R. Lopez-Olvera. 2013.

Mycoplasma conjunctivae in domestic small ruminants from high mountain habitats in Northern Spain. BMC veterinary research 9: 253.

Fernando, S. C., H. T. Purvis, F. Z. Najar, L. O. Sukharnikov, C. R. Krehbiel, T. G. Nagaraja, B. A. Roe, and U. DeSilva. 2010. Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet. Applied and Environmental Microbiology 76: 7482-7490.

Flint, H. J. 1997. The rumen microbial ecosystem some recent developments. Trends in Microbiology 5: 483-488.

Flint, H. J., and C. S. Stewart. 1987. Antibiotic resistance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides multiacidus. Applied Microbiology and Biotechnology 26: 450-455.

Forbes, J. M. 2007a. Appetites for specific nutrients Voluntary food intake and diet selection in farm animals. p 281. CABI.

Forbes, J. M. 2007b. Protein and other Nutrients Voluntary food intake and diet selection in farm animals. p 258. CABI.

Formenty, P., and J. Domenech. 1992. Une epidemie de keratoconjonctivite granuleuse ovine, d'origine chlamydienne, en Cote-d'Ivoire. Revue d'Elevage et de medecine veterinaire des pays tropicaux 45.

Freeman, K. S., S. M. Thomasy, S. D. Stanley, W. Van Bonn, F. Gulland, A. S. Friedlaender, and D. J. Maggs. 2013. Population pharmacokinetics of doxycycline in the tears and plasma of northern elephant seals (Mirounga angustirostris) following oral drug administration. Journal of the American Veterinary Medical Association 243: 1170-1178.

Fthenakis, G. C. 1994. Prevalence and aetiology of subclinical mastitis in ewes of Southern Greece. Small ruminant research 13: 293-300.

Fuller, M. F. 2004. Transit Time. In: M. F. Fuller (ed.) The encyclopedia of farm animal nutrition. p 554. CABI Publishing.

Gall, L. S., C. N. Stark, and J. K. Loosli. 1947. The Isolation and Preliminary Study of Some Physiological Characteristics of the Predominating Flora from the Rumen of Cattle and Sheep1. Journal of Dairy Science 30: 891-899.

Geier, M. S., L. L. Mikkelsen, V. A. Torok, G. E. Allison, C. G. Olnood, M. Boulianne, R. J. Hughes, and M. Choct. 2010. Comparison of alternatives to in-feed antimicrobials for the prevention of clinical necrotic enteritis. Journal of Applied Microbiology 109: 1329-1338.

Geier, M. S., V. A. Torok, G. E. Allison, K. Ophel-Keller, and R. J. Hughes. 2009. Indigestible carbohydrates alter the intestinal microbiota but do not influence the performance of broiler chickens. Journal of Applied Microbiology 106: 1540-1548.

George, L. W. 1985. Distribution of oxytetracycline into ocular tissues and tears of calves. Journal of Veterinary Pharmacology and Therapeutics 8: 47-54.

Georges, G., J. Evans, J. M. Hearn, D. B. Scott, D. Brownhill, B. Cooney, and J. R. Siddons. 1985. Export of Live Sheep from Australia. In: Senate Select Committee on Animal Welfare (ed.). Canberra publishing and printing, Canberra.

213

Gibson, G. G., and P. Skett. 2001a. The clinical relevance of drug metabolism. In: G. G. Gibson and P. Skett (eds.) Introduction to Drug Metabolism. p 203-242. Nelson Thornes.

Gibson, G. G., and P. Skett. 2001b. Pharmacological and Toxicological aspects of drug metabolism. In: G. G. Gibson and P. Skett (eds.) Introduction to Drug Metabolism. p 180-181. Nelson Thornes.

Goatcher, W., and D. Church. 1970a. Taste responses in ruminants. III. Reactions of pygmy goats, normal goats, sheep and cattle to sucrose and sodium chloride. Journal of Animal Science 31: 364-372.

Goatcher, W. D., and D. C. Church. 1970b. Taste Responses in Ruminants. I. Reactions of Sheep to Sugars, Saccharin, Ethanol and Salts. Journal of Animal Science 30: 777-783.

Gortazar, C., D. Fernandez-de-Luco, and K. Frolich. 1998. Keratoconjunctivitis in a free-ranging red deer(Cervus elaphus) population in Spain. Zeitschrift fÃ¼r Jagdwissenschaft 44: 257-261.

Graham-Smith, G. 1930. The Oscinidae (Diptera) as vectors of conjunctivitis, and the anatomy of their mouth parts. Parasitology 22: 457-467.

