Impact of mycotoxins and of a mycotoxin deactivatoron alpacas grazing perennial ryegrass infected with wildendophyte (Neotyphodium spp.)

K. F. M. ReedA,F, J. L. VaughanB, L. J. CumminsC and D. D. MooreD,E

AReed Pasture Science, Hamilton, Vic. 3300, Australia.BCria Genesis, PO Box 406, Ocean Grove, Vic. 3226, Australia.CIvanhoe, 559 Bulart Bridge Road, Cavendish, Vic. 3314, Australia.DBiomin Australia Pty Ltd, PO Box 2344, Carlingford, NSW 2118, Australia.EUniversity of Queensland, School of Animal Studies, Gatton College, Qld 4345, Australia.FCorresponding author. Email: rps@eftel.net.au

Abstract. Liveweight gain, animal health and the effectiveness of a mycotoxin deactivator were studied on an old pasturethat contained 61% perennial ryegrass. Sixty-seven percent of the ryegrass population was infected with endophyte(Neotyphodium spp.). The pasture was fenced into two halves and two groups of 28 alpaca male weaners were rotatedbetween the two plots. Nine to 10 Suris and 18–19 Huacayas were allocated to each group. One group was fed a concentratesupplement (100 g/head per day) and the other was fed the same supplement to which was added the toxin deactivator,Mycofix® Plus (5 g/100 g). Mean liveweight gain on the low-quality pasture over late summer and early autumn was notsignificantly (P> 0.05) different between the groups. For the control group it was 41 g/day but individual rates of gain rangedfrom 67 to 0 g/day, depending on the severity of signs of perennial ryegrass toxicosis (r = 0.82, P < 0.001). Liveweight gainwas independent of neurotoxic signs in the Mycofix® Plus treated group. Ergovaline concentration in perennial ryegrassvaried from0.43 to a peak in early autumn (March) of 1.05mg/kg.Meanurine lysergol alkaloid concentration peaked inmid-summer (January) at 109 ng/mg creatinine (control group) andwas consistently lower in theMycofix® Plus group, althoughthe difference approached significance (P = 0.06) only inMarch. LolitremB concentration in perennial ryegrass varied from0.78 to 1.57mg/kg. Neurotoxic signs in alpacas were observed throughout the study and peaked in early autumn, coincidingwith peak lolitrem B concentration; at this time, 84% of alpacas exhibited neurotoxic signs. Over the 145-day study, theMycofix® Plus treated group exhibited a lower mean rating of perennial ryegrass toxicosis signs (P < 0.05). Variation inliveweight gain and signs of toxicosis were not associated with significant differences in liver enzyme activity.

Additional keywords: creatine kinase, deoxynivalenol, neck tremors, ryegrass staggers.

Introduction

Many areas of pasture in south-eastern Australia containperennial ryegrass (Lolium perenne) infected with the wildtype of the naturally occurring endophyte, Neotyphodium lolii(Reed et al. 2000). This symbiotic association protects the plantfrom stress but as a consequence, some metabolites, e.g. lolitremBand ergovaline, that the infected plant produces cause perennialryegrass toxicosis (PRGT) in herbivores that graze the infectedplants (Easton and Fletcher 2005). Occasionally, when stronggrowth occurs in late spring–summer in southern Australia,these toxin levels rise sharply and remain high through latesummer–autumn, and, during the period of dry feed, perennialryegrass endophyte toxicosis may cause devastating losses ofsheep and cattle as a result of ill-thrift, heat stress ormisadventure(Butler 1987; Napthine and McLeod 1987; Jubb 2005). Duringsomeyears inVictoria andTasmania, severe epidemics have beenassociated with the death of deer, horses, tens of thousandsof sheep and hundreds of cattle (Reed et al. 2005). Clinical

signs of PRGT are common in exposed alpacas (Reed andCummins 2003), which appear likely to exhibit signs ofstaggers more readily than sheep and cattle grazing on thesame farm (Sampaio et al. 2008).

