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Published Quarterly:V O L U M E 6 5 n o . 3 2 0 1 0

Editor:T. Waner

Co. Editor: H. Barak

Associate Editors:M. Ballaiche A. Bomzon J. BrennerN. Galon S. Harrus E. Klement G. Segev R. Schahar A. Steinman

Editorial Board:G. Dank D. Elad S. FreidmanG. Leitner U. Orgad E. PipanoA. Rosner G. Simon A. Shimshony N. Speigel Z. Trainin

ISRAEL VETERINARY MEDICAL ASSOCIATIONP.O.BOX 22, 43100 Raanana, ISRAEL Tel: 09-7419929 Fax: 09-7431778E-mail :[email protected] Website: www.isrvma.org

I. Samina - President D. Dagan - Secretary A. Markovitz - Treasurer

AMERICAN VETERINARIANS FOR ISRAEL17 Cottage LaneSpringfield. N.J. USA 07081-2302

B. Bender - President S. Altman - Vice President A. Newman - Treasurer

ISSN 0334-9152

CONTENTS

EDITORIAL:GREATER VISIBILITYWaner, T.

REVIEWSTAPHYLOCOCCUS AUREUS MASTITIS:WHAT WE NEED TO KNOW TO CONTROL THEMZecconi, A.

ARTICLESINTOXICATION OF YOUNG CROCODILES IN CAPTIVITY DUE TO THE INGESTION OF DARKLING BEETLES BLAPS NITENS LAPORTEI ARDOIN (COLEOPTERA; TENEBRIONIDAE)Perelman, B. and Chikatunov, V.

BOVINE UROLITHS ANALYSIS: A REVIEW OF 30 CASES.Parrah, J.D., Hussain, S. S., Moulvi, B. A., Singh, M. and Athar, H. DIAGNOSIS OF MILK FEVER BY A WATER HARDNESS TEST KIT IN EWESAktas, M.S., Kaynar, O, Ozkanlar, S2, Ozkanlar, Y.

CANINE ORAL PAPILLOMAVIRUS INFECTION: CLINICAL COURSE, PATHOLOGY, L1 GENE AND NCR2 GENE SEqUENCINGJun, D., Yi, G., Na, T., Yipeng, J, Rui, Z, Degui, L. and Guozhong, Z.

CARDIOLOGY : WHAT IS YOUR DIAGNOSISECG OF THE MONTHGolani, Y. and Ohad D.

IMAGING: A DOG WITH ACUTE VOMITINGBibring, U. and Eizenberg, Z.

TOxIcOLOGY vIEwPOINTIMPROVED ANIMAL FEED CONTROL – "FARM TO FORK"Shlosberg, A.

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Arabian Horse(see next page)

Veterinary medicineisrael journal of

Formerly: reFuaH VeterinaritH

ISRAEL JOURNAL OF VETERINARY MEDICINE

INSTRUCTIONS TO AUTHORS

The Israel Journal of veterinary Medicine is the official publication of the Israel veterinary Medical Association. It is published quarterly and is devoted to all aspects of veterinary medicine with emphasis on research and events in the Middle East and Mediterranean Basin. All original articles pertaining to veterinary medicine and research are welcome and will be considered for publication.

Manuscripts will be accepted on the clear understanding that their contents have not been published previously and that they have not been submitted for publication elsewhere.

Short communications documenting important new findings that warrant rapid publication will also be considered.

Letters to the editor will be limited to comments on contributions already published in the journal; if a letter is accepted, a response for simultaneous publication will be invited from the author of the original contribution.

Adherence to principles outlined in the Guide for the Care and Use of Laboratory Animals, National Academy Press, washington, D.C. 1996, is implicit in animal experimentation. The journal requires written author verification of compliance with animal welfare and ethics policies. All material published in the Israel Journal of veterinary Medicine must adhere to high ethical and animal welfare standards.

Prior to acceptance of a manuscript, to verify compliance with theabove policies, the authors must

Sign a covering letter certifying that legal and ethical 1) requirements have been met with regards to the humane treatment of animals described in the study;Specify in the covering letter and in Materials and Methods 2) the international, national, and/or institutional guidelines followed;Provide evidence, such as a signed animal use form or protocol 3) number, of compliance with ethical review at the institution or practice;

The Editor retains the right to reject manuscripts on the basis of animal ethical or welfare concerns.

conflict of Interest:

All manuscripts are subject to editorial review by experts in the field who advise the editors of the manuscript's scientific quality. The Editor-in-chief will make the final decision regarding acceptability for publication.correspondence, exchange journals, books for review, etc. should be addressed to the Editorial Board, P.O. Box 22, 43100 Raanana, Israel.Manuscripts should be submitted by E-mail. E-mail addresses: [email protected] (Editor). confirmation of arrival of the manuscript will be sent to the author within one week.

we do not supply reprints. The author of each article will receive a copy of the issue in which his article was published.

Manuscripts preparation: The entire manuscript should be double-spaced on standard A4 or 8Ω x 11 inch paper, typed on one side only, with 3 cm margins. The editor reserves the right to change the style and grammar of the manuscript if necessary.Page numbers must be included in the upper right-hand corner of each page.Manuscripts must be formatted with line numbers in the left hand margin.Manuscripts must be submitted in English using English spelling and must be grammatically correct. Authors whose native language is not English are advised to seek assistance in manuscript preparation from someone fluent in written English.

PAPERS SHOULD BE SUBDIVIDED AS FOLLOWS:Title page: The first page of each paper should contain the title, in capital letters (short, specific and informative), followed by the name(s) with initials and affiliation(s) and professional degrees of the author(s), including address(es). Further, the complete mailing address of the person to whom correspondence should be sent (including Phone and Fax numbers, as well as E-mail address).

A short running title of maximum 4 words.

A minimum of 4 Keywords must be provided.

Abstract: the abstract should include a self-contained summary of the objectives, results and significance of the study. Uninformative sentences such as “the significance of the results is discussed” are not acceptable.

Introduction: A concise and clear statement of the background, purpose and significance of the work.

Material and Methods: The work and methodology used should be described and referenced, including the experimental design. Sufficiently informative protocols should be given to permit repetition of experimental work. Technical descriptions of methods

conflict of Interest: Authors of research articles must disclose any conflict of interests (e.g. financial arrangements) which they have with the company whose product features prominently in the submitted manuscript, or with a company making a competing product. Such information will be held in confidence.

Book references: Whole book: Author (or editor), Title, Publisher, city, Year.Example: clarke, E. G. J. and clarke, M. L.: veterinary Toxicology, Bailler Tindal, London, 1975.

Book chapter: Author: chapter title, Editor: book title, Publisher, city, pages, Year.Example: clarkson, T. B., Shively, c. A. and weingand, K. w.: Animals models of diet-induced atherosclerosis. In: Beynen, A. c. and west, c. E. (Eds.): Use of animal models in human nutrition. Karger, Basel, pp. 56-82, 1998.

Tables and figures: These must be intelligible without reference to the text and should be planned to fit the page size of the journal. Tables and figures should be numbered, in Arabic numerals, in the sequence in which they are mentioned in the text. The same data may not be reproduced in both table and figure format. Each table must have a title and on each column there should be a heading that clearly identifies the data therein. Illustrations and diagrams should be kept to a minimum; the figure number must appear only on the reverse side, together with the author’s name and an arrow marking to the top.

Units must conform to the International System of Units and should be expressed in metric units.Abbreviations: These should be used sparingly; they should be defined when first used in the text.Drugs: when referring to a drug, use the generic name. The trade name, manufacturer's name, city and state abbreviation should be provided. Equipment: When describing products or equipment, the generic name should be used in the text and the details of the product (brand name, manufacturer, city and state) should be provided.


should be detailed only when such methods are new. Sub-headings should be used for clarity.

- Common methods or procedures need not be described in detail, and where possible citation should be made to techniques that have been reported elsewhere.

- A statement of animal care must be made.

- A concise description of the statistical methods should be provided including analytical software and citation of sources for unusual methods.

Results: The statements should be presented concisely, with the aid of tables or figures where appropriate. Duplication of the text of this section and data presented in tables and figures should be avoided.

Discussion: This section must relate to the significance of the work to existing knowledge in the field and indicate the importance of the contribution of this study. Needless recapitulation of the results must be avoided. A comparison with related published studies should be made and conclusions drawn.

References: In the text, identify references by Arabic numbers (in brackets) in sequence of their appearance. Number references in the order in which they are first mentioned in the text. Material submitted for publication but not yet accepted should be noted as “unpublished data” and not be included in the reference list. The list of references should include only those publications, which are cited in the text.

Journal citations: Name(s) and initial(s), Title, Journal, Vol.,page(s), year.Example: Goodchild, w. M. and cooper, D. M.: Oviduct adenocarcinoma in laying hens. Vet. Rec. 82:389-390, 1968.

Cover image:Arabian HorseA breed of long standing, elegant and considered one of the most aristocratic horses among all the breeds of horses. They are used in shows, racing events and endurance competitions. They are generally very expensive, hot tempered and they have a light step which is somewhat bouncy. They are considered as very intelligent animals. Arabian horses are highly sought after especially in the Middle East. An ancient dynasty is attributed to the breed and the value of a horse is judged accordingly. They are characterized by their medium height, thin strong legs and small head with a depression on their forehead which enhances their aristocratic appearance. They are not recommended for long distance riding due to their jerky step but are most impressive in shows.

92 Volume 65 (3) 2010website: www.isrvma.org

EDITORIALvISIBILITY


An important aim which I have set for the Journal is acceptance to the Medline index, the bibliographic database of the National Library of Medicine for journal articles in life sciences and biomedicine. Medline is the index for about 100 veterinary science journals and is the database of choice by veterinarians. At the moment we are indexed by ISI web of Knowledge which also grades our citation index.

Improvement of our citation index will follow our acceptance to Medline. However the road is long and will necessitate substantial adjusts which we will have to undertake. To understand these adjustments I will review some of the requirements for our acceptance to Medline.

1. Scope of the Journal: we have now defined the Journal niche as the “Middle East and Mediterranean Basin” which to my mind a suitable scope which has not been covered by other veterinary journals.

2. Quality of content: Here we need to make an effort. we have all the potential. If only members of the academia would each devote one quality article a year to the effort we would be well on our way. we need to convince them that their academic careers will not be compromised and may even be enhanced by the fact that they will have in the long run a journal which is acceptable with a higher citation index. Not that we should only leave the work to the academic community: All of us must make every effort to improve our national journal.

3. The content of the articles: Here we need the full gamut of articles from both academics and those working in the field: Original research from experimentation, original clinical observations from both research and the field, critical reviews from veterinarians from all walks of life, case reports from both veterinary hospitals and clinics are all desirable and welcome..

4. Quality of editorial work: Every effort is being made to accept only good quality articles and the standard for acceptance is risings and will rise further with the acceptance of good quality articles. Furthermore the editorial board has laid emphasis on animal welfare where animal experiments are involved and the consideration of conflict of interests which must be declared by the authors. The peer review process, which is considered vital to the quality of the journal, is undergoing improvement and efforts are been made to enhance this aspect.

5. The production quality: The journal is produced both as a printed edition and also online, which is free of charge to users all over the world. The online version needs a lot of improvement in order to make the journal more “visible”. At the moment the articles from the journal can be found via internet search engines, however the site itself is in my opinion not optimal and requires improvement. The qualities of a good site should be ease of access, ease of use to download articles, the availability of a search engine within the site and an aesthetic appearance. This is being attended to at the moment. If anyone has ideas about this project please do not hesitate to contact me. A good internet site will make us more “visible” and that will take us a long way towards improving our status.

A special thanks to all those contributing articles. You are making the difference!wishing you all chag Samach and a Happy and Healthy New Year.

Trevor Waner

93Volume 65 (3) 2010 website: www.isrvma.org

STAPHYLOCOCCUS AUREUS MASTITIS:WHAT WE NEED TO KNOW TO CONTROL THEM

Zecconi, A.Department of Animal Pathology, Hygiene and Health. Università degli Studi di Milano, via celoria 10, 20133 Milano, Italy

INTRODUCTIONAmong bacteria causing mastitis, only Streptococcus

agalactiae, Staphylococcus aureus, Mycoplasma species, and Corynebacterium bovis are considered as fully contagious. Among these, S.aureus, is currently the most frequently isolated contagious pathogen in subclinical and chronic bovine mastitis worldwide (1).

The reasons for the large spread of S.aureus intramammary infections (IMI) worldwide are obviously related to bacteria characteristics, but also to a general misunderstanding of the epidemiology of S.aureus IMI, leading to inefficient control measures.

The description of all the factors involved in S.aureus IMI should include many different aspects, and it is not possible to cover all of them in a single review. Therefore, in this paper we reviewed the main information useful to develop control programs for S.aureus IMI under field conditions and it is focused on the following topics: diagnosis, major risk factors, therapy, vaccination and control methods.

Bacteria characteristicsS.aureus are described “ coagulase positive, β-haemolytic,

maltose and mannitol fermenting organisms, forming pigmented colonies” (2). Not all strains of S.aureus have characteristics that are consistent with the previous description. Indeed, some S.aureus are α-haemolytic, β-haemolytic, α+β-haemolytic, δ-haemolytic, non-haemolytic and even coagulase negative (3).

To cause mastitis S.aureus initially must gain access to the mammary gland through the teat canal and then has to avoid removal by the flushing of the fluids during the milking process. Therefore, the ability to adhere to the epithelial cells and extracellular matrix (EcM) proteins is instrumental to colonize the gland and develop the pathologic process. The adhesion mechanism of S.aureus is complex and includes multiple proteins able to specifically recognize components of the microbial surface that recognize adhesive matrix molecules (MScRAMM) (4), allowing bacterial anchorage in normal and inflamed tissues (5). Adhesive molecules are pivotal in the diffusion of S.aureus within and among herds, but they are only one of the several virulence factors involved in the pathogenesis of S.aureus infections. It is out of the scope of this review to describe all these virulence factors. However, assessing S.aureus genetic patterns is useful to understand its epidemiological features.

Several studies (6-8) indicate that virulence of S.aureus strains differ, an indication that strain typing is important. Similarly, transmissibility within a herd differs by strain type

(9). Thus, the importance of evaluating the combination of S.aureus virulence factors has been recently emphasized both in human and veterinary medicine (8, 10, 11).

DIAGNOSISSeveral factors have been shown to influence accuracy

for detecting S.aureus IMI using conventional bacteriological culture: i.e. sample type (quarter or composite), inoculum volume, sampling time and frequency. Hence, knowledge of the limits will guide the final decision on the method to be employed to detect S.aureus IMI at a cow or mammary quarter level.

It is generally accepted that there is relationship between bulk tank Scc (somatic cell count) and the proportion of cows with S.aureus in the herd (12). However, there is increasing evidence that a large proportion of cows in well-managed herds could be S.aureus positive, but with low SCC both at cow and bulk tank level (13, 14). This could be the result of recently acquired infections, reduced pro-inflammatory activity of strains or efficient host immune response (8, 15, 16).

Under field conditions, composite or quarter milk sampling can be applied. However, reliability of culturing composite milk samples compared with quarter samples for the diagnosis of S.aureus IMI 0.01 ml as inoculum volume showed a sensitivity and specificity respectively of 57.9 and 98% (17, 18). while composite milk sampling could be used for routine testing when the main interest is determining prevalence of infection at cow level, the use of this test in control programs, where the proper identification of infected cows is crucial, should be carefully evaluated.

Methods to increase diagnostic accuracyOne potential source of false positive results is the presence

of irregular shedding of bacteria (19). However, recent studies suggested that increasing the inoculum volume to 0.1 ml will significantly reduce the risk of false negative results (20, 21).

These latter observations are supported by the studies on the accuracy of diagnosis based on different inoculum volumes. Direct plating of 0.01 ml of milk from established S.aureus infections failed to detect these organisms in about 10% of the samples, while plating 0.05 resulted in 6% of false negative samples (22). Further studies, using 0.01 ml as inoculum volume (23), found a 94.2% agreement between duplicate quarter milk samples positive for S.aureus. These results confirmed earlier reports and suggested that the single quarter sampling method was capable of identifying 82-94% S.aureus quarter infections (23).

REVIEW


To overcome some of the diagnostic problems, “augmented methods” have been also proposed. Among them, freezing-thawing and centrifugation are considered the most interesting. Freezing may affect viability of organisms contained in milk samples taken for bacteriologic culture but it could increase the sensitivity of the test breaking the clusters. However, (24) found that the presence of S.aureus in subclinical and clinical milk samples kept frozen at about -20ºc for 4, 6 and 16 weeks did not differ significantly. The centrifugation of milk samples has been proposed to overcome the low-shedding cases (25). The results showed that cultures of the sediment significantly increased the number of positive outcomes, in comparison with conventional methods.

ANALYTICAL METHODSMilk culture could be performed either on blood-agar plate

or using selective media such as Baird-Parker or a modification of the latter technique. However, the different phenotypes could cause several problems in diagnostic laboratories when selective media are applied. In our laboratory for an internal comparison, 50 confirmed S.aureus strains were tested on 3 different media: (blood-agar, Baird-Parker -BP- and Baird Parker supplemented with rabbit plasma and fibrinogen-BPR-PF). The results showed that 31/50 hadn’t any surrounding clear zone on BP, 17/50 were coagulase negative on BPR-PF and 10/50 were only weakly positive in this latter medium. Therefore, milk culture on 5% blood agar plate still is the recommended method for S.aureus IMI diagnosis, followed by confirmation of the suspected colonies by other methods such as coagulase-test and biochemical tests. When selective media are applied, it is also recommended to confirm all the suspected colonies by other methods.