Grahn B.H., and R. L. Peiffer. 2007. Fundamentals of Veterinary Ophthalmic Pathology. In: K. N. Gelatt (ed.) Veterinary Ophthalmology No. 1. p 391-392. Blackwell.

Greeff, J., and L. Karlsson. 1997. Genetic relationships between faecal worm egg count and scouring in Merino sheep in a Mediterranean environment.

Greig, A. 1989. Ovine keratocon junctivitis. In Practice 11: 110-113. Groverman, F. A. 1992. The effects of disease and parasites on wool growth. Wool Production School,: 21. Grubb, T. L., J. R. Gold, J. W. Schlipf, A. M. Craig, K. C. Walker, and T. W. Riebold. 2005. Assessment of serum

concentrations and sedative effects of fentanyl after transdermal administration at three dosages in healthy llamas. American Journal of Veterinary Research 66: 907-909.

Hadani, Y., I. Lysnyansky, A. Bareli, M. Bernstein, D. Elad, R. Ozeri, D. Rotenberg, V. Bumbarov, S. Perl, and J. Brenner. 2013. An Unusual Widespread Outbreak of Blindness Caused by Mycoplasma conjunctivae on a Large Dairy Goat Farm. Israel Journal of Veterinary Medicine 68: 3.

Harrigan, W. F., and M. E. McCance. 2014. Laboratory methods in microbiology. Academic Press. Hayes Jr, J. E., and H. G. DuBuy. 1964. A simple method for quantitative estimation of tetracycline

antibiotics. Analytical Biochemistry 7: 322-327. Herdt, T. 2002. Digestion: the fermentative processes. In: J. G. Cunnigham (ed.) Textbook of Veterinary

Physiology. p 293-294. Higgs, A., R. Norris, R. Love, and G. Norman. 1999. Mortality of sheep exported by sea: evidence of similarity

by farm group and of regional differences. Australian Veterinary Journal 77: 729-733. Higgs, A., R. Norris, and R. Richards. 1993. Epidemiology of salmonellosis in the live sheep export industry.

Australian Veterinary Journal 70: 330-335. Hobson, P. 1969. Rumen bacteria. Methods in microbiology 3: 133-149. Hofland, G., P. Leeflang, and T. d. Vries. 1969. Contagious kerato-conjunctivitis of sheep in the Netherlands.

Tijdschr Diergeneesk. Hofmeister, V. 1881. Ueber Celluloseverdauung. Archival wissenschaftl. u. prakt. Tierheilkunde. Hopkins, J., E. Stephenson, J. Storz, and R. Pierson. 1973. Conjunctivitis associated with chlamydial

polyarthritis in lambs. Journal of the American Veterinary Medical Association. Horn, L. H., R. R. Snapp, and L. S. Gall. 1955. The Effect of Antibiotics upon the Digestion of Feed Nutrients

by Yearling Steers, with Bacteriological Data. Journal of Animal Science 14: 243-248. Hosie, B. 1989. Infectious keratoconjunctivitis in sheep and goats. Veterinary annual 1989. Hosie, B. D. 1988. Kertoconjuctivitis in a hill sheep flock. Veterinary Record 122: 40-43. Hosie, B. D. 1995. Role of oxytetracycline dihydrate in the treatment of mycoplasma associated ovine

keratoconjunctivitis in lambs. British veterinary journal 151: 83. Hungate, R. E. 1966a. Introduction. In: R. E. Hungate (ed.) The Rumen and its Microbes. p 1-7. Academic

Press Inc. Hungate, R. E. 1966b. The Rumen Bacteria. In: R. E. Hungate (ed.) The Rumen and its Microbes. p 8-81.

Academic Press Inc. Janovsky, M., J. Frey, J. Nicolet, L. Belloy, E. Goldschmidt-Clermont, and M. Giacometti. 2001. Mycoplasma

conjunctivae infection is self-maintained in the Swiss domestic sheep population. Veterinary Microbiology 83: 11-22.

Jones, G. E. 1983. Mycoplasmas of sheep and goats: a synopsis. Veterinary Record 113: 619-620. Jones, G. E., A. Foggie, A. Sutherland, and D. B. Harker. 1976. Mycoplasmasand ovinekeratoconjunctivitis.

Veterinary Record 99: 137-141.

214

Kamra, D. 2005. Rumen microbial ecosystem. Current Science 89: 124-135. Kang, S. H., P. Evans, M. Morrison, and C. McSweeney. 2013. Identification of metabolically active

proteobacterial and archaeal communities in the rumen by DNA- and RNA-derived 16S rRNA gene. Journal of Applied Microbiology 115: 644-653.