In years when clinical effects are infrequent, consultantshave estimated that the value of lost production in grazinglivestock, associated with a lifetime exposure to subclinicallevels of ryegrass endophyte toxins, may be of great economicsignificance (Lean 2005; Leng 2005; Sackett and Francis 2006).Such estimates may include effects on fertility (Foot et al. 1988;Cummins 2005). Many controlled experiments where animalproduction has been studied on perennial ryegrass pasturescomparing a select (low-toxin) endophyte/endophyte-freegrass with grass infected with the natural endophyte have beenconducted, mainly in New Zealand (Easton and Fletcher 2005).Such comparisonsprovide an indicationof subclinical productionlosses. With dairy cows, the elimination of toxin exposure hassignificantly increased milk production, e.g. by 14% in autumn

CSIRO PUBLISHING

Animal Production Science, 2010, 50, 902–908 www.publish.csiro.au/journals/an

� CSIRO 2010 10.1071/AN10068 1836-0939/10/090902

(Valentine et al. 1993) and by 9% over three lactations (Bluettet al. 2003).

Ruminants are less susceptible to fusario-mycotoxins thanare non-ruminants (Kurmanov 1977) and this is partly due tothe metabolism of mycotoxins by rumen microbes (McIntoshet al. 2002). Mycofix® Plus (Biomin, Herzogenburg, Austria)food additives have been developed to protect animal healthby deactivation of mycotoxins ingested with contaminated feed.Their dualmodeof action, including (1) adsorptionofmycotoxinsby selective blends of minerals that are effective for toxins thathave suitably located polar function groups (e.g. aflatoxins,ochratoxins and fumonisins), and (2) biological degradation byspecific enzymes of less- or even non-adsorbable mycotoxins(e.g. fusariotoxins such as zearalenone and tricothecenes), formsthe basis of their unique patented efficacies in non-ruminantand ruminant species. The latter constituents may include aEubacterium species that rapidly proliferates in the rumen ofcattle, producing enzymes for mycotoxin detoxification (Binderet al. 2001; Schatzmayr et al. 2006), and suppresses growth ofpathogenic bacteria. Mycofix® Plus also contains phytogenicsubstances and phycophytic constituents that strengthen theimmune response. Mycofix® Plus has proved effective inovercoming limitations to pigs, poultry and cattle, particularlywith regard to aflatoxin and zearalenone (Cheng et al. 2006),deoxynivalenol (Binder et al. 2001), fumonisins and ochratoxins(Hanif et al. 2008).

Mycofix® Plus was evaluated with Coopworth ewe lambsgrazing wild endophyte-infected perennial ryegrass at Hamilton,during a season when clinical signs of PRGT were not observed.The results indicated that the adsorption capability of Mycofix®

had highly significant effects on shade-seeking behaviour(Cummins and Reed 2008), presumably by alleviating impairedcognitive function. An on-farm experiment was conducted toinvestigate the potential of Mycofix® Plus 3e for its effectivenessin deactivating mycotoxins in young alpacas grazing wildendophyte-infected, perennial ryegrass-dominant pasture.

Materials and methods

PastureThe experiment was conducted at Wildflower Alpacas atMacarthur in south-western Victoria (average annual rainfall730 mm). The soil type was a brown chromosol, namely abasalt-derived duplex clay loam. The pasture had been top-dressed with ~200 kg/ha of superphosphate annually for25 years and muriate of potash had been applied occasionally.The pasture had been sown to Victorian perennial ryegrass in1988 and Fitzroy perennial ryegrass had been sod-seeded into itin autumn (May) 2007. Most seed of both these varieties isusually infected with wild endophyte. Perennial ryegrass wasdominant, with subterranean clover (Trifolium subterraneum,spp. yanninicum, cv. Trikala) making up most of the balance.Minor species present included some sweet vernal grass(Anthoxanthum odoratum) and annual grasses (Hordeum, PoaandVulpia spp.). Ryegrass staggers have been regularly observedin sheep grazing on this pasture over many years and then, onthe change of ownership and enterprises, neck tremors wereobserved amongst a few alpacas in autumn (March–April) 2008and again in (the same individuals) in spring (November) 2008.