Independently from the method applied, identifying a single colony of S.aureus is enough to define the quarter (cow) as infected.

Very recently, methods based on Real Time PCR have been proposed to improve mastitis diagnosis, including for S.aureus (26). However, data available until now are not sufficient to evaluate if the molecular approach will be useful to improve the accuracy of current diagnostic methods for S.aureus IMI.

RISK FACTORSTraditionally the prevalence of mastitis in heifers prepartum has been overlooked given the concept that heifers without a developed gland are not susceptible to IMI. Fox et al. (27) reviewed heifer mastitis, estimating prevalence between 1% and 10% of all heifers with S.aureus IMI at parturition. Therefore, heifers could be important risk factor acting both as a reservoir and as a vehicle for S.aureus.

waage et al.(28) found that S.aureus was the major cause of clinical mastitis in heifers, with clinical mastitis noted during the first 2 weeks of lactation. whether the heifer was raised on the farm or was imported into the herd did not

influence the odds of having clinical mastitis in early lactation. In an extended study by (29), there was a significantly greater percentage of S.aureus isolated from epidermal and mucosal sites of heifers in high prevalence herds. Overall, heifers colonized with S.aureus in the mammary gland area were 3.4 times as likely to calve with S.aureus IMI, and this was the largest risk factor of disease.

Once S.aureus enters into the herd, the major vehicle of diffusion among cows is milking machines and improper milking procedures. Indeed, several studies found that S.aureus on milking unit liners could have the same fingerprint as the isolates causing IMI (30-32). Additionally, S.aureus has been isolated from udder cloths (33), thus implicating their role as a fomite in the spread of S.aureus mastitis.

Davidson (34) in large study on sites of colonization and IMI found the udder and teat skin to be an important reservoir associated with S.aureus mastitis. Zadoks et al. (35) would suggest that the teat skin is not a likely reservoir for S.aureus IMI. Such an argument was partially supported by (36), but it is opposite to that reported by (29, 31, 37).

Matos et al. (38) suggested that there are several other potential sources of S.aureus in a dairy, as this pathogen could be isolated from bedding and air of the parlour. Flies were not a source of S.aureus in this study, which is in contrast to that reported by (39). Roberson et al. (29) showed that in low- prevalence herds (<3%), bedding, insects, and water did not yield S.aureus, but high-prevalence herds did have some of these samples that were positive.

There is not a broad consensus on the role played by external sources in S.aureus IMI, out of milking machine and, potentially, by heifers. Therefore, these two factors are the most important ones we should be considered when we decide to apply a control program.

THERAPYIn practice, the common opinion that S.aureus has a

relatively poor cure rate is based more on clinical impressions than on scientific evidences. Indeed, the cure rate for lactation therapy reported in literature has a very broad range (4-92%), a narrower, but still broad range has been reported also for dry-cow therapy (40). The recent debate on the increase in microbial resistance observed in human medicine, and particularly of methicillin-resistant S.aureus strains, increased the concerns on the use of antimicrobials for dairy cow therapy, particularly at drying-off. The discussion is still open, even if there are increasing evidences that the antibiotic therapy for mastitis and dry-cow treatment is not associated either with the development of methicillin-resistant strains or an increase of resistance (41). Despite this evidence, the application of antibiotic therapy following the principles of a prudent use of antibiotics, as proposed by various scientific organizations, should be recommended. To apply a prudent and efficacious protocol for mastitis treatment, different factors should be considered (42): type of pathogen involved, antibiotic

REVIEW


susceptibility patterns, severity of inflammatory response, duration of infection, stage of lactation and age of the cow.

Probably the most comprehensive analysis of the different factors affecting cure rate for clinical, subclinical mastitis and dry-cow therapy for S.aureus has been proposed by Sol and co-workers (43-45). Among the different factors, Scc showed to influence S.aureus cure rate after treatment of clinical and subclinical mastitis and at drying-off with lower cure rate when SCC were > 106/ml. Hind quarters showed a lower cure rate (subclinical and drying-off therapy), age and number of infected quarters showed to be significant only for drying-off therapy, with a decrease of the cure rate as age and number of infected quarters increased.

when the factors affecting cure rate are considered (i.e. age, Scc), the therapy is applied selecting molecules after susceptibility test and with a rational and efficient protocol, the cure rate could be higher than 75 % both for dry cow-therapy and for treatment of cows after calving (43-45).

VACCINATIONMuch interest has been devoted to the development of

a vaccine against bovine mastitis caused by S.aureus, but to date the results have not been fully convincing. Indeed, vaccination against S.aureus mastitis must take into account a large number of possible antigens and related interactions with the immune system. These different potential targets for an immune response are reviewed by (46) and these targets include capsules, adhesions, surface proteins and toxins.

Early vaccine formulations were composed of microorganisms that were cultured in vitro, killed, and injected systemically with or without toxoids and immunologic adjuvants as reviewed by (47, 48). Several such formulations were shown to increase the spontaneous cure rates of S.aureus IMI as well as to lessen the severity of infection but did not prevent new cases of mastitis, but none of them was broadly and consistently applied in dairy herds.

Heifer VaccinationNickerson et.al. (49) evaluated a polyvalent S.aureus

vaccine in heifers beginning at 6 months of age (with periodic 6-month boosters) to determine if vaccination reduced prevalence of S.aureus mastitis during pregnancy and at calving. Results demonstrated that the percentage of new S.aureus infections during pregnancy was lower in vaccinates than controls (14.3 vs. 25.9%), the percentage of quarters showing chronic S.aureus infection was lower in vaccinates than controls (10.7 vs. 18.8%), and at freshening, the percentage of quarters infected, with S.aureus was lower in vaccinates than controls (8.9 vs. 16.1%). The data demonstrated a positive effect of vaccination in increasing antistaphylococcal antibody titers and in preventing new S.aureus infections when the program was initiated at an early age in heifers raised in a herd with high exposure to this mastitis pathogen.

Edinger et al. (50) developed a herd-specific vaccine based on two strains of S.aureus previously isolated from cases of

clinical mastitis in the herd. Results showed that prevalence of S.aureus in quarter milk samples taken at calving and three to four weeks post partum did not differ significantly between the vaccine and control group. Regarding the development of clinical mastitis during the first three months after calving and the prevalence of S.aureus in quarter milk samples taken before the onset of treatment, there were no significant differences between the groups. The SCC was lower in vaccinated than in control heifers. However, the difference was only significant on the third milk test day.

Cow Vaccination In dairy cows, one of the first vaccines developed was

a heat-killed capsular type A and B S. aureus strains and capsular polysaccharide, and it was used in two herds. The results showed that the vaccinated animals had a decrease in mastitis incidence and higher milk yield in comparison with unvaccinated herd-mates (51). A vaccine containing whole inactivated bacteria with pseudocapsule and α-β toxoid was used in a field trial in Norway (52). The study showed that a significant increase in antibody was observed only for pseudocapsule and α toxoid. Moreover, vaccinated heifers showed a prevalence of S.aureus mastitis of 8.6%, while in control heifers it was of 16%.

One of the largest field trials on S.aureus vaccine efficacy was reported by (53). Results showed no overall reduction in the incidence of clinical mastitis, even though differences were observed within individual herds. Overall, the known commercially available S.aureus vaccines have shown limited efficacy under field conditions. A vaccine which would prevent the occurrence of bovine mastitis caused by S.aureus or a vaccine that would augment S.aureus control would be of benefit. More recently, two vaccines were commercially available in some countries, but only for one of these two data are available (54-56). This patented vaccine is derived from 3 field strains of S.aureus which contain a broad spectrum of antigenic and immunogenic properties. The composition enables the vaccine to induce a strong homologous as well as heterologous immune response. The results of the field trials showed a 70% specific protection from infection and almost complete protection from udder inflammation expressed by the low SCC (100x103 cell/ml) of the vaccinated cows. The effect of vaccination on subclinical udder infection revealed a significant clearance of S.aureus udder infection in comparison to the control, which was expressed also by a reduction in SCC to normal range. Because of low rate of spontaneous infection among the heifers in the field trial, no specific protection could be evaluated. However, a significant difference in Scc between the vaccinated and nonvaccinated heifers was found (108x103

and 178x103 cell/ml respectively). A significant difference in milk production (0.5 Kg per cow per day) was found as well.

Innovative vaccines developed by the means of molecular methods have been investigated by several research groups (57-60). Results are very encouraging, but none of these vaccines is available in dairy field.

Thus, even if S.aureus vaccine was the object of a large



number of studies, still a vaccine with proven efficacy in commercial dairy herds and in different areas is not available.

CONTROL PROGRAM Environmental sources and non-dairy animals do not

appear to be significant reservoirs and vectors for the disease. Therefore, a control/eradication program for this pathogen could be hypothesized. The frequency of infected herds and cows, and the high cost of the disease represent the reason to develop control programs. Moreover, the availability of accurate diagnostic procedures and efficacious therapeutical protocols allow for their implementation.

To develop a strategy to control the infection, the approaches currently applied are the test-and-cull strategy and control programs based on segregation. culling is often suggested as the only way to control S.aureus (61, 62). However, there is poor scientific evidence either on the efficacy or on the economic return of this approach. Indeed, (63) showed that at the end of a program based on test-and-cull strategies applied in three herds, all the three herds had a very similar culling rate, but only the third one, applying a well-managed program in addition, achieved the control of S.aureus IMI. Therefore, culling could be a component of a control program, mainly as the most efficient way to remove the chronic S.aureus cows. However, as a main method of control it showed to be poorly efficient and with a negative economic impact.

control programs based on segregation followed the general principles of contagious mastitis control (64) and was based on precise diagnostic procedures and strict control of segregation of infected cows.

we reported the epidemiologic pattern IMI in 9 commercial dairy herds after establishment of a standardized and detailed mastitis control program (65).

The main steps in the control program are (Figure 1): 1. Application of a precise and consistent milking procedure

that included use of a single-service towel to clean the teat, forestripping, and use of postmilking teat disinfectants of known efficacy.

2. Establishment of a milking sequence to reduce infection risk, by milking healthy cows first, then cows and heifers that had recently entered the herd either through purchase or freshening, and then milking cows with S.aureus IMI last.

3. After the first sampling of all lactating cows at the time of enrolment to segregate infected cows, a precise sampling schedule is used. Purchased cows are sampled 5-7 and 10-14 days after entry into the herd, and cows that had recently calved are sampled 5-7 and 10-14 days after calving. cows with S.aureus IMI are segregated and are not sampled again until they have calved. Non-infected cows are sampled again 2, 4, 7, 10, 14 and 18 months after the first sampling.

4. Diagnosis of S.aureus IMI is performed with mammary quarter milk samples by bacteriologic culture on 5% blood-agar media. Quarter samples are collected to increase the sensitivity of detection. Recovery of a single colony of S.aureus is considered a positive result indicating an IMI. All

mammary quarters of all cows are treated at drying-off with a commercial antimicrobial treatment. choice of the product is based on the susceptibility of the S.aureus strains isolated in the herd as determined by use of the disc diffusion method.

5. Treatment of infected lactating cows without clinical signs is restricted to those with ≤ 3 lactations and in their first 30 days of lactation, to avoid treatments with a poor cure rate. These cows are moved to a hospital pen to be sampled 5-7 and 10-14 days after the end of the antimicrobial withdrawal period. Only cows that are judged cured because of negative results of 2 consecutive bacteriologic cultures of milk samples are allowed to leave the hospital pen.

6. Antimicrobial and anti-inflammatory treatments are administered to cows that developed clinical mastitis, independently of their S.aureus infection status, and these cows are moved to the hospital pen to be sampled 7 and 14 days after the end of the withdrawal period. Only cows that were judged cured are allowed to leave the hospital pen to come back to the group of origin.

7. At the herd level, farm managers are advised to improve and keep proper bedding hygiene.

8. The control program is monitored directly by means of monthly visits by a trained practitioner and discussion of analysis results, and indirectly by a weekly check of samples sent to the laboratory, to ensure compliance.

Results of this field study suggested that a very low incidence rate can be achieved after about 10 months of the control program. Young cows and freshening cows are the most likely to develop new IMI among uninfected cows when segregation of infected cattle is used; uninfected cattle should be carefully checked and specific procedures should be applied to reduced the risk of infection. It could be suggested that heifers should be housed separately from older cows for at least 2 weeks before calving, to reduce the risk of becoming infected from older cows during periparturient period.

The proposed control/eradication program based on segregation showed, on average, to reduce the incidence rate of S.aureus IMI below 2% in 10 months and <1% in 18 months. cost/benefit analysis showed that a positive economic return could be achieved even when starting prevalence of S.aureus IMI are in the range 10-20% (66).

CONCLUSIONS Programs based on segregation has been applied, but on

a smaller scale when compared to Str.agalactiae. Diagnostic problems, poor cure rate, and unknown sources of infection are often the arguments advocated to refute the application of a control program. Moreover, the presence of intramammary infections, without large increase of Scc induces many practitioner and farmers to underestimate the impact on milk quality and yield.

However, based on the available scientific and practical evidences we can reasonably affirm that:

S.aureus • has a variable, but large economic impact on

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dairy herd;Diagnosis of • S.aureus IMI is feasible with conventional methods by experienced laboratories;Well-designed • S.aureus therapy protocols have a cure-rate not inferior to the one observed against other intramammary pathogens;S.aureus • is not an obligate parasite of the mammary gland, but the importance of potential reservoirs on other body sites or in the environment significantly decreases as the prevalence of IMI decreases in the herd. Therefore, they will not affect the control programs.

Therefore, when control programs are based on few important points such as the isolation or removal of reservoirs, the avoidance of S.aureus transmission during milking, a careful monitoring by a trained practitioner, they will be successful with positive economic returns for both the farmer and the practitioner.

REFERENCES Zecconi, A. Contagious mastitis control program: the 1. Staphylococcus aureus case. Cattle Practice 14, 67-76.2006.carter, G.R., cole, J.R. Diagnostic procedures in veterinary 2. bacteriology and mycology, 5th Edition. Academic Press, London, 1990.Fox, L.K., Besser, T.E., Jackson, S.M. Evaluation of a 3. coagulase-negative variant of Staphylococcus aureus as a cause of intramammary infections in a herd of dairy cattle. JAvMA 209, 1143-1146.1996.Patti, J., Bremell, T., Krajewska-Pietrasik, D., Abdelnour, A., 4. Tarkowski, A., Ryden, c., Hook, M. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect. Immun. 62, 152-161.1994.Foster, T., Hook, M. Surface protein adhesins of 5. Staphylococcus aureus. Trends Microbiol. 6, 484-488.1998.Younis, A., Krifucks, O., Fleminger, G., Heller, E.D., Gollop, 6. N., Saran, A., Leitner, G. Staphylococcus aureus leucocidin, a virulence factor in bovine mastitis. J. Dairy. Res. 72, 188-194.2005.Younis, A., Krifucks, O., Heller, E., Samra, Z., Glickman, A., 7. Saran, A., Leitner, G. Staphylococcus aureus exosecretions and bovine mastitis. J. vet. Med. B Infect. Dis. vet. Public Health 50, 1-7.2003.Zecconi, A., cesaris, L., Liandris, E., Daprà, v., Piccinini, R. 8. Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland. Microbial Pathogenesis 40, 177-183.2006.Sommerhauser, J., Kloppert, D., wolter, w., Zschock, M., 9. Sobiraj, A., Failing, K. The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme. vet. Microbiol. 96, 91-102.2003.Peacock, S.J., Moore, c.E., Justice, A., Kantzanou, M., Story, 10. L., Mackie, K., O’Neill, G., Day, N.P.J. virulent combinations of Adhesin and Toxin Genes in Natural Populations of Staphylococcus aureus. Infect. Immun. 70, 4987-4996.2002.von Eiff, c., Friedrich, A.w., Peters, G., Becker, K. Prevalence 11. of genes encoding for members of the staphylococcal leukotoxin family among clinical isolates of Staphylococcus aureus. Diagn. Microbiol. Inf. Dis. 49, 157-162.2004.

