37th Symposium of the Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Israel

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I SRAEL JOURNAL OF
VETERINARY MEDICINE
Formerly: Refuah Veterinarith
Published Quarterly:
Volume 70 • No. 1 • March 2015
Editor: T. Waner
Edi t or i al Boar d:
I. Aroch
G. Segev
J. Milgram
G. Dank
B. Perelman
I. Davidson
G. Kelmer
A. Steinman
J. Bojkovski
D. Elad
A. Markovitz
G. Leitner
S. Friedman
Y. Shimshoni
S. Pozzi
E. Lavi
Y. Stram
D. David
S. Perl
A. Shimshoni
Z. Trianin
G. Simon
Israel Veterinary Medical Association
P.O.Box 22, Raanana 4310001, Israel
Tel: 09
-
7419929 • Fax: 09
-
7431778
E-mail: ivma@zahav.net.il
I. Samina – President
D. Dagan – Secretary
A. Markovitz – Treasurer
American Veterinarians for Israel
125 Paterson Ave., Suite 1, Little
Falls, New Jersey 09424, USA
B. Bender – President
S. Altman – Vice President
G. Buchoff – Treasurer
Websi t e: www. i j vm. org. i l
Printed in Israel by E. Oren Publisher Ltd.
Email: oren.print@gmail.com
ISSN 0334
-
9152
Front Cover: DORPER SHEEP BREED
Te Dorper was developed in South Africa in the 1930’s by crossing the Dorset Horn with the Blackhead Persian. Te sheep is easy to care for
and is well adapted to survive arid conditions. Tey have a high fertility and maternal instinct along with a high growth rate and resilience. Tey
are outstanding browsers utilizing to the upmost the available pasture. Dorper sheep may have either black or white heads. Te name is derived
from the frst syllables of the parent breeds. Tis breed has been introduced into the Israeli herd and contributed its characteristics to the variety
of culinary treats in our country. Te two pictures were photographed in the Namaqualand region of South Africa in September 2014 during
the annual fowering season.
Contents
Edi tor i al – Waner, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Revi ew Ar t i cl e
Perioperative Antibiotics Prophylaxis in Equine Surgical Colic Patients –
Are We Doing the Right Ting?
Steinman A., Kelmer, G. Tatz, A. and Berlin, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Research Ar t i cl es
Translocation of Rabies Virus in Israel by Cattle: A Treat for the Public Health
David, D., Dveres, N., Yakobson, B.A. and Davidson, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Determination of Macroelement Parameters in Diferent Productive Stages
of Simmental Cows
Krsmanović, M., Djoković, R., Giadinis, N.D., Panousis, N., Bojkovski, D.,
Savić-Stevanović, V., Vasić, A., Zdravković, N., Korica, S. and Bojkovski, J. . . . . . . . . . . . . . . . . 12
Te Infuence of Subclinical Hypocalcemia on Production and Reproduction
Parameters in Israeli Dairy Herds
Gild, C., Alpert, N. and van Straten, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Reproductive Performance in Dairy Cows Synchronized with the Ovsynch
Protocol at Diferent Stages of the Estrus Cycle
Goshen, T., Tsitrin, K. and van Straten, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Detection of Toxoplasma gondii and Neospora caninum antibodies in Wild
Boars (Sus scrofa) in Eastern Turkey
Balkaya, I., Utuk, A. E., Babur, C., Beyhan, Y. E., Piskin, F. C. and Sozdutmaz, I. . . . . . . . . . . . 28
Retrospective Study of Disease Occurrence in Captive African Pygmy
Hedgehogs (Atelerix albiventris)
Gardhouse, S. and Eshar, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Diaphragmatic Hernia in Horses in Israel: A Case Series
Efraim, G. and Kelmer, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Pharmacokinetics of Enrofoxacin and its Metabolite Ciprofoxacin after
Intracoelomic administration in Tortoises (Testudo hermanni)
Salvadori, M.,De Vito, V., Owen, H. and Giorgi, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Case Repor t
Acute Pancreatitis in a Horse – a Case Report
Edery, N., Rosenbaum, A., Busnach, A., Steinman, A., Tirosh Levy, S. and Perl, S. . . . . . . . . . . 49
Concurrent Neosporosis and Hepatozoonosis in a Litter of Pups
Mazuz, L.M., Wolkomirsky, R., Sherman A., Savitzsky, I., Waner, T., Golenser, J. and Shkap, V. . . 53
37
th
Symposium of the Koret School of Veterinary Medicine,
Hebrew University of Jerusalem, Israel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 www.ijvm.org.il 2
Editorial
S
ince my commencement as editor of the Israel Journal for Veterinary Medicine (IJVM) in 2010 I have chosen a
species of animal each year to be portrayed on the cover of the journal. For example in the year 2010 diferent breeds
of horses appeared on the cover, in 2011 dogs and in 2013 cats. Te year 2015 has been declared the “Year of the
Sheep” by the Asian communities and it is just by chance that I also decided to choose this species. Te sheep ranks in the
eight position of animals in the Chinese zodiac and represents solidarity, harmony and calmness. I welcome all our readers
to submit pictures of sheep for inclusion on the cover of the journal during this year, 2015.
Te present issue presents four articles on bovine medicine and three on equine medicine. Tis is very pleasing and
I welcome more articles relating to these species. Two articles deal with exotic species medicine, both articles are from
overseas. One article from Italy through the gracious support of Prof. Mario Giorgi a good friend of Israel and the other
through Dr. David Eshar a former student of the Koret School of Veterinary Medicine. Dr. Monica Mazuz from the
Kimron Veterinary Institute presents an interesting frst case report of pups infected concurrently with Neospora and
Hepatozoon. Prof. Ibrahim Balkaya has presented a survey on the presence of Toxoplasma and Neospora in wild boars in
Eastern Turkey. So there is a lot for everyone to read.
Te endeavor of a number of countries to boycott Israeli academics is discussed frequently in the media. I want to
remind our Israeli veterinary scientists that this issue may one day afect all of us and also future veterinarians. I raise this
issue to emphasize the importance achieving a good professional Israeli veterinary journal of a high standard. In order to
achieve this, the journal must receive support from veterinarians in Israel.
Te status of the journal is improving annually and this year we have reached another record in the number of citations
which have been on the ascent since 2010 since I assumed the editorship of the IJVM. I have included a graph of the number
of citations per year from 2008 to demonstrate our achievements. Tis increase would have been even more signifcant if
only the Journal had the support of senior academic veterinarians, which I urge and for look forward to in the very near
future. Tis is not my frst request for this cooperation and a group-efort from academic veterinarians as documented in
many of my editorials will hopefully signal a change in their minds and attitude.
Te excellent standard of veterinary research in Israel is refected in the abstracts of the 37
th
Symposium of the Koret
School of Veterinary Medicine. I am proud to publish these abstracts and I congratulate the organizers in gathering such
admirable research presentations.
Te Editorial board of the IJVM wishes all its readers a pleasant and safe spring. To those going on holiday and attending
conferences, wishing you safe travels and successful conferences.
Chag Pesach Samach.
Trevor ( Tuvi a) Waner
Editor-in-Chief
Israel Journal of Veterinary Medicine
NUMBER OF CITATIONS OF ARTICLES BY YEAR
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 3 Perioperative Equine Antibiotics Prophylaxis
REVIEW
Perioperative prophylaxis is one of the most common
reasons for antimicrobial drug administration. Its goal is
to decrease the likelihood of infection after exposure to
bacteria during the surgical period (1). Te use of periop-
erative prophylaxis has evolved greatly in the last 30 years.
Improvements in the timing of initial administration, the
appropriate choice of antibiotic agents, and shorter dura-
tions of administration, defne its value in reducing surgical
site infections while minimizing the risks for side efects (2).
On the other hand, inappropriate use results in unnecessary
costs, increased antimicrobial resistance, and development
of superinfections (3). Whereas much information is avail-
able in human medicine, objective scientifc studies which
evaluate the use of perioperative prophylaxis in veterinary
medicine are limited and more studies are warranted.
An American College of Veterinary Internal Medicine
(ACVIM) consensus statement on antimicrobial drug use in
veterinary medicine was issued several years ago (4). In rec-
ommendation I, it is stated that the committee believes that
prophylactic and metaphylactic use of antimicrobial drugs is
appropriate for control and prevention of infectious diseases
Perioperative Antibiotics Prophylaxis in Equine Surgical Colic
Patients – Are We Doing the Right Ting?
Steinman A.,* Kelmer, G. Tatz, A. and Berlin, D.
Koret School of Veterinary Medicine, Te Hebrew University of Jerusalem, Israel.
*
Corresponding Author: Dr. Amir Steinman, DVM., Ph.D., MHA Director, Hospital Koret School of Veterinary Medicine – Veterinary Teaching Hospital,
Te Robert H. Smith Faculty of Agriculture, Food and Environment, Te Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel. Email:
amirst@savion.huji.ac.il
ABSTRACT
Perioperative prophylaxis is one of the most common reasons for antimicrobial administration. Its goal is
to reduce postoperative infection at the surgical site, thereby reducing morbidity, mortality and treatment
costs. When perioperative prophylactic treatment is used, several issues should be decided upon, including
the drugs that are used, timing of frst administration, re-administration after 2 half-lives of the drug if
surgery is still ongoing and duration of treatment. Equine colic surgery is typically an emergency procedure,
classifed in the best circumstances as a clean-contaminated surgical procedure and carries a high rate of
surgical site infection as an important short term complication. Information regarding the compliance with
prophylaxis guidelines in veterinary medicine is limited and is often not detailed enough. Te duration of
prophylactic treatment is a more complicated topic due to the high rate of post-operative complications in
horses undergoing colic surgery. Guidelines for the judicious use of antimicrobial therapy recommend that
antimicrobials should be administered for the shortest efective period possible to prevent the development
of resistant pathogens. A recent study in surgical colic patients has shown no diference in the rate of incision
infection with the use of perioperative antimicrobial therapy for 72 hours and for 120 hours, resulting in
the conclusion that there is no beneft for the longer duration of prophylactic antimicrobial administration.
Clinician’s awareness for guidelines or standard protocols for antimicrobial drug use for equine patients
undergoing surgery for colic is important. Implementing such guidelines, which should be reviewed and
updated regularly, and reducing the amounts of perioperative antimicrobials that are being used are important
goals which we all should strive for in order to reduce the emergence of resistant strains of bacteria that
could afect our patients.
Review Article
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Steinman A. 4
in animals. However, it is also stated that its use should be
conservative and should emphasize drugs assigned to the
primary use category. It is also stated that it is not necessary
to use antimicrobial drugs in all surgical cases to prevent
infections, which is especially true for clean surgeries, as
opposed to clean-contaminated or contaminated procedures
(4). Clean surgical wounds have an infection rate less than
5%, so antimicrobial prophylaxis is generally not neces-
sary (3). Clean-contaminated wounds are those in which
contaminated areas of the body (eg. gastrointestinal system,
genitourinary system) are entered under controlled condi-
tions without unusual contamination (3). Colic surgery in
horses is typically an emergency procedure, classifed in
the best circumstances as a clean-contaminated surgical
procedure (4). Surgical site infection (SSI) is one of the
most common short-term complications in colic patients
and can occur in as many as 22-40% of all cases (5, 6, 7).
When using prophylactic antibiotics, several aspects
should be taken into accounts including the selection of ap-
propriate agent, the timing of the frst dose, re-administration
of antibiotics during surgery after 2 half-lives of the drug
have passed, and duration of treatment. Regarding the timing
of the frst dose, prophylactic antimicrobials must be pres-
ent in appropriate levels at the surgical site during the time
of contamination (3). In human patients, administration
of the frst dose of antibiotics more than two hours before
surgery or three hours after surgery resulted in six times
higher complication rate as opposed to patients that were
treated 0-2 hours before surgery (8). Current guidelines for
use in horses suggest that prophylactic antibiotics should be
administered, preferably intravenously, within one hour of
frst incision (9). As for re-administration during surgery, in
human patients undergoing colorectal surgery, the incidence
of infection was decreased in patients that were administered
repeated intraoperative dosing of antibiotics (10). Regarding
re-administration of antimicrobials in veterinary patients if
surgery is ongoing after 2 half-lives of the drug have passed,
prophylactic beta-lactam antimicrobial administration should
be repeated during surgery (3).
Information regarding the compliance with prophylaxis
guidelines in veterinary medicine is limited. In a study report-
ing on prophylactic antimicrobial use in horses undergoing
elective arthroscopy, only 6.3% received preoperative antibiot-
ics within 60 minutes of the frst incision (11). Although it
is recommended that a second dose should be administered
in colic surgeries if the surgery is ongoing after 2 half-lives
of the drug have passed in order to assure adequate levels for
the duration of surgery, intraoperative antibiotic administra-
tion did not occur in any horse, in that study (11). Tese
discrepancies can be partially explained by logistical reasons
but it was suggested by the authors that adhering to these
recommendations is preferable (11). In a recent study that in-
vestigated antimicrobial use in horses undergoing emergency
colic surgery, only 88 (11.6%) horses received the appropriate
preoperative dose within 60 minutes of the start of surgery
and only 8 horses (1.8%) were re-dosed correctly (1).
Te duration of prophylactic treatment is a more com-
plicated topic due to the high rate of post-operative com-
plications in horses undergoing colic surgery. Guidelines
for the judicious use of antimicrobial therapy recommend
that antimicrobials should be administered for the short-
est efective period possible to prevent the development
of resistant pathogens (4). Continued use of prophylactic
antimicrobials beyond the conclusion of surgery contributes
to the development of resistant bacteria, superinfections
and nosocomial infections (3). While in horses undergoing
elective arthroscopic surgery, most (66%) were treated for 24
hours or less (11), this is clearly not the common practice
in colic patients. In 2006, Santschi suggested that equine
surgeons should seriously consider reducing the duration
of prophylactic antimicrobial use in their patients (9). Due
to various reasons, among which is the high post-operative
complication rate, it is still common practice in many equine
hospitals to administer prophylactic antimicrobial therapy
for 5 days, postoperatively, mostly aiming to reduce inci-
sional complications (6, 12). Tis is also the current protocol
at the Koret School of Veterinary Medicine – Veterinary
Teaching Hospital (KSVM-VTH). In a recent study, the
use of perioperative antimicrobial therapy for 72 hours and
for 120 hours was compared, in order to evaluate its efect
on the development of postoperative incisional infections.
High post-operative incisional complication rate (42.2%)
was found, but no diferences were seen between the two
tested groups, resulting in the conclusion that there is no
beneft for the longer duration of prophylactic antimicrobial
administration (6). Te authors of that study suggested that
further studies are warranted to identify the minimum ef-
fective duration of antimicrobial therapy as it is possible
that even a shorter course of 1-2 days would be equally
benefcial (6).
Review Article
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 5 Perioperative Equine Antibiotics Prophylaxis
Te last but not least decision when administering pro-
phylactic antimicrobial is the selection of appropriate agent.
As stated earlier, in the ACVIM consensus it is recommended
that prophylactic antimicrobial use should be conservative
and should emphasize drugs assigned to the primary use
category. Te most common antibiotic combination used in
adult colic patients is penicillin G and gentamicin (1) which
is in accordance with the ACVIM consensus. Tis is the stan-
dard antibiotic combination in the Royal Veterinary College
and at the Bell Equine Veterinary Clinic, both in the UK,
in colic patients (6). In a survey of 761 horses undergoing
emergency colic in the New Bolton Center of the University
of Pennsylvania, 89.3% were given potassium or procaine
penicillin G and gentamicin and very few were given other
drug combinations (1).
As stated earlier, inappropriate use of perioperative pro-
phylaxis results in unnecessary costs, increased antimicro-
bial resistance, and superinfections (3). We cannot conclude
inclusively, but it is possible that among other reasons, the
recent emergence of multi-drug resistant (MDR) bacteria in
the Large Animal Department of the KSVM-VTH is the re-
sult of such use of antimicrobials. During 2013 49% (27/55)
isolates from diferent body sites in hospitalized horses at
the KSVM-VTH were MDR (Berlin, personal communica-
tion). Multi-drug resistant bacteria are classifed as those that
are resistant to at least 3 groups of antimicrobials (13) and
included 6/12 Escherichia coli isolates, 4/7 Enterobacter iso-
lates, 7/8 Klebsiella isolates and 1/1 a Salmonella isolate which
further emphasizes the problem of nosocomial infection and
question the current prophylaxis antimicrobial use routine.
Methicillin resistant Staphylococcus aureus was also isolated
from both carrier horses and infected wounds during this
year. Strict guidelines for the use of prophylaxis antimicrobial
use in horses undergoing colic surgery is needed.
In a survey that was conducted little more than a de-
cade ago among diplomats of the American College of
Veterinary Surgeons, 28 of 32 respondents (88%) reported
that they were unaware of any written guidelines or stan-
dard protocols for antimicrobial drug use for equine patients
undergoing surgery for colic at their veterinary teaching
hospital (14). A decade later, in a survey among UK equine
surgeons, less than 1% of the practices had antimicrobial use
guidelines (15). Te authors emphasized the importance of
implementation of such guidelines in any institution where
antimicrobials are prescribed in order to maintain their
efectiveness (15). Implementing such guidelines, which
should be reviewed and updated regularly, and reducing the
amounts of perioperative antimicrobials that are being used
is an important goal which we all should strive for in order
to reduce the emergence of resistant strains of bacteria that
could afect our patients.
In conclusion, in horses undergoing colic surgery, it is
important to administer the frst dose of antibiotics, within
60 minutes of the frst incision. A second dose should be
administered if the surgery is ongoing after 2 half-lives of the
drug have passed. Prophylactic antimicrobial administration
should be limited to no more than 72 hours and further
studies are required to determine if even shorter duration
is accepted.
REFERENCES
1. Dallap Schaer, B.L., Linton, J.K. and Aceto, H.: Antimicrobial
use in horses undergoing colic surgery. J. Vet. Intern. Med. 26:
1449-1456, 2012.
2. Nichols, R.L.: Preventing surgical site infections, Clin. Med. Res.
2: 115-118, 2004.
3. Howe, L.M. and Boothe, H.W.: Antimicrobial use in the surgical
patients. Vet. Clin. Small Anim. 36: 1049-160, 2006.
4. Morley, P.S., Apley, M.D., Besser, T.E., Burney, D.P., Fedorka-
Cray, P.J., Papich, M.G., Traub-Dargatz, J.L. and Weese, S.: An-
timicrobial drug use in veterinary medicine. J. Vet. Intern. Med.
19: 617-629, 2005.