The4-hapaddockwasdividedon the east–west axis to providetwo 2-ha plots, similar in aspect and with similar access to shelterfrom a timber shelter belt on the western boundaries. Each plotwas provided with a water trough.

Pasture measurementsOn 28 September 2008, tillers of perennial ryegrass werecollected every 25 m along diagonal traverses of theexperimental area. Tillers were cut at ground level by scalpelblade, transferred to a portable freezer and then stored in afreezer until 30 tillers were selected at random and testedby Paratech Veterinary Services, Wickliffe, Victoria, forNeotyphodium endophyte infection frequency by using a solidphase, stacked immunoblot assay (Agrinostics Phytoscreen) thatprovides accurate and reliable detection of Neotyphodiumspp. endophytes (Hiatt et al. 1999).

The ergovaline and lolitrem B concentrations of the perennialryegrass population were determined by testing 50 randomlyscattered plants within the 4-ha area of the experiment. Perennialryegrass material was cut at ground level with a scalpel blade onfour occasions between November 2008 and May 2009.Harvested material was kept frozen. Alkaloid concentrationswere determined by Southern Scientific Services, Hamilton,Victoria, using high-pressure liquid chromatography forlolitrem B (Gallagher et al. 1984) and ergovaline. Ergovalinewas determined by a modification of the procedure used byShelby et al. (Shelby and Flieger 1997; Shelby et al. 1997) forscreening seed. Our modification as described by Reed et al.(2004) improved analyte selectivity in the extraction–clean-upphase and eliminated a problem of internal standard variability.

Pasture mass was measured on 10 December 2008 by cuttingto ground level with hand shears, in each of 12 ranked circles(diameter 290 mm) of 48 circles spread across the two plots.Pasture from each circle was weighed fresh and the samples werethen bulked, chopped and thoroughly mixed. Five subsampleswere taken to determine the following: dry matter content byoven-drying for 24 h at 60�C, botanical composition by hand-sorting, nutritive value by near-infrared reflectance spectroscopy(conducted by Department of Primary Industries, Victoria),mineral concentrations in oven-dried herbage by using radialCIROS inductively coupled plasma atomic emissionspectrometry (conducted by Waite Analytical services, SouthAustralia) and Fusarium mycotoxins, namely deoxynivalenoland zearalenone, by using high pressure liquid chromatography(conducted by Romer Laboratories, Singapore) with methodsdescribed by Reed and Moore (2009).

Alpaca managementMeasurements, observations, handling and management ofalpacas were carried out in accordance with best practice andthe approval of theVictorianWildlife andSmall InstitutesAnimalEthicsCommittee.Recentlyweaned alpacas born in autumn2008were collected from Victorian and South Australian studs. Fiveco-operating studs each delivered between 4 and 24 males byDay –14 of the experiment (28 November 2008). On Day –10(2 December), they were ear-tagged, weighed, vaccinated with5-in-1 clostridial vaccine and drenched with ivermectin. Theywere then allocated into one of two groups of 28, balanced for

Alpacas grazing wild-endophyte perennial ryegrass Animal Production Science 903

breed, bodyweight, farmof origin and staple length. In total, 9 and10 Suris were allocated to the treatment and control groups,respectively; the balance were Huacayas. The two groups weregrazed together for 10 days as they acclimatised to the perennial-ryegrass pasture and the daily supplement of a mash of crushedbarley (80%)andpeas, initially offeredat 50g/headand then, after4days, at 100g/head.The supplementwas spread out daily in fourtroughs per plot (10 cm/head). Alpacas were reweighed on Day 0(12 December 2008) of the trial, drafted into their group and thetwo groups were each put in one of the two plots to commencetreatment. The daily supplement was maintained, except that forthe treatment group the mash contained 5 g of Mycofix® Plus per100 g, mixed in by the feed processor. The control group and thetreatment group were moved every week, rotating between thetwo plots, so that the total pasture area was accessed equally byboth groups during the course of the experiment.