Kare, M. R., R. Black, and E. G. Allison. 1957. The Sense of Taste in the Fowl. Poultry Science 36: 129-138. Killinger, A., D. Valentine, M. Mansfield, G. Ricketts, G. Cmarik, A. Neumann, and H. Norton. 1977. Economic

impact of infectious bovine keratoconjunctivitis in beef calves. Veterinary Medicine and Small Animal Clinician.

Klopfenstein, T. J., D. B. Purser, and W. J. Tyznik. 1964. Influence of Aureomycin on Rumen Metabolism. Journal of Animal Science 23: 490-495.

Konig, C. 1983a. Review papers: Keratoconjunctivitis Infectiosa Ovis (KIO),'pink eye' or zere oogjes (a survey). Veterinary Quarterly 5: 127-130.

Konig, C. D. 1983b. 'Pink eye' or 'zere oogjes' or keratoconjunctivitis infectiosa ovis (KIO). Clinical efficacy of a number of antimicrobial therapies. Veterinary Quarterly 5: 122-127.

Kovac, G., O. Nagy, H. Seidel, E. Pilipcinec, J. Novotny, and R. Link. 2003. Infectious keratoconjunctivitis in sheep. Folio Veterinaria 47: 130-134.

Kruskal, J. B. 1964. Multidimensional scaling by optimizing goodness of fit to a nonmetric hypothesis. Psychometrika 29: 1-27.

Lafenetre, H. 1938. Existence de" Rickettsia conjunctivae" du mouton dans le midi de la France. Rev. med. vet., Toulouse 90: 323-324.

Le Jambre, L. F., S. Dominik, S. J. Eady, J. M. Henshall, and I. G. Colditz. 2007. Adjusting worm egg counts for faecal moisture in sheep. Veterinary Parasitology 145: 108-115.

Lee, G., W. McManus, and V. Robinson. 1982. Changes in rumen fluid composition and in the rumen epithelium when wheat is introduced to the diet of sheep: the influence of wheat and hay consumption. Australian Journal of Agricultural Research 33: 321-333.

Lee, S., B. Choi, S.-M. Yi, and G. Ko. 2009. Characterization of microbial community during Asian dust events in Korea. Science of The Total Environment 407: 5308-5314.

Leibowitz, H. M. 2000. The Red Eye. New England Journal of Medicine 343: 345-351. Levison, M. E., and J. H. Levison. 2009. Pharmacokinetics and Pharmacodynamics of Antibacterial Agents.

Infectious Disease Clinics of North America 23: 791-815. Levy, S. B., and B. Marshall. 2004. Antibacterial resistance worldwide: causes, challenges and responses. Nat

Med. Lindqvist, K. r. 1960. A Neisseria soecies associated with infectious keratoconjunctivitis of sheep-Neisseria

ovis nov. spec. Journal of Infectious Diseases 106: 162-165. Livingston.C. W, R. W. Moore, and W. T. Hardy. 1965. ISOLATION OF AN AGENT PRODUCING OVINE

INFECTIOUS KERATOCONJUNCTIVITIS (PINK EYE). American Journal of Veterinary Research 26: 295-&.

Lodge, J., J. Miles, N. Jacobson, and L. Quinn. 1956. Influence of Chlortetracycline on in Vitro Cellulose Digestion by Bovine Rumen Microorganisms. Journal of Dairy Science 39: 303-311.

Luthman, J., S. Jacobsson, B. Bengtsson, and C. Korpe. 1989. Studies on the biovailability of tetracycline chloride after oral administration to calves and pigs. Journal of Veterinary Medicine Series A 36: 261-268.

Lysnyansky, I., S. Levisohn, M. Bernstein, J. G. Kenigswald, S. Blum, I. Gerchman, D. Elad, and J. Brennee. 2007. Diagnosis of Mycoplasma conjunctivae and Chlamydophila spp in an episode of conjunctivitis/keratoconjunctivitis in lambs. Israel Journal of Veterinary Medicine 62: 79-82.

Mackie, R. I., C. S. McSweeney, and A. V. Klieve. 2002. Microbial ecology of the ovine rumen. In: M. Freer and H. Dove (eds.) Sheep Nutrition. p 71-94.

Maenz, D. D., J. F. Patience, and M. S. Wolynetz. 1993. Effect of water sweetener on the performance of newly weaned pigs offered medicated and unmedicated feed. Canadian Journal of Animal Science 73: 669-672.

Maggs, D. J., P. E. Miller, R. Ofri, and D. H. Slatter. 2008. Slatter's Fundamentals of Veterinary Ophthalmology. Saunders Elsevier.

Malbrant, R. 1939. Existence de Rickettsia conjunctivae du Mouton au Congo francais. Bull. Soc. path. exot 32: 907-908.

215

Matthews, A. G. 2009. Ophthalmic antimicrobial therapy in the horse. Equine Veterinary Education 21: 271-280.