Gonzalez, R.N., Jasper, D.E., Farver, T.B., Bushnell, R.B., 12. Franti, c.E. Prevalence of udder infections and mastitis in 50 california dairy herds, In: JAvMA, pp. 323-328 1988.Zecconi, A., Piccinini, R. Intramammary infections: 13. epidemiology and diagnosis. In: XXII World Buiatric Congress - Recent developments and perspectives in bovine medicine, Hannover 18-23/08/2002, pp. 346-359, 2002.Zecconi, A., Piccinini, R., Fox, L.K. Epidemiologic study of 14. intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds. JAvMA 223, 684-688.2003.Zecconi, A., Binda, E., Borromeo, V., Piccinini, R. Relationship 15. between some Staphylococcus aureus pathogenic factors and growth rates or somatic cell counts. J. Dairy Res. 72, 203-208.2005.Middleton, J.R., Fox, L.K., Gay, J.M., Tyler, J.w., Besser, 16. T.E. Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl-beta-D-glucosaminidase activity in cattle from eight dairies. J. Dairy Sci. 85, 1133-1140.2002.Morselt, M.L., Lam, T., vanwuijckhuise, L.A., Franken, P., 17. Hartman, E.G., Schukken, Y.H. The reliability of bacteriological culturing of composite milk samples in the diagnosis of sublinical udeder infections in dairy cattle. Tijdschr. Diergeneeskd. 120, 426-430.1995.Lam, T., vanwuijckhuise, L.A., Franken, P., Morselt, M.L., 18. Hartman, E.G., Schukken, Y.H. Use of composite milk samples for diagnosis of Staphylococcus aureus mastitis in dairy cattle. JAvMA 208, 1705-&.1996.Sears, P.M., Smith, B.S., English, P.B., Herrer, P.S., R.N., 19. G. Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J. Dairy Sci. 73, 2785-2789.1990.walker, J., Rajala-Schultz, P.J., DeGraves, F.J. The Effects of 20. Inoculum volume and Extended Sampling on the Microbiological Detection of Naturally Occurring Staphylococcus aureus Intramammary Infections In: 49th NMc Annual Meeting, Albuqyerque NM, pp. 230-231, 2010.Torres, A.H., Rajala-Schultz, P.J., DeGraves, F.J. Diagnosis of 21. intramammary infections at dry-off based on sampling strategy, epidemiology of pathogens, and agreement beyond chance. J. Vet. Diagn. Investig. 21, 427-436.2009.Neave, F.K. Diagnosis of mastitis by bacteriological methods 22. alone. In: IDF Seminar on the control of Mastitis, Reading (7-10/4/1975), pp. 19-36, 1975.Erskine, R.J., Eberhart, R.J., Hutchinson, L.J., Spencer, S.B., 23. campbell, M.A. Incidence and types of clinical mastitis in dairy herds with high and low somatic cell counts. JAvMA 192, 761-765.1988.Schukken, Y.H., Smit, J.A.H., Grommers, F.J., vandegeer, D., 24. Brand, A. Effect of Freezing on Bacteriologic culturing of Mastitis Milk Samples. J. Dairy Sci. 72, 1900-1906.1989.Zecconi, A., Piccinini, R., Zepponi, A., Ruffo, G. Recovery of 25. Staphylococcus aureus from centrifuged quarter milk samples. J. Dairy Sci. 80, 3058-3063.1997.Koskinen, M.T., Holopainen, J., Pyorala, S., Bredbacka, P., 26. Pitkala, A., Barkema, H.w., Bexiga, R., Roberson, J., Solverod, L., Piccinini, R., Kelton, D., Lehmusto, H., Niskala, S., Salmikivi, L. Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. J. Dairy Sci. 92, 952-959.2009.Fox, L.K., chester, S.T., Hallberg, J.w., Nickerson, S.c., 27.



Pankey, J.w., weaver, L.D. Survey of intramammary infections in dairy heifers at breeding age and first parturition. J. Dairy Sci. 78, 1619-1628.1995.Waage, S., Odegaard, S.A., Lund, A., Brattgjerd, S., Rothe, 28. T. case-control study of risk factors for clinical mastitis in postpartum heifers. J. Dairy Sci. 84, 392-399.2001.Roberson, J.R., Fox, K.L., Hancock, D.D., Gay, J.M., Besser, 29. T.E. Ecology of Staphylococcus aureus isolated from various sites of dairy farms. J. Dairy Sci. 77, 3354-3364.1994.Smith, T., Fox, L., Middleton, J. Outbreak of mastitis caused 30. by one strain of Staphylococcus aureus in a closed dairy herd. JAvMA. 212, 553-556.1998.Fox, L.K., Gershman, M., Hancock, D.D., Hutton, c.T. Fomites 31. and reservoirs of Staphylococcus aureus causing intramammary infections as determined by phage typing: the effect of milking time hygiene practices. Cornell Veterinarian 81, 183-193.1991.Zadoks, R.N. 2002. Molecular and mathematical epidemiology 32. of Staphylococcus aureus. Utrech University, Utrecht NL.Fox, K.L. Recovery of mastitis pathogens from udder cloths 33. following several laundering methods. Dairy Food and Environmental Sanitation 17, 14-19.1997.Davidson, I. Observation on the pathogenic staphylococci in 34. a dairy herd during a period of six years. Res vet. Sci. 2, 22-40.1961.Zadoks, R.N., van Leeuwen, w.B., Kreft, D., Fox, K.L., 35. Barkema, H.w., Schukken, Y.H., Belkum, A. comparison of Staphylococcus aureus isolates from bovine and human skin, milk equipment, and bovine milk by phage typing, pulsed-field gel electrophoresis, and binary typing. J. clin. Microbiol. 40, 3894-3902.2002.Piccinini, R., cesaris, L., Daprà, v., Borromeo, v., Picozzi, c., 36. Secchi, c., Zecconi, A. The role of teat skin contamination in the epidemiology of Staphylococcus aureus intramammary infections. J.Dairy Res. 76, 36-41.2008.Larsen, H.D., Sloth, K.H., Elsberg, c., Enevoldsen, c., Pedersen, 37. L.H., Eriksen, N.H.R., Aarestrup, F.M., Jensen, N.E. The dynamics of Staphylococcus aureus intramamammary infections in nine Danish dairy herds. vet. Microbiol. 71, 89-101.2000.Matos, J.S., white, D.G., Harmon, R.J., Langlois, B.E. 38. Staphylococcus aureus from sites other than the lactating mammary gland. J Dairy Sci 74, 1544-1549.1991.Owens, w.E., Nickerson, S.c. Role of horn flies (Haematobia 39. irritans) in Staphylococcus aureus-induced mastitis in dairy heifers. Am. J. vet. Res. 59, 1122-1124.1998.Leslie, K.E., Dingwell, R.T. Background to dry cow therapy: 40. what, where, why - is it still relevant ? In: NMc Annual Meeting Fort worth (Tx) 26-29/01/03, pp. 5-17, 2003.Erskine, R.J., cullor, J., Schaellibaum, M., Yancey, B., Zecconi, 41. A. Bovine mastitis pathogens and trends in resistance to antibacterial drugs. In: 43rd NMc annual meeting, charlotte (1-4/02/2004), pp. 400-414, 2004.Radostits, S.M. Herd Health: food animal production medicine, 42. 3rd Edition. W.B.Saunders Co., Philadelphia, 2001.Sol, J., Sampimon, O.c., Barkema, H.w., Schukken, Y. Factors 43. associated with cure after therapy of clinical mastitis caused by Staphylococcus aureus. J. Dairy. Sci. 83, 278-284.2000.Sol, J., Sampimon, O.c., Snoep, J.J., Schukken, Y. Factors 44. associated with bacteriological cure during lactation after therapy for subclinical mastitis caused by Staphylococcus aureus. J. Dairy. Sci. 80, 2803-2808.1997.Sol, J., Sampimon, O.c., Snoep, J.J., Schukken, Y.H. Factors 45. associated with bacteriological cure after dry cow treatment of

subclinical staphylococcal mastitis with antibiotics. J. Dairy. Sci. 77, 75-79.1994.Foster, T. Potential for vaccination against infections caused by 46. Staphylococcus aureus. Vaccine 9, 221-227.1991.Zecconi, A., Smith, K.L. Ruminant Mammary Gland Immunity. 47. FIL-IDF, Bruxelles, 2003.Middleton, J.R., Luby, c.D., Adams, D.S. Efficacy of vaccination 48. against staphylococcal mastitis: A review and new data. Vet. Microbiol. 134, 192-198.2009.Nickerson, S.c., Owens, w.E., Boddie, N.T. Efficacy of a 49. Staph.aureus mastitis vaccine in dairy heifers. In: International Symposium on Immunology of Ruminant Mammary Gland, Stresa IT 11-14 June 2000, pp. 426-431, 2000.Edinger, D., Tenhagen, B.A., Baumgartner, B., Heuwieser, w. 50. Efficacy of a herd vaccine against Staphylococcus aureus in dairy heifers. In: International Symposium on Immunology of Ruminant Mammary Gland, Stresa IT 11-14 June 2000, pp. 410-417, 2000.Yoshida, K., Ichiman, Y., Narikawa, S., Evans, w. Staphylococcal 51. capsular vaccine for preventing mastitis in two herds in Georgia. J. Dairy Sci. 1984. 67, 3, 620-627.1984.Nordhaug, M., Nesse, L., Norcross, N., Gudding, R. A field trial 52. with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 1. clinical parameters. J. Dairy Sci. 77, 1267-1275.1994.watson, D., Mccoll, M., Davies, H. Field trial of a staphylococcal 53. mastitis vaccine in dairy herds: clinical, subclinical and microbiological assessments. Australian vet. Journal 1996. 74, 6, 447-450.1996.Leitner, G., Yadlin, N., Lubashevsy, E., Ezra, E., Glickman, A., 54. chaffer, M., winkler, M., Saran, A., Trainin, Z. Development of a Staphylococcus aureus vaccine against mastitis in dairy cows. II. Field trial. Vet. Immunol. Immunopathol. 93, 153-158.2003.Leitner, G., Lubashevsky, E., Trainin, Z. 55. Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model. vet. Immunol. Immunopathol. 93, 159-167.2003.Leitner, G., Lubashevsky, E., Glickman, A., winkler, M., Saran, 56. A., Trainin, Z. Development of a Staphylococcus aureus vaccine against mastitis in dairy cows I. Challenge trials. Vet. Immunol. Immunopathol. 93, 31-38.2003.Kerro-Dego, O., Prysliak, T., Potter, A.A., Perez-casal, J. DNA-57. protein immunization against the GapB and Gapc proteins of a mastitis isolate of Staphylococcus aureus. Vet. Immunol. Immunopathol. 113, 125-138.2006.Shkreta, L., Talbot, B.G., Diarra, M.S., Lacasse, P. Immune 58. responses to a DNA/protein vaccination strategy against Staphylococcus aureus induced mastitis in dairy cows. Vaccine 23, 114-126.2004.Yin, R.L., Li, c., Yang, Z.T., Zhang, Y.J., Bai, w.L., Li, x., 59. Yin, R.H., Liu, H., Liu, S., Yang, Q., cao, Y.G., Zhang, N.S. construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18. Vet. Immunol. Immunopathol. 132, 270-274.2009.castagliuolo, I., Piccinini, R., Beggiao, E., Palù, G., 60. Mengoli, c., Ditadi, F., vicenzoni, G., Zecconi, A. Mucosal genetic immunization against four adhesins protects against Staphylococcus aureus-induced mastitis. Vaccine 24, 4393-4402 2006.Saperstein, G., Hinckley, L.S., Post, J.E. Taking the team 61. approach to solving Staphylococcal mastitis infection. vet. Med. 83, 939-947.1988.

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Stott, A.w., Jones, G.M., Gunn, G.J. chase-Topping, M., 62. Humphry, R.w., Richardson, H., Logue, D.N., Optimum replacement policies for the control of subclinical mastitis due to S. aureus in dairy cows. J. Agric. Econ. 53, 627-644.2002.Hoblet, K.H., Miller, G.Y. Use of partial budgeting to determine 63. the economic outcome of Staphylococcus aureus intramammary infection reduction strategies in three Ohio diary herds. JAvMA 199, 714-720.1991.white, G. An attempt to control the spread of staphylococcal 64. mastitis by segregation and culling. Vet.Rec. 77, 1384-1386.1965.

Zecconi, A., Piccinini, R., Fox, K.L. Epidemiologic study of 65. intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds. JAvMA 223, 684-688.2003.Zecconi, A., Piccinini, R., Romani, S. Results and cost-benefit 66. analysis of a Staph.aureus control program in commercial dairy herds. In: 2nd International Symp. on Mastitis and Milk Quality, vancouver (13-15 sep 2001), pp. 311-315, 2001.


FIGUREFigure 1 - Control program scheme


INTOXICATION OF YOUNG CROCODILES IN CAPTIVITY DUE TO THE INGESTION OF DARKLING BEETLES BLAPS NITENS

LAPORTEI ARDOIN (COLEOPTERA; TENEBRIONIDAE)

Perelman, B1*. and Chikatunov V2.1D.N.Negev P.O.Box 38, Beit Kama,Israel.

2Department of Zoology, The George S. wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel*Corresponding author:Dr. Benzion Perelman

D.N.Negev P.O.Box 38 Beit Kama, Israel

Email: [email protected]

ABSTRACTA sharp increase of mortality was reported among young crocodiles on a rearing farm in the south of Israel during the

summer months. clinical inspection of the animals in the rearing rooms revealed that about 20% of the young crocodiles, suffered from severely swollen expanded abdomens with clinical signs of dyspnea. Post mortem examination of affected animals revealed severe expansion of the stomach and extensive damage to the mucosa with the presence of partly digested traces of black beetles. visual inspection of the premises revealed very large numbers of black beetles. A tentative diagnosis of a potential poisoning or intoxication related to the ingestion of the beetles by the young crocodiles was suggested. Beetles were identified as Blaps nitens laportei belonging to the Tenebrionidae family. within a few days after the pens were cleaned from dead and live beetles and the windows covered with a nylon mesh, the morbidity and mortality began to decline strongly suggesting that the etiology was related to the ingestion of beetles by the young crocodiles. To the best of the knowledge of the authors this is the first case report on the potential poisoning of young crocodiles by the ingestion of the darkling beetle Blaps nitens laportei

INTRODUCTIONSince the late 80’s, some attempts have been carried in

Israel to develop a crocodile farming industry. At present there is only one small crocodile farm in the area of Fazael e Dead Sea where a small breeding colony with several adult crocodiles (Crocodylus niloticus) is maintained. The eggs are incubated using artificial incubation and the young hatchlings transported and reared in another farm located in the area of the Negev. Young crocodiles are reared under intensive conditions in small greenhouse buildings in separated rooms with concrete pools and temperature controlled water. Every hatch of crocodiles is located in a different pool in groups containing 100 to 200 crocodiles per room. The rearing farm had a capacity to rear about 2000 young crocodiles up to the age of about 2 years.

During the summer season, large numbers of darkling beetles where observed in the surroundings and inside the crocodile rearing facilities. A sudden increase in mortality among the youngest crocodiles was tentatively related to a possible intoxication due to the ingestion of darkling beetles. In this case report we describe the clinical and pathological findings of the intoxication of young crocodiles after ingestion of darkling beetles Blaps nitens laportei (1).

CASE REPORTYoung crocodile hatchlings (Crocodylus niloticus) where

transported from the hatchery to the rearing farm in the area of the Negev in Israel. The facilities at the farm include two green-houses like buildings with concrete walls and floor and green polycarbonate ceilings that enables light to enter the building. Each building is separated into 5 rooms by concrete walls of about 1.2 meters high, the concrete floor has a slope that allows for filling with water for about 50% of the area of the floor, creating a pool for the crocodiles as well as a dry area to rest.

The young crocodiles were fed with minced fresh ostrich meat from a nearby ostrich slaughter house, the meat was mixed with a special premix containing calcium, vitamins and microelements in order to provide a balanced diet to the young crocodiles.

The temperature of the water in the pools was controlled to avoid hypothermia of the young reptiles, and changed every 3 days to avoid excessive contamination of the water.

A sharp increase of mortality was reported among the youngest crocodiles in some of the rooms in one of the rearing houses.

clinical inspection of the animals in the rearing rooms revealed that about 20% of the young crocodiles in some of the rooms suffered from severely swollen expanded abdomens; the affected animals held their heads high and the mouths open

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indicative of dyspnea (Fig 1).Post mortem examination of affected animals revealed

severe expansion of the stomach (Fig. 2). Opening of the stomach revealed extensive damage to the mucosa with traces of ostrich meat, a gelatinous content and partly digested traces of black beetles (Fig. 3).

visual inspection of the premises revealed very large numbers of black beetles all over the facility including on the floor of the rearing areas (Fig 4). Young crocodiles were observed to feed on live beetles falling on the water of the pools or crawling on the floor while the older one year old crocodiles did not appear to touch the live or dead beetles.

A tentative diagnosis of a potential poisoning or intoxication related to the ingestion of the beetles by the young crocodiles was suggested as other etiologies were discarded such as addition of water treatments, use of disinfectants or any other toxic product used accidentally. The measures taken to reduce the presence of black beetles inside the rearing areas included physical cleaning of the rearing areas from any dead or live beetle and location of a nylon mesh on the windows of the buildings to avoid further entrance of the beetles to the rearing areas. within a ten days period, all the affected young crocodiles showing bloated bellies died and general mortality within the affected groups started to reduce, but many of the less affected animals showed apathy and reluctance to eat. Pathologic examination of some of these crocodiles, revealed extensive damage of the mucosa of the stomach and secondary development of some fungal infection probably due to the extensive primary damage to the epithelium of the stomach, and the accumulation of organic debris due to stomach stasis.

Treatment with copper sulphate at ½ kg /1000 liters of water was added to the ponds. within a week, mortality decreased significantly and no new cases of bloated bellies were observed.

DISCUSSIONDistention of the stomach or “bloating” has been reported

in crocodiles by Huchzermeyer as result of over feeding (2). crocodiles feed after long periods of food withdrawal may eat large quantities of meat and develop stomach stasis, bacterial decomposition of the stomach content that may cause toxemia and mortality.

In this case the young crocodiles where fed every second day just enough meat to feed all the crocodiles in the pen. The development of bloating and mortality was acute and many of the affected crocodiles had only some residues of meat from their last meal. All the crocodiles in this case report examined showed swollen bellies and had residues of black beetles in their stomachs. The internal mucosa of the stomach was eroded and severe desquamation of the epithelium was observed in all the affected crocodiles.