Alpaca measurementsAlpacas were mustered, weighed and rated for signs of PRGT atabout monthly intervals and on most occasions, urine sampleswere collected from as many alpacas as possible during an hourof observation with the aid of funnels fixed to the end of long(2-m) rods. Urine was kept frozen pending analysis of ergolinealkaloids, namely the lysergol : creatinine ratio (Hill et al. 2000).The analysis was conducted by Paratech Veterinary Services,Wickliffe, Victoria. Each animal was rated for PRGT signs whilethe animals were yarded, using the scale for neurotoxic signsdescribed (Table 1).

On Day 91 (13 March 2009), a blood sample was collectedfrom each alpaca following jugular venipuncture. Plasma washarvested and stored frozen before analysis. Fourteen samplesfrom each group were analysed for liver enzymes by RegionalLaboratory Services, Benalla, Victoria, to provide an indicationof hepatic function and muscle damage. The 14 samplesrepresented two subgroups per group; Subgroups 1 and 2represented alpacas with a relatively high or low liveweightgain, respectively. At the completion of the experiment, faecalsamples were collected from all alpacas in each group and ratedfor consistency (Table 1). Theywere thenoven-dried at 60�C.Drysamples (1.5 g) from each of about five alpacas were pooled toprovidefive replicate samples per group for analysis of ergovaline

and lolitrem B by Southern Scientific Services, Hamilton,Victoria.

StatisticsByusing theunpaired t-test, results from the twogroupsof alpacaswere tested for significant differences by using the two-tailedP value. Linear relationships between liveweight gain and PRGTratings were tested by regression analysis in Microsoft Excel(Microsoft Corporation, Redmond, WA, USA). For each day onwhich PRGT signs were rated, mean rating was modelled overtime separately for each animal by using a random coefficientregression including a cubic spline for time (Verbyla et al. 1999).The final model fitted was as follows:

PRGT rating ¼ mþ dayþ treatmentþ animalþ animal:day

þ spline ðdayÞ þ animal:spline ðdayÞ:The terms ‘day’ and ‘treatment’were fitted as fixed factors or

covariates, whereas all other terms were fitted as random effects,with a covariance between the animal intercept (animal) andslope (animal.day). The likelihood ratio test was used to assessany spline effects after the previously mentioned terms (day,treatment, animal and animal.day) had been fitted.

Results

Pasture

Neotyphodium spp. endophyte infection was confirmed in theperennial ryegrass population that was sampled in September2008. Sixty-seven percent of the 30 perennial ryegrass plantstested were found to be infected. From the pasture assessmentcarried out on 10 December, the pasture mass was 7.80(�3.21) t DM/ha, with a botanical composition (DM basis) of60.7% perennial ryegrass, 32.7% barley grass (Hordeumleporinum), 5.8% other annual grasses and 0.8% subterraneanclover. The pasture had a dry matter (DM) content of 43.6%,crude protein was 7.5%, neutral detergent fibre was 70.0%,digestible organic matter in dry matter (DOMD) was 52% andthemetabolisable energywas7.6MJ/kgDM.Onadrymatterbasis,the pasture contained 0.36% Ca, 0.15%Mg, 0.21%Na, 0.96% K,0.19% P, 0.14% S, 210 mg/kg Fe, 183 mg/kg Mn, 5.3 mg/kg B,2.8mg/kgCu,<0.9mg/kgMo,<0.9mg/kgCo,13mg/kgNi,17mg/kgZn, 300 mg/kg Al, <0.2 mg/kg Cd, <2 mg/kg Pb, <8 mg/kg Se,2.1 mg/kg Ti and 1.6 mg/kg Cr. Aflatoxin, ochratoxin, fumonisinand zearalenonewere not detected in the pasture; deoxynivalenolwas detected, with a concentration of 682 mg/kg DM.