McConnel, C., L. Shum, and J. House. 2007. Infectious bovine keratoconjunctivitis antimicrobial therapy. Australian Veterinary Journal 85: 65-69.

McEwen, S. A., and P. J. Fedorka-Cray. 2002. Antimicrobial use and resistance in animals. Clinical Infectious Diseases 34: S93-S106.

McKellar, Q. 1994. Therapy: Chemotherapy - Problems and Solutions. Proceedings of the Sheep Veterinary Society 17: 87.

Michael. 1953. Ovine ophthalmia: some observations - response to riboflavin. Veterinary Record 65: 88. MLA. 2013. Australia's sheep meat industry No. 2014. MLA, and Livecorp. 2014. Australian livestock export industry statistical review 2013 No. 2014. Moir, D. 2010. Farm note : 430 Transporting livestock: when are animals fit to load? In: D. o. A. a. Food

(ed.), Perth. Moore, C., and R. Whitley. 1984. Ophthalmic diseases of small domestic ruminants. The veterinary clinics of

North America 6. Motha, M. X. J. 2003. Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and

infectious keratoconjunctivitis. New Zealand veterinary journal 51: 186-190. Motha, M. X. J., J. Frey, M. F. Hansen, R. Jamaludin, and K. M. Tham. 2003. Detection of Mycoplasma

conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis. New Zealand veterinary journal 51: 186-190.

Mould, F. L., and E. R. Orskov. 1983. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry matter degradation and the rumen microflora of sheep offered either hay or concentrate. Animal Feed Science and Technology 10: 1-14.

Munch-Petersen, E., and J. Armstrong. 1958. The influence of orally administered oxy-tetracycline on the rumen bacteria of sheep. Aust J Exp Biol Med 36: 77-82.

Murdoch, F. R., G. L. Maker, I. Nitsos, G. R. Polglase, and G. C. Musk. 2013. Intraperitoneal medetomidine: a novel analgesic strategy for postoperative pain management in pregnant sheep. Laboratory Animals 47: 66-70.

Naglic, T., B. Seol, D. Hajsig, K. Busch, J. Frey, and M. Lojkic. 2000. Epidemiological and microbiological study of an outbreak of infectious keratoconjunctivitis in sheep. Veterinary Record 147: 72-75.

Naglic, T., B. Seol, Z. Kelneric, and K. Busch. 1999. In vitro susceptibility of field isolates of Mycoplasma conjunctivae to some antimicrobial agents.

Nanda, P., and M. Abdussalam. 1943. Observations on a contagious-keratitis of Indian sheep. Ind. J. Vet. Sci 13: 228-230.

National Research Council. 2007. Anatomy, Digestive Physiology, and Nutrient Utilization. In: P. T. Whitacre (ed.) Nutrient Requirements of Small Ruminants. p 5-25.

Neu, H. 1978. A symposium on the tetracyclines: a major appraisal. Introduction. Bulletin of the New York Academy of Medicine 54: 141.

Neumann, A., R. Snapp, and L. Gall. 1951. The long-time effect of feeding aureomycin to fattening beef cattle, with bacteriological data. In: Journal of Animal Science. p 1058-1059.

Neuschl, J. 2000. Biological availability of chlortetracyclinium chloride in sheep after administration of Vubivet C prm. auv in milk or water. Acta veterinaria Brno 50: 331.

Nicolet, J. 1975. Isolation of mycoplasma conjunctivae from Chamois and sheep affected with kerato-conjunctivitis. Zentralblatt fur Veterinarmedizin. Reihe B. Journal of veterinary medicine. Series B 22: 302-307.

Norris, R., R. Richards, and R. Dunlop. 1989a. Pre-embarkation risk factors for sheep deaths during export by sea from Western Australia. Australian Veterinary Journal 66: 309-314.

Norris, R. T., R. B. Richards, and R. H. Dunlop. 1989b. An epidemiological study of sheep deaths before and during export by sea from Western Australia. Australian Veterinary Journal 66: 276-279.

Nossa, C. W., W. E. Oberdorf, L. Yang, J. r. A. Aas, B. J. Paster, T. Z. DeSantis, E. L. Brodie, D. Malamud, M. A. Poles, and Z. Pei. 2010. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World journal of gastroenterology: WJG 16: 4135.

Nouws, J. F. M., and C. D. W. Konig. 1983. Penetration of some antibiotics into the lacrimal fluid of sheep. Veterinary Quarterly 5: 114-121.

216

Nouws, J. F. M., C. A. M. Van Ginneken, and G. Ziv. 1983. Age-dependent pharmacokinetics of oxytetracycline in ruminants. Journal of Veterinary Pharmacology and Therapeutics 6: 59-66.