5. Mair, T.S. and Smith, L.J.: Survival and complication rates in 300
horses undergoing surgical treatment of colic. Part 2: Short-term
complications. Equine Vet. J. 37: 303-309, 2005.
6. Durward-Akhurst, S.A., Mair, T.S., Boston, R. and Dunkel, B.:
A comparison of two antimicrobial regimens on the prevalence of
incisional infections after colic surgery. Vet. Rec. 172: 287, 2013.
7. Colbath, A.C., Patipa, L., Berghaus, R.D. and Parks, A.H.: Te
infuence of suture pattern on the incidence of incisional drain-
age following exploratory laparotomy. Equine Vet. J. 46: 156-160,
2014.
8. Classen, D.C., Evans, R.S., Pestotnik, S.L., Horn, S.D., Menlove,
R.L. and Burke, J.P.: Te timing of prophylactic administration
of antibiotics and the risk of surgical-wound infection. N. Engl. J.
Med. 30: 281-286, 1992.
9. Santschi, E.M.: Prevention of postoperative infections in horses.
Vet. Clin. Equine.22: 323-334, 2006.
10. Morita, S., Nishisho, I., Nomura, T., Fukushima, Y., Morimoto,
T., Hiraoka, N. and Shibata, N.: Te signifcance of the intraop-
erative repeated dosing of antimicrobials for preventing surgical
wound infection in colorectal surgery. Surg. Today. 35: 732-738,
2005.
11. Weese, J.S. and Cruz, A. Retrospective study of perioperative anti-
microbial use practices in horses undergoing elective arthroscopic
surgery at a veterinary teaching hospital. Can. Vet. J. 50: 185-188,
2009.
Review Article
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Steinman A. 6
12. Wormstrand, B.H., Ihler, C.F., Diesen, R. and Krontveit, R.I.:
Surgical treatment of equine colic – a retrospective study of 297
surgeries in Norway 2005-2011. Acta Vet.Scand. 56: 38, 2014.
13. Magiorakos, A.P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas,
M.E., Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Ols-
son-Liljequist, B., Paterson, D.L., Rice, L.B., Stelling, J., Struelens,
M.J., Vatopoulos, A., Weber, J.T., and Monnet, D.L: Multidrug-
resistant, extensively drug-resistant and pan drug-resistant bacteria:
an international expert proposal for interim standard defnitions
for acquired resistance. Clin. Microbiol. Infect. 18: 268-281, 2012.
14. Traub-Dargatz, J.L., George, J.L., Dargatz, D.A., Morley, P.S.,
Southwood, L.L. and Tillotson, K.: Survey of complications and
antimicrobial use in equine patients at veterinary teaching hos-
pitals that underwent surgery because of colic. J. Am. Vet. Med.
Assoc. 220: 1359-1365, 2002.
15. Hughes, L.A., Pinchbeck, G., Callaby, R., Dawson, S., Clegg, P.
and Williams, N.: Antimicrobial prescribing practice in UK equine
veterinary practice. Equine Vet. J. 45: 141-147, 2013.
Review Article
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 7 Translocation of Rabies Virus by Cattle
INTRODUCTION
Rabies virus is a member of the Rhabdoviridae, genus
Lyssavirus. All mammals are susceptible to rabies infection.
Rabies is enzootic throughout the Middle East, including
Israel. Rabies is also a serious enzootic disease in Jordan,
Syria, Lebanon and Iran and Irak where stray dogs maintain
rabies virus in circulation, with frequent spillover to wildlife,
including jackals, squirrels, stone-martens, foxes, wolves and
infection of domestic animals and human (1, 2, 3, 4, 5, 6).
Since 1979, red foxes (Vulpes vulpes) have become the
most important reservoir of rabies virus in Israel (7). An
oral vaccination (ORV) program directed at wild animals
has been implemented since 1998 in northern Israel (8). In
2004 the program was extended and it currently covers all the
territories controlled by Israel and the Palestinian Authority.
Israel is the only country in the Middle East that imple-
ments ORV program. To insure its success, extensive rabies
surveillance along Israel’s borders is carried out. Despite the
efcacy of the ORV in controlling fox rabies in north Israel, a
new outbreak occurred in 2004 in this region, in which stray
dogs (Canis familiaris) where shown to be main reservoir and
transmitter of genetic variant V7 (9, 10).
Molecular epidemiological studies of rabies in Israel be-
tween 1993 and 1998 revealed four fox strain genetic lineages
(V1-V4), were distributed within four geographical regions
(11). Molecular analysis of rabies isolates on the borders of
Israel and neighboring countries revealed the presence of
three genetic variants V5, V6 and V7 (12). Due to the ORV
program, fox rabies genetic variants V1 and V2 specifc to
northern Israel region were eliminated in 2003, while fox
strain genetic variants V3 and V4 enzootic to central and
southern of Israel were eliminated by 2005 (9).
However, incursions of fox rabies variant infected ani-
mals persisted across Israel’s borders. Between 2002 to 2013
about 32 isolates of fox rabies virus belonging to the genetic
variant V1, were detected within Israel and its neighboring
countries.
All mammals are susceptible to rabies virus. Bovine rabies
has a direct economic impact (13) on the livestock industry
and represents a public health threat in rabies endemic area.
In Israel between the years 1996-2014, 104 rabies cases
were reported in cattle, most of them on the borders with
neighboring countries.
Recently two human rabies mortalities, one in Iran and
Translocation of Rabies Virus in Israel by Cattle:
A Treat for the Public Health
David, D.,* Dveres, N., Yakobson, B.A. and Davidson, I.
Kimron Veterinary Institute, Bet Dagan 50250, POB 12, Israel.
*
Corresponding Author: Dr. D. David, Tel :+972-506241984, Fax: +972-3-9681721. Email: davidd@int.gov.il
ABSTRACT
Rabies is endemic in Israel, the only country in the Middle East that implements a nation-wide anti-rabies
campaign. However, between 2002 and 2013 about 32 rabies virus isolates belonging to genetic variant V1
were recovered within Israel. Te present study describes for the frst time the translocation of the rabies
virus strain, fox V1, by infected cattle born on “Tzfon HaGolan dairy farm” located in Kibbutz Ortal on the
Golan Heights to two farms situated in the western Yezre’el Valley, emphasizing that cattle may serve also
a source of human rabies infection.
Keywords: Rabies; Cattle; Translocation; Diagnosis; Post exposure vaccination.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 David, D. 8
the second in Brazil were reported from human handling ra-
bies infected livestock (14, 15). Te two veterinarians involved
were not vaccinated against rabies and did not received post
exposure prophylaxis.
Tere are several examples of long distance transmis-
sion translocation of rabies virus by human mediated ani-
mal movements and trade. Recently there was a number of
reported translocations of infected bovine from Romania
to Croatia (16), and in Ohio, USA (17). Translocation of
dog rabies from northern Israel to Jerusalem (18) and to the
center of the country was also reported (19).
In the present report, we describe for the frst time rabies
translocation by infected cattle from a dairy herd on the
Golan Heights to two farms located in the western part of
the Yezre’el Valley, as well as a rabies infected case in cattle
in Israel. In addition to the economic damage caused to the
dairy industry by rabies infection, the zoonotic aspects are
discussed.
CASE REPORT
On September 29, 2013, two 11 weeks old calves one from a
village Bet Zaid and the other form a village, Kfar Yehushua
in the western Yezre’el valley showed clinical signs of change
in behavior, salivation, difculty in swallowing, vocalization
and recumbence. Rabies was suspected and the two calves
were diagnosed rabies positive by direct immunofuorescence
assay (DFA) on the brain tissue, at the Israeli National Rabies
Laboratory, Kimron Veterinary Institute (KVI), Bet Dagan,
Israel.
In addition on November 1
st
2013, two more calves, one
8 months old, from Kibbutz Ortal, and the second from Kfar
Yehushua were rabies positive by DFA (Fig. 1).
A case investigation revealed that on August 26, a Jackal
had entered an orchard near the Kibbutz Ortal it was shot
and the carcass sent to National Rabies Laboratory (20).
Te Ortal dairy farm is one of the largest and most modern
in Israel. Male calves are sold at a few weeks of age to other
Figure 1: Succession of events following entry of a rabid jackal into Kibbutz Ortal dairy farm.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 9 Translocation of Rabies Virus by Cattle
10 20 30 40 50 60
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus TAAAGGCTGGTCATCCTTTTGACGCTTCAAGTCCTGAAGATCGCCTCCCCTTGGGGTTGG
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus TAAAGGCTGGTCATCCTTTTGACGCTTCAAGTCCTGAAGATCGCCTCCCCTTGGGGTTGG
70 80 90 100 110 120
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus GTAGAATCTCTGGGTTCAATAGTCCTCCTTGAACTCCATGCAACAGGGTAGATTCAAGAG
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus GTAGAATCTCTGGGTTCAATAGTCCTCCTTGAACTCCATGCAACAGGGTAGATTCAAGAG
130 140 150 160 170 180
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus TCATGAGATTTTCATTAATCATCTCAGTTGATCAAACTAGATCATGTAGATTCTCATAAT
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus TCATGAGATTTTCATTAATCATCTCAGTTGATCAAACTAGATCATGTAGATTCTCATAAT
190 200 210 220 230 240
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus ACGGGAAATCTTCTAGCAGTTTCAGTGACCAACGGTGCTTTCATTCTCCAGGAACTGGTA
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus ACGGGAAATCTTCTAGCAGTTTCAGTGACCAACGGTGCTTTCATTCTCCAGGAACTGGTA
250 260 270 280 290 300
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus CCAAAGGCTGTGGACGGGTCGAGAGGTGTTTCGGATGACTCCGTACTAGGGCACGGACAG
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus CCAAAGGCTGTGGACGGGTCGAGAGGTGTTTCGGATGACTCCGTACTAGGGCACGGACAG
310 320 330 340 350 360
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus AGGTCATGGTGCGTCCCATGATAGCAAACTCAGCATGAGTTTATTGAGAAAGGCAATTTG
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus AGGTCATGGTGCGTCCCATGATAGCAAACTCAGCATGAGTTTATTGAGAAAGGCAATTTG
370 380 390 400 410 420
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus CCTCCCATGAGGGACATAAGCAATAGATCATGATCATCTCGCATTTCAGCAAAGTGTGCA
780/2013/Jackal/OR7353/ORTAL ............................................................
783/Cattle/2013/Bet Zaid ............................................................
786/cattle/2013/Kfar Yehosua ............................................................
787/Cattle/2013/Ortal ............................................................
784/cattle/2013/Kfar Yehosua ............................................................
Consensus CCTCCCATGAGGGACATAAGCAATAGATCATGATCATCTCGCATTTCAGCAAAGTGTGCA
430 440 450 460 470 480
----:----|----:----|----:----|----:----|----:----|----:----|
Consensus CAATTATAAAGGGCTGGGTCATCTAAGCTTTTCAGTCGAGAAAAAAACT
780/2013/Jackal/OR7353/ORTAL .................................................
783/Cattle/2013/Bet Zaid .................................................
786/cattle/2013/Kfar Yehosua .................................................
787/Cattle/2013/Ortal .................................................
784/cattle/2013/Kfar Yehosua .................................................
Consensus CAATTATAAAGGGCTGGGTCATCTAAGCTTTTCAGTCGAGAAAAAAACT
Figure 2: Comparison of 469 base pairs of the G-L intergenic fragments from the sequences of
the four calves and the Jackal isolates.
locations throughout Israel for fattening.
A group of 54 rabies unvaccinated calves
was transported form Kibbutz Ortal on
September 10, 2013 to a cattle holding
farm in Bet Zaid and then distributed to
several farms in Kfar Yehushua.
Four calves and the jackal were diag-
nosed rabies positive by DFA. Diagnosis
was confrmed by virus isolation in tissue
culture, and its inoculation into suckling
mice (21). Reverse transcriptase – PCR
and direct sequencing were applied to a
469 base-pair (bp) G-L intergenic region
fragment (19). A phylogenetic tree was
constructed by the neighbor–joining
method, with the distance calculated
using the Kimura-2 parameter with the
computer program MEGA, version 4.1
(22). Te reliability of the phylogenetic
groupings was evaluated using bootstrap-
ping with 1000 replicates.
Te molecular analysis showed that
the viral sequence obtained from the 4
calves and the Jackal belonged to the V1
genetic variant (Fig.2). Following the
diagnosis of rabies, post-exposure vacci-
nation was administered to a group of 12
people, who had come into contact with
the three calves and the jackal (Fig. 3).
DISCUSSION
Rabies is endemic in Israel and since
2004 stray dogs were found to be the
main reservoir and transmitter (7, 10).
However the ORV program of wildlife
eliminated the fox rabies variants V1 to
V4 from almost all rural areas in Israel.
Nevertheless, there is still incursion of
fox genetic variants V1 across the borders
with neighboring countries. Te genetic
variant V1 caused mortality of 11 cattle,
2 sheep and 2 horses during the last 12
year on the border but was never detected
in the Yezre’el valley region (Fig. 3).
Based on molecular and epidemio-
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 David, D. 10
logical analysis the 4 calves reported here were most likely
bitten by the rabid Jackal. We therefore assumed that the
calves were infected by the rabid jackal that had entered
Kibbutz Ortal. Tis notion is supported by the chain of events
described above and the identical molecular fndings in the
infected calves and Jackal.
Te incubation period of rabies virus in cattle varies from
20 to 165 days (23). In our case the period of incubation was
between 1 to 3.5 months. Various sanitary measures were
implemented in Ortal and two farms in Emek Yezre’el as a
result of this case: quarantine of cattle at the Ortal dairy farm
and quarantine and vaccination of all the calves at the two
farms in Emek Yezre’el.
Vaccination of cattle is not mandatory in Israel however,
this case illustrates the importance of the current recommen-
dation of the Israeli Veterinary Services and Animal Health
to vaccinate cattle in rabies areas, where a high risk exists of
rabies-infected wildlife penetrating its borders.
ACKNOWLEDGEMENTS
Te authors thank the veterinary ofcer of Afula district, for his
help. Te authors are indebted Dr. Abed Hamaysi the for providing
information about the cases.
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Figure 3: Map of Israel showing the area of northern Israel where 32
rabies virus belonging to the VI genetic variants were isolated during
2002 through 2012 on the borders with neighbors countries; the three
positive calves isolated in Emek Yezre’el the Jackal and the fourth calf
in Kibbutz Ortal.
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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 11 Translocation of Rabies Virus by Cattle
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Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Krsmanović, M. 12
INTRODUCTION
Deviation from the normal values of the inorganic blood
parameter status of cows in early lactation, as well as their
defciency in the diet can lead to subclinical or clinical
manifestations (puerperal paresis, tetany), which have a
negative impact on health and fertility (1, 2, 3, 4, 5). Late
pregnancy and the beginning of lactation present a burden
for the cow’s physiology, as it has increased demands in
calcium, which is necessary for the construction of the fetal
skeleton. Phosphorous represents the second most important
macro-element for bone tissue production. In addition, it
participation in the cellular process of phosphorylation and
energy production, afects acid-base balance and plays a role
in the detoxifcation process (6, 7).
Determination of calcium, magnesium and inorganic
phosphorus in the blood serum is of important diagnostic
value in preventing puerperal paresis and other diseases (8,
9). However, their values can difer in cattle for many rea-
sons, among which is the breed (10). Te values of the above
mentioned macro-minerals in Simmental cows particularly
at diferent production stages are lacking in the literature.
Tis study was aimed at determining and comparing levels
of serum calcium, inorganic phosphorus and magnesium pre-
parturienly, peri-parturiently and during the peak of lactation
in Simmental dairy cattle.
MATERIALS AND METHODS
Animal selection and study design
Te study was conducted in a dairy cattle farm consisted of
150 Simmental cows. In total, 45 clinically healthy cows of
3 diferent productive stages were selected. Te animals of
the 1
st
group (15 cows - group A) were close-up cows, at the
Determination of Macroelement Parameters in Diferent
Productive Stages of Simmental Cows
Krsmanović, M.,
1
Djoković, R.,
1
Giadinis, N.D.,
2
Panousis, N.,
2
Bojkovski, D.,
3
Savić-Stevanović, V.,
4
Vasić, A.,
4
Zdravković, N.,
4
Korica, S.
5
and Bojkovski, J.
4
*
1
Agricultural Faculty, University of Kragujevac, Cara Dušana 34, 32000 Čačak, Serbia.
2
Faculty of Veterinary Medicine, Aristotle University of Tessaloniki, University Campus, 54124 Tesaloniki, Macedonia, Greece.
3
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
4
Faculty of Veterinary Medicine, University of Belgrade, Bul.Oslobodjenja 18, 11000 Belgrade, Serbia.
5
University Union, Faculty for Ecology and Environmental Protection, Cara Dušana 62-64, 11000 Belgrade, Serbia.
*
Corresponding Author: Jovan Bojkovski, DVM. PhD, Associate Professor, Department for Farm Animal Disease, Faculty of Veterinary Medicine,
University of Belgrade, Belgrade, 11.000 Belgrade, Serbia. Tel: +381113615436. Email:bojkovski@vet.bg.ac.rs
ABSTRACT
Te results of inorganic blood parameters are presented in the periparturient period and during lactation of
Simmental dairy cows. Serum calcium and inorganic phosphorus in blood of dairy cows in puerperium were
signifcantly lower (p<0.05) compared to the values in the blood of dairy cows in advanced pregnancy and
during peak of lactation, probably indicating the increased use of these macro-elements by the Simmental
cows’ mammary gland at the early stages of lactation. When the cows were in the periparturient period
serum magnesium levels were signifcantly lower (p<0.05) compared to the values of dairy cows during their
maximal lactation probably indicating the increased use of magnesium by the Simmental dairy cow during
the periparturient period.
Keywords: Simmental cows; Blood Serum; Calcium; Phosphorus; Magnesium.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 13 Macroelement Parameters of Simmental Cows
last 15 days before the expected parturition. Te animals of
the 2nd group (15 cows - group B) were fresh cows 1 to 15
days after calving, while the 3rd group (15 cows - group C)
consisted of high-production cows, from the 90
th
to 100
th
day
of lactation. All the included cows were in their 3
rd
lactation
and their average milk production was 6,825± 305 liters per
lactation period.
Feeding and housing were in accordance with the in-
tended use of the animals. Diet composition was adjusted to
the requirements of cows during pregnancy and lactation. Te
feeding regime of the cows at diferent production stages is
presented in Table 1.
Cows were judged to be clinically healthy for the duration
of the study.