The ergovaline concentration in perennial ryegrass exceededthe tolerance level at which cattle exhibit clinical signs (namely0.4 mg/kg DM) for the duration of the experiment (latespring to late autumn) and exceeded the tolerance level forsheep (0.8 mg/kg) in early autumn. The concentration of theneurotoxin lolitrem B remained below the tolerance level(1.8 mg/kg) for sheep and cattle at all times (Table 2).

Liveweight gain

The alpacas gained weight gradually over the 135-day period ofevaluation, with no significant differences between the twogroups (Fig. 1). The rate of liveweight gain slowed late in thedry period, and rose when green pick became available followingautumn rain.

Table 1. Rating scales for signs of toxicosis and faecal consistency

Score Description

Clinical signs of perennial ryegrass toxicosis0 No clinical signs0.5 Slight head wobble1 Head wobble2 Head, neck and torso tremors; wide-based stance; bunny-hop3 Fall down with legs splayed

Appearance of faeces1 Loose pellets2 Fused pellets; pellets structured3 Fused pellets; pellets losing structure4 Soft5 Diarrhoea

904 Animal Production Science K. F. M. Reed et al.

Toxicosis

A few alpacas exhibited signs of PRGT in December and beforethe commencement of the treatment. The frequency of signs roseto a peak on Day 84 (6 March), by which time 84% of alpacasshowed some signs (Fig. 2). Within the Suri alpacas, signs ofPRGT were observed in five of nine individuals treated with

Mycofix® Plus, compared with 9 of 10 in the control group; theeffect of breed was not significant (P > 0.05).

Over 145 days, the mean rating for PRGT signs wasconsistently lower for the Mycofix® Plus treated group(P < 0.05). It peaked in mid-March (Day 91) when threealpacas (one from the control and two from the treated group)were rated as 3; these were removed from the toxic pasture for3 weeks and fed lucerne hay. Subsequently, with the onset of rainand the opportunity to select green material, the frequency andseverity of signs began to decline (Fig. 3).

The total score for individual toxicosis ratings taken betweenDay –8 (4 December 2008) and Day 145 (6 May 2009) wascalculated (SPRGT). For alpacas that were removed, theirsubsequent ratings were not included in the total. Within thecontrol group (range 0–15.5), SPRGT was correlated withliveweight gain over the experimental period on a linear basis(r = 0.82, P < 0.001) (Fig. 4), as follows:

LWG ðkgÞ ¼ 8:97 ð�0:625Þ � 0:622 ð�0:086Þ PRGT; ð1Þwhere LWG = liveweight gain, and SPRGT = sum of PRGTratings.

Table 2. Ergovaline, lolitrem B and dry matter (DM) content ofperennial ryegrass before, during and at the end of the grazing period

Day 0 = 12 December 2008

Day ofexperiment

Ergovaline(mg/kg DM)

Lolitrem B(mg/kg DM)

%DM

Day –14 (28 Nov.) 0.76 0.78 40.8Day 28 (9 Jan.) 0.43 1.01 40.4Day 84 (6 Mar.) 1.05 1.57 58.8Day 145 (6 May) 0.43 1.19 36.4

33

34

35

36

37

38

39

40

41

–50 0 50 100 150 200

Time (days)

Live

wei

ght (

kg)

Fig. 1. Mean liveweight for control (square) and Mycofix® Plus treated(circle) groups of alpaca (n = 28).

0

5

10

15

20

25

30

–50 0 50 100 150 200

Time (days)

Num

ber

of a

lpac

as w

ith

clin

ical

sig

ns

Fig. 2. Incidence of perennial ryegrass toxicosis in control (square) andMycofix® Plus treated (circle) groups of alpaca (n = 28).