O'Connor, A., H. Shen, C. Wang, and T. Opriessnig. 2012. Descriptive epidemiology of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in beef calves with naturally occurring infectious bovine keratoconjunctivitis (Pinkeye). Veterinary Microbiology 155: 374-380.

O'Doherty, J. V., and T. F. Crosby. 1998. Blood metabolite concentrations in late pregnant ewes as indicators of nutritional status. Animal Science 66: 675-683.

O'Donovan, M., E. Lewis, and P. O'Kiely. 2011. Requirements of future grass-based ruminant production systems in Ireland. Irish Journal of Agricultural and Food Research: 1-21.

O'Flynn, M. A., and J. D. O'Dea. 1996. The present state of national animal welfare policy. Australian Veterinary Journal 73: 121-124.

Oka, H., Y. Ikai, Y. Ito, J. Hayakawa, K.-i. Harada, M. Suzuki, H. Odani, and K. Maeda. 1997. Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B: Biomedical Sciences and Applications 693: 337-344.

Osborne, V. R., K. E. Leslie, and B. W. McBride. 2002. Effect of supplementing glucose in drinking water on the energy and nitrogen status of the transition dairy cow. Canadian Journal of Animal Science 82: 427-433.

Osuagwuh, A., and J. Akpokodje. 1979. Infectious keratoconjuctivitis in goats and sheep in Nigeria. Veterinary Record 105: 125-126.

Page, S. W. 1991. Chloramphenicol 1. Hazards of use and the current regulatory environment. Australian Veterinary Journal 68: 1-2.

Palgrave, T. 1910. Ophthalmia. Agricultural Gazette of New South Wales 21. Pankey, G. A., and L. D. Sabath. 2004. Clinical Relevance of Bacteriostatic versus Bactericidal Mechanisms of

Action in the Treatment of Gram-Positive Bacterial Infections. Clinical Infectious Diseases 38: 864-870.

Papich, M. G., and J. E. Riviere. 2009. Tetracycline Antibiotics. In: M. G. Riviere, M. G. Papich and H. R. Adams (eds.) Veterinary Pharmacology and Therapeutics. p 895-913.

Pasteur, L. 1863. Examen du role attribue au gaz oxygene atmospherique dans la destruction des matieres animales et vegetales apres la mort. Mallet-Bachelier.

Patamasucon, P., P. J. Rettig, K. L. Faust, H. T. Kusmiesz, and J. D. Nelson. 1982. Oral v topical erythromycin therapies for chlamydial conjunctivitis. American Journal of Diseases of Children 136: 817-821.

Patwardhan, W., M. Jangnure, and R. Andhari. 1975. An OUtbreak of infectious keratitis in sheep in Maharashtra due to Colesiota conjunctivae. Indian Veterinary Journal 52: 526-530.

Paull, D. R., C. Lee, S. J. Atkinson, and A. D. Fisher. 2008. Effects of meloxicam or tolfenamic acid administration on the pain and stress responses of Merino lambs to mulesing. Australian Veterinary Journal 86: 303-311.

Pe'er, J., and D. BenEzra. 1988. COrneal damage following the use of the pediculocide a-200 pyrinate. Archives of Ophthalmology 106: 16-17.

Perry, T. W., W. M. Beeson, E. C. Hornback, and M. T. Mohler. 1954. Aureomycin for Growing and Fattening Beef Animals. Journal of Animal Science 13: 3-9.

Pigoury, L. 1937. Existence de Rickettsia conjunctivae du mouton au Levant. Bull. Soc. path. exot 30: 621-624.

Pijpers, A., E. J. Schoevers, H. van Gogh, L. A. van Leengoed, I. J. Visser, A. S. van Miert, and J. H. Verheijden. 1991. The influence of disease on feed and water consumption and on pharmacokinetics of orally administered oxytetracycline in pigs. Journal of Animal Science 69: 2947-2954.

Purser, D., T. Klopfenstein, and J. Cline. 1965. Influence of tylosin and aureomycin upon rumen metabolism and the microbial population. Journal of Animal Science 24: 1039-1044.

Radostits, G., Hinchcliff, Constable. 2007a. Diseases associated with bacteria - V Veterinary Medicine. p 1142-1143. Saunders Elsevier.

Radostits, G., Hinchcliff, Constable. 2007b. Metabolic Diseases Veterinary Medicine. p 1668-1671. Saunders Elsevier.

Rajaian, H. a. S. M., E. 2007. Pharmacokinetics of tetracycline hydrochloride in fat-tailed sheep. Iranian Journal of Veterinary Research 8: 138-143.