Beetles were identified by Dr. v. chikatunov from the University of Tel Aviv as Blaps nitens laportei belonging to the Tenebrionidae family (1) (Fig 5). It has been reported that some members of tenebrionid beetles, have defensive

glands able to produce a mixture of p-benzoquinones and hydrocarbons such as 1,4-benzoquinone, 2-methyl-1,4-benzoquinone and2-ethyl-1,4-benzoquinone (3, 4, 5). These toxic products are produced and contained in small glands in the abdominal cavity of the insects and are released from small openings at the tip of the abdomen when the beetles are threatened. Some Tenebrionid beetles posses special glands able to produce at least three types of 1,4-benzoquinones (5, 6) and hydrocarbons (4). One of the most effective mechanisms of protection among some insects is the production of caustic or toxic repellent products against predators.

Another possibility, quinones which are very reactive and toxic compounds are able to cause severe membrane damage, enzyme destruction and cell death, and are mutagenic and carcinogenic (Ollinger and Brunmark 1991). The toxicity of the benzoquinones is related to their capacity to produce free oxygen radicals able to severely affect cellular components.

The fact that the number of new cases of bloating among the young crocodiles started to decrease within a few days after the pens were cleaned from dead and live beetles and the windows covered with a nylon mesh, strongly suggest that the bloating and severe damage observed was related to the ingestion of beetles by the young crocodiles. For ethical reasons we did not try to reproduce the clinical signs and pathology of the intoxication by artificially feeding healthy young crocodiles, but the clinical and pathological picture are highly suggestive of an intoxication caused by the toxic benzoquinones contained in the poison glands of the beetles released in the stomach of the young crocodiles.

To the best of our knowledge this is the first case report on the potential poisoning or young crocodiles by the ingestion of the darkling beetle Blaps nitens laportei

REFERENCES Ardoin, P. contribution à l’étude des Tenebrionidae (coleoptera) 1. de Sardaigne. Ann. de la Soc. Ent. de France (N.S.) 9: 257-307, 1973.Huchzermeyer, F.w. crocodiles: Biology, Husbandry and 2. Diseases. cABI Publishing, Oxon, United Kingdom. 2003. Eisner, T., Mc Henry, F., and Salpeter, M.M. Defense 3. mechanisms of arthropods-xv. Morphology of the quinine-producing glands of a tenebrionid beetle (Eleodes longicollis lec.) J.Morphology.115,355-399, 1964.Happ M G. Quinone and Hydrocarbon production in the 4. defensive glands of Eleodes longicollis and Tribolium castaneum (coleopteran), tenebrionidae. J. Insect Physiology. 14:1821-1837, 1968.Eisner, T., Eisner M., and Siegler M. Secret weapons: Defenses 5. of Insects, Spiders, Scorpions and Other Many-Legged creatures. Harvard University Press, cambridge. 2005Eisner, T . and Meinwald, J. 1966. Defensive secretions of 6. arthropods. Science, N.Y. 153, 1341-1350, 1966.Ollinger, K., and Brunmark, A. Effect of hydroxyl substituents 7. position on 1,4-naphtoquinone toxicity to rat hepatocytes. J..Biol.chem. 266:21496-21503, 1991.



Figure 4 - Large quantities of black beetles were found covering the floor of the crocodile rearing pens

Figure 5 - Blaps nitens laportei 1973 - darkling beetle, Negev, Israel

LEGENDS FOR FIGURES

Figure 1 - A young crocodile showing extensive distention of the abdominal cavity “bloating”.

Figure 2 - Post mortem examination of a young affected crocodile showing severe distention of the stomach.

Figure 3 - Open stomach showing bleeding and desquamation of the epithelium of the stomach, a large amount of a gelatin like content and semi-digested parts of the black beetles.

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BOVINE UROLITHS ANALYSIS: A REVIEW OF 30 CASES.Parrah, J.D., Hussain, S. S., Moulvi, B. A., Singh, M. and Athar, H

Division of veterinary Surgery and RadiologyFaculty of veterinary Sciences and Animal Husbandry, SKUAST –K Shuhama Aulsteng, Kashmir, India .

Key words: Urolithiasis, Calculi, Calf

Corresponding author:Dr. J.D. Parrah

[email protected]

ABSTRACTcalculi obtained from 30 clinical cases of obstructive urolithiasis in male calves during surgery were subjected to

complete analysis including physical, microscopic and chemical examination. Cystic lumen and neck, jointly, were the commonest site of calculi retrieval (47%) cases, followed by cystic neck (33%) cases and cystic lumen (20%) cases. In majority (90%) of the cases small multiple calculi were retrieved. The calculi retrieved were usually as free sandy material mixed with blood and other tissue debris but in 3 cases a mass comprising of calculi embedded in blood clot and tissue debris was retrieved from cystic lumen. The urethral calculi were either loop shaped or impacted sandy material. Microscopic examination revealed that one or more well defined nuclei (nidus) were found in each concretion. The nuclei and the surrounding concentric layers of laminae were enclosed by a single capsule. The calculi were composed of magnesium ammonium phosphate, calcium phosphate, calcium carbonate, calcium oxalate, hippuric acid, tyrosine and uric acid. Twenty three (77%) calculi samples were composed of magnesium ammonium phosphate only.

INTRODUCTIONUrolithiasis in countries like India presents an important

economic repercussion where cattle - based agriculture is strongly linked with the livelihood of an important segment of the population. The primary objective of urinary calculus analysis was to determine the qualitative composition, as the prevention of urolithiasis and its treatment depends on a detailed knowledge of the composition and structure of the calculi (1). For complete analysis of calculi, a combination of methods was adopted: Microscopic, spectroscopic, chemical and x-ray diffractometry are complementary for the analysis of calculi and no one method is sufficient, as quantitative analysis are best obtained by x-ray diffractometry, while qualitative identification of depositional sequence and the quantitative determination of minor constituents can be determined by microscopic and chemical methods (2). The chemical composition of urinary calculi varies and depends largely on the dietary composition of individual elements, the geographical location and local management practices (3). Obstructive urolithiasis is very common in ruminants of Kashmir valley, however the highest incidence is found in calves. No report is available about the chemical composition of uroliths retrieved from the obstructive urolithiasis cases in the valley of Jammu and Kashmir state. This study was thus undertaken to understand the chemical composition of the uroliths retrieved from clinical cases so as to suggest the remedial measures for preventing the disease and its effective treatment.

MATERIALS AND METHODSThirty male cattle calves, suffering from complete retention

of urine, presented for treatment at Teaching veterinary clinical Services complex, Faculty of veterinary Sciences and Animal Husbandry (F. v. Sc & A. H.), Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Srinagar, formed the material of the study. Ten bovine clinical cases of obstructive urolithiasis each were subjected to tube cystostomy using polyvinylchloride urinary catheter, tube cystostomy using Foleys catheter and cystotomy and normograde cystourethral catheterization (cystotomy with indwelling urethral catheterization).

Every effort was made to retrieve all the uroliths from cystic lumen in a sterile jar containing dextrose saline solution. For this purpose, bladder and its neck were irrigated many times to collect all the uroliths. The uroliths were cleaned and cleared of all tissue and blood, and dried in an incubator at 45oC for 24 hours. The uroliths were preserved at 4oc for further examination. During the operation, calculi were removed from urethra and/or bladder for re-establishing the patency of the urinary tract using different surgical techniques. These calculi were subjected to following examinations:

1. Physical characteristics calculi removed from the urethra and/or bladders were

observed for their types (hard/pasty/sandy), shapes (round/irregular etc.), number and anatomical position. After washing the calculi and drying, the calculus mass was weighed and the largest ones measured.

2. Microscopic examinationThe representative calculi were examined under dissection

microscope. The number and geometrical location of the nidus



within the calculus was recorded. 3. Chemical analysisAfter recording physical characteristics, the calculi were

stored at 4oc in the refrigerator untill further analysis of their composition. The chemical composition of the calculi was determined by the standard procedures (4).

RESULTS 1. Urolith analysiscalculi of different number, shape and size were

retrieved from various locations in the urinary tract of all the animals subjected to various surgical techniques including tube cystostomy and cystotomy with indwelling urethral catheterisation for correction of obstructive urolithiasis. These calculi were observed for their location, number, shape and composition (Table 1).

1.1 Location of calculiAlmost in all the cases calculi were retrieved simultaneously

from multiple sites. cystic lumen and neck jointly was the commonest site of calculi retrieval {14/30 (47%)} cases, followed by cystic neck {10/30 (33%)} cases and cystic lumen 6/30 (20%) cases. calculi were retrieved from urethra from one site only in 8/10 (80%) cases and from 2 sites simultaneously i.e. pre- and post-scrotal region in 2/10 (20%) cases only.

1.2 Number of calculi In one case only a single calculus (Fig 1) was retrieved

from cystic lumen, and only two calculi were retrieved from cystic neck in 2 cases. Among the remaining 27/30 (90%) cases multiple small calculi (Fig 2) were retrieved from the cystic lumen (5), cystic neck (8) and cystic lumen and neck jointly in 14 cases.

1.3 Gross morphology of calculiThe calculi retrieved from the cystic lumen and neck were

usually in the form of free sandy material (Fig 2) mixed with blood and other tissue debris but in 3 cases a mass comprising of calculi embedded in blood clot and tissue debris was retrieved from the cystic lumen. These masses measured from 3.7 – 5.9 cm and weighed between 35 to 65 gm (Fig 3). On ultrasonographic examination these calculus masses yielded acoustic shadows typical for sonographic evaluation of bladder stones.

calculi from most of the cases {20/30 (67%)} were sandy, irregular in shape with smooth surfaces and edges. Of these, calculi were creamy white in 17 cases and off-white in 3 cases (Fig 2). All these calculi were soft and easily broken. calculi from seven cases were pasty in nature and dark brown in colour. The individual calculi (Fig 1) single or double obtained from 3 cases were dendritic in shape, white in colour and hard to break.

The urethral calculi were either loop shaped (Fig 5) or impacted sandy material (Fig 4). These calculi were yellow in colour and easy to break.

1.4 Microscopic examination of calculiSurface morphology of intact representative calculi was

studied under dissection microscope. One or more well defined nuclei (nidus) were found in each concretion. The nidus appeared dense and homogenous, and was not necessarily in the geometrical centre of the uroliths. The nidus was surrounded by concentric layers of crystals from precipitating minerals without clear demarcation between the adjacent layers of

concentric laminae. The nuclei and the surrounding concentric layers of laminae were enclosed by a single capsule (Fig 6).

1.5 Chemical composition of calculiThe calculi were composed of magnesium ammonium

phosphate, calcium phosphate, calcium carbonate, calcium oxalate, hippuric acid, tyrosine and uric acid. Twenty three (77%) calculi samples were composed of magnesium ammonium phosphate only; while in other (23%) samples magnesium ammonium phosphate (major component) was accompanied by any one of the other chemical components listed above.

DISCUSSION1.1 Location of calculiThe calculi may be lodged in any part of the urinary

tract i.e., starting from renal pelvis to glans penis. But the lodgement of the urolith in the bladder neck and urethra may lead to complete obstruction to urine flow thereby enhancing the acuteness and severity of the condition. The longer length of urethra and presence of sigmoid flexure make the urethra more prone to the lodgement of calculi as compared to other parts of the urinary tract in ruminants. In this study cystic lumen and neck jointly was the commonest site of calculi retrieval, followed by the cystic neck. calculi were retrieved from the sigmoid flexure of urethra in 60% cases of animals where cystotomy with indwelling urethral catheterization was performed. The findings are in agreement with those of other researchers (5) who recovered about 68% of calculi in the sigmoid flexure of the bovines. In another study (6) a high incidence of bovine urinary calculi were found in the distal portion of the sigmoid flexure. Distal sigmoid flexure in cattle and the urethral process in sheep are the commonest sites of urethral obstruction by urolith, as the diameter of lumens at these sites are the narrowest in the urethral canal, thus calculi could easily be trapped at these sites (7).

1.2 Number of calculiIn most of the cases (90%) multiple small calculi without

any distinct morphology were seen, while in 3.3% cases single and in 6.6% cases two distinct calculi were found. Generally a single distinct calculus is responsible for urethral obstruction in cattle (8), which is in agreement with the findings of the present study.

1.3 Gross morphology of calculiPhysical characteristics, including size, shape, colour and

texture of uroliths, may serve as a preliminary and tentative indicator of the composition of the calculi and thereby assist in establishing the aetiological factors (9). calculi may be of varying sizes, commonly described as sand, gravel or stone. Most common type of calculi was sandy (66%), pasty (23%) or assuming the shape of urethra. These findings are in accordance with those of other researchers, who reported that phosphate calculi e.g. calcium phosphate and triple phosphate are usually white, smooth, numerous, chalky and friable (6). Hard and discrete uroliths in cattle but friable and sandy masses in fattening lambs are usually present (10). The individual single or double struvite calculi retrieved from the clinical cases of obstructive urolithiasis in calves were usually white but dendritic in shape. These findings are in total consonance with those of the previous studies, who reported that struvite may also occur as a single urocystolith with sharp

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facets traumatizing the bladder wall and causing the marked haematuria (11).

1.4 Microscopic view of calculiMicroscopic examination clearly showed a central

homogenous nidus easily differentiated from the outer concentric laminae. Formation of nidus is usually the first phase in the formation of the urolith. Any foreign material or cellular debris may act as nidus, however the nidus may be formed spontaneously due to supersaturation and oversaturation of the urine with lithogenic crystalloids (12). Further precipitation of crystalloids around the central nidus lead to the formation of concentric laminae. There were no clear demarcations between adjacent layers as also reported by other researchers (13, 14).

Slight eccentric location of the nidus suggested that the calculus was not equally accessible to precipitating minerals from all sides so that the growth proceeded at variable rates around the calculus. The nucleus represents the starting point of the stone and it need not be in its geometrical centre (15). Moreover, after formation of the nidus, calculi may grow into a urolith of same or different composition depending on the condition of urine saturation. The presence of a second eccentric nidus might be due to abrupt changes in the conditions of supersaturation of the urine thus resulting in precipitation of another nidus.

1.5 Chemical composition of calculiMagnesium ammonium phosphate was present in

every urinary calculus either alone or with other chemicals. Magnesium ammonium phosphate was alone in 77% of cases and as major component in combination with other chemicals in 23% of cases. The findings seem to fall in line with those of previous studies, who reported 61% of calculi were composed of a single mineral substance (16).

calculi composition is affected by the factors like geography, species, age, sex, composition of feed, pH of urine, urinary tract infection, etc. During this study, composition of feed and urine pH seemed to be the profound predisposing factors, as wheat bran alone or in combination with other feeding stuffs was given to the maximum number of calves (77%). Rations high in grain but with limited amount of roughage leads to ammonium phosphate urolithiasis in feedlot cattle. considering the feeding habits of calves of this study mostly phosphate calculi observed, were as expected. The findings are in agreement with those of previous studies, who reported that highly digestible, low roughage ration having more phosphorus than calcium (i.e. high grain feeding) leads to the formation of insoluble struvite calculi (17, 18, 19). These observations also in agreement with those of other workers, who observed that diet having more wheat bran, predisposed animals to phosphate stones (20, 21).

Phosphate calculi are formed rapidly in alkaline urine but are more soluble in acidic urine (22). In the present study, base values of urine pH were alkaline in all the groups, so the formation of phosphate calculi was to be expected. The findings substantiate the observations of previous researchers who found the most common pH for precipitation of magnesium ammonium phosphate, calcium phosphate and ammonium urate crystals to be 7 (13, 23).

Presence of calcium phosphate deposits between the struvite crystals represented the epitaxial growth, which signifies the growth of one type of crystal upon another type (24). chances of occurrence of struvite and calcium phosphate occurring

together is more likely as both types of crystals are formed and precipitated at alkaline urine pH (25). Epitaxial growth of calculus may provide a plausible explanation of why uroliths are frequently of mixed composition. It could also explain a heterogenous form of nucleation.

CONCLUSIONS From the findings of the present study, it is evident

that gross morphology besides chemical and microscopical examination aids in the identification of calculi, thereby assisting in the establishment of the etiological factors. Furthermore, the feeding habits have been found to have a profound predisposing effect on the development of particular calculi. The incidence of obstructive urolithiasis, is mostly found in winter under temperate conditions, pointing towards the inadequate water intake as another predisposing factor for the development of disease. Obstructive urolithiasis, a dreadful disease in ruminants especially in cattle, can therefore be prevented if precautionary measures like balanced feeding and encouraging the animals to take adequate amounts of water in order to induce diuresis, by addition of sodium chloride to their feed especially during the chilly winter season.