–2

0

2

4

6

8

10

12

0 5 10 15 20

Total toxicosis score

Live

wei

ght g

ain

(kg)

Fig. 4. Individual liveweight gain (LWG) in relation to the total ratingsof perennial ryegrass toxicosis (SPRGT) for the control group of alpacas.LWG (kg) = 8.97 (�0.625) – 0.622 (�0.086) SPRGT.

–0.2

0.2

0.4

0.6

0.8

1.0

1.2

200

Control

Mycofix

Time (days)

Mea

n ra

ting

for

sign

s of

P

RG

toxi

cosi

s

–50 50 100 150

Fig. 3. Severity of perennial ryegrass toxicosis; mean rating for controland Mycofix® Plus treated groups of alpaca (P < 0.05) across 145 days oftreatment. Refer to Table 1 for a key to ratings. PRG, perennial ryegrass.

Alpacas grazing wild-endophyte perennial ryegrass Animal Production Science 905

The relationship for animals within the treatment group(SPRGT range 0–11) was negative, but not significant(r = 0.33, P = 0.08), as follows:

LWG ðkgÞ ¼ 5:17 ð�1:106Þ � 0:304 ð�0:168ÞSPRGT ð2ÞHaving been trained to daily feeding in the pre-experimental

period, alpacas converged rapidly around feed troughswhen theirdaily supplement was distributed; it was consumed within a fewminutes. Over the last fortnight of the experiment, however, twoor three alpacas in each group (the worst PRGT-affected onesincluding individuals that had been temporally removed frompasture) lost interest in consuming the supplement and remainedseated by the water for much of the day.

Blood sampled on Day 91 (early autumn) indicated that nineindividuals showed slight hypoproteinemia (five control and fourtreatment animals). Generally, tests were normal (Table 3), apartfrom the slightly lowered albumin : globulin ratio, and treatmenteffects were not different (P > 0.05). Slightly elevated creatinekinase (CK) levels (341 and 411 U/L) were observed for twoindividuals, with a PRGT rating of 2; the mean CK values forthe combined poor subgroups (i.e. low liveweight gain) werenot significantly different (P = 0.09) from those for the better-performing subgroups.

Mean urine lysergol alkaloid concentration peaked in Januaryat 109 ng/mg creatinine (control group) and was consistentlylower in the Mycofix® Plus group; the difference approachedsignificance in March (Table 4) when the control group had anaverage concentration of 48 ng/mg creatinine, i.e. 1.9 times thatmeasured in theMycofix® Plus group. During late summer–earlyautumn, the urine lysergol content declined from the Januarypeak, whereas in May, the content increased when the differencebetween the groups in urine lysergol concentration declined.

The mean concentration of lolitrem B in faeces was 0.57 and0.59 mg/kg dry matter for control and Mycofix® Plus groups,respectively; the difference was not significant. On a scale of1–5, the mean moisture/softness of faeces sampled on Day 107was 2.27 for the control group and 2.75 for those receiving

Mycofix®Plus (5 g/head per day). This difference was notsignificant (P = 0.08).

Discussion

Considerable variation in the liveweight gain of individualswithin the control group was observed, depending on theseverity of their neurotoxic signs. The apparently resistant/susceptible individuals in the control group achieved a rate ofliveweight gain that varied from 67 g/day to 0 g/day, overthe range 0–15.5 for total perennial-ryegrass toxicosis rating.The group’s mean liveweight gain of 41 g/day was 61% ofthat exhibited by the resistant individuals. This considerableproduction loss occurred on pasture where the neurotoxinconcentration in perennial ryegrass never exceeded therecognised tolerance level for sheep and cattle (1.8 mg/kg) andconfirmed the observations of surveyed producers that alpacasare more sensitive than sheep and cattle to the ryegrassendophyte toxins inducing staggers (Sampaio et al. 2008).