Table 1: Feeding cows before and after calving
Cows before the calving were
fed daily
Cows after calving and during
lactation consumed daily
3 kg alfalfa hay 1,800 Kg alfalfa hay
Wheat straw 3 kg, 10 kg 15 kg of corn silage
(30% dry weight)
Corn silage (30% dry weight ) 8 kg of alfalfa silage
4 kg alfalfa haylage 4 kg of maze ear silage
(68% dry weight)
2 kg maize ear silage
(68% dry weight)
2 kg of dry sugar beet pulp
0.5 kg dry sugar beet pulp 2 kg exstruded soybeans
1.5 kg of additional mixtures
(30% of total protein)
4.5 kg additional mixtures
(30% of total protein)
Blood sampling
Blood sampling of all the tested cows was conducted once,
by jugular veni-puncture into 20 ml sterile vacuum glass
tubes. All samples were drawn between 10:00-12:00 a.m. Te
samples were placed in ice and were forwarded to the labora-
tory within an hour; sera were separated by centrifugation at
2,500 × g for 15 min. Tereafter the sera were placed in plas-
tic tubes (1.5 ml) (Eppendorf, Germany) and stored at -20°C
until analysed. Te concentration of calcium and magnesium
was determined by atomic absorption spectrophotometry
(AAS), and inorganic phosphorus by a spectrophotometrical
assay kit (Inorganic Phosphorus Assay Kit, Bioo Scientifc
Corporation, USA) using the spectrophotometer, UV-1800
(Shimadzu, Japan). Serum levels of calcium, magnesium and
phosphorus in sera were determined by the standard method
of by atomic absorption spectrophotometry (Serbian SRPS
ISO 6869 2002 using UNICAM 969, USA).
Statistical analysis
Te ANOVA procedure followed by LSD post hoc was used to
analyze the diference between analyzed groups of Simmental
cows. Te diferences with p-values of p<0.05 were considered
signifcant (95% signifcance). Statistical analysis was carried
out by Ver.5.0 Microsoft Statistical Stat.Soft.Inc.1995.
Ethical compliance
Statement of ethical compliance: Te experiment was
done in compliance with Serbian Law on Animal Welfare
(Ofcial Gazette of the Republic of Serbia No 41/09) and
Ordinance on the conditions for registration for experimental
animals and the keeping of such register, training programs
on welfare on experimental animals, request forms for ap-
proval of conducting experiments on animals, standing,
treatment and killing experimental animals and reproduc-
tion, circulation, or implementation experiments on animals
(Ofcial Gazette of the Republic of Serbia No 39/10).
RESULTS
Results of the concentration of inorganic phosphorus in
serum of tested Simmental cows are shown in Table 2.
Signifcantly higher (p<0.05) levels of phosphorus were in
Table 2: Serum inorganic phosphorus concentrations (mmol/l) from
all the sampled cows (groups A, B and C)
Productive stage Group N X SD CV P<0.05
Close-up dry cows A 15 2.09 0.43 32.55
A:C
B:C
Fresh cows B 15 1.97 0.39 30.98
High-production cows C 15 2.40 0.38 18.93
Table 3: Serum magnesium concentration (mmol/l) from all the
sampled cows (groups A, B and C)
Productive stage Group N X SD CV P<0.05
Close-up dry cows A 15 1.02 0.26 43.25
A:C Fresh cows B 15 1.09 0.32 44.76
High-production cows C 15 1.28 0.20 29.23
Table 4: Serum calcium concentrations (mmol/l) from all the
sampled cows (groups A, B and C)
Productive stage Group N X SD CV P<0.05
Close-up dry cows A 15 2.35 0.24
21.98
B:C Fresh cows B 15 2.17 0.22
23.56
High-production cows C 15 2.53 0.26 19.23
N = Number of cows
X = Mean
SD = Standard deviation
CV = Coefcient of variance
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Krsmanović, M. 14
group C compared to group B. Te mean serum phosphorus
concentration of close-up dry cows of group A was signif-
cantly higher (p<0.05) compared to fresh cows of group B.
Variations in serum inorganic phosphorus concentration was
not high within tested groups; the most pronounced vari-
ability was recorded in the fresh cows group (CV = 32.55%).
Results of serum magnesium concentration are shown
in Table 3.Te highest mean magnesium concentration
was found in high-production lactating Simmental cows
(1.28±0.20 mmol/l). Mean magnesium values were sig-
nifcantly higher (p<0.05) in high-production lactating
cows (group C) compared to close-up dry cows (group A).
Magnesium levels in the blood of tested cows had relatively
high variations within tested groups: in range from 0.20 to
0.32 mmol/l and the coefcient of variation varied from
29.30% to 44.76%.
Results of serum calcium concentrations are shown in
Table 4. Mean calcium concentration of fresh cows (group
B) was signifcantly lower in comparison with those of
high-production cows of group C (p<0.05). Te variability
of serum calcium concentrations among the three groups of
cows was not high, being most pronounced in the fresh cows
(CV=23.56%).
DISCUSSION
Lactating dairy cows use large quantities of calcium for
milk synthesis (11). Hypocalcemia in intensively managed
dairy cows causes periparturient paresis and contributes
to “downer cow syndrome”, while during lactation it can
also cause fertility disorders (2,6). Te results of the pres-
ent study indicate that in a group of Simmental dairy cows
in puerperium, lowest calcium levels were found, and these
values were signifcantly lower than the level of calcium in
the blood in Simmental cows in advanced pregnancy and
during maximum lactation. Tese fndings are similar to the
fndings of other researchers, who suggest that a sudden loss
of calcium from the body occurs in the puerperal cows, as for
every liter of colostrum 1.0-2.0 g of calcium is consumed (7),
while a physiological decline of calcium in the blood is held
steady for several days and on account of intensive produc-
tion, serum calcium decrease is correlated with the appear-
ance of puerperal paresis (7). Calcium metabolism is closely
related with phosphorus metabolism and is responsible for
the proper metabolism of vitamin D (12, 13).
Inorganic phosphorus levels in the blood of cows are at
low concentrations in the range of 2.1-3.0 mmol/l, while
signifcantly more phosphorus is bound to organic com-
pounds such as phospholipids (14, 15, 16). Similarly to serum
calcium, inorganic phosphorus values were signifcantly lower
in Simmental cows in puerperium compared to the values
of inorganic phosphorus in the blood of cows in advanced
pregnancy and during maximal lactation. Grünbergand co-
workers (17) found that hypophosphatemia is present in the
blood of highly productive Holstein-Friesian cows through-
out the postpartum period which is associated with the entry
of glucose in the glycolytic path of peripheral tissues, as well
as to supply the necessary quantities of mammary gland
phosphorus. Te authors believe that hypophosphatemia
allows proper glucose metabolism which prevents ketosis im-
mediately after calving (6, 18). Te sudden loss of phosphorus
from the body occurs in the puerperal cows when 0.8-1.9 g
phosphorus per liter of colostrum is spent. Tis physiological
decline of phosphorus in the blood is held steady for several
days and the intensity of phosphate fall is correlated with
previous fndings that phosphate blood level falls in older
animals (19).Te results of these tests show a decrease in
serum calcium and inorganic phosphorus in the blood of
Simmental cows in early lactation, suggesting increased use
of these macroelements by the mammary glands. Our results
show slightly higher calcium, phosphorus and magnesium
blood serum levels than in Jersey cows but similar to levels
of Holstein breed cows (12, 20), on the other hand calcium
and magnesium blood sera levels were found to be lower
than in beef cattle such as Angus, Charolais and Hereford,
or Limousin (10).
Magnesium, like calcium reduces neuro-muscular irrita-
bility and a drop in its concentration in the blood results in
spontaneous muscle contractions or tetany (1). Te results
of this study indicate that the lowest magnesium levels were
detected in Simmental cows in advanced pregnancy and that
greater values of magnesium in the blood were determined in
cows in puerperium. However the diferences of these values
were not signifcant. Te highest values of magnesium in the
blood were detected in high production Simmental cows
during the peak of lactation which were signifcantly higher
compared to the values of the cows in advanced pregnancy
and the puerperium. Te results obtained in this study in-
dicate that the magnesemia was within the normal range in
all tested cows, and lower values were found in a group of
cows in the periparturient period. Tis probably points to the
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 15 Macroelement Parameters of Simmental Cows
increased use of magnesium during negative energy balance
in early lactation.
CONCLUSION
Serum calcium and inorganic phosphorus in the blood of
Simmental dairy cows in puerperium were signifcantly
lower (p<0.05), compared to the values of cows in advanced
pregnancy and during maximum lactation, indicating the
increased use of these macro-elements by the mammary
gland of cows in early lactation. For the cows in the peri-
parturient period, values of magnesium in the blood were
signifcantly lower (p<0.05), compared to the values during
the periparturient period.
ACKNOWLEDGMENTS
Ministry of science, education and technological development of
the Republic of Serbia, projects number ID P 46002, TR 31071
and TR 31079.
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11. Tilsing, T., Larsen, T., Jorgensen, R.J. and Houe, H.: Te efect
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14. Arnaud, M.J.:Update on the assessment of magnesium status. Br.
J. Nutr.,99, supplement 3:S24-S36,2008.
15. Bojkovski, J., Borozan, S., Jozef, I. and Šamanc, H.: Colostrum
ingredients before and after calving in Holstein-Friesian Cows.
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17. Grunberg,W., Staufenbiel, R., Constable, PD., Dann,
H.M.,Morin, D.E. and DrackleyJ.K.:Liver phosphorus content
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19. Wagner, W. and Stoft, G.: Serum minerals, leucocyte, profles and
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Holstein Cows During Early Lactation, phD thesis, Virginia State
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Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gild, C. 16
INTRODUCTION
Parturient paresis is a metabolic disorder occurring close
to parturition especially in high producing dairy cows. Te
disease is characterized by a rapid decline in blood calcium
(Ca) concentrations. Nearly all mature cows experience some
degree of hypocalcemia during the frst day after calving as
the intestine and bone adapt to the Ca demands of lacta-
tion (1). In some cows, the mammary drain of Ca causes
extracelluar and blood Ca concentrations to decline to levels
that disrupt neuromuscular function, resulting in the clinical
syndrome of “Milk Fever”. Tis Ca decline lasts in some cases
for several days postpartum (2).
Post-parturient hypocalcemia is divided into clinical
and subclincal forms (3, 4). Te literature indicates that the
clinical form is associated with an increase in post-parturient
diseases (5, 6, 7, 8). In large parts of the United States and
some European countries it is assumed that the subclinical
form is also related to post parturient diseases (5, 9, 10, 11)
and has a negative impact on proftability. Terefore, many
dairies use expensive feed additives to reduce the incidence
of this form (12, 13). Fatty acid metabolism might difer
between cows with subclinical hypoclacemia and their nor-
mocalcemic counterparts (14) however, there is conficting
evidence regarding the impact of subclinical blood calcium
levels on milk production, reproduction parameters and
post-parturient disorders (10, 14, 15). Most of the research
Te Inf luence of Subclinical Hypocalcemia on Production and
Reproduction Parameters in Israeli Dairy Herds
Gild, C.,
1
* Alpert, N.
2
and van Straten, M.
3
1
HaChaklait, Kfar Tavor Hashkedim, Israel.
2
HaChaklait, Kibbutz Degania, Israel.
3
HaChaklait, Moshav Klahim, Israel.
*
Corresponding Author: HaChaklait, Kfar Tavor Hashkedim 50, P.O. Box 484, 15241 Israel. Email: gild@hachaklait.co.il
ABSTRACT
A large percentage of mature dairy cows experience some degree of hypocalcemia during the frst days
post-calving. In some cases calcium concentrations decline to levels that disrupt neuromuscular function,
resulting in the clinical syndrome known as parturient paresis or milk fever. Post-parturient hypocalcemia
is divided into clinical and subclinical forms. It has been established that cows sufering from clinical milk
fever are susceptible to a variety of secondary conditions, however to the best of the authors’ knowledge there
has been no evaluation of the impact of the subclinical form on production and reproductive parameters.
Te objective of this study was to investigate the association between subclinical hypocalcemia and post-
parturient disorders, production and reproductive parameters in Israeli dairy herds. Blood results for corrected
calcium concentrations were analyzed from 634 mature cows from 5 farms. Te subclinical hypocalcemic
cows produced 3.2, 2.7 and 1.9 kg more milk in the frst three milk recordings than the normocalcemic cows.
Subclinical hypocalcmic cows did not show an increased risk for post-parturient diseases nor compromised
reproduction parameters in comparison to normocalcemic cows. It was concluded that there was no negative
impact of subclinical hypocalcemia on production and reproductive parameters in Israeli dairy cows after
parturition.
Keywords: Bovine; Milk Fever; Calcium; Hypocalcemia; Milk Production.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 17 Subclinical Hypocalcemia in Dairy Cows
published targets methods to improve calcium homeostasis
through manipulations of dietary cationic anionic difer-
ence or through calcium binders (16, 17, 18, 19) however to
the best knowledge of the authors’ the direct impact of the
subclinical form on production and reproductive parameters
has not been adequately investigated. Te objective of this
study to investigate whether the subclinical hypocalcemia
state has an infuence on post-parturient diseases, reproduc-
tive parameters and milk production parameters in Israeli
dairy herds.
MATERIALS AND METHODS
Animals and study design
Te study was comprised of two separate entities: Te frst
study, the preliminary study was conducted on a 60 cow dairy
farm in the northern part of Israel. Cows were housed in large
covered loose housing systems and fed dry cow total mixed
ration (TMR) pre-calving and a standard milking TMR
post-calving both manufactured by Givaat Yoav Feeding
Center located in Moshav Givaat Yoav, Israel. Blood was
drawn from the coccygeal vein of 11 mature Holstein cows
at a 4 hour interval from the beginning of frst stage of la-
bor and up to 12 hours postpartum. Blood was drawn once
more at 24 hours postpartum. Samples were immediately
centrifuged and serum was harvested and frozen at -20°C
for laboratory analysis at the Kimron Veterinary Institute,
Beit Dagan. According to these results a post-calving calcium
curve was prepared to determine the time of nadir levels of
serum calcium.
Te second study, the main study was conducted on 4
commercial Israeli dairy herds of 200-450 milking cows each
between June 2006 and July 2007. Blood was drawn from
633 mature Holstein cows between 8-20 hours postpartum
when serum Ca levels were expected to be the lowest based
on the results of the frst preliminary study. Samples were
immediately centrifuged and serum was harvested and frozen
at -20°C for further laboratory analysis.
All cows were either housed in free stalls or large covered
loose housing systems. All herds were fed dry cow TMR pre-
calving and a standard milking TMR post-calving. All farms
received their feed from diferent feed manufactures (Table 1).
In all herds cows were milked 3 times daily identifed
by ear tags and freeze marking in computer controlled
milking parlors. Te annual milk production was 10,000-
12,500 kg/cow. Te herds were within the practice area of
the “HaChaklait”, a mutual society for veterinary medicine
which provides a complete herd-health service. Visits are
conducted by a veterinarian to the farms during the study
period at least twice weekly.
All cows were examined after calving by trained veteri-
narians who diagnosed, treated and recorded all the peripar-
turient disease conditions. Cases of retained fetal membranes
(RFM) were defined as the presence of placental tissues 24
hours or more after calving as observed by trained farm em-
ployees or the attending veterinarian. Animals with observed
or suspected RFM were submitted for veterinary examination
on the next routine veterinarian visit (1-4 days postpartum).
Animals without a history or diagnosis of RFM were submit-
ted for examination between 6 and 9 days postpartum. At this
examination, body condition scoring (BCS) of all animals
was recorded and cows were comprehensively examined by
intravaginal palpation after thoroughly cleaning the perineal
area. Te diagnosis of clinical endometritis (CEM) was based
on the combined characteristics of vaginal discharge obtained
by manual examination of the vagina. Afected cows with
CEM had a watery or purulent, fetid vaginal discharge as
previously described (20).
All cows were examined for ketosis by placing a drop of
urine obtained with a sterile disposable plastic catheter on
a reagent strip (Ketostix, Bayer, Germany). Te color reac-
tion was compared to the standardized color chart after 15
seconds. Cows with urine aceto-acetate concentrations above
15 mg/dl were recorded as ketotic (3). Cows with lower than
expected milk production and poor appetite were examined
for displacement of the abomasum (LDA) by auscultation
and percussion. BCS was further recorded approximately
40-60 days after calving and before the dry-of period. All
animals not observed in estrus by the end of the voluntary
waiting period at approximately 60 days postpartum were
recorded and submitted for examination.
Table 1: Farm name and feed manufacture
Farm Feed manufacturer
Kibbutz Geshur
Givaat Yoav Feeding Center, Moshav Givaat
Yoav, Israel
Kibbutz Beit Zera
Amabar Feeding Center, Moshav Kefar
Yehezkel, Israel
Kibbutz Givaat
Haim Meuhad
Amatz Feeding Center, Moshav Amatz, Israel
Kibbutz Afkim Kibbutz Afkim, Israel
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gild, C. 18
Clinical, reproductive, production and management
data were computer recorded by the herd manager and the
attending veterinarians. Cows not observed in estrus were
recorded for further reproduction calculation as cows not
showing heat. Once a month, each cow’s milk was sampled
and analyzed for fat, protein, lactose and somatic cell count
by the Central Laboratory for Milk Recording at the national
service for udder health and milk quality located in Caesarea
industrial park, Israel.
Reproductive management was solely based on artifcial
insemination performed by trained technicians employed by
“Sion” Israeli Company for artifcial insemination and breed-
ing, Migdal Ha’emek, Israel. In all herds, cows were mainly
inseminated on observed estrus or computerized pedometry
system. Conception rates and cumulative pregnancy were
based on pregnancy diagnosis performed by rectal palpation
of the uterus and its contents 40-50 days post-insemination.
Blood analysis
Total serum calcium levels were determined using Arsenazo
III method and calcium levels were corrected for serum al-
bumin which was determined using the Bromocresol Green
method. Correction was done using the following equation (3).
Corrected Calcium (mg/dl) = Measured Calcium (mg/dl) –
Albumin (g/dl) + 3.5
Statistical analysis
All data editing and analysis were performed using SAS
version 9.0 (21). Results were considered to be of statistical
signifcance if the relevant P-value was < 0.05.
In general, data analysis followed a 3 step approach: (1).
Descriptive statistics which included calculation of the mean,
standard deviation and histogram for continuous variables,
and frequency tables for other variables (2). Bivariate analysis
in which associations between a dependent variable and an
independent variable were assessed using the chi-square
test for categorical variables and t-tests when one of the
variables was on a continuous scale. For time to event data,
i.e. days from calving to conception, survivor functions were
compared using the Kaplan-Meier method and log-rank test
(3). Multivariable analysis in which associations between the
dependent variable and two or more independent variables
were assessed. Multivariable analysis was only performed
if, for the same dependent variable, two or more signifcant
associations were found in the bivariate analysis. In our case,
this only occurred in the analysis of milk production data.