REFERENCESUlrich, L.K., Kathleen, A.B., Koehler, L.A. and Swanson, L. 1. Urolith analysis. Submission, methods and interpretation. vet. clin. North Amer. Small Anim. Pract. 26:393-400. 1996.Otnes, B. and Montgomery, O. Method and reliability of 2. crystallographic stone analysis. Invest. Urol. 17: 314-319. 1980.Singh, J. and Singh, K. Obstructive urolithiasis and uraemia in 3. cattle and buffalo-a review. Indian J. vet. Surg. 11: 1-20. 1990.varley, H. Practical clinical biochemistry. cBS Publishers and 4. distributors. 1988.Gera, K.L. and Nigam, J.M. Urolithiasis in bovines (a report of 5. 193 clinical cases). Indian vet. Journal. 56: 417-423. 1979.Loretti, A.P., Oliveira-Lo,de., Cruz, C.E.F., Driemeier, D. and 6. de-Oliveira, Lo. clinical and pathological study of an outbreak of obstructive urolithiasis in feedlot cattle in southern Brazil. Pesquisa Veterinaria-Brasileira. 23:61-64. 2003.Tiruneh, R. A retrospective study on ruminant urethral obstruction 7. in Debre Zeit area, Ethiopia. Revue-de-Medecine-veterinaire. 151: 855-860. 2000.Radostits, O.M., Blood, D.c., Gay, c.c. and Hinchcliff, K.w. 8. veterinary Medicine: A Textbook of the Diseases of cattle, Sheep, Pigs, Goats and Horses. Bailliere Tindall, London. pp. 493-498. 2000.Lavania, J.P. and Angelo, S.J. Studies on the physical analysis 9. of bovine nephroliths. Indian vet. Med. Journal. 1: 35-37. 1977.Hawkins, w.w. Experimental production and control of 10. urolithiasis. JAvMA. 147 1321-1323. 1965.Guthrie, S. cystic calculi in cats. vet. Rec.120:416-417. 1987.11. Osborne, c.A., Lulich, J.P., Bartges, J.w., Unger, L.K., 12. Thumchai, R., Koehler, L.A., Bird, K.A., and Felice, L.J. canine and feline urolithiasis: relationship of aetiopathogenesis to treatment and prevention In: Canine and Feline nephrology and urology by Osborne, C. A. and Finco, D.R. Williams and wilkins. pp. 798-888. 1995.Osborne, c.A., clinton, c.w., Moran, H.c. and Bailie, N.c. 13.



comparison of qualitative and quantitative analyses of canine uroliths. vet. clin. North Amer. Small Anim. Pract. 16:317-323. 1986.Osborne, c.A., Lulich, J.P., Polzin, D.J., Sanderson, S.L., Koehler, 14. L.A., Ulrich, L.K., Bird, K.A., Swanson, L.L., Pederson, L.A. and Sudo, S.Z. Analysis of 77,000 canine uroliths. Perspectives from the urolith centre. vet. clin. North Amer. Small Anim. Pract. 29:17-38. 1999.Khan, S.R. and Hackett, R.L. Role of organic matrix in urinary 15. stone formation: an ultrastructural study of crystal matrix interface of calcium oxalate monohydrate stones. J. Urol. 150:239-245. 1993.Ling, G.v., Franti, c.E., Johnson, D.L. and Ruby, A.L. 16. Urolithiasis in dogs. Iv: Survey of interrelations among breed, mineral composition, and anatomic location of calculi, and presence of urinary tract infection. Amer.J.vet. Res. 59: 650-660. 1998.Munakata, K., Ikeda, K., Tanaka, K. and Suda, H. Urolithiasis 17. syndrome in beef cattle in Japan. National Institute of Animal Health, Quarterly, Japan. 14: 17-28. 1974.Munakata, K., Suda, H. and Ikeda, K. Induction of urolithiasis 18.

syndrome in cattle. National Institute of Animal Health, Quarterly, Japan14: 31 -32. 1974.Ahmed, A. S., Amer, H.A. and Ibrahim, I.M. Influence of dietary 19. mineral imbalance on the incidence of urolithiasis in Egyptian calves. Arch. Exp. Veterinarmed. 43: 73-77. 1989.Anjaria, J.v. Observations on bovine urethral calculosis. Indian 20. vet. Journal. 46: 449-453. 1969.Kataria, R.S. and Rao, U.R.K. chemical composition of some 21. bovine nephroliths with special reference to silica nephroliths. Indian vet. Journal. 46: 848-854. 1969.Prien, E.L. and Prien, E.L. composition and structure of urinary 22. stone. Amer. J. Med. 45(5): 654-672. 1968.Osborne, c.A., O, Brien, T.D., Ghobrial, H.K., Meihak, L. and 23. Stevens, J.B. crystalluria, observations, interpretations and misinterpretations. vet. clin. North Amer. Small Anim. Pract. 16:45-65. 1986.Finlayson, B. Symposium on renal lithiasis. Urol. clin. North 24. Am. 1: 181-0212. 1974.Klausner, J.S. and Osborne, c.A. canine calcium phosphate 25. uroliths. vet. clin. North Amer. Large Anim. Pract. 16:171-184. 1986.

TABLE AND FIGURES

Table 1: Distribution of urinary calculi in calves of different groups

Location of calculiGroup AI Group AII Group B

Total %age No. ofanimals

No. ofcalculi

No. ofanimals

No. ofcalculi

No. ofanimals

No. ofcalculi

Cystic lumen 1 1 - - 5 M 6/30 20

Cystic neck3 M 3 M 2 M

11/30 371 2 1 2 0 0

Cystic lumen andneck 5 M 6 M 3 M 14/30 47

Urethral lumen - - - - 10 10/10 100

DSF - - - - 4 4/10 40

PSF - - - - 2 2/10 20

Pre and post scrotal region - - - - 2 2/10 20

Between DSF & Glans penis - - - - 2 2/10 20

DSF = distal sigmoid flexure, PSF = proximal sigmoid flexure

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Figure 1 - Single dendritic calculus

Figure 2 - Off white multiple concretionsFigure 3 -

Figure 3 - Single calculus mass

Figure 5 - Single calculus mass

Figure 6 - Single loop shaped calculus in urethra

Figure 5 - calculi showing nidus in the centre



DIAGNOSIS OF MILK FEVER BY A WATER HARDNESS TEST KIT IN EWES

Aktas, M.S1., Kaynar, O2, Ozkanlar, S2., Ozkanlar, Y1.1Department of Internal Medicine, Faculty of veterinary Medicine, Ataturk University, Erzurum/Turkey

2Departments of Biochemistry, Faculty of veterinary Medicine, Ataturk University, Erzurum/Turkey

Corresponding author: Dr. Mustafa Sinan Aktas

Ataturk University Faculty of Veterinary Medicine

Department of Internal Medicine Erzurum/Turkey

E-mail: [email protected]: 0904422315530

ABSTRACT The aim of the study was to evaluate the measurement of total calcium levels in sera of sheep with and without milk

fever by using a commercial water hardness test kit. Thirty sheep with findings of milk fever from 9 different farms were used in the present study. Total serum calcium concentrations were determined by using a commercial water hardness test kit and a laboratory automated biochemical analyzer. Results of the test kit and laboratory methods were significantly (P < 0.001) correlated (Spearman’s p = 0.896). In conclusion, it has been determined that total calcium levels in sheep sera may be determined with a water hardness test kit as used in this study, and that data are in concordance with the clinical findings and the other laboratory results.

INTRODUCTION Milk fever - parturient paresis or hypocalcaemia - is a

metabolic disorder that occurs around the time of parturition or during early lactation in sheep (1). The disease commonly occurs in outbreaks, in groups of ewes exposed to forced exercise, long-distance transport, and sudden deprivation of food and grazing on oxalate-containing plants or green cereal crops (2). Although it is may be seen occasionally as epidemic in the herd where up to 25% of the herd may be affected, its course is mostly sporadic and generally affects less than 5% of the herd (1, 2, 3, 4).

The disease is characterized by a low serum calcium (Ca) level (2, 5, 6). As a result of inadequate calcium intake during the last periods of pregnancy or first periods of lactation, the body may meet the required need of calcium through calcium mobilization from bones. If hormonal mechanisms are inadequate, for example in the case of an inactive parathyroid gland, mobilization is delayed and blood calcium concentration is reduced with the resultant development of milk fever. In small ruminants, hypocalcaemia may develop secondarily during the course of other periparturient diseases, particularly in pregnancy toxemia (7). The diagnosis of the disease is based on clinical or laboratory methods or based on the presence of risk factors which have been reported (8). Therefore, monitoring the calcium level in sheep during periparturient period may be useful for the identification of the development risk of both milk fever and other hypocalcaemia related periparturient diseases. The definitive diagnosis is made through measurement of total and /or ionized serum calcium concentration (7). Serum total or ionized calcium levels may be measured by laboratory and/or portable biochemical analyzers. In veterinary practice chemistry analyzers are often unavailable in the field. Therefore, the aim of the present study

was to measure the total calcium levels in sheep sera with and without milk fever using a commercial water hardness test kit (wHTK) compared to a routine laboratory method.

MATERIAL AND METHODSAnimals

Thirty ewes were used in this study had at least one clinical findings of milk fever such as depression, increased respiration, muscular tremor, weakness, sternal recumbence and extended or twisted head. Clinical examinations were made by the same veterinarian. The sheep were 3.3±1 years of age (range 2 and 5 years) were taken from 9 different farms. Fifteen sheep were pregnant and 15 sheep were lactating.

Approximately 20 ml blood was collected from the jugular vein. Blood samples were allowed to clot for minimum 30 minutes at ambient temperature. The samples were then centrifuged and sera were collected. The sera were kept at -20°C until the analysis was performed. All samples were analyzed within 4 weeks.Serum calcium analyses

Total serum calcium concentrations were determined by spectrophotometric method using an automatic biochemical analyzer (Cobas 6000 analyzer, Roche, Switzerland). All samples were analyzed at the same time. Based on the results obtained with the automatic chemical analyzer the values were found to be in the range of 6.1 to 10.1 mg/dl.

Total calcium concentrations in the sera samples of all sheep were determined using a commercial wHTK which was designed for measurement of calcium carbonate concentrations in water samples (cHEMetrics. Inc Cat. No: K-1705. Calverton, VA, USA). The test was carried out as follows: Calcium in the test sample was allowed to react with a zinc salt in the presence of a color indicator. The determined blue and blue-

Key words: diagnostic, milk fever, sheep, water hardness test kit.

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green color of the resulting zinc-zincon complex was assigned as the endpoint of the reaction. According to the manufacturer the test range for the kit was between 2 to 20 mg/dl. Briefly, all samples were thawed at room temperature (approx. 20°C) during the test period. In order to achieve proper concentration, 1 drop of indicator solution were dropped in 6.7 ml of sera sample and mixed. The content turned an orange color. This sample was drawn into an ampoule by using a titrator apparatus and the content of the ampoule was shaken gently back and forth. This application was performed until the orange color of the ampoule content was transformed into blue or blue-green color. The calcium carbonate concentration (ppm) was inversely correlated to volume of sera reaching the endpoint titration on the scale of the ampoule. The result was further multiplied by 0.04 to calculate total calcium concentration in mg/dl. The samples were assayed with the wHTK in a blind manner without prior knowledge of the laboratory results. Statistical analyses

Linear regression test was performed in order to determine the relationship between the sera total calcium levels obtained from the laboratory versus the wHTK analyses. Sperman’s rank correlation test was applied to calculate the correlation between the results obtained by these two methods. Two samples were excluded from statistical analysis due to hemolysis.

In order to evaluate the wHTK capacity to diagnose milk fever, calcium concentrations of less than 8 mg/dl was determined as a laboratory-derived value for defining milk fever. The cutoff reference value was used according to results of Bickhardt et al. (9).

Sensitivity and specificity were calculated on the basis of actual test kit measurements using the defined total ca concentration for milk fever, the cutoff value and predicted total ca concentrations, derived from the regression equation. All statistical analyses were performed with computer based software for statistics (Sigma Stat).

RESULTS Based on the results obtained in the laboratory, sera total

calcium values were between 6.1 mg/dl and 10.1 mg/dl (median, 8.5 mg/dl) and the concentration in 6 of 30 samples was below 8.0 mg/dl.

There was a significant relationship between the laboratory results and wHTK results in the 28 samples tested (R2= 0.7743; Figure). The regression formula was: laboratory value = 0.4324 + (0.953 x test kit value). The 95% confidence intervals for the regression equation were 0.4 to 1.6, the intercept and slope and 0.8 to 1, respectively. Results of the wHTK and laboratory methods were found to be significantly (P < 0.001) correlated (Spearman’s p = 0.896).

when comparing the two methods, 6 samples of the laboratory results were evaluated to fall into the range designated for the diagnosis of milk fever (ca< 8 mg/dl), while only 5 samples analyzed by the wHTK method were found to fall into this range (sensitivity = 83.3%). Laboratory results showed that when 22 samples which were found to be greater than 8 mg/dl sera total calcium concentration were compared to the wHTK method results, all samples were found to be above the cutoff value of 8 mg/dl, therefore giving a specificity of 100% for this range. Thus, predictive value of a negative test result was 100% (22/22) and predictive value of a positive test result was 83% (5/6). The accuracy of wHTK to indicate the diagnosis of milk fever was verified by the predicted total calcium concentration of < 8 mg/dl and the regression equation results.

Analysis of two the hemolyzed serum samples did not react with the wHTK giving no color change where laboratory method was able to calculate a calcium value.

DISCUSSIONMilk fever in sheep develops mostly in the last 4-6 weeks

of pregnancy and the first 6 weeks of lactation (2, 4). The most frequently observed clinical findings of the disease involve ataxia, tremor, tetania, constipation and reduced ruminal motility, increased respiration and pulsation, regurgitation, tympani, depression, sternal recumbency and extended or twisted head (2, 5). In the study presented, 30 sheep, with at least one clinical finding of milk fever, were tested during the periods between the last 4 weeks of pregnancy and the first 4 weeks of lactation.

The clinical signs of milk fever are nonspecific and therefore cannot be relied upon. The differential diagnosis of milk fever includes hypomagnesemia, osteomalacia, pregnancy toxemia and enterotoxemia (2, 10).

In cows, milk fever may coexist with hypermagnesemia or hypomagnesemia. Relative hypermagnesemia may occur by shifting the ratio of ca:Mg from 6:1 to 2:1. Hypophosphatemia in milk fever may be secondary to the hypocalcemia rather than being a concurrent event. woldemeskel et al. (5) found that serum while total calcium may be decreased phosphorus and magnesium were normal in sheep with milk fever. In contrast, El-Khodery et al. (1) reported both total calcium and magnesium levels were low in milk fever.

The clinical pathological diagnosis of milk fever is based on the measurement of the total serum calcium concentration which is comprised of the sum of ionized calcium and the calcium bound protein. Ionized calcium is important for immediate metabolic functions however the analysis of ionized calcium in the field is not practical due to the unavailability of equipment and the high expense involved (2). Practically this is not required due to the correlation between the concentrations of ionized and total sera (2, 4, 11). In the last decade, there have been many reports of diagnosis of milk fever using total serum total calcium level in cows (12, 13, 14), buffalo (15), and sheep (1, 5, 6, 16). Bickhardt et al. (9) showed that serum total calcium concentrations are less 8 mg/dl in sheep with milk fever.

In the field the drawing blood from the animal with suspected milk fever, delivering the sample to the laboratory and waiting for the result lead to a loss of valuable time which is disadvantageous to the health of the patient. In the

Figure -Linear regression of total serum Ca concentrations derived by use of a laboratory based-method and by use of wHTK for 28 sheep.



1990’s, portable biochemical analyzers developed for humans were used in animals, particularly in cats, dogs and horses providing more accessible results in a shorter time frame (18). Later, portable biochemical analyzers were produced specific for animals including sheep, which are still being used successfully. The disadvantage of these analyzers are their high costs.

In this study, sera total calcium concentrations were determined by using a commercial water hardness test kit which is highly affordable and the results correlate well with the standard laboratory method (P < 0.001) (Spearman’s p = 0.896). Previous studies have been undertaken to determine the calcium levels in sera using commercial water hardness test kits. A commercial kit designed for water quality analysis was used by Ley et al. (17) in order to obtain the quantitative value of calcium concentration in mare milk. Matsas et al. (16) further determined sera total calcium levels in dairy cattle by using such a kit in order to obtain a diagnosis of milk fever. They found sensitivity of 100% and specificity of 73% when comparing the laboratory method with the data obtained by using wHTK indicating that there was a significant correlation between two methods (P<0.001). Comparing the laboratory results and the results obtained by using wHTK in the present study we found a sensitivity of 83.3% and a specificity of 100%. There was a significant correlation (P<0.001) between two methods as was shown Matsas et al. (16) working with dairy cattle.

The wHTK lacks the ability to determine magnesium and phosphorus concentration which is a disadvantage. The use of non-hemolysed blood is a prerequisite for using the wHTK analysis. The advantages of the kit lies in the fact that the test is a rapid (less than 5 minutes) and inexpensive method for measuring serum total ca concentrations in sheep. This offers a benefit ovine practitioners and veterinarians who do not own portable clinical analyzers or blood chemistry machines, allowing diagnosis of milk fever in individual sheep and monitoring of postpartum ca concentrations of a herd. The assay range of wHTK (2 to 20 mg/dl) is wide enough to measure total calcium level in serum samples. It can be assumed that this test may be used in other situation other than milk fever which has been correlated in this study with a standard laboratory method.

In conclusion, it has been determined that total serum calcium levels in sheep may be measured with the water hardness test kit used in this study, and that data are in concordance with standard laboratory results.

REFERENCES El-Khodery, S., El-Boshy, M., Gaafar, K. and Elmashad, A.: 1. Hypocalcaemia in Ossimi Sheep Associated with Feeding on Beet Tops (Beta vulgaris). Turk. J. vet. Anim. Sci. 32: 199-205, 2008.Radostits, O.M., Gay, c.c. and Hinchcliff, K.w., constable, P.D.: 2. Parturient paresis (milk fever).veterinary Medicine. 10th Ed., Elsevier Saunders, London. pp. 1626-1644, 2007.Sweeney, H. J. and cuddeford, D.: An outbreak of hypocalcaemia 3. in ewes associated with dietary mismanagement. Vet. Rec. 120:114, 1987.Hunt, E. and Blackwelder, J.T.: Disorders of ca metabolism. In: 4.