The fact that 90%of alpacas in the control group showed signsof PRGT may seem high, given that the recent Australia-widetelephone survey on alpaca staggers (Sampaio et al. 2008)reported that alpaca producers observed that 12% and 9% oftheir alpacas grazingperennial ryegrasswere affected byPRGT in2004 and 2005, respectively. However our study was on youngalpacas and the survey did note that the young portion were themost likely to exhibit neck-tremor signs of PRGT. A PRGT scoreof 1 (Table 1), simply a slight neck tremor, can be seen whilethe alpaca is at rest. PRGT scores of 0.5 and 1 were observedafter careful and prolonged observation andwould not be obviousto the untrained observer. Some producers surveyed may haveoverlooked such slight effects. However, given the relationshipwith liveweight gain (Eqn 1), they can be taken as a usefulindication that production losses are being incurred, and thesemay possibly include a lowered reproductive performance(Sampaio et al. 2008). No difference in susceptibility to PRGTwas observed between the two breeds represented and thissupports the survey findings reported by Sampaio et al. (2007).

Table 3. Mean liveweight gain (LWG), perennial ryegrass toxicosis (PRGT) rating andblood enzyme levels (mean� s.d.)for four groups of alpacas (n = 7)

AST, aspartate transaminase; GGT, gamma glutamyl transpeptidase; GLDH, glutamate dehydrogenase

Measurement Normal Control group Mycofix® Plus treatedrangeA Subgroup 1 Subgroup 2 Subgroup 1 Subgroup 2

Day 0 (12 December 2008) – Day 84 (6 March 2009)LWG (kg) 4.93 –0.07 6.36 0.50

Day 91 (13 March 2009)PRGT score (rated 0–5) 0 0.36 1.79 0.50 1.21GGT (U/L) <60 29.0 ± 8.1 16.4 ± 6.5 19.1 ± 6.8 18.3 ± 7.5GLDH (U/L) <20 22.1 ± 38.9 16.1 ± 5.4 17.9 ± 8.1 15.6 ± 10.0AST (U/L) <320 211.6 ± 52.3 220.1 ± 26.7 196.0 ± 23.7 224.3 ± 31.5Bilirubin (mmol/L) 0.1–3.2 2.6 ± 2.8 1.6 ± 0.6 2.1 ± 0.5 2.0 ± 0.9Creatine kinase (U/L) <750 91.6 ± 13.3 134.6 ± 126.1 82.9 ± 29.7 143.6 ± 104.9Protein (g/L) 54–75 55.4 ± 3.7 55.1 ± 2.7 57.4 ± 7.1 55.5 ± 4.2Albumin (g/L) 25–45 29.7 ± 2.4 30.1 ± 2.6 31.4 ± 0.1 29.0 ± 2.7Globulin (g/L) 15–41 25.6 ± 2.1 25.0 ± 2.7 26.0 ± 4.0 26.5 ± 2.0Albumin/globulin 1.3–3.3 1.17 ± 0.16 1.23 ± 0.21 1.21 ± 0.11 1.09 ± 0.07

ARegional Laboratory Services, Benalla, Vic.

906 Animal Production Science K. F. M. Reed et al.

In studies on animal selection, aspartate transaminase and CKlevels were 30% greater in sheep susceptible to ryegrass staggersthan those in the resistant ones (Morris et al. 2007). No suchvariation was recorded in our work with unselected animals.Possibly, this could be because the most-affected alpacasprobably consumed less pasture? Sheep can detect endophytetoxins in their diet and will limit their intake when given choiceof such contrasting pastures (Cosgrove et al. 2002). The elevationof CK levels in the serum of animals with a PRGT score of 2was not unexpected as elevated serum CK is associated withmuscle damage. Mycofix® Plus did not eliminate the staggerssigns of PRGT. The slight difference between groups inliveweight gain at 107 days was not significant (P = 0.25) andwas not maintained after pasture growth resumed in autumn. Theindependence of liveweight gain with staggers scores for thetreatment group suggests that, for susceptible alpacas at least,either the physical ability to graze, the drive to eat and/or theefficiency of utilisation of digested nutrients, was improved byMycofix® Plus. However, the lower intercept reflects the fact thatthe liveweight gain of themore resistant individualswas lower forthis group (Eqn 2) so that the effects of the feed additive may nothave all been positive. The lack of difference between the groupsin faecal lolitremBcontrastswithour studyof lambs, although thesofter faeces we observed in treated alpacas is consistent withobservations made in that previous experiment (K. F. M. Reed,L. J. Cummin and D. D. Moore, unpubl. data).