For the latter, average test-day milk (kg) was estimated from
monthly test-day data using a linear model with a marginal
efect to account for repeated measurements from the same
cow. A maximum of 10 test-days was allowed per cow.
Lactation number was grouped into four categories, ie.
second, third, fourth and ffth, or greater lactation. Summer
months were considered June to September, inclusive.
Subclinical hypocalcemia was defned in a cow with a
corrected serum Ca level of < 7.5 mg/dl and without clini-
cal hypocalcemia 12-24 h postpartum. Somatic cell counts
(cells/mL) were grouped in 4 categories: ≤ 100,000; 101,000-
200,000; 201,000-400,000 and > 400,000. Farms were mod-
eled as a fxed efect and the correlation matrix used for R
was autoregressive. Te model we used was:
Y = farm (4 index variables) + summer (2 index variables)
+ lactation (4 index variables) + MIM (10 index variables) +
HCOR (2 index variables) + MIM + HCOR*MIM + SCCL
(4 index variables) + CEM (2 index variables) + e.
Y was test-day fat percentage, summer represents test
days occurring in the summer months, lactation was lacta-
tion group, MIM was month in milk, HCOR was sub-
clinical hypocalcemia, SCCL was somatic cell count level,
and “e” a complex error term representing the within-cow
correlation of test-day fat percentage and the residual error.
Signifcance of the fxed efects was determined using the
F-test (21).
RESULTS
Preliminary study
Corrected serum calcium levels obtained from all 11 cows
were combined into an average level for each 4 hour inter-
val. Tese average levels were plotted by time from calving.
Calcium levels decreased between calving and reached a nadir
at 8 hours-calving and stayed low until 20 hours post-calving.
Although not statistically signifcant, based on these results,
8-20 hours post-calving was chosen as the period of the nadir
of serum calcium levels for the main study. (Figure 1).
Main study
Data sets included measurements from 634 cows. 247, 186,
103 and 97 cows from second, third, fourth, ffth or greater
lactations, respectively. Tere was missing data on calving dis-
ease incidence for one cow and 16 cows sufered from clinical
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 19 Subclinical Hypocalcemia in Dairy Cows
milk fever. Terefore a total of 617 cows were included in the
fnal data set.
Of all cows 18.9% sufered from subclinical hypocalcae-
mia. Milk fever incidence was 7.63% and 1.36% for the sub-
clinical hypocalcemic and normocalcemic cows respectively.
Te probability for development of clinical milk fever was
found to be statistically higher in the subclincial hypocalce-
mic group as opposed to the normocalcemic group of cows.
(P < 0.0007) (Table 2).
Te probability for a cow developing subclinical hypocal-
cemia increased with lactation number (P < 0.0001) (Table 3).
Table 2: Milk Fever by Subclinical hypocalcaemia
Subclinical
Hypocalcemia
Milk Fever
Total
No Yes
No 508 7 515
% 98.64 1.36
Yes 109 9 118
% 92.37 7.63
Total 617 16 633
% 97.47 2.53 100
Table 3: Subclinical hypocalcaemia by lactation
Lactation
Number
Subclinical Hypocalcemia
Total
No Yes
2 235 12 247
% 95.14 4.86
3 164 22 186
% 88.17 11.83
4 63 40 103
% 61.17 38.83
>=5 53 44 97
% 54.64 45.36
Total 515 118 633
% 81.36 18.64 100
Calving diseases
Of the normocalcemic cows, 12% versus 11% of subclinical
hypocalcemic cows sufered from retained placenta. Tere was
no statistical diference between normocalcemic and subclini-
cal hypocalcemic cows (P < 0.897). Of the normocalcemic
27.6% cows versus 19.3% of subclinical normocalcemic
cows sufered from metritis however there was no statistical
diference in the probability of a subclinical hypocalcemic
cow sufering from metritis (P < 0.087). 21.3% of normo-
calcemic cows versus 25.7% of subclinical normocalcemic
cows sufered from ketosis however there was no statistical
diference in the probability of a subclinical hypocalcemic
cow sufering from ketosis (P < 0.376). Only 2 cows from
the normocalcemic group sufered from an LDA. 5.7% of
normocalcemic cows versus 3.7% of subclinical hypocalcemic
cows had stillbirths. Tere was no statistical diference in
the probability of a subclinical normocalcemic cow to have
stillbirths (P < 0.533).
Although subclinical milk fever (defned as corrected
serum calcium below 7.5 mg/dl) was associated with milk
fever incidence no association was found between serum
calcium and calving diseases.
Reproduction
Tirty-two cows received a “do not breed” code or were culled
before frst insemination and therefore 585 cows were in-
cluded in the reproduction analysis study.
Tere was no statistical diference (P < 0.755) between
groups for the frst artifcial insemination (AI) conception
rate. 29.7% vs. 27.6% for the normocalcemic and subclini-
cal hypocalcemic cows respectively. Tere was no statistical
diference (P < 0.453) between groups for cows not showing
heat. 33.7% vs. 38.1% for the normocalcemic and subclini-
cal hypocalcemic cows respectively. Tere was no statistical
diference (P < 0.958) in the cumulative conception until 180
days in milk between both groups (Figure 2).
No associations were found between subclinical milk
fever (defned as corrected calcium below 7.5 mg/dl) and
reproduction parameters.
Milk production
Subclinical hypocalcemic cows produced signifcantly more
milk when compared with normoclacemic cows. 3.17, 2.71
and 1.90 kg more milk was produced on the frst, second and
third test days, respectively (Table 4).
Figure 1: Corrected serum calcium (mg/dl) by time from
calving (hours)
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gild, C. 20
Table 4: Milk production by calcium group.
Test day Subclinical
Hypocalcemia
Estimate
Kg
Standard
error
P-value
1
st
test day No -3.1673 1.1607 0.0064
1
st
test day Yes 0
2
nd
test day No -2.7079 1.1508 0.0187
2
nd
test day Yes 0
3
rd
test day No -1.8951 1.1368 0.0956
3
rd
test day Yes 0
Subclinical hypocalcemic cows produced more milk in
the frst 6 milk test days (Figure 3).
DISCUSSION AND CONCLUSIONS
Many physiological pathways are dependent on blood ionized
calcium levels. Decreased ionized calcium levels have been
found to be associated with increased fat mobilization around
calving (14) and these decreased levels could presumably
infuence the gastrointestinal track motility (22) leading to
decreased feed intake and as a result an increased prevalence
of metabolic disorders and other post-parturient diseases. It
has also been shown that decreased levels of calcium stores
in peripheral blood mononuclear cells precedes measurable
hypocalcemia and that hypocalcemia at parturition further
exacerbates the ability of these cells to release intracellular
calcium in response to intracellular signals therefore impair-
ing these cells’ ability to be activated (8). Tese changes col-
lectively could probably contribute to the impaired immune
system of the periparturient cow and its increased susceptibil-
ity to infectious diseases (5, 6, 7, 8).
It would be reasonable to assume that these cows would
show impaired reproductive performance and decreased
milk production later in their lactation. However, our re-
sults demonstrate that in the Israeli high producing cow
this subclinical hypocalcemia is not a risk factor for the
development of postpartum diseases, furthermore we have
demonstrated that subclinical hypocalcemic cows produce
more milk. Tese results agree with previous studies which
showed that hypocalcemia at calving is not a signifcant
risk factor for decreased milk yield (14, 23). Our results of
higher milk production for the hypoclacemic cows could
be related to a higher genetic merit causing these cows to
produce more milk and subsequently exhibit lower circulating
calcium levels. On the other hand it must be pointed out that
these milk parameters do not rule out the possible damage
caused by these low circulating blood calcium levels. It has
been demonstrated that lame cows produce more milk in
comparison to their non-lame herd mates (25, 26). Despite
these fndings it is clear that lameness is a risk factor for
decreased milk production. When comparing the impact of
these two diseases it could be that the hypocalcemic cows in
our study, although higher in milk than their normoclacemic
herd mates could have produced even more milk had we cor-
rected their circulating blood calcium levels. Furthermore, we
did not analyze the quality of milk and whether calcium level
had any efect on the immunoglobulins or other parameters
of milk quality.
Our study had several limitations. First, due to the num-
ber of cows in the frst study we made a subjective decision on
the time of sampling which could have infuenced the number
of cows being defned as subclinical hypocalcemic. Second,
we could not measure ionized calcium and therefore had to
Figure 2: Cumulative conception until 180 days in milk Figure 3: Milk production (Kg) by month
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 21 Subclinical Hypocalcemia in Dairy Cows
use the correction equation according to albumin levels. It
is possible that this calculation of corrected calcium resulted
in an underestimation of the true level of ionized calcium
in the blood and that some cows in the subclinical calcium
group should have actually been included as normocalcemic
cows. On the other hand our study was done on several farms
feeding from diferent rations, feeds and feed suppliers which
gave substantial power to our results.
Taking into consideration other research done in this
feld, our results should be interpreted with caution and
ideally should be validated by future large scale studies and
further research.
In conclusion, subclinical hypoclacemia in the Israeli
dairy herd does not seem to impair production and repro-
duction parameters. Terefore the authors do not fnd it
reasonable or necessary to incorporate feed additives that
reduce the incidence of this disorder as long as milk fever
incidence remains in the normal range for Israeli dairy herds.
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15. LeBlanc, S.J., Leslie, K.E. and Dufeld, T.D.: Metabolic predictors of
displaced abomasum in dairy cattle. J. Dairy Sci. 88:159-170, 2005.
16. Tilsing-Hansen, T. and Jørgensen, R. J.: Prevention of Parturient
Paresis and Subclinical Hypocalcemia in Dairy Cows by Zeolite
Administration in the Dry Period. J. Dairy Sci. 84:691-693, 2001.
17. National Research Council: Nutrient Requirements of Dairy
Cows.7
th
rev. ed. Natl. Acad. Sci., Washington, DC, 2001.
18. Roche, J. R., Morton, J. and Kolver, E. S.: Sulfur and chlorine play a
non- acid base role in periparturient calcium homeostasis. J. Dairy
Sci. 85:3444-3453, 2002.
19. Gof, J. P.: Te monitoring, prevention, and treatment of milk fe-
ver and subclinical hypocalcemia in dairy cows. Te Vet. Journal
176:50-57, 2008.
20. Sheldon, I.M., Lewis, G.S., LeBlanc, S.J. and Gilbert, R.O.:
Defning postpartum uterine disease in cattle. Teriogenology.
65:1516-1530, 2006.
21. SAS Institute User’s Guide Version 9.3: Statistics. SAS Institute,
Cary, NC. 2006.
22. Hansen, S. S., Norgaard, P., Pedersen, C., Jorgensen, R. J., Mellau,
L. S. B., and Enemark, J. D.: Te efect of subclinical hypocalcae-
mia induced by Na2EDTA on the feed intake and chewing activity
of dairy cows. Vet. Res. Communic. 27:193-205, 2003.
23. Østergaard, S. and Larsen, T.: Associations between Blood Cal-
cium Status at Calving and Milk Yield in Dairy cows. J. Dairy
Sci. 83:2438-2440, 2000.
24. Carrier, J., Stewart, S., Godden, S., Fetrow, J. and Rapnicki, P.:
Evaluation and Use of Tree Cow side Tests for Detection of
Subclinical Ketosis in Early Postpartum Cows. J. Dairy Sci.
87:3725-3735, 2004.
25. Green, L.E., Hedges, V.J., Schukken, Y.H., Blowey, R.W. and
Packington, A.J.: Te Impact of Clinical Lameness on the Milk
Yield of Dairy Cows. J. Dairy Sci. 85:2250-2256, 2002.
26. Bicalho, R.C., Warnick, L.D. and Guard, C.L.: Strategies to Ana-
lyze Milk Losses Caused by Diseases with Potential Incidence
throughout the Lactation: A Lameness Example. J. Dairy Sci.
91:2653-2661, 2008.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Goshen, T. 22
INTRODUCTION
Reproductive efcacy is a key element in dairy farm manage-
ment. A major component in reproductive management is
estrus detection and artifcial insemination (AI) of the cow at
the correct time in the estrus cycle. In Israel, the vast majority
of large dairy farms rely mainly on electronic estrus detection
by pedometry or activity meters (1, 2). Recently, automated
estrus detection aids have become more available and ad-
opted worldwide (3-5). In farms without automated estrus
detection, timed AI (TAI) has become widely accepted as a
reproductive management tool. Te Ovsynch protocol, frst
described by Pursley et al. in 1995 (6), is most widely used
either on its own or in combination with pre-synchronization
(7-10) or post insemination protocols (11, 12).Te Ovsynch
program consists of 2 injections of Gondotropin-releasing
hormone (GnRH), 7 days before, and 48 hours after an injec-
tion of Prostoglandin F2 alpha (PGF
2α). Cows are insemi-
nated 16 to 25 hours after the second injection of GnRH (6).
Te advantages of Ovsynch include the lack of need for estrus
detection by visual or by expensive electronic means, high
insemination rates, reduced losses due to trauma or mastitis
that are estrus related, and fexibility in the number of cows
to be synchronized. Additionally, AI at a fxed times allows
for on farm concentrated work efort for hormone injections,
for AI in a predicted time, and Ovsynch was also described
as a treatment for acyclic cows (13-15). It has been reported
that cows inseminated using the Ovsynch protocol conceived
in comparable rates to the conception rates (CR) of cows
Reproductive Performance in Dairy Cows Synchronized with the
Ovsynch Protocol at Diferent Stages of the Estrus Cycle
Goshen, T.,
1, 2
* Tsitrin, K.
1
and van Straten, M.
1, 2
1
Te Koret School of Veterinary Medicine, the Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
2
“HaChaklait” Veterinary Services, P.O. Box 3039 Caesarea Industrial Park 38900, Israel.
*
Corresponding Author: Dr. Tamir Goshen, Te Koret School of Veterinary Medicine, Te Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100,
Israel. Tel: +972 8 9489730. Email: goshen@agri.huji.acl.il
ABSTRACT
Te objectives of the study were to evaluate reproductive performance in cows synchronized with the Ovsynch
protocol at diferent stages of the estrus cycle as detected by pedometry, and to compare the reproductive
performance of cows inseminated by Ovsynch in diferent stages of the estrus cycle (no observed estrus, 0-4,
5-12, 13-17 and 18-24 days after estrus) to cows inseminated by pedometry in Israel. Timing the beginning
of Ovsynch protocol did not afect frst artifcial insemination (AI) conception rate, proportion of cows empty
at 150 days in milk (DIM) or the time from calving to conception. Cows without observed estrus 25 days or
more before initiation of the Ovsynch protocol had rate of cows not pregnant by 150 DIM in comparison to
the control group (Odds Ration (OR) = 1.885, P = 0.013) and longer calving to conception interval than the
control group (155.4 days and 135.7 respectively, P = 0.018). It was concluded that reproductive performance
cannot be improved by combination of pedometry and timed artifcial insemination (TAI); Ovsynch has
low efcacy as a treatment for anestrus. However, other anestrus treatment options should be compared to
Ovsynch.
Keywords: Bovine; Reproduction; Ovsynch; Anestrus; Estrus Detection; Reproduction; Timed
Artifcial Insemination; Pedometry; Ovsynch; Dairy; Anestrus.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 23 Ovsynch Protocol at Diferent Stages of the Estrus Cycle
detected in estrus, but with higher pregnancy rates (PR) due
to the higher insemination rates of the Ovsynch treated cows
(1, 6, 10, 16-18).. Te best conception rates of synchronized
insemination are achieved when the Ovsynch protocol is
initiated at days 5-12 of the estrus cycle. Pre-synchronization
protocols evolved to improve conception rates with the aim
of the most commonly used pre-synchronization protocol,
Presynch, to achieve higher proportions of cows that are 5-12
days post estrus at the beginning of the Ovsynch protocol.
Tis is done by injecting PGF
2α twice 14 days apart and ini-
tiation of the Ovsynch protocol 11-14 days after the second
PGF
2α treatment, thus contributing to higher conception
rates at TAI (7, 9).
Te extensive use of estrus detection systems in Israeli
dairy farms makes the sweeping use of TAI protocols redun-
dant. However, cows not detected in heat or repeat breeders
are synchronized in some farms. Te intelligent use of TAI
in cows that are detected in estrus and are not inseminated
due to technical reasons (religious considerations – Saturdays
and holidays, or other reasons) according to the known estrus
date may yield better reproductive performance. Knowledge
of the estrus date (recorded by pedometry) could be used as
a substitute to the Presynch protocol in order to time the
beginning of the Ovsynch protocol to days 5-12 in the estrus
cycle.
Te objectives of this study were to compare conception
rates between cows treated with Ovsynch protocols initiated
at diferent periods of the estrus cycle detected by pedometry
and to compare these to conception rates in cows inseminated
by pedometry alone. Finally, we attempted to evaluate the
efcacy of Ovsynch as a treatment for anestrus.
MATERIALS AND METHODS
Study design and animals
Te study was designed as a retrospective cohort study in
a sample of cows that were inseminated for the frst time
in lactation between January 2006 and December 2007.
Te study was conducted in two commercial Israeli dairy
herds consisting of 450 and 550 Israeli Holstein cows. Cows
were housed in loose housing systems in large, completely
covered open sheds and fed total mixed ration (TMR)
ad libitum. In both herds, cows were milked three times
daily in computer controlled milking parlors. Mean 305
day milk production was approximately 11,500 kg per cow.
All cows were identifed by ear tags, electronic identifca-
tion tags (SAE, Afkim, Israel) and freeze marking. Te
herds were within the practice area of the Ambulatory
Clinic of Te Koret School of Veterinary Medicine, which
provided a complete herd-health service. Both herds were
visited at least three times weekly during the trial period.
Clinical, reproduction, production and management data
were recorded to the farm management software (“NOA”,
Israeli Cattle Breeders Association). Both farms performed
monthly milk recordings through the Central Laboratory
for Milk Recording. Reproductive management was solely
based on AI performed by highly trained technicians em-
ployed by the Artifcial Insemination Service of the Israel
Cattle Breeders Association (ICBA). In both herds, cows
were equipped with computerized pedometry systems
(SAE, Afkim, Israel). Pregnancy diagnosis was performed
by transrectal palpation of the uterus and its content 45-51
days post insemination.
Clinical Examination
All cows were examined routinely between 5 and 12 days
after calving by trained veterinarians, who diagnosed, treated
and recorded periparturient diseases as described previously
(19).