Smith, B.P. (ed.): Large animal internal medicine, 3th Ed., Mosby copyright, california. pp. 1248-1252, 2002. woldemeskel, M., Eneyew, M. and Kassa, T.: Study on ovine 5. hypocalcaemia in ewes in central Ethiopia. Revue. Med. vet. 151: 345-350, 2000.Gurbulak, K., Pancarcı, S. M., Gungor, O., Kacar, c., Oral, H., 6. Kırmızıgul, A.H., Kamiloglu, N.N., Karapehlivan, M. and Kaya D.: Postpartum Uterine Involution in winter-Lambing Tuj Breed Sheep and Effects of Subclinical Hypocalsemia on Uterine Involution in Tuj Breed Sheep. Kafkas Univ.vet. Fak. Derg. 11: 55-59, 2005.Saun, R. v.: Nutritional Diseases of Small Ruminants: diagnosis, 7. treatment and prevention. http://vbs.psu.edu/ext/resources/pdf/small-ruminant/Sm%20Rum%20nutr%20disease.pdf 2010.Houe8. , H., Ostergaaard, S., Thilsing-Hansen, T., Jorgensen RJ., Larsen T., Sorensen JT., Agger JF. and Blom JY.: Milk fever and subclinical hypocalcaemia-an evaluation of parameters on incidence risk, diagnosis, risk factors and biological effects as input for a decision support system for disease control. Acta. Vet. Scand. 42: 1-29, 2001.Bickhardt, K., Henze, P. and Gander, M.: clinical findings and 9. differential diagnosis in ketosis and hypocalcaemia in sheep. Dtsch. Tiarariztl. Wochenschr. 105: 413-419, 1998.Thomas, H. H.: Metabolic Diseases. In: Jimmy, L. H. and Robert, 10. A. S. (eds.): Current Veterinary Therapy 4 Food Animal Practice, w.B. Saunders company, Philadelphia. pp. 215-218, 1999.Lincoln, S. D. and Lane, v. M.: Serum ionized ca concentration 11. in clinically normal dairy cattle, and changes associated with Ca abnormalities. J. Am. vet. Med. Assoc. 197: 1471-1474, 1990.Zadnik, T., Staric, J., Klinkon, M. and Sorsak, B.: Impact of Two 12. Different Preventive Treatments on Milk Fever Incidence in Dry Dairy cows. Krmiva. 6: 349-355, 2006.Kojouri, GH. A.: Parturient Paresis and its Relationship with 13. Hypophosphatemia Acta vet. Scand. 44:126, 2003.Sakha, M. and Jamshidian, M.: Evaluation of Bovine Parturient 14. Paresis in 64 cows with Respect To Preventive Methods. Acta Vet. Scand. 44: 136, 2003.Saeed, M., Khan, M. S., Avais, M., Ijaz, M., Mahmood, A. K. and 15. Ur-Rehman, Z.: A Study on Serum ca, creatine Phosphokinase and Lactate Dehydrogenase Concentrations in Post Parturient Hypocalcemic Nili- Ravi Buffaloes. Pakistan J. Zool. Suppl. Ser. 9: 357-359, 2009.Matsas, D. J., warnick, L. D., Mechor, G. D., Seib, L. N., Fatone, 16. S., white, M. E. and Guard, c. L.: Use of a water hardness test kit to measure serum ca concentration in cattle. J. Am. vet. Med. Assoc. 214: 826-828, 1999.Ley, w. B., Bowen, J. M., Purswell, B. J., Irby, M. and Greive-17. crandell, K.: The sensitivity, specificity and predictive value of measuring Ca carbonate in mares’ prepartum mammary secretions. Theriogenology 40: 189-198, 1993.Grosenbaugh, D. A., Gadawski, J. E. and Muii, w. w.: Evaluation 18. of a portable clinical analyzer in a veterinary hospital setting. J. Am. vet. Med. Assoc. 213: 691-694, 1998.

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CANINE ORAL PAPILLOMAVIRUS INFECTION: CLINICAL COURSE, PATHOLOGY, L1 GENE AND NCR2 GENE

SEqUENCINGJun, D.1,†, Yi, G.1,†, Na, T.1,†, Yipeng, J1, Rui, Z2, Degui, L1,*, Guozhong, Z2,*

1 Department of Small Animal clinical Sciences, college of veterinary Medicine, china Agricultural University, Beijing 100193, P.R.china

2 Department of Preventive veterinary Medicine, college of veterinary Medicine, china Agricultural University, Beijing 100193, P.R.china

*Corresponding author: Zhang Guozhong; Lin DeguiTelephone: +86-10-62733660

Fax: +86-10-62732984E-mail: [email protected] (G.Z. Zhang); [email protected] (D.G. Lin)

Postal address: College of Veterinary Medicine, China Agricultural University, Beijing100193, P.R. China

ABSTRACTcanine oral papillomatosis is a self-limiting and spontaneous-regressing neoplastic disease caused by canine oral

papillomavirus (cOPv). In this report, five warts from the oral mucosa of five dogs suffering from canine oral papillomatosis were removed surgically for histopathologic examination, immunohistochemical analysis, L1 gene and NcR2 gene sequencing. Histopathology revealed various degrees of epithelial hyperplasia, hyperkeratosis and basophilic keratohyalin granules in the stratum lucidum and granular layer. cOPv-L1 gene was highly conserved in all the five samples and several mutations of non-coding region (NcR2) gene were detected in three out of five samples. Immunohistochemistry revealed that cell division took place from stratum basale to stratum spinosum, even reaching the granular layer with cOPv-L1 protein mainly distributed in stratum corneum and adjacent granular layer. The tendency of canine oral papillomatosis to transform into a squamous cell carcinoma is also discussed.

INTRODUCTIONcanine oral papillomatosis is a self-limiting neoplastic

disease caused by canine oral papillomavirus (COPV)

(1). Major lesions are observed in the canine oral cavity characterized by the formation of papillary or cauliflower-like tumors. The most significant cytopathic feature caused by cOPv is the vacuolization of cells in the granular layer and prickle cell layer (2).

cOPv is a double-stranded DNA virus of 8,607 base-pairs (bp), and its genome contains several major early (E6, E7, E1, E2 and E4) and late (L2 and L1) open reading frames, and two non-coding regions (NcR1 and NcR2). The early viral proteins E1 and E2 play a role in replication of the viral genome. E4 may play some part in viral DNA replication or release of viral particles from infected cells. E6 and E7 control cell growth and the cell cycle which maximizes viral DNA replication, and the late proteins L1 and L2 form the viral capsid and package viral DNA. The function of two non-coding regions is not known (3,4,5,6,7,8,9).

In the study, five cases from canine oral papillomatosis were chosen for histopathologic examination, immunohistochemical analysis, L1 gene and NcR2 gene sequencing. Our aim was to provide evidence for the clinical diagnosis from a pathological view point with reference to the relationship between the proliferation of papillomavirus, the structure of the histiocyte, the development and regression of the tumor, and its tendency of inducing carcinoma. To the best of the authors knowledge this is the first time that research on the pathology with

reference to the viral gene of canine oral papillomatosis has been undertaken in China.

MATERIALS AND METHODSPatient selection

The 5 samples in this study were obtained from 5 domestic dogs of different ages and breeds. They were all diagnosed with canine oral papillomatosis at the Animal Hospital of the china Agricultural University (Table 1).Histopathology

The papilloma tissue were collected by biopsy, fixed in 10% neutral buffered formalin solution and processed routinely in paraffin wax. Sections (5 μm) were cut and stained with haematoxylin and eosin (H&E).Electron microscopy

The papilloma tissue were collected by biopsy, immersed in 2.5% glutaraldehyde solution, washed with 0.1M PBS buffer (pH7.2), fixed by 1% osmic acid solution, dehydrated and embedded. Ultrathin slides were prepared and double-stained with uranyl acetate and lead citrate, and examined under a JEM-1230 electron microscope (Jeol, Tokyo, Japan).Immunohistochemistry

Papilloma and normal canine oral mucosa epithelial tissue were treated following routine process, fixed in 4% paraformaldehyde, embedded and cut at 3μm. The samples were dewaxed, the antigens repaired and the endogenous biotin removed by 3% H2O2. Immunohistochemical staining of proliferating cell nuclear antigen (PcNA) was performed

Keywords: Canine oral papillomatosis, histopathology, immunohistochemistry, sequencing.


using a mouse anti-PcNA antibody (wuHan Boster Bio-engineering co. Ltd.) as the first antibody and a horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG as the second antibody. The staining intensities were observed and photographed microscopically. The positive signal was dark brown and the negative signal colorless to very light yellow. The same method was used to perform cOPv L1 gene immunohistochemical staining (rabbit anti-human HPv L1 antibody as the first antibody and HRP-conjugated sheep anti-rabbit IgG as the second antibody).

For the control groups, slides were taken from the papillomatous lesions and PBS was used to replace the first antibody. Tissue from normal oral epithelium was used as a negative control.Polymerase chain reaction and sequencing

viral DNA was extracted with DNAvzol kit (vigorous Biotechnology Beijing Co. Ltd., China) according to the manufacturer’s instructions. The primers for L1 gene and NcR2 sequence were designed based on sequenced cOPv genome (GenBank Accession No.D55633) as shown in Table 2. Specific polymerase chain reaction (PcR) was performed using primers described in Table 2. Briefly, a 20µl reaction system was composed which included the template DNA 3µl, 0.5µl of upstream and downstream primer, 10µl of PcRmix (Mylab corporation, Beijing, china) and 6µl tri-distilled water. This was uniformly mixed and then started with an initial heating step of 5 minutes at 940c. Further cycles consisted of a denaturation step of 45 seconds at 940c, followed by 45 second primer annealing at 59.90C, and 90 second primer extension at 720c. After 35 cycles, the final extension step was set at 10min/ 720c. The amplified products were examined and target DNA were recovered using the Axygen kit, then directly sequenced at a commercial sequencing facility (Beijing Sunbiotech co. Ltd., Beijing, China).Phylogenetic analysis

The available sequences of cOPvs and other major Pvs were downloaded from NcBI to compare their relationship with the determined sequences in the study. Multiple sequence alignments were performed with DNAman computer software and phylogenetic trees were constructed with MEGA 4.0 program.

RESULTSClinical aspects

On visual inspection single or multiple pink filiform papillae-like growths were seen in or around the lip, tongue, buccal mucosa and hard palate. The diameter of the growths was often less than 1 cm (Fig.1A). Highly mature tumors could be removed easily. All five dogs had undergone surgery to remove their warts; no recurrences were observed.Histological examination

Under light microscopy, hyperplasia of the epithelium, hyperkeratosis, basophilic keratohyalin granules in stratum lucidum and granular layer were observed. Large koilocytes were found in the granular layer and prickle cell layer. compared with the three dogs which had tumors for only a short time, koilocytes in the two dogs with tumors of 5 and 6 weeks were larger in both number and size. Among tumor cells, exposed intercellular bridges were significantly elongated. Mitotic figures and intranuclear inclusion bodies formed by virus could also be seen in cells from dogs which had had

tumors of 3 or 4 weeks (Fig.1B).Electron microscopy (EM)

Under electron microscope, small polygonal particles aggregated in the nucleus were observed in tumor cells of all cases. Other changes noted were swelling of organelles, vacuolar degeneration, distortion of nuclear appearance, shrunken, margination and dissolution of chromatin, microfilament aggregated in the cytoplasm or connections with abnormal hyperplasic desmosomes around the cell membranes (Fig.2).PCNA immunohistochemical staining

After PcNA staining, positive cells were detected only in stratum basale of normal canine oral epithelial tissue (Fig.3c), while in all of the oral papillomatosis sections of dogs, positive cells were distributed from stratum basale to stratum spinosum, and even to granular layer (Fig.3A). This demonstrated that the prickle cell layer and granular layer both were undergoing cell division in addition to the stratum basale in the case of oral papillomatosis. This phenomenon might have resulted from virus proliferating in tumor cells.COPV L1 protein immunohistochemical staining

No positive signal was found in control group, while in each section of the experimental group positive signals were present mainly distributed in the stratum corneum and adjacent granular layer. In the koilocytes of the granular layer and prickle cell layer, only in those cells which were close to the stratum corneum could a positive signal be detected. Furthermore, the closer the cells were to the surface layer, the easier it was to detect a positive signal. Under higher magnification, a greater number of deeply stained irregular particles could be seen. Thin strong-positive deposit areas could often be seen in parakeratotic area of some dogs. Positive cells of all cases in the non-stratum corneum mostly showed a weak positive diffusively stained reaction with the cytoplasm, however some cell presented with a strong positive nuclear reaction (Fig.3B,D).PCR Amplification and SequencingCOPV L1 gene

In all five cases, the same size L1 DNA sequence could be amplified by PcR, as shown in Fig.4A confirming the presence of cOPv. All the L1 genes were sequenced and submitted to the GenBank. GenBank accession numbers were HM054511, HM054512, HM054513, HM054514 and HM054515. compared with all other cOPv sequence loaded in GenBank by DNAman computer software there was no variation among L1 gene sequences.Specific NCR2 sequence

All the NcR2 sequences were obtained and submitted to the GenBank. GenBank accession numbers were HM054516, HM054517, HM054518, HM054519 and HM054520. The amplified NcR2 sequence had a length of 849bp (Fig.4B) and was rich in base A and base T (about 65%, which was about 56% in the entire genome of cOPv). compared with the isolate Y62 (GenBank Accession No.D55633) (10), point mutations existed in sample 2, sample 3 and sample 4 in the NcR2 gene (Table 3).Phylogenetic analysis

The L1 gene sequences determined in the study were compared and phylogenetically analyzed with all other PVs’ L1 gene sequence obtained from GenBank (Fig.5). compared with other viruses from papillomavirus family, cOPv

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had the most closely evolutionary relationship with feline papillomavirus (FPV). COPV was also evolutionary related to the Canis familiaris papillomavirus type 2 (cfPv-2) which is also a variety of canine papillomavirus (11).

DISCUSSIONGenerally, clinical signs of canine oral papillomatosis

appear 1 to 4 weeks after infection and disappear 6 to 12 weeks later (12). All five dogs used in the study had tumors for more than 3 weeks of tumor growth indicating that the tumors were in the maturation period.

Observation with light microscopy revealed that koilocytes were larger than normal cells. In affected cells the cytoplasm was found to be hollow and transparent, the nucleus pyknotic and the nucleolus heavily stained. Under electron microscopy, koilocytes were pyknotic and the nuclear membrane severely wrinkles with halos appearing around the nucleus. At different growth stages the appearance of the tumor changed. In the cytoplasm, vacuoles of different sizes replaced swelling denatured organelles occupying a major portion of the cell. compared with the disappearance of many inherent structures in the cytoplasm, microfilament increased and aggregated. In more mature papillomas, koilocytes were filled with homogeneous rarefied substances which obliterated the nucleus. A few cells even appeared to undergo apoptosis, which may represent the spontaneous regression of the tumor.

The formation of koilocytes may have resulted from the joint change of nucleus and cytoplasm infected by cOPv. Reproduction of virus possibly compromised normal cell activities resulting in changes in material exchange and energy consumption appearing as an enlarged cell with pathological changes in mitochondria and endoplasmic reticulum (13). Changes in the nucleus presented as nuclear membrane wrinkling and perinuclear space broadening. with growth of the papilloma, cells infected by virus gradually underwent necrosis resulting in the disappearance of all kinds of structures and finally presenting as a completely homogeneous cavitation-like framework.

Sundberg et al. (8) reported that squamous cell carcinoma might occur at injection site in dogs which had been vaccinated for cOPv , and that in some dogs with some degree of immunodeficiency, benign canine oral papillomatosis could transform into squamous cell carcinoma (14). This suggests that canine oral papillomatosis and squamous cell carcinoma might have a closer relationship than previously thought (15, 16). An increase in microfilaments and apparent intercellular bridge and multi-layer keratinized keratin pearls has been reported in carcinogenic tissues of squamous cell carcinoma type-1 (17). In this study we found similar characteristics as mentioned above. Electron microscopic observation showed an abundance of stacking desmosomes, step-type intercellular bridges and related microfilament aggregates which stratified inside the cell membrane. Large blocks or concentric circular multi-layer structures composed of keratin microfilamentthere were also seen (Fig.6). The findings mentioned above illustrated that the development of canine oral papillomatosis has caused damage to microfilaments and intercellular bridges, which resulted in abnormal hyperplasia of these structures. Under the situation of immunosuppression or immunodeficiency, where the papilloma could not regress normally these hyperkeratotic structures could possibly become induced to develop into

squamous cell carcinomas.Results of cOPv L1 protein staining showed that a strong

positive signal was distributed in the stratum corneum. L1 protein is one of Pvs’ major proteins and its appearance suggests that the virus is reproducing. Former research has shown that only the stratum corneum and granular layer have the essential materials for virus assembly, so that the stratum corneum and granular layers are the major sites of for reproduction and assembling of Pv (18). Only when the infected cells differentiated to hornification did the late protein L1 of cOPv began to appear and complete the final assemblage (19, 20). During this period, stratum corneum cells full of virus particles can exfoliate easily and infect other healthy dogs thus starting a new round of infection.

As mentioned above, the distribution of L1 protein positive signal and long fine cosh-like positive particle sedimentation area detected in parakeratotic areas have been previously reported in HPv research (21). Therefore, we concluded that cOPv and HPv had a few similarities in their pathogenic processes.

The comparison of gene sequences showed no variation among different strains of cOPv, indicating that L1 gene is quite conserved. L1 gene of cOPv had a relatively higher homology with FPv (22), HPv-1a and HPv-63 (23, 24). The phylogenetic tree indicated further that cOPv has quite close evolutionary relationship with HPv. we also successfully detected cOPv L1 protein using rabbit HPv L1 antibody in an immunohistochemical test. Therefore, it is possible that canine infected by cOPv can be used as an animal model for human infection by HPv.