The saprophyte-produced deoxynivalenol detected in thesummer pasture may be expected where mature herbagehas accumulated (Reed and Moore 2009) and could haveboth influenced the impact of the endophyte mycotoxins onthe alpacas and contributed to the effects of Mycofix® Plus(Binder et al. 2001). During late summer–early autumn(February–March) when the pasture was predominantly dry-standing feed, the rate of liveweight gain fell and, possiblyreflecting reduced nutritive value and intake, the urine lysergolcontent declined from the January peak. It was elevatedagain after rain generated green feed in April and the rate ofliveweight gain increased. This suggests an increasing intake oftoxin with the onset of pasture growth and is reflected by theincrease in urinary lysergol alkaloids. Investigation into severePRGT has previously shown that the alkaloid concentration ofseedlings/fresh shoots is 5–10 times higher than that in matureherbage (Aasen et al. 1969).

We conclude that PRGT signs in young alpacas are associatedwith a significant loss of liveweight gain and that most young

alpacas may be expected to exhibit PRGT signs when toxinconcentrations in perennial ryegrass exceed 0.9 mg/kg lolitrem B.This level is half the concentration atwhich clinical signs in sheepand cattle are observed; however, we note that this observationwas associatedwith the concurrence of 0.4–1.1mg/kg ergovalinein the perennial ryegrass and 0.7 mg/kg deoxynivalenol in thepasture. Relative to unaffected individuals in the control group,the mean liveweight gain of the clinically affected animalswas 39% lower. The mycotoxin deactivator, Mycofix® Plus,administered once daily at 5 g/head per day did not eliminatesigns of PRGT, although it reduced the frequency and severity ofclinical signs and reduced lysergol alkaloids in urine. A higherdose or more even administration of the deactivator throughoutthe day, as distinct from the once-a-day intake of deactivator asused in our study, may increase the time it is present in the rumenand thus extend its efficacy.

Acknowledgements

We thank Steve and Sue Ridout,Wildflower Alpacas, Macarthur, Vic., for theuse of their facilities and alpacas and Sue Ridout for her careful assistancewith the feeding and care of the experimental groups.We also thankMatthewand Cathy Lloyd, EPCambridge Alpacas, Oakbank, SA, Peter Kennedyand Robert Gane, Canchones Alpacas, Taggerty, Vic., Julie Wilkinson andRussell Synnot, Baarrooka Alpacas, Strathbogie, Vic., and Ian and AngelaPreuss, Surilana Alpacas, Strathbogie, Vic., for the use of their alpacas.We thank Dr Robert Cunningham of Tarrington, Vic., for secure storage ofconcentrate and Biomin Australia Pty Ltd for supporting the study.

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Table 4. Concentration of lysergol alkaloids in urine (ng/mg creatinine)Day 0 = 12 December 2008; no. of individuals tested is given in parentheses

Day of trial Control Mycofix® Plus P

Pretreatment periodDay –10 10.2 (10) 19.6 (7) n.s.Day 0 43.6 (11) 41.5 (7) n.s.

Treatment periodDay 28 (9 Jan. 2009) 108.9 (11) 50.3 (14) n.s.Day 84 (6 Mar. 2009) 47.9 (13) 25.7 (13) 0.06Day 145 (6 May 2009) 97.6 (14) 68.1 (13) n.s.

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Manuscript received 3 May 2010, accepted 29 July 2010

908 Animal Production Science K. F. M. Reed et al.

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