Body condition scoring (BCS scale: 1 = thin, 5 = obese)
was recorded before dry-of, at the routine post-partum
examination (5-12 days after calving) and between 40 to 60
days after calving (peak yield BCS).
Ovsynch and treatment groups
Te Ovsynch group (OS) included cows treated with the
Ovsynch protocol. Hormonal synchronization was used on
the farms as part of a study evaluating the efcacy of Ovsynch
in comparison to pedometry, or for re-synchronization of
animals detected in heat during Saturdays or holydays (the
farms did not inseminate cows during Saturdays for religious
reasons). OS Cows were treated with IM injection of 150
µg Gonadorelin Acetate (Gonabreed, Parnell Laboratories,
New Zealand) followed seven days later by IM injection of
500 µg Cloprostenol Sodium (Estrumate, Schering-Plough,
Germany), followed two days later with another IM injection
of 150 µg Gonadorelin Acetate. OS cows were insemination
16-24 hours after the last GnRH injection regardless of heat
signs.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Goshen, T. 24
Cows from the OS group were individually matched
to the control cows by farm, calving year and parity. When
possible, the calving season, calving diseases and calving to
frst AI interval (voluntary waiting period) of the matched
control cow were also similar.
In the second part of the study the cows were divided
into six groups:
1. Control (CO) – cows inseminated at the detected
heat.
2. Anestrus (AN) – cows inseminated after treatment
with Ovsynch protocol with no detected estrus in the previ-
ous 24 days.
3. Ovsynch at days 0-4 (OS4) – cows inseminated after
treatment with Ovsynch protocol 0-4 days after detected
estrus.
4. Ovsynch at days 5-12 (OS12) – cows inseminated
after treatment with Ovsynch protocol 5-12 days after de-
tected estrus.
5. Ovsynch at days 13-17 (OS17) – cows inseminated
after treatment with Ovsynch protocol 13-17 days after
detected estrus.
6. Ovsynch at days 18-24 (OS24) – cows inseminated
after treatment with Ovsynch protocol 18-24 days after
detected estrus.
Statistical analysis
Computerized data were retrieved from the farm comput-
ers and ICBA central computer and analyzed using Excel
(version 2010, Microsoft, Redmond, WA) and SPSS 17.0.1
(SPSS Inc., Chicago IL, USA). Lactation Incidence Risk
(LIR) for all recorded diseases and reproductive performance
parameters were evaluated for Control and Ovsynch cows.
Te same evaluation was done for cows in the diferent
Ovsynch timing groups.
Reproductive performance parameters included frst arti-
fcial insemination conception rate (1
st
AI CR), the interval
from calving to pregnancy (Days Open), time from 1
st
AI
to conception (Waste days) and the proportion of cows not
pregnant > 150 days after calving (Empty at 150 DIM ie.
Days in Milk = Days from Calving).
Te efect of Ovsynch on dichotomous outcome variables
was evaluated using conditional multivariable logistic regres-
sion modeling.
Separate analysis was done for Ovsynch versus Control
cow and for Ovsynch timing groups as an independent vari-
able. Various parameters and disease conditions were tested
in the models as possible confounders. Tese included: farm,
calving season (May to September = Summer; October to
April = Winter), twin calving, retained placenta (RP, retained
fetal membranes ≥ 24 hrs. post calving), clinical metritis
(CM), ketosis, displaced abomasum, body condition loss
≥0.5 between calving and peak milk yield, mastitis at calving,
mammary edema at calving, stillborn calves and induction of
calving. Crude bivariate associations of outcome and potential
confounding variables with treatment were initially assesses
by use of Pearson χ
2
asymptotic 2-sided tests of signifcance
for reproductive dichotomous parameters and student’s T test
for waiting period and days empty. To build the models, the
Control versus Ovsynch timing group variables were forced
into the model and subsequent covariates with signifcance
of P ≤ 0.25 were included in primary multivariate models. Te
fnal models were built with entry criteria set at P < 0.05 and
exit criteria set at P > 0.10. For all analysis, values of P < 0.05
were considered signifcant. Te Odds Ratios (OR) were
calculated for all variables in the model.
Te conditional logistic multivariate analysis was done
using the Cox regression procedure for dichotomous out-
come variables with pairing as strata, and linear regression
procedure was used for scale outcome variables. Te efects of
Ovsynch treatment and its timing in the estrus cycle on days
open were evaluated using Kaplan-Meier survival analysis.
As the segmentation of the waste days is not equal, there is
an association between specifc period of times and elevated
risk for conception. Also the risk of conception is higher on
the frst AI and declining later. To compensate for these facts,
the efect of the treatment was analyzed using a Proportional
Odds model as proposed by Cox.
For all survival analyses, days open were limited to 200
days, cows not pregnant at 200 DIM were considered as empty.
RESULTS
Descriptive statistics
During the study period 267 cows were treated by Ovsynch
in the study farms. Control cows were matched and difered
from the Ovsynch cows only in waiting days (Table 1).
Reproductive performance
Te Ovsynch protocol and the timing of its beginning in the
estrus cycle did not afect signifcantly the 1
st
AI CR (Table 2).
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 25 Ovsynch Protocol at Diferent Stages of the Estrus Cycle
Te control cows Empty at 150 DIM rate was lower
than in the Ovsynch cows (36.7% and 44.9 respectively;
OR = 0.817, P = 0.0321, table 2). Te timing of the Ovsynch
protocol in the estrus cycle afected the probability of groups
1 and 5 to be Empty at 150 DIM.
Only group 1 (Anestrus group) difered from the other
Ovsynch groups. For further analysis of cows Empty at 150
DIM, the cyclic Ovsynch groups (3-6) were united. Te cyclic
status of the cows at the initiation of the Ovsynch protocol
afected signifcantly the Empty at 150 DIM rates. Cows in-
seminated by Ovsynch protocol without a detected estrous 24
days before the initiation of the protocol had higher Empty
at 150 DIM rate in comparison to the control group (OR
to be empty at 150 DIM = 1.885, P = 0.013, table 3). Cyclic
cows inseminated by Ovsynch protocol did not difer from
cows inseminated in pedometry detected estrus (Table 3).
Cows inseminated by Ovsynch had longer open peri-
ods than control cows (mean days open = 146.1 and 135.7
respectively, P = 0.017). Tis diference was mainly due to
the cyclic status of the cows. Cyclic cows inseminated by
the Ovsynch protocol did not difer from the control (mean
days empty = 141.2, Figure 1), while the anestrus cows had
signifcantly longer empty period (mean days empty = 155.4,
Figure 1).
Table 1: Descriptive statistic of the calving diseases LIR and waiting
period of the Ovsynch and Control cows.
Parameter Ovsynch Control Overall P value
Farm:
A 197 197
394
(73.8%)
1
B 70 70
140
(26.2%)
1
Parity :
1 95 95
130
(35.6%)
1
2 61 61
122
(22.8%)
1
3+ 111 111
222
(41.6%)
1
Summer Calving’s 37.1% 35.2% 36.1% 0.652
RP
1
13.9% 17.2% 15.5% 0.282
CM
2
37.1% 36.3% 36.7% 0.857
Ketosis 19.1% 16.5% 17.8% 0.428
Waiting period
3
100.7 95.9 98.3 0.007
1 – Retained placenta, retained fetal membranes ≥ 24 hrs.
2 – CM – Clinical/Puerperal metritis.
3 – Waiting period – calving to 1
st
service interval.
Table 2: Descriptive statistics of diferent reproductive parameters
in the control (Inseminated at estrus) and the diferent study groups.
Ovsynch Group*
Over
all**
Control 2 3 4 5 6
N 267 93 32 98 25 19 267
1
st
AI CR
1
39.7% 35.5% 46.9% 34.7% 40% 31.6% 36.7%
Empty >150
DIM
2
36.7% 54.8% 46.9% 36.7% 48% 31.6% 44.9%
Days empty
3
144.9 169.4 163.1 147.5 161.5 145 158.1
Waste days
4
27.1 28.9 33.6 23.5 30.5 24.1 27.3
* group 2 – Ovsynch protocol started ≥25 days from estrus or no estrus
before Ovsynch, group 3 – Ovsynch protocol started 0-4 days after
estrus, group 4 – Ovsynch protocol started 5-12 days after estrus,
group 5 – Ovsynch protocol started 13-17 days after estrus and group
6 – Ovsynch protocol started – 18-24 days after estrus.
** Over all – all Ovsynch cows.
1
1
st
AI CR – the proportion of cows conceived from 1
st
artifcial
insemination.
2
Empty >150 DIM – the proportion of cows not pregnant > 150 d after
calving.
3
Days empty – Mean calving to conception interval. Maximum value is
250 days.
4
Waste days – Mean 1
st
AI to conception interval.
Table 3: Logistic regression model summaries of the relationship
between the cyclic status of the cows when Ovsynch is initiated
and the Empty at 150 DIM in cows inseminated by pedometry or
by Ovsynch.
Variable β S.E. df Sig. O.R.
95% CI for O.R
Lower Higher
Group:
Control
2 0.047 Reference
Anestrus
Ovsynch
1
0.634 0.256 1 0.013 1.885 1.141 3.113
Cyclic
Ovsynch
2
0.170 0.209 1 0.416 1.185 .787 1.786
Parity :1 2 <0.001 Reference
2 -0.045 0.278 1 0.871 1.885 1.141 3.113
3+ 0.861 0.232 1 <0.001 1.185 0.787 1.786
Calving
Diseases
3
0.353 0.202 1 0.081 1.423 0.957 2.117
Long Waiting
period
4
1.073 0.222 1 <0.001 2.923 1.890 4.520
Constant -1.403 0.266 1 <0.001 0.246
1 – Cows inseminated after treatment with Ovsynch protocol with no
detected estrus in the previous 24 days.
2 – Groups 3-6 combined. Te cows had a detected estrus 24 days or less
prior to the beginning of the Ovsynch protocol.
3 – Retained placenta, clinical metritis and ketosis.
4 – Calving to 1
st
AI interval ≥108 days (higher quartile).
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Goshen, T. 26
Higher lactation numbers and lower body condition
scores at peak yield were associated with reduced pregnancy
rates. Post-partum endometritis was associated with reduced
pregnancy rates, but the efect declined as DIM increased.
Summer calving tended to reduce the pregnancy risk
(OR = 1.079, P = 0.0607). Te OR to become pregnant of
cows inseminated by Ovsynch at 0-4 or 13-17 days after
estrus was reduced (OR = 0.706, P < 0.0001 and OR = 0.881,
P < 0.0001 respectively).
DISCUSSION
Te novelty of this study is in combining the TAI with
automated estrus detection systems, and by comparing the
conception rate of cows inseminated by Ovsynch protocol
started at diferent times of the estrus cycle. Te main fnding
in this study was that the timing of the beginning of the
Ovsynch protocol in the estrus cycle had no efect on 1
st
AI
conception rate. Tis fnding was unexpected as Presynch
protocols are widely used for CR improvement (7, 8, 20,
21). Te purpose of the Presynch protocols is to lower the
proportion of anovular cows (22), and to time the beginning
of the Ovsynch protocol to the period of time where maximal
reproductive efcacy can be achieved at days 5-12 of the
estrus cycle (20, 21). Te start of the Ovsynch protocol at
this timing is supposed to ensure the ovulation of smaller
follicles at the timed AI, with higher CR (20). Modifcation
of the Ovsynch protocol by adding presynchronization had
improved pregnancy rates in previous studies (7, 9, 10, 23).
However, the results of this study did not demonstrate advan-
tage (as refected in the measured reproductive parameter) to
the starting of synchronization protocol at days 5-12 of the
estrus cycle or to any other diferent timing. Te benefcial
efects of presynchronization with prostaglandin might be
due to efects on pyometra and subclinical endometritis (24),
hence not present in this study.
Te anestrus cows that were treated by Ovsynch did not
difer signifcantly from the other study groups in 1
st
AI CR.
Te Anestrus cow group was actually composed of two dif-
ferent populations of cows: cyclic cows which the pedometry
system failed to detect in estrus and acyclic cows. In some
of previous studies, cows with anovular follicles had low CR
when inseminated by Ovsynch (13, 25). As the anestrus cows
group included the cyclic cows not detected by pedometry
system, this sub-population may confound the efect of the
acyclic cow population on 1
st
AI conception.
Tis confounding efect was limited to the 1
st
AI, as
anestrus cows treated by Ovsynch, had longer calving to
conception interval (Figure 1), a fact that can be explained
by the continues efect of the acyclic cows sub-population on
pregnancy rate of the anestrus cows. Te longer calving to
conception interval demonstrates a low efcacy of Ovsynch
as a treatment for ovarian inactivity as was documented be-
fore (13, 26). However, as the control group was composed
of cyclic cows detected in estrus by pedometry, the efcacy
of Ovsynch as a treatment for ovarian inactivity should be
compared to other treatment options.
Over all, cows inseminated based on Ovsynch or by
pedometry did not difer in the 1
st
AI conception rate. Tis
fnding is similar to fndings in previous studies in Israel (1)
and worldwide (5, 6, 10, 17), suggesting that Ovsynch can
replace pedometry in farms without pedometers or activity
meters, or in farms where management conditions limits
the efcacy of estrus detection by electronic aids. Recently,
Neves et al. (5) found that in north American conditions
automated estrus detection systems are at least equal to TAI
in the reproduction efcacy, despite the fact that this study
was conducted in free stall commercial dairy herds (as op-
posed to the loose shade Israeli system). Tey also found
Figure 1: Survival plot of pregnancy rate in Control cows inseminated
at estrus detected by pedometry (

), Cyclic cows inseminated by
Ovsynch protocol (
....
) and Anestrus cows inseminated by Ovsynch (—).
Te mean survival time of the Anestrus cows difered signifcantly from
the Control cows in the Log Rank test (P = 0.018, mean survival time
of 155.4 and 135.7 respectively). Te Cyclic cows did not difer from
the Control group (mean survival time of 141.2 and 135.7 respectively).
Days In Milk
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 27 Ovsynch Protocol at Diferent Stages of the Estrus Cycle
that herd level management may confound the efects of the
reproductive management system.
CONCLUSIONS
Timing Ovsynch with estrus detection did not improve the
reproductive performance of treated cows. In farms with con-
ditions allowing for high efcacy of estrus detection systems,
and where such systems are available, Ovsynch protocols may
be redundant as a reproduction management tool for all cows.
Te reproductive performance of anestrus cows treated
by Ovsynch is not comparable to the cyclic cows; however
the efcacy of Ovsynch as a treatment of anestrus should be
compared to other treatment options.
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12. Green, J.C., Newsom, E.M. and Lucy, M.C.: Incorporation of a
rapid pregnancy-associated glycoprotein ELISA into a CIDR-
Ovsynch resynchronization program for a 28 day re-insemination
interval. Teriogenology. 75:320-328, 2011.
13. Lopez-Gatius, F., Santolaria, P., Yaniz, J., Rutllant, J. and Lopez-
Bejar, M.: Persistent ovarian follicles in dairy cows: a therapeutic
approach. Teriogenology. 56:649-659, 2001.
14. Shephard, R.W.: A comparison of performance of the Ovsynch
treatment program between cycling and non-cycling cows within
seasonally-calving dairy herds. Aust.Vet. J. 83:751-757, 2005.
15. Cordoba, M.C. and Fricke, P.M.: Initiation of the breeding season
in a grazing-based dairy by synchronization of ovulation. J Dairy
Sci. 85:1752-1763, 2002.
16. Pursley, J.R., Kosorok, M.R. and Wiltbank, M.C.: Reproductive
management of lactating dairy cows using synchronization of
ovulation. J. Dairy Sci. 80:301-306, 1997.
17. Pursley, J.R., Wiltbank, M.C., Stevenson, J.S., Ottobre, J.S.,
Garverick, H.A. and Anderson, L.L.: Pregnancy rates per artifcial
insemination for cows and heifers inseminated at a synchronized
ovulation or synchronized estrus. J. Dairy Sci. 80: 295-300, 1997.
18. Miller, R.H., Norman, H.D., Kuhn, M.T., Clay, J.S. and Hutch-
ison, J.L.: Voluntary waiting period and adoption of synchronized
breeding in dairy herd improvement herds. J. Dairy Sci. 90:1594-
1606, 2007.
19. Goshen, T. and Shpigel, N.Y.: Evaluation of intrauterine antibiotic
treatment of clinical metritis and retained fetal membranes in dairy
cows. Teriogenology. 66:2210-2218, 2006.
20. Vasconcelos, J.L., Silcox, R.W., Rosa, G.J., Pursley, J.R. and Wilt-
bank, M.C.: Synchronization rate, size of the ovulatory follicle,
and pregnancy rate after synchronization of ovulation beginning
on diferent days of the estrous cycle in lactating dairy cows. Te-
riogenology. 52:1067-1078, 1999.
21. Moreira, F., de la Sota, R.L., Diaz, T. and Tatcher, W.W.: Efect
of day of the estrous cycle at the initiation of a timed artifcial
insemination protocol on reproductive responses in dairy heifers.
J. Anim. Sci. 78:1568-1576, 2000.
22. Stevenson, J.S., Pulley, S.L. and Mellieon, H.I., Jr.: Prostaglandin
F2alpha and gonadotropin-releasing hormone administration
improve progesterone status, luteal number, and proportion of
ovular and anovular dairy cows with corpora lutea before a timed
artifcial insemination program. J. Dairy Sci. 95:1831-1844, 2012.
23. El-Zarkouny, S.Z., Cartmill, J.A., Hensley, B.A. and Steven-
son, J.S.: Pregnancy in dairy cows after synchronized ovulation
regimens with or without presynchronization and progesterone.
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24. Galvao, K.N., Frajblat, M., Brittin, S.B., Butler, W.R., Guard, C.L.
and Gilbert, R.O.: Efect of prostaglandin F2 alpha on subclinical
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25. Bisinotto, R.S., Chebel, R.C. and Santos, J.E.: Follicular wave of
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26. De Rensis, F., Bottarelli, E., Battioni, F., Capelli, T., Techakum-
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Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Balkaya, I. 28
INTRODUCTION
Toxoplasma gondii is one of the most important zoonotic
protozoa that infect a variety of birds and mammals, includ-
ing humans. Felids are the defnitive hosts and many animal
species are the intermediate hosts. Among these intermediate
hosts, feral pigs are infected with T .gondii have been found
to be infected at diferent rates between 4.4 to 45.9% (1).
