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Professionnel Documents
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Veterinary medicine
Formerly: RefuaH Veterinarith
Published Quarterly:
CONTENTS
VOLUME 65
no. 3
2010
Editor:
T. Waner
Co. Editor:
H. Barak
Associate Editors:
M. Ballaiche
A. Bomzon
J. Brenner
N. Galon
S. Harrus
E. Klement
G. Segev
R. Schahar
A. Steinman
Editorial Board:
G. Dank
D. Elad
S. Freidman
G. Leitner
U. Orgad
E. Pipano
A. Rosner
G. Simon
A. Shimshony
N. Speigel
Z. Trainin
I. Samina - President
D. Dagan - Secretary
A. Markovitz - Treasurer
Website: www.isrvma.org
EDITORIAL:
Greater visibility
Waner, T.
92
REVIEW
STAPHYLOCOCCUS AUREUS MASTITIS:
WHAT WE NEED TO KNOW TO CONTROL THEM
Zecconi, A.
93
Articles
INTOXICATION OF YOUNG CROCODILES
IN CAPTIVITY DUE TO THE INGESTION OF
DARKLING BEETLES BLAPS NITENS LAPORTEI
ARDOIN (COLEOPTERA; TENEBRIONIDAE)
Perelman, B. and Chikatunov, V.
100
103
108
111
117
121
Toxicology viewpoint
Improved Animal Feed Control
"Farm to Fork"
Shlosberg, A.
123
17 Cottage Lane
Springfield. N.J. USA 07081-2302
B. Bender - President
A. Newman - Treasurer
ISSN 0334-9152
Published by:
Giraffica Studio - Graphic design for Magazines
www.giraffica.com
Arabian Horse
(see next page)
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 the
above policies, the authors must
1) Sign a covering letter certifying that legal and ethical
requirements have been met with regards to the humane
treatment of animals described in the study;
2) Specify in the covering letter and in Materials and Methods
the international, national, and/or institutional guidelines
followed;
3) Provide evidence, such as a signed animal use form or protocol
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:
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.
Cover image:
Arabian Horse
A 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.
EDITORIAL
visibility
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
92
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REVIEW
INTRODUCTION
DIAGNOSIS
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93
REVIEW
To overcome some of the diagnostic problems, augmented
methods have been also proposed. Among them, freezingthawing 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 -20C 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 Methods
Risk Factors
Therapy
website: www.isrvma.org
Vaccination
Heifer Vaccination
Nickerson 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
Volume 65 (3) 2010
website: www.isrvma.org
95
REVIEW
number of studies, still a vaccine with proven efficacy in
commercial dairy herds and in different areas is not available.
Control Program
96
CONCLUSIONS
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REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
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97
REVIEW
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
98
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
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FIGURE
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99
ARTICLES
Abstract
A 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
Introduction
100
Case report
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REFERENCES
1.
Discussion
2.
3.
4.
5.
6.
7.
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101
ARTICLES
102
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Corresponding author:
Dr. J.D. Parrah
drjdparrah@yahoo.co.in
Abstract
Calculi 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.
Key words: Urolithiasis, Calculi, Calf
Introduction
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103
ARTICLES
within the calculus was recorded.
3. Chemical analysis
After 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 analysis
Calculi 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 calculi
Almost 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 calculi
The 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 calculi
Surface 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
104
Discussion
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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.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
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ARTICLES
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Group AII
Group B
Total
%age
6/30
20
11/30
37
14/30
47
10
10/10
100
4/10
40
2/10
20
2/10
20
2/10
20
No. of
animals
No. of
calculi
No. of
animals
No. of
calculi
No. of
animals
No. of
calculi
Urethral lumen
DSF
PSF
Between DSF
& Glans penis
Cystic lumen
Cystic neck
106
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ARTICLES
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 (Spearmans 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.
Key words: diagnostic, milk fever, sheep, water hardness test kit.
was to measure the total calcium levels in sheep sera with and
INTRODUCTION
Milk fever - parturient paresis or hypocalcaemia - is a without milk fever using a commercial water hardness test kit
metabolic disorder that occurs around the time of parturition (WHTK) compared to a routine laboratory method.
or during early lactation in sheep (1). The disease commonly
occurs in outbreaks, in groups of ewes exposed to forced MATERIAL AND METHODS
exercise, long-distance transport, and sudden deprivation of Animals
Thirty ewes were used in this study had at least one clinical
food and grazing on oxalate-containing plants or green cereal
crops (2). Although it is may be seen occasionally as epidemic findings of milk fever such as depression, increased respiration,
in the herd where up to 25% of the herd may be affected, its muscular tremor, weakness, sternal recumbence and extended
course is mostly sporadic and generally affects less than 5% of or twisted head. Clinical examinations were made by the same
veterinarian. The sheep were 3.31 years of age (range 2 and
the herd (1, 2, 3, 4).
The disease is characterized by a low serum calcium (Ca) 5 years) were taken from 9 different farms. Fifteen sheep were
level (2, 5, 6). As a result of inadequate calcium intake during pregnant and 15 sheep were lactating.