Based on the coherence of molecular mass measurement, identity of restriction enzyme patterns between sequenced cloned cOPv and different wild-type cOPv (10, 25), we may conclude that NcR2 was essential DNA part of wild-type cOPv rather than artificial cloned product. Retrieval in GenBank and EMBL database revealed no similarity between NcR2 and known DNA sequence. Therefore, we considered the detection of NcR2 as basis for identification of cOPv.

As opposed to the conservative L1 gene, NcR2 sequence has a relatively stronger variability. Compared with the isolate Y62 (26), 3 of the 5 samples demonstrated point mutation in different degrees (Table 1). The significance of these mutations needs further study.

REFERENCESNicholls, P.K. and Stanley, M.A.: canine papillomavirus-A 1. centenary review. J. comp. Pathol. 120: 219-233, 1999.Bell, J.A., Sundberg, J.P., Ghim, S.J., Newsome, J., Jenson, A.B. 2. and Schlegel, R.A.: A formalin-inactivated vaccine protects against mucosal papillomavirus infection: a canine model. Pathobiol. 62: 194-198, 1994.Birnstiel, M.L., Busslinger, M. and Strub, K.: Transcription 3. termination and 3′ processing: the end is in site! cell. 41: 349-359, 1985.Delius, H., van Ranst, M.A., Jenson, A.B., zur Hausen, H. and 4. Sundberg, J.P.: canine oral papillomavirus genomic sequence: a unique 1.5-kb intervening sequence between the E2 and L2 open reading frames. virology. 204: 447-452, 1994.Goldstein, D.J., Finbow, M.E., Andresson, T., McLean, P., 5. Smith, K., Bubb, v. and Schlegel, R.: Bovine papillomavirus E5 oncoprotein binds to the 16K component of vacuolar H(+)-



ATPases. Nature. 352: 347-349, 1991.Masterson, P.J., Stanley, M.A., Lewis, A.P. and Romanos, M.A.: 6. A c-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit. J. virol. 72: 407-7419, 1998.Proudfoot, N.: Poly (A) signals. cell. 64: 671-674, 1991.7. Sundberg, J.P., O’Banion, M.K., Schmidt-Didier, E. and 8. Reichmann, M.E.: cloning and characterization of a canine oral papillomavirus. Am. J. vet. Res. 47: 1142-1144, 1986.Teifke, J.P., Lohr, c.v. and Shirasawa, H.: Detection of 9. canine oral papillomavirus-DNA in canine oral squamous cell carcinomas and p53 over expressing skin papillomas of the dog using the Polymerase chain reaction and non-radioactive in situ hybridization. vet. Microbiol. 60: 119-130, 1998.Sandburg, J.P., Reszka, A.A., williams, E.S. and Reichmann, 10. M.E.: An oral papillomavirus that infected one coyote and three dogs. Vet. Pathol. 28: 87-88, 1991.Yuan11. , H., Ghim, S., Newsome, J., Apolinario, T., Olcese, V., Martin, M., Delius, H., Felsburg, P., Jenson, B. and Schlegel, R.: An epidermotropic canine papillomavirus with malignant potential contains an E5 gene and establishes a unique genus. Virology. 359: 28-36, 2007.Calvert12. , C.A. Canine viral papillomatosis. In: Greene CE, eds. Infectious Diseases of the Dog and Cat. Philadelphia: WB Saunders, 288. 1990.Ling13. , Y. P. and Yu, Z.: Cell ultrastructure and electron microscopy, Fu Dan University Press, Shanghai, 2004.Watrach14. , A.M., Small, E. and Case, M.T.: Canine papilloma: progression of oral appaloosa to carcinoma. J. National Cancer Institute. 45: 915-920, 1970.Nespeca15. , G., Grest, P., Rosenkrantz, W.S., Ackermann, M. and Favrot, C.: Detection of novel papillomaviruslike sequences in paraffin-embedded specimens of invasive and in situ squamous cell carcinomas from cats. Am. J. Vet. Res. 67: 2036-2041, 2006.Zaugg16. , N., Nespeca, G., Hauser, B., Ackermann, M. and Favrot, C.: Detection of novel papillomaviruses in canine mucosal,

cutaneous and in situ squamous cell carcinomas. Vet. Dermatol. 16: 290-298, 2005.Liu17. , F.S. and Liu, T.H.: Pathology of Tumours, United Press of Beijing Medical University and China Union Medical University, Beijing, 1997.Tori18. , T.: Immunoperoxidase demonst rat ion of papillomavirus antigen in dysp lasia of the uterinecervix. Nippon Sanka Fujinka Gakkai Zassbi. 37: 411, 1985.Chen19. , B.P., Fang, P. and Dong, C.Y.: Observation of Human Papillomavirus Morphogenesis in Warts. Acta Acad. Med. Hubei. 18: 21-24, 1997.Doorbar20. , J.: The papillomavirus life cycle. J. Clin. Virol. 32: S7-15, 2005.Yu21. , L., Peng, J., Peng, J.Q., Bao, J.Y., Wu, Q.H. and Liu, X.H.: Study on condyloma acuminatum by in situ hybrihistochemistry compared with immunohistochemistry. Chin. J. Dermatovenereology. 18: 233-234, 2004.Terai22. , M. and Burk, R.D.: Felis domesticus papillomavirus, isolated from a skin lesion, is related to canine oral papillomavirus and contains a 1.3 kb non-coding region between the E2 and L2 open reading frames. J. Gen. Virol. 83(Pt 9): 2303-2307, 2002.Danos23. , O., Katinka, M. and Yaniv, M.: Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among Papovaviridae. The EMBO Journal. 1: 231-236, 1982.Egawa24. , K., Delius, H., Matsukura, T., Kawashima, M. and De Villiers, E. M.: Two Novel Types of Human Papillomavirus, HPv 63 and HPv 65: Comparisons of Their Clinical and Histological Features and DNA Sequences to Other HPv Types. Virology. 194: 789-799, 1993.Sundberg25. , J.P., Smith, E.K., Herron, A.J., Jenson, A.B., Burk, R.D. and Van Ranst,M.: Involvement of canine oral papillomavirus in generalized oral and cutaneous verrucosis in a Chinese Shar Pei dog. Vet. Pathol. 31: 183-187, 1994.Isegawa26. , N., Nakano, K., Ohta, M., Shirasawa, H., Tokita, H. and Simizu, B.: Cloning and sequencing of the L1 gene of canine oral papillomavirus. Gene. 146: 261-265, 1994.

TABLES

Table 1 - Information of dogs

No. Breed Age (Month) Sex Weight (kg) Wart existence (week)

1 German Shepherd Dog 6 Male 20 6

2 Beagle 4 Female 8 3

3 Siberian Husky 6 Female 13 5

4 Great Pyrenees 4 Male 18 4

5 Caucasian Sheepdog 5 Female 20 5

Table 2 - The primers used in the study

Target gene Primers (5’-3’) Predicted product

L1P1:cAcAGcccAGcAccAAGP2:TGcGTTTGcGTTTcAcA

1464 bp including nt 6879-8343

NcR2P1:GAcAAGTccGAcAGTccAAcP2:GGTcAGATAAGcGGGTAGG

916 bp including nt 4218-5134

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FIGURES

Figure 1 - clinical and histopathological examination of canine oral papillomatosis. (A) Appearance of dog’s oral mucosa (German Shepherd Dog). (B) Transverse section of canine oral papillomatosis, H.E staining, 100×. This picture displays each layer of epithelium thickened around vascular connective tissue axis, hyperkeratosis. Koilocytes in different sizes distribute in prickle cell layer.

Table 3 - Mutations in specific NcR2 sequence of cOPv

Sample No. Mutation sites Changes

COPV T2 nt 4254 ”T” was replaced by ”c”

COPV T3 nt 4252-4253 ”AT” were replaced by ”AcT”

COPV T4 nt 4379 ”A” was replaced by ”T”

nt 4937-4938 ”TG” were replaced by ”TTG”

nt 4992 ”A” was replaced by ”G”

Figure 3 - Results of PcNA and cOPv L1 protein immunohistochemical staining (100×). (A) PcNA positive cells distributed widely from stratum basale to granular layer of the epithelium. (B) Strong positive signal of cOPv L1 protein mainly distributed in stratum corneum while weak positive signal could be seen in granular and prickle cell layer. (c) The control group of PcNA presented no positive signal. (D) The control group of L1 protein immunohistochemical staining presented no positive signal in any layer.

Figure 2 - Koilocytes. (A) Panorama of a typical koilocyte. cell nucleus is extruded to side, the nuclear membrane is wrinkled, the chromatin is marginated and the cytoplasm is filled with rarefied substance (Scale=2000nm). Inset: small particles in cell nucleus (Scale bar=500nm). (B) Mitochondria in koilocyte are swollen with vacuolization. (Sale bar=500nm). (c) Microfilament-like substances aggregated in koilocyte; with slight swelling mitochondria. (Scale bar=500nm).



Figure 4 - Electrophoretogram of cOPv L1 and NcR2. (A) Amplified products of cOPv L1. (B) Amplified products of NcR2. 1-5: Sample No. of amplification. M: DNA Marker III. The length of L1 gene amplification is about 1386bp and the length of NcR2 amplification is about 849bp.

Figure 5 - Phylogenetic tree of Pvs’ L1 gene. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (only>50% is shown). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum composite Likelihood model as described in the methods section, and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the data set (complete deletion option). Phylogenetic analyses were conducted in MEGA4. cOPv’s Accession No. are D55633, L22695, Nc_001619 and D26115, the other 6 kinds of Pvs were taken from their standard strain.

Figure 6 - Intercellular bridge and unknown multi-layer keratinized structure. (A) Elongated step-type intercellular bridges between cells with intermediates desmosomes. (Scale bar=1000nm). (B) Concentric circular multi-layer hornification structure in mature papilloma prickle cell layer, about ten times the size of koilocytes. Several organelles arescattered in the center while a few completely vacuolized cells (K) are present in the surrounding around (Scale bar=5000nm).

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WHAT IS YOUR DIAGNOSIS?

cARDIOLOGYECG OF THE MONTH

Golani, Y. and Ohad D.

cardiology Unit The Koret School of veterinary Medicine

The Robert H. Smith Faculty of Agriculture, Food and EnvironmentThe Hebrew University of Jerusalem, Israel.

CASE REPORTA 10 year old spayed female, mixed brachycephalic dog, weighing 25 kg was presented for routine annual vaccination. The owners described a healthy energetic dog with a good appetite and normal bowel movements. On physical examination the heart rate was noted to be irregular and on femoral artery palpation an occasional pulse deficit was detected. The first pulse palpated after the pulse deficit appeared to be much stronger than others. The rest of the physical examination was unremarkable.The concern generated by these findings led the clinician to carry out an EcG and to request a blood panel consisting of complete blood count, electrolytes and biochemistry. All clinical pathology results were within normal limits. A 30-second-long Lead-II rhythm strip was recorded (Figure 1A). A short strip of all 6 leads at a paper speed of 50 mm/sec was also recorded (Figures 1B and 1C).What is your diagnosis and why? What are the possible etiologies? How do you propose to manage this case?

Figure 1

LEGENDS FOR FIGURESFigure 1: Electrocardiograms recorded from a 10 year old spayed female, mixed brachycephalic dog. A: a 30 second continuous recoding of Lead II divided into three continuous rows, each representing 10 seconds recording. Calibration is 10mm=1mV and paper speed is 25mm/sec. B: A short recording of six frontal plane leads, calibrated at 2.5mm=1mV (note the difference in calibration between Figure1A and 1B causing the amplitudes to appear different). Paper speed is 50mm/sec, which causes waves and complexes to appear wider than those in Figure 1A. C: This section represents an average of one beat per lead for each of the six leads. Calibration: 10mm=1mV, paper speed 50mm/sec. This calibration and paper speed is the most useful for measuring intervals such as P, PR (PQ), QRS, and QT. Turn the following page to read the interpretation and diagnosis


What are the ECG findings? Should this patient be treated based on these finding, and if it should, what is the best treating strategy?

It is recommended to examine the EcG systematically in order to collect and then integrate all findings, prior to concluding a final diagnosis. The following features should be examined:

Is1. the ventricular rate normal? Determine whether Tachyarrhythmia or Bradyarrhythmia is present taking note of the paper speed.Is2. the rhythm regular? If not, define the rhythm changes.Try3. to find at least one ”gold standard” (sinus) beat which can be used as a reference of comparison to other beats. Are all P-QRS-T complexes identical to this specific beat, in shape and character?Are4. there any QRS complexes that do not have a P wave preceding them? Are there any P waves that are not followed by a QRS complex?Is5. the P-R interval repeatable and constant? Measure6. the width of P wave, P-R interval (from the beginning of P wave till the beginning of QRS), the width of QRS complex, the Q-T interval (from the beginning of QRS complex till the end of the T wave), and the amplitudes of P, R, and S waves. (See Table 1 for normal values).Calculate7. the Mean Electrical Axis (MEA) using the algebraic sum of the predominant QRS deflections in any two leads. Using leads I and aVF may often be the most convenient, but nit necessarily the only pair for this purpose. Are8. there any pre-mature complexes? Note their timing, width and morphology.

ANSWERS:The1. calculated heart rate was 90 beats per minute (bpm). The2. rhythm was irregular. The third row is the most useful to observe in this respect. The R-R interval appears to change in a predictive periodical manner which can be defined as ”regularly-irregular”. This finding is characteristic of Sinus Arrhythmia, indicating that the arrhythmia originates from the Sino-Atrial node (SA node) and is the result of regular and periodic changes in the autonomic input to that main pacemaker. Sinus arrhythmia confirms the presence of an arrhythmia but also establishes that the irregularity is not pathologic. The presence of sinus arrhythmia is incompatible with heart failure as it indicates a sufficiently high parasympathetic tone. On physical examination, one might have noticed a cyclic change of heart rate related to respiration, with an increase during inspiration and decrease during expiration. If so, that is also a normal finding characteristic, termed Respiratory Sinus Arrhythmia.There3. were many ”gold standard” P-QRS-T complexes (see the complex labeled ”A” in Figure 2, as an example).There4. is a QRS complex after each and every P wave. There is a P wave before every QRS complex, except for the complexes marked ”B” and ”C” in Figure 2The5. P-R interval was constant, indicating a causative relationship between the P wave and the subsequent QRS complex. This finding confirms the presence of sinus

rhythm.Measurements6. of waves and intervals should be performed in Lead II. (Table 1)

Normal findings:The amplitude of1. the P wave was seen to change in a cyclic way. This is considered a normal finding referred to as a “wandering pacemaker” which is commonly seen with sinus arrhythmia. Although P wave width was longer than the normal range (P>0.04sec) and might have indicated the presence left atrial enlargement, this is considered a non-specific finding and may be of little significance where there is no other supporting evidence such as radiographic or sonographic left atrial enlargement.R wave amplitude was normal2.

Q-T interval was3. normal

Figure 2Abnormal findings:

The1. P-R interval was longer than the normal range at 0.14 ms (see Table 1), indicating a first degree AV block. This conduction disturbance derives from a slow conduction of the AV-node. Possible etiologies of prolonged conduction across the AV-node may be associated with disorders that cause an increased parasympathetic tone. It may be an incidental finding or resultant to anti-arrhythmic drugs therapy, which was not administered to this patient). There are no immediate hemodynamic consequences for this condition and therefore there is no indication for treatment.Prolongation2. of the QRS complex (0.09 sec). Possible etiologies: Severe hyperkalemia, ventricular escape beats due to severe bradycardia, an extremely sick myocardium (e.g. cardiomyopathy or sub-aortic stenosis with secondary myocardial fibrosis) (which are unlikely as dog seems clinically healthy and has a high vagal tone), ventricular premature complexes (VPC) or a bundle branch block (BBB).

Although there was a slight conduction disturbance through the AV-node, there was still a constant P-R interval with sinus rhythm at a normal rate of 90 bpm. All these findings rule out ventricular escape complexes or VPCs (as opposed to the 2 complexes labeled B and C; see below) and indicates that the cause for the prolonged QRS can only be derived from a ventricular conduction disturbance, i.e. a BBB.

In a normal heart, the activation of the ventricles takes place nearly simultaneously thanks to the fast conduction



through the Purkinje fibers. These fibers are located at the left and right branches of the bundle of His that runs through the sub-endocardium of the inter-ventricular septum. Each branch passes the electrical impulse arriving from the atria to the ventricles through the AV-node, to its corresponding single ventricle. With a (left or right) BBB, there is a lesion in one of the bundle branches that completely blocks the conduction down this branch to the ventricle. The result is a blockade of the ”fast” conduction to the affected ventricle. The depolarization process in this ventricle, therefore, propagates very slowly through contracting myocytes that are not specialized in fast conduction. As a result, a slow ”bypass” is formed in one ventricle while the other still does go through a normal ”fast” propagation process that ends much earlier. The graphic result is a wider (slowly inscribed) QRS.

What kind of a Bundle Branch Block does this dog have? Right (R-) or Left (L-) BBB?

A left-BBB can often be recognized based on a supra-ventricular origin (i.e. having a P-wave in front of it, with a constant PR interval), a wide QRS, a left-sided ventricular MEA and a predominant R wave on Lead I. Often, a right BBB is similarly of a supra-ventricular origin with a wide QRS, a right ventricular MEA and a predominant S wave in Lead I.