Antibodies to T. gondii have been reported in wild boars
from Austria, Brazil, Czech Republic, France, Germany, Italy,
Japan, Slovak Republic, Spain, United States (Georgia and
South Carolina) (1, 2). Te meat of wild boars with tissue
cysts of T. gondii may be an important source for human
toxoplasmosis when people consume these meats under-
cooked or without control (3).
Neospora caninum, an important parasitic disease of
cattle, causes reproductive failure, especially abortion (4).
Neosporosis is primarily a disease of cattle and dogs but
antibodies have been detected a series of species, including
domestic, wildlife and zoo animals (5). Dogs can acquire
infection by ingestion of infected tissues; and intermedi-
ate hosts can be infected either by horizontal, postnatal or
by vertical transmission (6). Due to the economic impact
of neosporosis, existing studies are concentrated on farm
animals and there is limited serological data about wild life
(4-6). Anti-N. caninum antibodies have been reported in wild
boars from Spain and the Czech Republic (7, 8).
For both T. gondii and N. caninum wild pigs may be
important indicators for following environmental contamina-
Detection of Toxoplasma gondii and Neospora caninum antibodies in
Wild Boars (Sus scrofa) in Eastern Turkey
Balkaya, I.,
1
* Utuk, A. E.,
2
Babur, C.,
3
Beyhan, Y. E.,
4
Piskin, F. C.
5
and Sozdutmaz, I.
6
1
Ataturk University, Faculty of Veterinary Medicine, Department of Parasitology, Erzurum, Turkey.
2
Cukurova University, Faculty of Veterinary Medicine, Department of Parasitology, Adana, Turkey.
3
Refk Saydam National Hygiene Center, Communicable Diseases Research Department, Parasitology Laboratory, Ankara, Turkey.
4
Yuzuncu Yil University, Faculty of Medicine, Department of Medical Parasitology, Van, Turkey.
5
Veterinary Control Central Research Institute, Parasitology and Bee Diseases Laboratory, Ankara, Turkey.
6
Erciyes University, Faculty of Veterinary Medicine, Department of Virology, Kayseri, Turkey.
*
Corresponding Author: Ibrahim Balkaya, Address: AtaturkUniversity, Faculty of Veterinary Medicine, Department of Parasitology, Erzurum, Turkey.
Tel: +90-0442-2315532. Email: balkayaibrahim@hotmail.com
ABSTRACT
Te aim of this study was to examine for the presence of anti-Toxoplasma gondii and Neospora caninum
antibodies in wild boars and to study the impact of infection between the sylvatic and domestic life cycles
of these apicomplexan parasites. For this purpose, sera were collected from hunter-killed wild boars (Sus
scrofa) during the winter period of 2011 from Erzurum province of Turkey. Collected sera were examined for
antibodies against T. gondii and N. caninum by Sabin Feldman dye test (SFDT) and competitive-enzyme-
linked immunosorbent assay (c-ELISA), respectively. Out of 12 collected samples, 4 (33.3%) of sera were
found to be seropositive at the dilution of 1:16 for T. gondii however no seropositivity in any of the samples
was detected against N. caninum. To the best knowledge of the authors, this is the frst serologic study to
detect anti-T. gondii antibodies in wild boars in Turkey.
Keywords: Wild Boar; Sus scrofa; Eastern Turkey; Toxoplasma gondii; Neospora caninum;
Sabin-Feldman; c-ELISA.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 29 Toxoplasma gondii in Wild Boars in Turkey
tion since they are omnivorous animals obtaining infection
from their local environment (9). Serologic tests (IFA, MAT,
LAT, DT and c-ELISA) are used to monitor anti-T. gondii
and N. caninum antibodies in wild pigs (1, 8, 9).
Te aim of this study was to investigate the presence of
anti-T. gondii and N. caninum antibodies in wild boars and to
examine the impact between the sylvatic and domestic life of
pigs in relation to these apicomplexan parasites.
MATERIAL AND METHODS
Sample collection
Blood samples were collected from 12 wild boars that were
provided by certifed hunters from Horasan and Pasinler
regions of Erzurum province in Turkey in the year 2011.
From information provided by hunters, the majority of these
wild boars were adult in the age range 1-3 years, and all of
them were female. Te blood samples were collected for
serological studies from the heart, following the death of
animals after shooting. Te blood samples were transferred
into vacuum tubes, allowed to clot and then centrifuged at
4000 rpm for 10 minutes at room temperature. Subsequently
they were placed into eppendorf tubes and stored at -20°C
until use.
Serologic examination
All sera were examined for T. gondii antibodies using the
SF dye test (SFDT) as described (10). Te examinations
were carried out at the National Reference Laboratory for
Parasitology, Public Health Institution of Turkey. Te pro-
cedure included two steps in preparation for performing the
test. Healthy 3-4 week old white Swiss albino mice were
injected with the virulent RH strain of T. gondii. T. gondii RH
antigen was collected from the peritoneal fuid of mice after
48 hrs post injection. As an activator serum, human serum
seronegative for T. gondii was used including factors such as
magnesium, properdin, C
2
, C
3
, C
4
. Alkaline methylene blue
dye was prepared with 9.73 ml of 0.53 % Na
2
CO
3
(Sigma,
Seelze, Germany), 0.27 ml of 1.91 % Na
2
B4O
7
.10H
2
O
(Merck, NJ, USA) and 25 mg of methylene blue (Difco,
Detroit, MI, USA). Following inactivation of complement
at 56°C for 30 minutes, 25 µl of test sera were prepared
with normal saline in dilutions of 1:4, 1:16, 1:64, 1:256 and
1:1024. Te antigen was added to the sera preparations of
25 µl activator serum at approximately 25 T. gondii tachyzo-
ites observed in a microscopic feld of 40X magnifcation,.
Te mixture was incubated in a water-bath at 37°C for 50
minutes. 25 µl of alkaline methylene blue was added to the
mixture and kept in 4°C for 10 minutes. Examination was
carried out using light microscopy with 40X objective to
gauge whether T. gondii tachyzoite were stained. If more
than 50 % of tachyzoites on one microscopic feld were not
stained, this dilution step was accepted as positive. Titers of
1:16 and greater were considered as positive (11). Positive and
negative controls, which were confrmed by IFAT method,
were included in the above procedure.
Serologic examination (c-ELISA)
Antibodies to N. caninum were detected by using a commer-
cially available competitive enzyme-linked immunosorbent
assay (c-ELISA) kit (VMRD, USA). Te test was done by
following the instructions of manufacturer. Te mean optical
density (OD) at 630 nm was determined for all wells using
a microplate reader (ELx 800 UV, Universal Microplate
Reader, Bio-Tek Instruments, Invc., Winooski, VT, USA).
Te percent inhibition for each test sample was determined
using the below mentioned formula:
Inhibition (%) = 100 - [( Sample O.D. X 100)
÷ (Mean Negative Control O.D.)]
Te samples with values of ≥ 30% inhibition were re-
garded as positive and those with the values < 30% inhibition
were regarded as negative (12).
RESULTS
Anti-T. gondii antibodies in SFDT test were detected in 4 of
12 (33.3%) wild boar sera to be seropositive at 1:16 dilution
and, no seropositive cases (0%) were detected for N. caninum
antibodies.
DISCUSSION
Te wild boar is the only member of the suidae family present
in Turkey and bioecological data about this animal are limited
(13). Except in touristic regions, pork meat consumption is
restricted in Turkey; however, wild boars are important game
species and have the potential to cause damage to agricultural
crops in Turkey (13). When the wild boar population in-
creases, hunting organizations are arranged to cull wild boars
and in so doing decrease the crop damage. Due to restricted
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Balkaya, I. 30
meat consumption, hunted animals are often left behind,
close to the farm lands and consumed by domestic and wild
carnivores. Tis scenario leads wild boars into importance
in the sylvatic and domestic life cycles of T. gondii and N.
caninum.
Studies concerning N. caninum infections in wild boars
is limited (8, 14). Almería et al. studied 298 wild boars in
Spain and found a prevalence of 0.3% (14). Bártova et al.
studied on 565 wild boars and they detected the prevalence
as 18.1% in Czech Republic (8). Researchers used c-ELISA
for monitoring and IFAT for confrmation. In our study we
could not detect any seropositivity with c-ELISA. In do-
mestic pigs, experimental and natural infections have shown
a low N. caninum incidence that suggests that environmental
exposure to N. caninum is rare. Tis opinion may explain why
we could not detect seropositivity for wild boars in our survey
(14). Terefore, it appears that wild boar may not play an
important role between the sylvatic and domestic life cycle
of N. caninum infection.
Ranucci et al. studied 400 wild boars in Italy and found
the prevalence of T. gondii as 14% using IFAT (2). Gauss et
al. (15) studied on 507 wild boars and found the prevalence
of T. gondii 38.4% with MAT in Spain. Richommeet et al.
studied 148 wild boars and found the prevalence 17.6% with
MAT in France (16). Hejlícek et al. (17) studied on 124 wild
boars and found a prevalence of 15% with SFDT in the
Czech Republic. Furthermore the prevalence rates of T.gondii
were found as 18.2% (n=170) and 34.4% (n=257) in Georgia
and South Carolina provinces of United States respectively
with MAT, 4.4% (n=90) and 5.6% (n=108) in Kukamoto and
Iriomote Island of Japan respectively with LAT (1).
In our study we detected T. gondii antibodies in 4 out of
12 (33.3%) animals with SFDT. Te diferences between the
results may be associated with diferent climatic conditions,
sample sizes, serological tests used and diferent species of
fnal hosts, their population density and their feeding behav-
iors. Although our sample size is small, 33.3% prevalence may
be important for the transition between sylvatic to domestic
life cycles by domestic and wild felids.
Limitations of this study include the small sample size
and the fact that only female wild pigs were tested. In this
regard this study only gives an indication of the distribution
of these diseases among wild pigs may be considered a pre-
liminary study to further larger and wider ranging studies in
this area of Turkey. Diferences in infection rates between the
sexes has not been extensively studied for either Toxoplasma
gondii and Neospora caninum although a study in dogs has
demonstrated a greater prevalence of N. caninum antibodies
in female dogs than in males (18, 19).
In conclusion, it is hoped that the results of this pre-
liminary study will contribute to the understanding of these
apicomplexan parasites’ epidemiology in the wild life. Further
studies are required for developing efective control programs
and a clear understanding of these diseases both in Turkey
and worldwide.
REFERENCES
1. Dubey, J.P.: Toxoplasmosis of Animals and Humans. Second Edi-
tion, CRC Press, Taylor & Francis Group, p: 159, 2010.
2. Ranucci, D., Veronesi1, F., Moretti, A., Branciari, R., Miraglia,
D.,Manfredi, M.T. and Fioretti, D.P.: Seroprevalence of Toxo-
plasma gondii in wild boars (Sus scrofa) from Central Italy. Parasite.
20: 48-48, 2013.
3. Jones, J.L and Dubey, J.P.: Foodborne Toxoplasmosis. Food Safety.
55: 845-851, 2012.
4. Dubey, J.P.: Review of Neospora caninum and neosporosis in ani-
mals. Korean. J. Parasitol. 41: 1-16, 2003.
5. Almeria, S.: Neospora caninum and Wildlife. Hindawi Publish-
ing Corporation, ISRN Parasitology, Volume 2013, Article ID
947347, http://dx.doi.org/10.5402/2013/947347.
6. Anderson, M.L., Andrianarivo, A.G. and Conrad, P.A.: Neospo-
rosis in Cattle. Anim. Rep. Sci. 60-61: 417-431, 2000.
7. Sobrinoa, R., Dubey, J.P., Paboonc, M., Linarez, N., Kwokb, O.C.,
Millan, J., Arnal, M.C., Lucoe, D.F., Lopez-Gatius, F., Tulliez,
P., Gortazar, C. and Almeria, S.: Neospora caninum antibodies in
wild carnivores from Spain. Vet. Parasitol. 155: 190-197, 2008.
8. Bártová, E., Sedlák, K. and Literák, I.: Prevalence of Toxoplasma
gondi iand Neospora caninum antibodies in wild boars in the Czech
Republic. Vet Parasitol. 142: 150-153, 2006.
9. Beral, M., Rossi, S., Aubert, D., Gasqui, P., Terrier, M.E., Klein,
F., Villena, I., Abrial, D., Gilot-Fromont, E., Richomme, C., Hars,
J. and Jourdain, E.: Environmental factors associated with the
seroprevalence of Toxoplasma gondii in Wild Boars (Sus scrofa),
France. Ecohealth. 9: 303-309, 2012.
10. Sabin, A. B. and Feldman, H. A.: Dyes as microchemical indica-
tors of a new immunity phenomen afecting a proton parasite
(Toxoplasma). Science. 108: 660-663, 1948.
11. Balkaya, I., Aktas, M.S., Ozkanlar, Y., Babur, C. and Celebi, B.:
Seroprevalence of Toxoplasma gondii in dogs in Eastern Turkey.
Isr. J. Vet. Med. 65: 58-61, 2010.
12. Piskin, F.C. and Utuk, A.E.: Prevalence of Neospora caninum in
cows with stillbirth and abortion. Te Journal of Etlik Veterinary
Microbiology. 20: 23-26, 2009.
13. Inci, S., Albayrak, I. and Wilson, C.J.: Bioecology of the Wild Boar
(Sus scrofa Linnaeus 1758) in Kirikkale Province, Turkey. Hacettepe
J. Biol. & Chem. 41: 143-150, 2013.
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M.I.G., Ruiz-Fons, F., Alzaga, V., Marco, I., Calvete, C., Lavin,
S., Gortazar, C., López-Gatius, F. and Dubey, J.P.: Seroprevalence
of Neospora caninum in non-carnivorous wildlife from Spain. Vet.
Parasitol. 143: 21-28, 2007.
15. Gauss, C., Dubey, J., Vidal, D., Ruiz, F., Vicente, J., Marco, I.,
Lavin, S., Gortazar, C. and Almeria, S.: Seroprevalence of Toxo-
plasma gondii in wild pigs (Sus scrofa) from Spain. Vet. Parasitol.
131: 151-156, 2005.
16. Richomme, C., Aubert, D., Gilot-Fromont, E., Ajzenberg, D.,
Mercier, A., Ducrot, C., Ferte, H., Delorme, D. and Villena, I.:
Genetic characterization of Toxoplasma gondii from wild boar
(Susscrofa) in France. Vet. Parasitol. 164: 296-300, 2009.
17. Hejlícek, K., Literák, I. and Nezval, J.: Toxoplasmosis in wild mam-
mals from the Czech Republic. J. Wildlife Dis. 33: 480-485, 1997.
18. Goździk, K., Wrzesień, R., Wielgosz-Ostolska, A., Bień, J., Kozak-
Ljunggren, M. and Cabaj, W.: Prevalence of antibodies against
Neospora caninum in dogs from urban areas in Central Poland.
Parasitol. Res. 108: 991-996, 2011.
19. Wouda, W., Dijkstra, T., Kramer, A.M., van Maanen, C. and
Brinkhof, J.M.: Seroepidemiological evidence for a relationship
between Neospora caninum infections in dogs and cattle. Int. J.
Parasitol. 29: 1677-82, 1999.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gardhouse, S. 32
INTRODUCTION
Te African Pygmy hedgehog (Atelerix albiventris) is a mem-
ber of the family Erinaceidae, order Insectivora (1). African
hedgehogs are native to the savannah and steppe regions of
central and eastern Africa (1). In the wild, African hedgehogs
are nocturnal and spend the daytime in burrows (1). Tey are
insectivores and their diet consists mainly of invertebrate
predators such as earthworms, slugs, and snails (1, 2). Wild
African hedgehogs reach sexual maturity at 1 year of age;
however, there are reports of hedgehogs reproductively active
prior to this age, particularly in captivity (3). Hedgehogs are
popular as pets and are also common in zoological collections
(1, 4).
Due to the popularity of this captive species, several ret-
rospective studies have been previously reported in hedgehogs
but focused mainly on neoplasia. Other diseases have only
been briefy or anecdotally described in the literature (1, 4-7).
Te goal of this review is to present disease occurrence in
captive African Pygmy hedgehogs presented to the veterinary
teaching hospital at the Kansas State University.
MATERIALS AND METHODS
Animals
Medical records of privately owned and zoologically kept cap-
tive African Pygmy hedgehogs were reviewed. Tese animals
were presented for varying signs of disease or regular health
checks and were examined by the Exotics and Zoological
Medicine Service, College of Veterinary Medicine, Kansas
State University, between 1994 and 2013.
Retrospective Study of Disease Occurrence in Captive African
Pygmy Hedgehogs (Atelerix albiventris)
Gardhouse, S.* and Eshar, D.
College of Veterinary Medicine, Department of Clinical Sciences, Kansas State University, Manhattan, Kansas, 66506, USA.
*
Corresponding Author: Dr. Sara Gardhouse, Departments of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis,
Davis, CA 95616, USA. Email: saragardhouse@yahoo.com
ABSTRACT
African Pygmy hedgehogs (Atelerix albiventris) are commonly kept in zoological collections and as pets.
Te aim of this study was to present disease occurrence in captive African Pygmy hedgehogs. Medical
records of 106 male and female African Pygmy hedgehogs, both privately owned and from zoological
collections, presented to a veterinary teaching hospital from 1994 to 2013 were reviewed. Te most common
diseases found in captive African Pygmy hedgehogs in this study were dermatological diseases (66.04%),
gastrointestinal diseases (33.02%), and skeletal diseases (15.09%). Other identifed disorders included
neurologic disease, obesity, and renal disease. Tis is the frst retrospective study to describe occurrence
of diseases observed in captive African Pygmy hedgehogs. Te most common clinical fnding in captive
hedgehogs was dermatological disease, specifcally acariasis. Based on this study, it is recommended that all
captive hedgehogs be examined regularly for timely diagnosis and treatment of common diseases of this
species.
Keywords: Captive African Pygmy Hedgehogs; Atelerix albiventris; Common Diseases;
Dermatology; Gastroenterology; Orthopedics.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 33 African Pygmy Hedgehogs (Atelerix albiventris)
History and Clinical Examination
Each African Pygmy hedgehog’s history, including any previ-
ous diagnoses or treatments, was obtained from its medical
record. Depending on the nature of the hedgehog, most of
the physical examinations were performed under general
anesthesia.
Laboratory Testing
In some cases, blood was obtained from the jugular vein
under short-term isofurane gas anesthesia. Occasionally,
when only a very small amount of blood was required, the
peripheral vessels (cephalic vein, lateral saphenous vein) were
used. Blood samples were then submitted for complete blood
count and plasma biochemical analysis. When indicated,
tissue samples were submitted for cytology, bacteriology,
mycology and/or histology tests. Fecal parasitological testing
was performed in the cases of diarrhea, progressive weight
loss, and some annual health examinations.