217

Rake, G. 1948. Colesiota. In: R. S. Breed, E. G. D. Murray and A. P. Hitchens (eds.) Bergey's Manual of Determinative Bacteriology. p 1119-1120. Balliere Tindall and Cox, London.

Rang, H. P., M. M. Dale, and J. M. Ritter. 2000. Antibacterial Drugs: Beta-lactam antibiotics Pharmacology. p 690-691. Churchill Livingstgone.

Rang, H. P., M. M. Dale, J. M. Ritter, and R. J. Flower. 2007a. Absorption and Distribution of Drugs. In: H. P. Rang, M. M. Dale, J. M. Ritter and R. J. Flower (eds.) Pharmacology. p 102-106. Elsevier.

Rang, H. P., M. M. Dale, J. M. Ritter, and R. J. Flower. 2007b. Drug Elimination and Pharmacokinetics. In: H. P. Rang, M. M. Dale, J. M. Ritter and R. J. Flower (eds.) Pharmacology. p 122-123. Elsevier.

Rang, H. P., M. M. Dale, J. M. Ritter, and R. J. Flower. 2008. Absorption and distribution of drugs. In: H. P. Rang, M. M. Dale, J. M. Ritter and R. J. Flower (eds.) Pharmacology. p 106-110. Churchill Livingstone Elsevier.

Regnier, A. 2007. Clinical Pharmacology and Therapeutics. In: K. N. Gelatt (ed.) Veterinary Ophthalmology No. 1. p 271-285. Blackwell.

Regnier, A., V. r. Laroute, A. Gautier-Bouchardon, V. r. Gayrard, N. Picard-Hagen, and P.-L. Toutain. 2013. Florfenicol concentrations in ovine tear fluid following intramuscular and subcutaneous administration and comparison with the minimum inhibitory concentrations against mycoplasmal strains potentially involved in infectious keratoconjunctivitis. American Journal of Veterinary Research 74: 268-274.

Rehbinder, C., and A. Nilsson. 1995. An outbreak of kerato-conjunctivitis among corralled, supplementary fed, semi-domestic reindeer calves. Rangifer 15: 9-14.

Richards, R., M. Hyder, J. Fry, N. Costa, R. Norris, and A. Higgs. 1991. Seasonal metabolic factors may be responsible for deaths in sheep exported by sea. Crop and Pasture Science 42: 215-226.

Richards, R., R. Norris, R. Dunlop, and N. McQuade. 1989. Causes of death in sheep exported live by sea. Australian Veterinary Journal 66: 33-38.

Riley Jr, W., and R. Barner. 1953. Treatment of infectious ovine keratitis. Journal of the American Veterinary Medical Association 123: 434-435.

Rowland, M., and T. N. Tozer. 1995a. Absorption. In: M. Rowland and T. N. Tozer (eds.) Clinical Pharmacokinetics, Concepts and Applications. p 119-136. Williams and Wilkins.

Rowland, M., and T. N. Tozer. 1995b. Extravascular Dose. In: M. Rowland and T. N. Tozer (eds.) Clinical Pharmacokinetics Concepts and Applications. p 34-50. Williams and Wilkins.

Rusoff, L., M. Haq, C. Branton, T. Patrick, and G. Darensbourg. 1952. Report on feeding aureomycin supplement to mature dairy bulls and lactating dairy cows. In: Journal of Animal Science. p 774-775.

Russel, A., J. Doney, and R. Gunn. 1969. Subjective assessment of body fat in live sheep. The Journal of Agricultural Science 72: 451-454.

Saeed, A., R. Pir-Mohammadi, and F. Pour-Hasani. 2007. A Two-Stage Rumen Cannulation Technique in Sheep. Journal of Animal and Veterinary Advances 6: 29-32.

Scott, L., P. Menzies, R. J. Reid-Smith, B. P. Avery, S. A. McEwen, C. S. Moon, and O. Berke. 2012a. Antimicrobial resistance in fecal generic Escherichia coli and Salmonella spp. obtained from Ontario sheep flocks and associations between antimicrobial use and resistance. Canadian Journal of Veterinary Research 76: 109.

Scott, L., P. Menzies, R. Reidâ€•Smith, B. Avery, S. McEwen, C. Moon, and O. Berke. 2012b. Antimicrobial resistance in Campylobacter spp. isolated from Ontario sheep flocks and associations between antimicrobial use and antimicrobial resistance. Zoonoses and Public Health 59: 294-301.

Shahzad, W. 2013. Prevalence, molecular diagnosis and treatment of Mycoplasma conjunctivae isolated from infectious keratoconjunctivitis affected Lohi sheep maintained at Livestock Experiment Station, Bahadurnagar, Okara, Pakistan. Tropical animal health and production 45: 737-742.