According to Lead I (at the lower right corner Labeled C in Figure 1), this dog had a left BBB. The normal ”fast” propagation of the right ventricle ended much earlier than the left ventricular one. The graphic result was that the S wave (RV propagation) was ”swallowed” and offset by the R wave (reflecting mostly the LV propagation), which is more dominant because by the time it completes its course there

is no longer any negation coming from the faster-conducting right ventricle.

In a healthy myocardium, there is only little hemodynamic impact as a result of a BBB. Nonetheless, there may be some prognostic value to this diagnosis since a left BBB is typically expected to be less reversible than a right BBB. No treatment is indicated, nor is one possible (other than via invasive electrophysiological intervention which is not yet available to veterinary patients).

7) The mean electrical axis of the ventricles was calculated using the complexes illustrated in Figure 1C: the QRS complex in Lead I was mostly positive. In Lead aVF it was also positive. The MEA is aimed, therefore, towards the left ventricle (Figure 3). When combined with a sinus rhythm demonstrating wide QRS complexes, this finding is compatible with a left BBB.

Figure 3Two pre-mature complexes were seen on the Lead II

strip, labeled B and C in Figure 2. They were both identical to each other, but morphologically very different from the ”gold standard”. Their timing was early, and they begin at the very end of the preceding T wave with a direction opposite to the other complexes. The origin of a pre-mature complex that looks different form the ”gold standard” is usually ventricular (e.g. a VPC) as opposed to an atrial pre-mature complex (APC) which looks identical (albeit early in timing) to the ”gold standard”. The VPCs’ morphology is different, because there is an ectopic ventricular origin to this beat, that forces the ventricular depolarization front to propagate between myocytes in an abnormal course which does not utilize the



fast conducting Purkinje fiber network. This course does not allow for rapid conduction, hence is slower than usual and is inscribed as a wide QRS complex. Note that the T wave of this VPCs might be mistakenly identified as an R wave, appearing almost the same as the R wave of the preceding complex. Although its morphology is almost identical, there is an essential difference between the two: the R wave represents ventricular depolarization as opposed to the T wave that represents ventricular repolarization. This difference can be picked up by carefully paying attention to timing: in order to be sure a suspected wave is a T wave it is necessary to measure the Q-T interval, which is typically longer than a QRS complex. In this case, Q-T=0.22sec, which indicates that this is truly a T wave rather than an R wave.

Why do these VPCs seem so narrow? In fact, they are not narrow at all, as their width is above normal range at 0.09sec. Their appearing narrow is only an optical illusion because of a relative (rather than absolute) difference: while the VPCs are composed of 2 waves with a total duration of 0.09sec, the ”gold standard” complex is composed of one single (R) wave of an identical duration, and therefore appears wider.

What’s the location of the ectopic focus or foci? This question cannot be answered judging a single lead. In Lead II, the (negative) S wave in complexes B and C is dominant. Hence the depolarization front propagates away from the positive electrode of this particular lead, i.e. moving toward the right ventricle. Based on this one can only suspect that the origin may be left ventricular. Another VPC labeled ”D” in Figure 2 is different in shape and direction from the other complexes. It has a dominant S wave in Leads II (D4) and aVF (D3), and a positive R wave in Leads aVR (D1) and aVL (D2). Because it is taller in aVR than it is in aVL, this indicates that the depolarization not only propagates towards the head, but also moves towards the right. It is now possible to confirm that Complexes B and C in Figure 2 are definitively left ventricular VPCs.

The hemodynamic consequence is only momentary. The pre-mature beat doesn’t allow the heart to fill properly during the short preceding diastole, thus causing a transiently


decreased stroke volume (SV) following this specific beat. This is the reason for the pulse deficit. The volume of the next beat will be above normal because there is a prolonged pause after the VPC (labeled ”Z” in Figure 2), resulting in the diastole that immediately follows the VPC being longer and leading to a higher end-diastolic volume and an increased SV (according to the Frank-Starling law). This is felt as the single strong femoral pulse that follows the pulse deficit, as described earlier.

There is no treatment indicated for these two VPCs, as the dog is stable and asymptomatic, based on the history, physical examination, and background sinus arrhythmia attesting to a high vagal (parasympathetic) tone. There is probably no myocardial pathology and the VPCs will unlikely increase the risk of secondary complications. However, it would be advisable to consider an echocardiogram to confirm the condition of the heart, especially since there are two separate findings involving the left ventricle.Summary of Findings:

Sinus1. arrhythmia with a wandering pacemaker, reflecting a high enough vagal tone.A2. first degree AV Block, probably reflecting a conduction anomaly.A3. Left BBB.Left4. ventricular VPCs

The main finding of this case is the combination of two types of conduction disturbances in two different cardiac locations (one is supra-ventricular and the other is intra- ventricular). This is probably not coincidental and may be indicative of a rather diffuse conduction disturbance. Nonetheless, the conduction disturbances did not appear to have hemodynamic consequences at the time of EcG recording and the presence of a high vagal tone indicated there was no distress at that time. The VPCs occurred at a low frequency and therefore there appeared to be no need for treatment at that point in time. It is recommended, however, to follow up the case with a periodic EcG, to make sure the AV block does not degenerate to a higher degree. Also, in light of the findings echocardiography should be considered.

Table 1

Width (sec)Amplitude (mV)Normal Reference RangeWave

0.06 Changes along with the sinus arrhythmia up to a

maximum of 0.3

< 0.04 sec < 0.4mVP

1.5> 2.5mV (> 3.0 mV in Giant Breeds)

R

0.09< 0.06sec;(< 0.065 sec inGiant Breeds)

qRS

0.140.06-0.13 secP-R

0.220.15-0.25 secq-T



RADIOLOGYA DOG WITH ACUTE VOMITING

Bibring, U. and Eizenberg, Z.Imaging Unit

The Koret School of veterinary MedicineThe Robert H. Smith Faculty of Agriculture, Food and Environment

The Hebrew University of Jerusalem, Israel

HISTORYLady, an eleven year-old, spayed golden retriever, was presented

to the Koret School of veterinary Medicine, Hebrew University of Jerusalem Teaching Hospital for evaluation for acute vomiting. Lady was fully vaccinated and dewormed and lived in a private house with free access to a garden. The owner reported that during the morning Lady vomited several times. They reported that the vomitus consisted of a large amount of watery yellow fluid with digested food. She also had soft stool with a normal brown color. Since then, Lady has been lethargic. For the last 2 weeks Lady has been on cortisone (Prednisone) due to pruritis. Physical Examination

Lady weighed 33 kg (BcS=6/9). Her demeanor was bright, alert and responsive. She was adequately hydrated and her mucous membranes were hyperemic. Her body temperature was 41.4oC; pulse 180 beats per minute and on examination she was panting. Her cRT was less than 2 seconds. On palpation she exhibited a distended and painful abdomen. Abnormalities detected on the Complete Blood Count (CBC) included a leucopenia (WBC = 4.09 x103/µL (normal range 5.2-13.9 x103/µL)) and thrombocytopenia (PLT = 88 x103/µL (normal range 143-400 x103/µL)). Examination of the blood smear revealed a left shift and toxicity.

Blood Chemistry abnormalities were limited to increased activities of alkaline phosphatase (ALP = 309 U/L (normal range 4-140 U/L)) and alanine aminotransferease (ALT = 497 U/L (normal range 5-103 U/L)).Thoracic radiographs were performed (Figures 1 and 2)

what are your radiographic findings?1. Make a list of differential diagnoses for each abnormal 2. finding.Make a radiographic diagnosis.3. Decide whether additional imaging studies are required.4.

SEE THE FOLLOwING PAGE FOR THE DIAGNOSIS AND ExPLANATION.

Table 1

Width (sec)Amplitude (mV)Normal Reference RangeWave

0.06 Changes along with the sinus arrhythmia up to a

maximum of 0.3

< 0.04 sec < 0.4mVP

1.5> 2.5mV (> 3.0 mV in Giant Breeds)

R

0.09< 0.06sec;(< 0.065 sec inGiant Breeds)

qRS

0.140.06-0.13 secP-R

0.220.15-0.25 secq-T


WHAT IS YOUR DIAGNOSIS?RADIOGRAPHIC FINDINGS

The overall opacity of the thorax was increased. On the lateral view, there was border effacement of the cardiac silhouette (mainly at the ventral margins). Fluid opacity was observed at the ventral thorax-dorsal to the sternum and there was loss of visibility of the pulmonary parenchyma as well as the diaphragmatic outline at this area. “Skin fold artifact” was also visible at the ventral thorax.

within the viewable abdomen, there was a decrease of peritoneal detail, and multiple poorly defined radiolucent gas shadows were visible at the area of the ventral liver parenchyma (Figure 3-black arrows). The liver margins extends beyond the costal arch.

On the dorsoventral view, numerous interlobar fissure lines were observed and the lung lobes were displaced away (retracted), from the thoracic wall. Fluid opacity was visible between the lung lobes and the thoracic wall. The cardiac and diaphragmatic silhouettes were completely obscured. Loss of peritoneal detail was again evident within the viewable abdomen.

Differential diagnoses for increased radio-opacity of the pleural cavity and lung lobe retraction1. Pleural effusion2. Pleural fat deposition 3. Diaphragmatic hernia

Differential diagnoses for border effacement in the thorax1. Pleural effusion or pleural masses2. Alveolar or severe interstitial lung pattern3. Pulmonary or large mediastinal masses4. Diaphragmatic hernia5. Artifactual due to technical factors (e.g., underexposure)

Differential diagnoses for decreased peritoneal detail1. Peritoneal effusion (e.g., ascites, peritonitis, uroabdomen)2. Emaciation or normal puppy/kitten (due to lack of abdominal fat)3. Artifactual due to technical factors (e.g., underexposure)

Differential diagnoses for focal irregular gas lucencies within the liver1. Hepatic abscess (penetrating injury or hematogenous)2. Infection with gas-producing organisms

RADIOGRAPHIC DIAGNOSIS1. Pleural effusion2. Abdominal effusion3. Hepatomegaly and suspected hepatic abscessationSince Lady’s condition deteriorated quickly, abdominal ultrasound was undertaken (Figure 4).

Abdominal ultrasound-findings1) Free abdominal fluid (Figure 5- arrows)2) Hepatic gas pockets with ecogenic shadows (reverberation artifacts). (Figure 5-arrow heads)

These findings were consistent with abdominal effusion and liver abscessation.Needle aspiration of the abdominal free fluid was performed. Neutrophils, and many cocci and rod bacteria were observed. One liter of pleural fluid was drained.

The assessment was that Lady suffered from septic peritonitis

due to rupture of hepatic abscess. On surgery, a large focal hepatic abscess was observed. Hepatic lobectomy was made and the abdominal cavity flushed.

IMAGING DISCUSSIONThis case emphasizes the significance of a thorough and

systematic radiographic evaluation. Even-though the initial aim of the radiographs were to evaluate the thoracic cavity, it is very important to also evaluate systematically the extra thoracic structures such as the peripheral soft tissues, skeletal structures (vertebrae, ribs etc.) and the viewable abdomen. The small part of the viewable cranial abdomen that was seen at the radiograph provided essential information (i.e. irregular gas lucencies, decreased peritoneal details) for further diagnostic work-up. Abdominal ultrasound findings were suggestive of hepatic abscessation and abdominal effusion. The ultrasonographic examination and the aspiration results provided a definitive diagnosis. These results, combined with the poor clinical state of the patient, brought the clinician to decide that emergency procedures were required.

REFERENCES1. Thrall, D.E. (2007) veterinary Diagnostic Radiology, fifth

edition.2. Dennis, R., Kirberger, R.M., wrigley, R.H. and Barr, F.J. (2001)

Small Animal Radiological Differential Diagnosis, first edition.


TOXICOLOGY VIEWPOINT

IMPROVED ANIMAL FEED CONTROL AND "FARM TO FORK" FOOD SAFETY POLICY

Shlosberg, A.Department of Toxicology, Kimron veterinary Institute, 50250 Bet Dagan, Israel

Email: [email protected]

As part of the policy of the Israeli Governmental aimed at improving the safety of food for human consumption, it was decided in the Ministry of Agriculture (MOAG) in the last few months to gradually move the control of animal feed within MOAG to the Veterinary Services and Animal Health (vSAH). In so doing, it was understood that vSAH will implement a considerable upgrade in the present feed control systems. The changes and improvements will be made concomitantly with the introduction of a new Feed Law and many accompanying new Regulations. In addition, this whole process will be done in parallel with an 18 months-long ”Twinning” project supplied by the European Union (EU). The EU adopted in 2003 a new framework for its relations with its neighbours, including Israel, the so-called European Neighbourhood Policy (ENP), whose overall goal is to foster economic reform processes, promote closer economic integration, legal and technical approximation and sustainable development. The ENP is backed by financial and technical assistance to bring about reforms that will bring benefits in terms of economic and social development with the potential of greater trade and other access to the EU. This is done through approximation or harmonisation with the relevant acquis communautaire (the body of EU laws and policies already in place), that may be required in order to be capable of fully reaping the benefits of such participation. For instance, for more efficient trade, it is necessary to harmonise with parts of EU labelling rules and with food safety (veterinary and phytosanitary) standards and to use EU customs procedures. ”Twinning” is a project tool within the ENP, enabling the EU to send out officials from EU Member State administrations to work together with their counterparts in the administration of a partner country, so as to prepare together for the implementation of the acquis in a particular sector. Such a Twinning Project is paid for by the EU and demands both thorough preparation and a long-term official commitment from the partner to act according to the protocols of the Project. In the next few months a Twinning Project will be agreed upon and signed between the EU and its partner, the Government of Israel, the overall objective being to strengthen the means of the vSAH/MOAG to implement the ”Farm to Fork” food safety policy. This food safety policy will be within a new legislative framework compliant in quality with EU provisions and international standards allowing for the enhancement of food and feed quality. The Twinning Project aims at multiplying the chances for Israeli agricultural products to land on EU and international markets. In order to reach this goal, improvements in the fields of animal feed, animal health, and animal welfare will be approximated or harmonized to the EU acquis, institutional capacity will be strengthened, training will be instigated, and information recording and registration systems developed. The Twinning Project, which will start most probably in January 2011, will mainly comprise the planning, establishment and training of

an Animal Feed Control Unit (AFcU) within vSAH/MOAG at the Bet Dagan campus, which will be responsible for all aspects of feed control, as detailed below. AFcU will comprise mainly field inspectors highly-trained in regulatory inspection and sampling, a well-equipped toxicology laboratory with a well-qualified staff to enable analysis for the necessary natural and synthetic toxicants at the level of a National Feed Reference Laboratory, and a small administrative staff. Other tasks of the AFcU will be the registration and control of medicated feeds, feed additives, and companion animal feeds. A major role will be the much-improved control of the very large amounts of imported feedstuffs arriving at the port terminals of Ashdod and Haifa, including state of the art sampling techniques of immense cargoes. The AFcU, as envisaged by the Twinning Project, will comprise a total of up to about 20 permanent staff members in all of these activities. Although some compulsory fees will be charged by the AFcU, the Government of Israel will have the overall responsibility for providing funding to employ the recommended staff and to acquire the analytical equipment needed for the toxicological examinations. The new legislation, the training and guidance in the Twinning Project, and the consequent development of the AFcU, will bring about marked changes in animal feed control in Israel, to ensure maximal food and feed safety. These changes will address various obligations (taken from EU food and feed legislation), still to be finalized, but basically comprising:-

Responsibility1. . Feed operators will be registered and are responsible for the safety of the feedstuffs which they import, produce, transport, store, sell or provide without cost. Farmers are responsible for the feedstuffs that they use in animal production. This is the basic principle of Feed Control. In order to ensure food and feed safety, feed suppliers and producers will be responsible for examining feedstuffs for potential toxicants. AFcU will periodically sample and analyze feedstuffs from all feed operators to ensure compliance with the responsibilities of the feed operators;Prevention2. . Feed operators will identify and regularly review the critical points in their processes and ensure that pre-determined controls are applied at these points;Safety3. . Feed operators will not supply any feed thought, known or found to be potentially harmful. The new Feed Regulations that will be part of the Feed Law will list all the feedstuffs regarded as being suitable and safe. Any substance not in that list, including “non-conventional” feedstuffs, will have to be shown to be suitable and safe, as they are supplied, and also during conditions of storage;Traceability4. . Feed operators will have to be able to rapidly identify the source of any feedstuff and to ascertain the route of supply to all recipients. This will be done mainly by identification / marking of feedstuffs, and computer-enabled tracking;


Transparency5. . Feed operators will immediately inform the competent authorities if they have any reason to believe that any of their feed is potentially unsafe. Feed operators will be encouraged to ensure availability of feedstuff control practises and results to their clients through newsletters and the internet;Emergency6. . Feed operators will immediately inform the competent authorities and all recipients if they suspect, know or find that a feedstuff is potentially unsafe. They will immediately start a withdrawal of feedstuffs (recall) from all supply routes and recipients; Cooperation7. . Feed operators will cooperate with the competent authorities in actions planned and taken to avoid or reduce risks.

Development and approval of the Feed Regulations and the implementation of the Feed Law and Regulations will take many months to complete. There will be time to invest in comprehensive discussion and interaction with other government bodies and feed operators, and to ensure that whilst food and feed safety is the prime objective, there may be approximation in some obligations to suit the unique facts of feedstuff supply in Israel.

Overall, this is a very positive step that the Government of

Israel has committed itself to take and our veterinary profession will be at the forefront of both bringing the changes into action and in benefiting from the improved animal health that they will doubtless engender. As citizens, we shall have an optimal feed control system to ensure that food of animal origin will be both wholesome and safe to eat.

TOXICOLOGY VIEWPOINT

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