Imaging Methods
When clinically indicated, abdominal ultrasonography and
two-view (dorsoventral and lateral) whole body radiographs
were obtained under general isofurane gas anesthesia.
Computed tomography scan was performed in one case with
skull pathology. Echocardiography was performed in one case
with suspected cardiac disease.
RESULTS AND DISCUSSION
A total of 106 individual cases were reviewed, out of which
47 animals were intact females and 59 were intact males.
Te mean age was 26 months (range 1 month to 9.0 years)
and mean weight was 348 g (range 112 g to 786 g). Te goal
of this study was to determine disease occurrence in cap-
tive African Pygmy hedgehogs by retrospectively reviewing
medical records of cases admitted to a veterinary teaching
hospital for clinical evaluation over a time period of 9 years.
Description of the observed disease conditions in African
Pygmy hedgehogs revealed in this study are summarized in
Table 1.
In this study, the oldest animal was 9 years old and 9
other hedgehogs were older than 4 years of age. Te expected
life span of wild African Pygmy hedgehogs is reported to
be up to 4 years, though it has been noted they can live up
to 10 years in captivity due to advances in veterinary care
(2, 6). In this study, adult hedgehogs weighed 361 g mean
for males (range 112 g-770 g) and 369 g mean for females
(range 124 g-670 g). Te weight of adult hedgehogs has been
Table 1: Prevalence of health disorders in 106 captive African pygmy
hedgehogs from 1994-2013 presented to the College of Veterinary
Medicine, Kansas State University.
a
Organ/tissue disease All hedgehogs % (N=106)
Dermatological disease 66.04
Ectoparasites 46.22
Pododermatitis 3.77
Dermatophytosis 5.66
Otitis externa 2.83
Neoplasia 4.72
Gastrointestinal disease 33.02
Oral squamous cell carcinoma 9.43
Tooth root abscess 4.72
Dental calculus/gingivitis/periodontitis 4.72
Liver disease 3.77
Musculoskeletal disease 15.09
Degenerative joint disease 1.89
Spondylosis 2.83
Annular pedal constriction 4.72
Neurologic disease 11.32
Wobbly hedgehog disease 8.49
Obesity 10.38
Renal disease 9.43
Hematuria 4.72
Interstitial nephritis 0.94
Glomerulopathy 0.94
Multifocal interstitial fbrosis 1.89
Reproductive disorders 8.49
Mammary carcinoma 1.89
Uterine neoplasia 3.77
Endometrial polyps 0.94
Pyometra 0.94
Respiratory disease 7.55
Pneumonia 1.89
Ocular disease 5.66
Cataracts 3.77
Globe proptosis 1.89
Cardiac disease 1.89
Congestive heart failure 1.89
Endocrine disease 1.89
Tyroid gland hypertrophy 0.94
Tyroid carcinoma 0.94
Status ante fnem (diagnosis not known) 8.49
Healthy animals 11.32
a
Some animals displayed more than one symptom or disease and were
included in more than one group.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Gardhouse, S. 34
reported to be 400-600 g for males and 300-400 g for females
(1).
Dermatologic disease was the most common health
disorder seen in hedgehogs, being recorded in 66.04% (70
of 106) of all hedgehogs examined in this study. Te most
common dermatological disease seen was mite infestation
(acariasis) identifed in 65.71% (46 of 70) of the dermato-
logical cases. Te species of mites was not recorded in every
case, but when recorded (5 of 46), the species observed were
Chorioptes, 60% (3 of 5) and Caparinia, 40% (2 of 5). Two
cases with feas and one case with lice were also found.
External parasites (feas, ticks, and mites), as well as a va-
riety of dermatophytes were commonly seen in hedgehogs in
this study and have been previously reported in the literature
(8). Acariasis is commonly reported in pet hedgehogs (1).
Mites are normal inhabitants of hedgehogs in the wild, but
infestation can become severe in ill or immunocompromised
animals (7). Similar to what was observed in this study, the
most common mite of hedgehogs is Caparinia spp. (non-
burrowing mite), with reports of Chorioptes spp. (burrowing
mite) also being implicated (1).
However, in many of the
cases of mites that were identifed in this study, the species
was not recorded.
Dermatophytosis was found in hedgehogs in this study
with all cases diagnosed as Trichophyton spp. based on fungal
culture. Te most commonly identifed organisms in the lit-
erature include Trichophyton spp. and Microsporum spp. (1, 9).
In 4.72% of cases in this study, dental disease, including
calculus, gingivitis, and periodontitis was noted, which has
commonly been reported in the literature in hedgehogs (1).
Various skeletal diseases have been recorded in hedgehogs
(10). Spondylosis was noted on radiographs of three hedge-
hogs in this study. A case series (n = 4) of intervertebral disc
disease in hedgehogs noted spondylosis as a radiographic
fnding in three hedgehogs, hypothesized to be a result of
abnormal movement between vertebrae, degeneration of the
annulus fbrosis and infammation with bony proliferation
from the protruding disc (10). Annular pedal constriction, as
noted in this study in fve hedgehogs, causes an interference
with the blood supply leading to local congestion, edema,
hypoxia, and/or avascular necrosis (11). Tese injuries can be
caused by cage wire, fabric, string, hair, or other fbers (11).
Hedgehogs appear to be particularly prone to this injury
of a leg, foot or digit, often requiring amputation (11). In
this study, removal of the foreign material resulted in return
to normal function of the limb in all but one case which
required amputation.
A common presenting complaint of neurologic hedge-
hogs in this study was ataxia. Neurological disease is reported
commonly in hedgehogs in the literature (1). Common causes
of ataxia in hedgehogs include torpor, Wobbly Hedgehog
Syndrome (WHS), intervertebral disc disease, trauma, toxins,
infarcts, malnutrition, and neoplasia (1, 12-13). WHS was the
most common cause of ataxia found in this study (8.49%).
WHS (as a result of demyelination) is a progressive paralysis
in hedgehogs that begins with ataxia and ends in complete
paralysis within 15 months of the onset of clinical signs (12,
14). However, intervertebral disc disease (IVDD) can also
present with similar signs as WHS (1, 13).
Brain tumors
have also been reported to present with similar signs as WHS
and IVDD with reports of astrocytomas, gemistocytic as-
trocytomas and microgliomas (12). One report of anaplastic
astrocytoma in the spinal cord of a hedgehog has also been
reported (15). Additionally, circling, as seen in hedgehogs in
this study, may be caused by otitis media/interna or primary
neurologic disease (1).
Obesity was found in 10.38% of cases in this study, which
is refective of a previous report (16). Obesity is commonly
reported in pet hedgehogs as a result of improper husbandry,
including overeating, incorrect diet, lack of exercise, and an
ambient temperature that is too cold (16).
Renal diseases in this study were found in 9.43% of cases,
but many of the hedgehogs with suspected renal disease were
not necropsied to allow a defnitive diagnosis. A retrospective
study of post mortem fndings in 14 African hedgehogs re-
vealed renal disease in 50% of the hedgehogs including tubu-
lointerstitial nephritis, chronic renal infarcts, glomerulopathy,
and tubular nephrosis (17).
When necropsy was performed
in hedgehogs in this study, similar histopathological fndings
were also described.
In this study, cardiac disease was clinically diagnosed in
just 1.89% of cases and necropsy fndings from 13.21% of
cases revealed no evidence of cardiomyopathy. In a study
published in 2000, 38% (16 of 42) of hedgehogs examined
on necropsy, were noted to have cardiomyopathy (18).
Cases of leiomyoma and endometrial polyps were found
in this study, however some of the suspected cases of uterine
disease in this study did not have necropsy or other diagnostic
tests performed. Uterine disease in African hedgehogs has
been reported in the literature (5, 13, 19-20). Te types of
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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 35 African Pygmy Hedgehogs (Atelerix albiventris)
tumors of the uterus reported in hedgehogs in the literature
includes adenoleiomyosarcoma, adenosarcoma, endometrial
stromal cell sarcoma, endometrial polyps, adenoleiomyoma,
uterine adenocarcinoma, carcinosarcoma and uterine spindle
cell tumor (5, 13, 18-19).
One case of pyometra was found in
this study. Te predominant cause of abnormal vaginal dis-
charge is reported to be neoplasia but pyometra and metritis
have been reported (1, 20, 21).
Neoplasia was the third most common general pathology
in hedgehogs observed in 20.75% (22 of 106) of cases in
this study, when incorporating all body systems. Te most
common neoplastic process observed in this study was oral
squamous cell carcinoma 45.45% (10 of 22 neoplasias).
Mammary gland adenocarcinoma was seen in 9.09% (2 of
22 neoplasias) of cases. Dermal neoplasia was reported in
22.73% (5 of 22) of neoplasia cases.
Oral squamous cell carcinoma was commonly diagnosed
in hedgehogs ranging from 1-6 years of age. Oral neoplasms,
particularly squamous cell carcinomas are commonly reported
in hedgehogs in the literature (1). Oral squamous cell carci-
noma was reported as the third most common tumor of the
hedgehog preceded by mammary gland adenocarcinoma and
lymphoma in a retrospective study, in hedgehogs ranging
from 2-5.5 years (4).
In this study, oral squamous cell carcinoma was the most
commonly reported neoplasm in 45.45% (10 of 22 neopla-
sias) of cases, mammary gland adenocarcinoma was seen in
9.09% (2 of 22 neoplasias) of cases, and lymphosarcoma was
not reported. Dermal neoplasia was reported in 22.73% (5
of 22) of neoplasia cases, and is also considered relatively
common (4). Neoplasia is reported commonly in African
hedgehogs in the literature (1, 4-6). Tere are various reports
of tumors and disseminated neoplastic processes in hedge-
hogs, afecting many body systems (4-6). Several retrospective
studies reported various neoplasms in African hedgehogs (1,
4-6). In one retrospective study of 66 hedgehogs, the most
common tumors included mammary gland adenocarcinoma,
lymphosarcoma, and oral squamous cell carcinoma (4).
Limitations of this study include the accuracy of informa-
tion available in the record, which is inherent in any retrospec-
tive study. Te age of the animals was reported by the owners
and may not be accurate. Te reproductive status was also
reported by the owners and verifed by the attending clinician.
Te population of hedgehogs used in this study is a
good representation of the captive hedgehog population,
from both zoologically kept and privately owned hedge-
hogs, since both primary care and secondary referral cases
are included. Additionally, given the large time scale, there
was a decreased chance of disease grouping. However, the
localized geographical area may represent a localized gene
pool which may result in diferent disease prevalence in other
areas of the world.
CONCLUSION
Te most common disease in hedgehogs found in this
study is dermatological disease, particularly ectoparasites.
Gastrointestinal disease, skeletal disease, and neurologic
disease were all relatively common presentations. Tis retro-
spective study may assist in extending our clinical knowledge
of disease prevalence in captive African Pygmy hedgehogs.
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5. Mikaelian, I. and Reavill, D.R.: Spontaneous proliferative lesions
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hogs (Atelerix albiventris). Proc. Pre-Conf. AAV. 65: 2010.
13. Garner, M.M.: Diseases of pet hedgehogs, chinchillas, and sugar
gliders. Proc. AAV. 405-407, 2011.
14. Graesser, D., Spraker, T.R., Dressen, P., Garner, M.M., Raymond,
J.T., Tarwilliger, G., Kim, J. And Madri, J.A.: Wobbly Hedgehog
Syndrome in African pygmy hedgehogs (Atelerix spp.). J. Exot.
Pet. Med. 15:59-65, 2006.
15. Gibson, C.J., Parry, N.M.A., Jakowski, R.M. and, Eshar, D.:
Anaplastic astrocytoma in the spinal cord of an African Pygmy
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2008.
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A, Redrobe S, eds. BSAVA Manual of Exotic Pets. 4th ed. British
Small Animal Veterinary Association, Quedgeley, Gloucester. p.
110. 2010.
17. Raymond, J.T. and White, M.R.: Necropsy and histopathologic
fndings in 14 African hedgehogs (Atelerix albiventris): a retrospec-
tive study. J. Zoo. Wildl. Med. 30:273-277, 1999.
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Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 37 Diaphragmatic Hernia in Horses
INTRODUCTION
Diaphragmatic hernias (DH) are classifed as true, having a
hernia sac, or false where this is a lack of a hernia sac. False
hernias can probably be better defned as a diaphragmatic
defect, rupture or tear (1). Nevertheless, in order to conform
to the current literature the term DH will be used throughout
the manuscript for consistency. A further classifcation takes
into account the etiology of DH, namely congenital and
acquired. Te congenital form is mostly associated with an
abnormal development of the various parts of the diaphragm,
which do not fuse together, while the acquired form is usu-
ally caused by trauma or increased intra-abdominal pressure
(e.g. parturition (1, 2)). Congenital as well as acquired DH
are typically left-sided (1, 3), though a right sided congenital,
Morgagni type DH has been also reported (4). Interestingly,
there is an in-between form that can be defned both as
traumatic-acquired and as congenital. Tis form of DH oc-
curs upon parturition in which the foal’s ribs fracture and
tear its diaphragm upon passing through the birth canal (5).
Te location of the defect in the diaphragm varies, but it
most commonly occurs at the junction between the tendine-
ous and the muscular parts of the diaphragm. Te location of
Diaphragmatic Hernia in Horses in Israel: A Case Series
Efraim, G.* and Kelmer, G.*
Large Animal Department, Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Te Robert H. Smith Faculty
of Agriculture, Food and Environment, Te Hebrew University of Jerusalem, Israel.
*
Corresponding Author: Dr. Gal Kelmer, Large Animal Department, Koret School of Veterinary Medicine, Te Robert H. Smith Faculty of Agriculture,
Food and Environment, Te Hebrew University of Jerusalem, PO Box 12, 76100, Rehovot, Israel.Tel: +972-(3)-9688588, Fax: +972-(3)-9688525, Email:
gal.kelmer@mail.huji.ac.il
ABSTRACT
Diaphragmatic hernia (DH) is possibly more frequent in the horse than typically reported in the literature.
Since DH is not as rare as perceived, reporting on its occurrence is critical in order to increase awareness
of this problem. DH typically presents as an emergency situation, whose prompt treatment infuences the
prognosis. Tus, it should be included in the diferential diagnosis of horses presenting with signs of colic
or of respiratory distress, or with a combination of the two. Te aims of this study were to determine the
prevalence of DH cases at the Veterinary Teaching Hospital of the Koret School of Veterinary Medicine
and to describe the clinical signs, case management and outcome of these cases. Te equine medical records
of our hospital for January 2008 – August 2012 were reviewed. Te information retrieved included chief
complaints, mode of diagnosis, physical characteristics of the hernia, organs found in the thorax, treatment,
results and, where relevant and pathology report. Te number of DHs was compared to the hospital’s caseload
and colic surgeries during the reference period. Four horses were presented with DH in the reference period
constituting 0.46% of all surgeries and 1.29 % of all colic surgeries in the reference period, and 0.14% of the
annual referral fgure. Tree of the four cases showed signs of colic with respiratory symptoms, whereas the
fourth presented only acute respiratory distress. Te survival rate was 25%, one patient being successfully
treated. Te prevalence of DH was found to be more frequent at the KVSM-VTH than previously reported.
Early diagnosis and a suitable surgical approach proved essential to the successful surgical repair of DH, but
the overall survival rate remained low.
Keywords: Diaphragmatic Hernia; Equine; Prevalence; Respiratory Distress; Colic.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Efraim, G. 38
the lesion is probably afected by the nature of the etiologic
factor (1). Te organs that are usually incarcerated in DH
are the small and large intestines (1, 2, 6), but the spleen, the
stomach and the liver can also be involved (2).
All types of DH can remain subclinical for prolonged
periods and clinical signs typically appear acutely upon
incarceration or strangulation of intestine. Clinical signs
are typically signs of colic, but may include and limited to
respiratory distress (7). Symptoms however may be mild,
such as lethargy and exercise intolerance alone, up to a stage
when organs migrate into the thoracic cavity. Initial suspicion
of DH can be established following physical examination,
when signs of colic are accompanied by respiratory distress.
Other, inconsistent, non-specifc, signs include resistance
upon nasogastric intubation and a sensation of emptiness
upon rectal palpation. Useful diagnostic techniques include
thoracic radiographs and ultrasonographic evaluation (1).
Notwithstanding, diagnosis is unfortunately often reached
only by exploratory celiotomy or post-mortem examination
(2).
Treatment is exclusively surgical and aims at removing the
herniated bowel from the thorax, with or without resection,
and repairing the diaphragmatic defect (8). Post-operative
treatment should comprise management of pneumothorax
and pleuritis in addition to the typical post-operative colic
treatment regimen.
Tis report describes the diagnostic procedures and
the treatment of four cases of DH which were recorded at
the Veterinary Teaching Hospital of the Koret School of
Veterinary Medicine (KVSM-VTH) during January 2008
– August 2012. Te study was carried out in order to better
understand DH prevalence and possibly assist improve future
treatment of similar cases.
MATERIALS AND METHODS
Tis article is a retrospective cohort study that was carried out
in the KVSM-VTH between January 2008 and August 2012.
Te clinical and clinical pathology records of the DH cases
recorded in the reference period were reviewed. Te clinicians
involved in the cases also assisted in the retrieval of informa-
tion regarding the clinical history, signalment, medical and
surgical treatment, and outcome for their respective cases.
Records were found for two fllies, a mare and a stallion,
aged 10 days, 7 months, 18 years and 17 years respectively.
Te main equine population seen in the hospital com-
prised riding and breeding horses. Te total number of cases
for each year was retrieved and an average case load per year
was computed. Te same calculation was performed for
the number of surgeries and colic surgeries at the hospital.
Te prevalence of DH was calculated in percentage terms,
along with the percentage of DH in total surgeries and colic
surgeries.
RESULTS
During the reference period, the KSVM-VTH received an
average of 67 surgical colics per year, and DH comprised
1.29% of our surgical colic caseload. Te KVSM-VTH, with
an average of 637 referrals per year over the reference period
( January 2008 –August 2012), received four horses with DH
during this time which amounted to 0.14% of the total refer-
rals. Only one of the four cases with DH survived (25%) to
discharge with a good long-term outcome.
CLINICAL CASES
CASE 1
Clinical History
A 10-day-old Arabian flly was presented to the KVSM-
VTH, after uneventful pregnancy and parturition. Te own-
ers reported having noticed signs of weakness and carpal
swelling on the right forelimb the following day. On arrival to
the hospital the complete blood count (CBC) revealed, WBC
of 6.3x10
9
/L, Reference Range (RI): 5.6-12.1x10
9
/L), packed
cell volume (PCV 37%, (RI): 27-43%) and total solids (TS)
of 6g/dl, (RI): 6-8g/dl) were within the reference ranges.