Approximately 20 ml blood was collected from the jugular
the last periods of pregnancy or first periods of lactation,
the body may meet the required need of calcium through vein. Blood samples were allowed to clot for minimum 30
calcium mobilization from bones. If hormonal mechanisms minutes at ambient temperature. The samples were then
are inadequate, for example in the case of an inactive centrifuged and sera were collected. The sera were kept at
parathyroid gland, mobilization is delayed and blood calcium -20C until the analysis was performed. All samples were
concentration is reduced with the resultant development of analyzed within 4 weeks.
milk fever. In small ruminants, hypocalcaemia may develop Serum calcium analyses
Total serum calcium concentrations were determined by
secondarily during the course of other periparturient diseases,
particularly in pregnancy toxemia (7). The diagnosis of the spectrophotometric method using an automatic biochemical
disease is based on clinical or laboratory methods or based analyzer (Cobas 6000 analyzer, Roche, Switzerland). All
on the presence of risk factors which have been reported samples were analyzed at the same time. Based on the results
(8). Therefore, monitoring the calcium level in sheep during obtained with the automatic chemical analyzer the values were
periparturient period may be useful for the identification of the found to be in the range of 6.1 to 10.1 mg/dl.
Total calcium concentrations in the sera samples of all
development risk of both milk fever and other hypocalcaemia
related periparturient diseases. The definitive diagnosis is made sheep were determined using a commercial WHTK which was
through measurement of total and /or ionized serum calcium designed for measurement of calcium carbonate concentrations
concentration (7). Serum total or ionized calcium levels in water samples (CHEMetrics. Inc Cat. No: K-1705. Calverton,
may be measured by laboratory and/or portable biochemical VA, USA). The test was carried out as follows: Calcium in
analyzers. In veterinary practice chemistry analyzers are often the test sample was allowed to react with a zinc salt in the
unavailable in the field. Therefore, the aim of the present study presence of a color indicator. The determined blue and blue108
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RESULTS
DISCUSSION
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109
ARTICLES
1990s, 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) (Spearmans 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.
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2.
3.
4.
110
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8.
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18.
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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
hypocalcaemia in ewes in central Ethiopia. Revue. Med. Vet. 151:
345-350, 2000.
Gurbulak, K., Pancarc, S. M., Gungor, O., Kacar, C., Oral, H.,
Krmzgul, 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,
treatment and prevention. http://vbs.psu.edu/ext/resources/pdf/
small-ruminant/Sm%20Rum%20nutr%20disease.pdf 2010.
Houe, 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
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,
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
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
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
Hypophosphatemia Acta Vet. Scand. 44:126, 2003.
Sakha, M. and Jamshidian, M.: Evaluation of Bovine Parturient
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
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,
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 GreiveCrandell, 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
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*Corresponding author:
Zhang Guozhong; Lin Degui
Telephone: +86-10-62733660
Fax: +86-10-62732984
E-mail: zhanggz@cau.edu.cn (G.Z. Zhang); csama@sina.com (D.G. Lin)
Postal address: College of Veterinary Medicine, China Agricultural University, Beijing100193, P.R. China
ABSTRACT
Canine 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.
Keywords: Canine oral papillomatosis, histopathology, immunohistochemistry, sequencing.
Introduction
Patient 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 m) 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 3m. 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
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using a mouse anti-PCNA antibody (WuHan Boster Bioengineering 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 antirabbit 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
manufacturers 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 20l reaction
system was composed which included the template DNA 3l,
0.5l of upstream and downstream primer, 10l of PCRmix
(Mylab Corporation, Beijing, China) and 6l 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.
Results
Clinical 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
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Discussion
References
1.
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6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Tables
Table 1 - Information of dogs
No.
Breed
Age (Month)
Sex
Weight (kg)
Male
20
Beagle
Female
Siberian Husky
Female
13
Great Pyrenees
Male
18
Caucasian Sheepdog
Female
20
Primers (5-3)
Predicted product
L1
P1:CACAGCCCAGCACCAAG
P2:TGCGTTTGCGTTTCACA
NCR2
P1:GACAAGTCCGACAGTCCAAC
P2:GGTCAGATAAGCGGGTAGG
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Mutation sites
Changes
COPV T2
nt 4254
T was replaced by C
COPV T3
nt 4252-4253
COPV T4
nt 4379
A was replaced by T
nt 4937-4938
nt 4992
A was replaced by G
Figures
Figure 1 - Clinical and histopathological examination of canine
oral papillomatosis. (A) Appearance of dogs 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.
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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.
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Cardiology
ECG 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 Environment
The Hebrew University of Jerusalem, Israel.
Case report
Figure 1
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Answers:
3.
4.
5.
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rhythm.
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Table 1
120
Wave
Amplitude (mV)
Width (sec)
0.06
> 2.5mV
(> 3.0 mV in Giant Breeds)
1.5
QRS
< 0.06sec;
(< 0.065 sec in
Giant Breeds)
0.09
P-R
0.06-0.13 sec
0.14
Q-T
0.15-0.25 sec
0.22
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RADIOLOGY
A DOG WITH ACUTE VOMITING
Bibring, U. and Eizenberg, Z.
Imaging Unit
The Koret School of Veterinary Medicine
The Robert H. Smith Faculty of Agriculture, Food and Environment
The Hebrew University of Jerusalem, Israel
History
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Radiographic findings
Imaging discussion
REFERENCES
1.
2.
Radiographic diagnosis
1. Pleural effusion
2. Abdominal effusion
3. Hepatomegaly and suspected hepatic abscessation
Since Ladys condition deteriorated quickly, abdominal ultrasound
was undertaken (Figure 4).
Abdominal ultrasound-findings
1) 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
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Toxicology viewpoint
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Toxicology viewpoint
6.
7.
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