Shepard, R. N. 1962a. The analysis of proximities: Multidimensional scaling with an unknown distance function. I. Psychometrika 27: 125-140.

Shepard, R. N. 1962b. The analysis of proximities: Multidimensional scaling with an unknown distance function. II. Psychometrika 27: 219-246.

Short, C. R. 1994. CONSIDERATION OF SHEEP AS A MINOR SPECIES - COMPARISON OF DRUG-METABOLISM AND DISPOSITION WITH OTHER DOMESTIC RUMINANTS. Veterinary and Human Toxicology 36: 24-40.

Simpson, B., and D. Wright. 1988. The use of questionnaires to assess the importance of clinical diseases in sheep. Ministry of Agriculture and Fisheries, Palmerston North, New Zealand.

218

Sinclair, K. B. 1955. An outbreak of ovine contagious ophthalmia treated with Chloramphenicol. British veterinary journal 3: 507.

Slatter, D. 2001a. Basic Diagnostic techniques. In: D. Slatter (ed.) Fundamentals of Veterinary Ophthalmology. p 88. Saunders.

Slatter, D. 2001b. Cornea and Sclera. In: D. Slatter (ed.) Fundamentals of veterinary ophthalmology. p 260, 293. Saunders.

Spradbrow, P. 1968. The bacterial flora of the ovine conjunctival sac. Australian Veterinary Journal 44: 117-118.

Squires, V., and A. Wilson. 1971. Distance between food and water supply and its effect on drinking frequency, and food and water intake of Merino and Border Leicester sheep. Crop and Pasture Science 22: 283-290.

Stasiak, K. L., D. Maul, E. French, P. W. Hellyer, and S. Vandewoude. 2003. Species-specific assessment of pain in laboratory animals. Journal of the American Association for Laboratory Animal Science 42: 13-20.

Stevenson, E. 1936. Cod Liver Oil as Local Treatment for External Affections of the Eyes. The British journal of ophthalmology 20: 416.

Stewart, C. S., H. J. Flint, and M. P. Bryant. 1997. The Rumen Bacteria. In: P. N. Hobson and C. S. Stewart (eds.) The Rumen Microbial Ecosystem. p 10-55. Blackie Academic and Professional.

Suijdendorp, P., Evans, D. 2005. Farmnote 41/1995 Buying healthy sheep at saleyards No. 2014. Department of Agriculture and Food, Western Australia.

Surman, P. 1968. Cytology of" pink-eye" of sheep, including a reference to trachoma of man, by employing acridine orange and iodine stains, and isolation of Mycoplasma agents from infected sheep eyes. Australian journal of biological sciences 21: 447-467.

Surman, P. G. 1973. Mycoplasma aetiology of keratoconjunctivitis (Pink Eye) in domestic ruminants. Aust J Exp Biol Med 51: 589-607.

Symons, T. 1912. Ophthalmia. Agricultural Gazette of New South Wales 23: 507-509. Tajima, K., R. I. Aminov, T. Nagamine, K. Ogata, M. Nakamura, H. Matsui, and Y. Benno. 1999. Rumen

bacterial diversity as determined by sequence analysis of 16S rDNA libraries. FEMS Microbiology Ecology 29: 159-169.

Tappeiner, H. 1884. Untersuchungen uber die Garung der Cellulose, insbesondere uber deren Losung im Darmkanale. Zeitschrift fur Biologie 20: 52-134.

Taylor, P. W., P. D. Stapleton, and J. Paul Luzio. 2002. New ways to treat bacterial infections. Drug Discovery Today 7: 1086-1091.

Tejedor-Junco, M. T., C. Gutierrez, M. Gonzalez, A. Fernandez, G. Wauters, T. De Baere, P. Deschaght, and M. Vaneechoutte. 2010. Outbreaks of keratoconjunctivitis in a camel herd caused by a specific biovar of Moraxella canis. Journal of Clinical Microbiology 48: 596-598.

Ter, E. A. 1988. The occurrence of Mycoplasma conjunctivae in The Netherlands and its association with infectious keratoconjunctivitis in sheep and goats. The Veterinary quarterly 10: 73-83.

Theodorides, V. J., C. J. DiCuollo, J. R. Guarini, and J. F. Pagano. 1968. Serum concentrations of chloramphenicol after intraruminal and intra-abomasal administration in sheep. Am J Vet Res 29: 643-645.

Toop, C. 1964. Contagious Ophthalmia (Pink Eye). Journal of Agriculture, Western Australia 5: 955-957. Torok, V. A., G. E. Allison, N. J. Percy, K. Ophel-Keller, and R. J. Hughes. 2011. Influence of Antimicrobial Feed

Additives on Broiler Commensal Posthatch Gut Microbiota Development and Performance. Applied and Environmental Microbiology 77: 3380-3390.

Torok, V. A., K. Ophel-Keller, M. Loo, and R. J. Hughes. 2008. Application of Methods for Identifying Broiler Chicken Gut Bacterial Species Linked with Increased Energy Metabolism. Applied and Environmental Microbiology 74: 783-791.

Toutain, P.-L., A. Ferran, and A. Bousquet-Melou. 2010. Species differences in pharmacokinetics and pharmacodynamics Comparative and Veterinary Pharmacology. p 19-48. Springer.

Townsend, W. M. 2007. Food and Fiber-Producing Animal Ophthalmology. In: K. N. Gelatt (ed.) Veterinary Ophthalmology No. 2. p 1308-1311. Blackwell Publishing.

Townsend, W. M. 2010. Examination Techniques and Therapeutic Regimens for the Ruminant and Camelid Eye. Veterinary Clinics of North America: Food Animal Practice 26: 437-458.

219

Trufelli, H., P. Palma, G. Famiglini, and A. Cappiello. 2011. An overview of matrix effects in liquid chromatography mass spectrometry. Mass spectrometry reviews 30: 491-509.

Tryland, M., C. G. Das Neves, M. Sunde, and T. Mork. 2009. Cervid Herpesvirus 2, the Primary Agent in an Outbreak of Infectious Keratoconjunctivitis in Semidomesticated Reindeer. Journal of Clinical Microbiology 47: 3707-3713.

Tschopp, R., J. Frey, L. Zimmermann, and M. Giacometti. 2005. Outbreaks of infectious keratoconjunctivitis in alpine chamois and ibex in Switzerland between 2001 and 2003. Veterinary Record 157: 13-18.

Van Halderen, A., W. Van Rensburg, A. Geyer, and J. Vorster. 1994. The identification of Mycoplasma conjunctivae as an aetiological agent of infectious keratoconjunctivitis of sheep in South Africa.

Wald, E. R. 1997. Conjunctivitis in infants and children. The Pediatric Infectious Disease Journal 16: S17-S20. Wall, A. 1982. Breed susceptibility to infectious keratoconjunctivitis. Veterinary Record 110: 457. Walridge B.M. , C. C. M. H. 2002. Diseases of the Eye. In: D.G.Pugh (ed.) Sheep and Goat Medicine. p 317-

339. W.B.Saunders Company. Ward, D. A. 1991. Ocular pharmacology. The Veterinary clinics of North America. Food animal practice 7:

779-791. Webber, J. J., L. D. Edwards, and I. K. McLeod. 1988. Topical treatment of ovine keratoconjunctivitis.

Australian Veterinary Journal 65: 95-97. Wegener, H. C. 2003. Antibiotics in animal feed and their role in resistance development. Current opinion in

microbiology 6: 439-445. Whitley, R., and C. Moore. 1984. Ocular diagnostic and therapeutic techniques in food animals. The

Veterinary clinics of North America. Large animal practice 6: 553-575. WHO. 2015. Worldwide country situation:response to antimicrobial resistance, World Health Organisation,

Geneva. Wilsmore, A., G. Dagnall, and R. Woodland. 1990. Experimental conjunctival infection of lambs with a strain

of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis. Veterinary Record 127: 229-231.

Wirbelauer, C. 2006. Management of the Red Eye for the Primary Care Physician. The American Journal of Medicine 119: 302-306.

Witte, W. 1998. Medical Consequences of Antibiotic Use in Agriculture. Science 279: 996-997. Wood, M. 1999. Conjunctivitis: diagnosis and management. Community Eye Health 12: 19-20. Wyman, M. 1983. Eye diseases of sheep and goats. The Veterinary clinics of North America. Large animal

practice 5: 657. Ziemer, C. J., R. Sharp, M. D. Stern, M. A. Cotta, T. R. Whitehead, and D. A. Stahl. 2000. Comparison of

microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes. Environmental Microbiology 2: 632-643.

Ziv, G., and F. G. Sulman. 1972. Binding of Antibiotics to Bovine and Ovine Serum. Antimicrobial Agents and Chemotherapy 2: 206-213.

Ziv, G., and F. G. Sulman. 1974. ANALYSIS OF PHARMACOKINETIC PROPERTIES OF 9 TETRACYCLINE ANALOGS IN DAIRY-COWS AND EWES. American Journal of Veterinary Research 35: 1197-1201.

Zuntz, N. 1879. Geschichtspunkte zum kritischen Studium der neueren Arbeiten auf dem Gebiete der Ernahrung.

the treatment of infectious ovine keratoconjunctivitis in ...infectious ovine keratoconjunctivitis...

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