Sample of synovial fuid taken from the right inter-carpal
joint was consistent with synovial infection (TS 7g/dL, (RI):
2-3.5g/dl; lactate 8 mmol/L, (RI): 0-2 mmol/L; glucose 19
mg/dL, (RI): 76-130 mg/dL).
Case Management
Amikacin (20 mg/kg s.i.d., Vetmarket, Shoaham, Israel),
ampicillin (20 mg/kg q.i.d., Penibrin, Sandoz GmbH, Kundl,
Austria), funixin meglumine (0.5 mg/kg b.i.d., Norbrook
laboratories Ltd, Newry, N .Ireland) and LRS (Teva Medical,
Petah Tikva, Israel) were administered intravenously and
ranitidine (7 mg/kg t.i.d., Dexcel Pharma, Jerusalem, Israel)
was administered orally. Te next day the flly underwent
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 39 Diaphragmatic Hernia in Horses
arthroscopic surgery under general anaesthesia. In both
the inter-carpal and the radio-carpal joints, pannus, syno-
vial edema and discoloration were found, consistent with
synovial sepsis. In both joints, pannus was removed, partial
synovectomy was performed, followed by generous lavage (12
liters per joint) and amikacin (1g) was injected. Te joints
were lavaged daily under heavy sedation (butorphanol (3
mg, Morphasol, Animedica GmbH, Senden, Germany),
diazepam (10 mg, Teva Medical, Petah Tikva, Israel) and
xylazine (10 mg, Sedazine, AST Farma, Oudewater, Te
Netherlands)) using teat cannulas, for 10 days. Since clini-
cal signs persisted despite the aggressive management, joint
lavage was replaced by regional limb perfusion (RLP) with
imipenem (Merck Sharp & Dohme, Chibret, France) alter-
nating with intra-articular injections with imipenem. Te
same day the flly exhibited difculty in expiration subsequent
to tranquilization. Dyspnea recurred the following day and
lateral thoracic radiographs taken in recumbent position
showed no evidence of a respiratory pathology.
After another RLP procedure carried out the same week,
the patient was kept under observation, with daily bandage
changes, as there was improvement in the condition of carpus.
During the third week of hospitalization, the flly’s lameness
and general condition improved, whereas acute respiratory
distress continued to fare up occasionally.
On day 23 of hospitalization, the flly developed severe re-
spiratory distress accompanied by tachycardia and tachypnea
(heart rate 120 beats/min and respiratory rate 56 breaths/
min). At that stage, the flly had a severe bout of coughing
and then collapsed in agonal breathing. An attempt was made
to insert an endotracheal tube, but the flly collapsed and
stopped breathing. Intubation was successfully performed
but resuscitation eforts were attempted to no avail, until
death ensued.
Post-mortem examination
Te post-mortem examination revealed: yellowish liquid
flling the thoracic cavity, the lung lobes were collapsed, and
discoloured, with consolidation in ~30% of the lung feld. In
addition, there was a 15 cm long defect in the right crus of
the mid-diaphragm at the border of the muscular and fbrous
portions of the diaphragm. Te margins of the opening were
thick. Multiple loops of the small intestine were found in
the thoracic cavity. Te dorsal loops appeared thickened and
compromised (Figure 1).
CASE 2
Clinical History
A 7 months old Arabian flly had had a history of severe
abdominal pain. Two days earlier the owner called the refer-
ring veterinarian to treat the flly for a ventral abdominal
swelling. Te veterinarian detected that the swelling included
the right-side thorax, and administered anti-infammatory
medication. Several hours before arriving to the hospital,
the flly showed severe signs of abdominal pain, which were
unresponsive to analgesics, and was referred to the hospital.
On arrival to the hospital Te flly was severely painful
and required potent analgesia in order to tolerate the initial
evaluation. A swelling was detected on the right hemi-
thorax and on the ventral part of the abdomen. Blood tests
included CBC: ( WBC of 4.2x10
9
/L, Reference Range (RI):
5.6-12.1x10
9
/L), packed cell volume (PCV 45%, (RI): 27-
43%) and total solids (TS 8.2g/dl, (RI): 6-8g/dl) were con-
sisted with mild dehydration and leukopenia. On ultrasound
examination, a portion of the small intestine with peristaltic
movement, and fractured ribs were visible in the swelling on
the right hemi-thorax. At that stage, the flly was submitted
for urgent abdominal exploration under general anaesthesia.
Case Management
Preoperative medication was administered intravenously as
follows: benzylpenicillin sodium (20,000 IU/kg, Norbrook
laboratories Ltd, Newry, N. Ireland), gentamicin (6.6 mg/kg,
Figure 1: Necropsy photograph of case 1, depicting diaphragmatic
hernia in a 10 days old Arabian flly. Vertical white arrow points to the
herniated small intestine while the horizontal white arrow points to
the torn diaphragm. Vertical black arrow points to the stomach while
the horizontal black arrow points to the collapsed lungs.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Efraim, G. 40
Gentaveto, Eurovet, Netherlands) and funixin meglumine
(1.1 mg/kg). Te patient was then premedicated intravenously
with 100 mg xylazine, and induction was performed with 220
mg of ketamine (Clorketam, Vetquinol, Paris, France) and 10
mg of diazepam. Te flly was positioned in dorsal recum-
bency and isofurane (Piramal Critical Care, Inc, Bethlehem,
Pennsylvania, USA) was used to maintain anaesthesia.
A ventral abdominal midline approach was performed,
and small intestine entrapped in a diaphragmatic tear,
were observed. Te diaphragmatic tear was about 10 cm
long, located on the right ventral muscular portion of the
diaphragm (Figure 2). After carefully reducing the incarcer-
ated intestine into the abdominal cavity, it was found to be
non-viable. Over 70% of small intestine was discolored and
had no pulse or motility with several tears in the mesentery
(Figure 3). In addition, the right hemi-thorax had a large
defect that contained some of the damaged intestine, and four
sharp-edged fractured ribs 12-15. Te ribs were fractured at
the costo-chondral junction. Tere was no external wound,
neither at the thorax nor caudally, but the ribs lacerated the
thoracic wall allowing several loops of small intestine to
migrate subcutaneously. Due to poor prognosis the flly was
euthanized at the owner’s consent.
CASE 3
Clinical history
An 18 year-old mixed breed mare sufered from colic which
had begun about twelve hours earlier and had responded well
to the initial analgesic therapy in the feld. Nevertheless, a
few hours later the mare showed severe signs of colic and was
referred to the hospital. On arrival at the hospital the mare
had severely compromised cardiovascular status with marked
tachycardia (100 beats per minute), hyperemic mucous mem-
branes and cold extremities. Auscultation revealed decreased
borborygmus and nasogastric intubation produced 13 liters of
refux. On rectal examination, dry feces and a gas-distended
large colon were felt. A CBC revealed a high WBC count
(15.4x10
9
/L, RI: 5.6-12.1x10
9
/L) and elevated PCV, indicative
of hemoconcentration (PCV 48%, RI: 27-43%), while TS was
low (4.7 mg/dL, RI: 6-8mg/dL), and lactate concentration was
elevated (5.1 mmol/L, RI: < 2 mmol/L). During examination,
the mare showed signs of uncontrollable pain and immediate
exploratory celiotomy was therefore uninitiated.
Case Management
Preoperatively the mare was given sodium penicillin G
(20,000 IU/kg) and gentamicin (6.6 mg/kg) intravenously.
Te mare underwent induction and isofurane and anaesthe-
sia. With the mare in dorsal recumbency, a ventral midline
abdominal approach was performed. Exploration revealed a
defect in the diaphragm, about 22-cm long, situated in the
left dorsal part of the diaphragm, at the musculo-tendinous
junction. Te stomach, several meters of the small intestine,
the pelvic fexure and a left lobe of the liver were incarcerated
in the thorax. Furthermore, a large colon volvulus was found
(360° counter-clockwise). Te cecum and the large colon were
edematous and purple. Part of the omentum was adhered
Figure 2: Intra-operative photograph of case 2, depicting a
diaphragmatic tear in a 7 months old Arabian flly. Te image is viewed
through an abdominal approach for exploratory celiotomy.
Figure 3: Intra-operative photograph of case 2, depicting torn
mesentery and damaged small intestine in a 7 months old Arabian
flly sufering from diaphragmatic hernia. Te arrows points to the
extensive tear in the small intestine mesentery.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 41 Diaphragmatic Hernia in Horses
to the lateral border of the hernia. Te abdominal organs
were retrieved and returned in their normal position in the
abdominal cavity. Te volvulus was manually corrected, which
led to an improvement in color and motility of the large
colon. Te omentum was ligated and resected. A blood gas
analysis performed an hour after induction showed respira-
tory acidosis (with values of 102.9 mm Hg for PaO
2
, (RI:
100-500 mm Hg) 50 mm Hg for PaCO
2
(RI: 30-45 mm Hg)
and 7.26 for blood pH (RI: 7.35-7.45), which was resolved by
having the mare tilted 30 degrees head up (reverse trendelen-
burg position) for an hour in order to decrease the pressure
exerted on the lungs by the abdominal organs. Attempts to
correct the hernia through the abdominal cavity failed. Te
deep dorsal location and considerable tension on the sutures,
due to the large gap in the diaphragm; combined with the
friable diaphragmatic muscular tissue; hindered the repair.
Te abdomen was lavaged and closed in a routine fashion.
At that stage the mare was positioned in right lateral recum-
bency as the left hemithorax was prepared for surgery. An
incision was made in the left hemithorax over the 11
th
rib,
approximately 30cm length of rib was removed using a gigli
wire saw (Narang Medical Limited, Delhi, India) and the
space between the 10
th
and the 12
th
rib was expanded using
a Finochietto rib spreader. Te tear was located and an atel-
ectatic left lung lobe was detected. Suturing proved difcult
using this approach. A polypropylene mesh was attached to
cover the diaphragmatic defect using surgical skin staples
(Figure 4). At termination of the procedure two 32 French
drains (Well Lead Medical, Panyu district, Guangzhou city,
China) were inserted, one dorsally at the 13
th
intercostals
space and one ventrally at the 8
th
intercostal space.
Postoperatively the mare received intensive treatment
including: intravenous fuid therapy consisting of crystal-
loids (LRS) and colloids (Hetastarch, Fresenius Kabi AG,
Badhomburg, Germany)), fresh frozen plasma, antibiotic
therapy (penicillin 22.000 IU/kg q.i.d. IV and gentamicin
6.6 mg/kg s.i.d. IV), analgesics (funixin 1 mg/kg b.i.d.
IV) and anti-endotoxin therapy (polymyxin B 6000 IU/kg
b.i.d., IV, X-GEN Pharmaceuticals Inc, Northport, NY).
Ice therapy applied on all four extremities to prevent for
laminitis. Initially the horse received a nasal oxygen supply
(15 liters/min).
Post-operative radiographs confrmed good lung infa-
tion and verifed that the mesh was intact at its location.
Te drains were removed the day after surgery following a
signifcant decrease in the amount of blood and air that was
drained. Two days after surgery signifcant edema developed
at both incisions but both remained dry. At that stage, oral
antimicrobials (enrofoxacin (7.5 mg/kg s.i.d., Phibro Animal
Health Corporation, Petah Tikva, Isreal) and metronidazole
(25 mg/kg t.i.d., Vetmarket, Shoham, Israel)) and gastric
mucosal protectants (omeprazole 4 mg/kg s.i.d., Nature Vet
Pty Ltd., Glenorie, NSW, Australia) and ranitidine 6.6 mg/
kg q.i.d.) were added. Te mare recovered smoothly and was
discharged 12 days after surgery. Te mare resumed her career
as an endurance horse, with no further respiratory or colic
problems in a fve-year post-operative follow-up.
CASE 4
Clinical History
A 17-year-old male Quarter horse was referred to the
KVSM-VTH with clinical signs of acute colic, which lasted
for 12 hours. He was unresponsive to analgesics or seda-
tives and was referred to the hospital. Te owners reported
weight loss and occasional breathing difculties in the last
few months.
Te horse presented to the hospital with a heart rate of
40 beats/minute, a respiratory rate of 30 breaths/minute with
the presence of a heart murmur. On rectal examination cecal
impaction containing hard fecal material was palpated. Te
horse was treated with mineral oil (Vetmarket, Shoaham,
Figure 4: Intra-operative photograph of case 3, depicting a
polypropylene mesh stapled to a tear in the diaphragm in an 18 year-
old mixed breed mare. Long vertical arrow points to the fnochietto
rib retractors, short vertical arrow points to the polypropylene mesh
and the oblique arrow depicts the skin staples used to attach the mesh
to the diaphragm.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Efraim, G. 42
Israel) and water through the nasogastric tube and LRS
intravenously. Tough the horse received potent analgesia
several times, pain recurred and emergency celiotomy was
recommended.
Case management
Preoperative medication was administered intravenously as
follows: benzylpenicillin sodium (20,000 IU/kg), gentamicin
(6.6 mg/kg), and funixin meglumine (1.1 mg/kg). Te horse
was placed under general anesthesia, positioned in dorsal
recumbency and isofurane anesthesia was used. On explor-
atory celiotomy a 6 cm x 6 cm right ventral diaphragmatic
tear was found. Te cecum was incarcerated in the hernia
and it was adhered to the pleura in several places. During
manipulation of the cecum signifcant bleeding occurred in
the thorax. Te hernia was closed by direct suturing, and a
drain was inserted into the mid-height, right hemi-thorax at
the 12
th
inter-costal space. Following recovery the horse had
respiratory distress and thoracic radiographs revealed large
intestine in the thorax, along with an old fracture in the right
fourteen rib. Te horse was taken to surgery in an attempt to
repeat the repair of the diaphragm, however death occurred
during induction.
DISCUSSION
Diaphragmatic hernia is commonly referred to as a rare lesion
in the horse (9), however it is probably more common than
previously considered (1). In one recent study, DH made up
to 1% of all colic surgeries performed between 1998 and 2005
at a university referral hospital (1). Tis fgure is comparable
with the percentage of surgeries for ileocecal intussusception
or gastrosplenic entrapment (1.3%, 0.3%, respectively) – two
conditions that are not considered rare (10). Te incidence
of DH reported in the KVSM-VTH in this study (1.3 %) is
similar to the results mentioned above. In another retrospec-
tive study from a single center, over two cases of DH per
year were recorded (6). Tus, according to this and to our
experience, DH is quite uncommon but defnitely not rare.
Te four cases making up the current series presented
exclusively diaphragmatic ruptures or defects, none of which
had a hernia sac. Tis is in accordance with previous studies,
confrming that true DHs, containing a hernia sac, are indeed
rare (1). With regard to etiology, it is reasonable to assume
that all our cases had a traumatic origin, in two cases, the
trauma was recent and in the other two, it occurred months
prior to the acute presenting episode. In several reported
cases, there was a history of trauma months to years prior
to an acute episode of colic. It is likely that the damage to
the mare’s diaphragm occurred at the time of the trauma/
parturition, however the acute episode was probably triggered
by the sudden incarceration of viscera in the thoracic cavity.
In case 1 (the 10-day old foal) the respiratory clinical
signs were mild but evident since arrival and most likely had
existed since parturition. It is likely that during parturition,
while passing in the birth canal, the flly fractured her ribs,
which tore the diaphragm. Tis case falls into the mixed cat-
egory of congenital DH with a traumatic cause. In congenital
DH, the defect is located mainly on the left side, owing to
the incomplete fusion of the pleuroperiontal folds that is
secondary to the slower development of the left lung (1).
Acquired DH also seems to be predominantly left-sided,
probably because the liver acts as a protection on the right
side (1, 2). In contrast to the predominance of left-sided
DH in the literature (1, 2, 6), in three of the four cases that
make up our series, the lesion was located on the right side
of the diaphragm. In three of the presented cases, the DH
appeared on the ventral aspect, which is consistent with their
traumatic history combined with the evidence of ipsilateral
rib fractures. Overall, two cases in this series are presumed to
be related to parturition associated trauma (cases # 1 and #3),
while two were likely due to direct external thoracic trauma
(cases # 2 and #4).
Te diagnosis of DH is typically difcult to make even
during pre-operative evaluation. Te clinical manifestations
of DH typically include acute abdominal signs or colic; colic
and dyspneic signs may be consecutive (11) or simultaneous,
and only seldom do respiratory signs occur alone. Our frst
case presented with exclusive respiratory signs for several days
prior to a fatal abdominal crisis episode. Te respiratory signs
can be attributed to the defect in the diaphragm leading to
a loss of negative pressure in the thoracic cavity resulting in
atelectatic lungs. Tis may be supported by a report describ-
ing a mare sufering from a diaphragmatic rent without any
herniation and showing respiratory signs only (12). Te fatal
episode could be attributed to the migration of viscera into
the thoracic cavity. It is interesting that case 4 had history of
respiratory problems, demonstrated as episodes of dyspnea,
months prior to the abdominal crises that led to the refer-
ral. Cases #2, #3 in our series did show signs of abdominal
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 43 Diaphragmatic Hernia in Horses
pain, involving respiratory distress as well, however, at least
initially, respiratory distress was interpreted as part of the
abdominal crisis. Tis is consistent with the reported concern
that respiratory signs in foals with DH, are often masked by
the acute abdominal crises and attributed to abdominal pain
and stress (11).
Most commonly, DH is diagnosed either at abdominal
exploration or at necropsy. Radiographs and ultrasonography
are considered the most useful diagnostic aids for attaining
ante-mortem or pre-operative diagnosis of DH (1). In the
current series, ultrasonographic evaluation was useful though
not defnitive in the diagnosis of DH in one horse; in two
cases DH was diagnosed by abdominal exploration, and in
one case only at necropsy. Ultrasonographic evaluation can
nevertheless easily lead to false positive diagnosis due to
the bell-shaped diaphragm and the cranial migration of the
small intestine. In our experience, when used judiciously, both
ultrasound and radiograph can be helpful in the diagnosis of
DH. An efort should be made to incorporate these modali-
ties in the workup of any colic case where clinical signs are
indicative of respiratory compromise, or where the history
or initial evaluation may lead the clinician to suspect DH
(1, 12). In case #1 in this series clinical signs were exclusively
of a respiratory nature, radiographs however revealed no DH.
Te difculty in sometimes detecting DH should be borne
in mind and one should not rely on any single negative di-
agnostic test to rule out diagnosis of DH.
Te only defnitive treatment for DH is surgical repair.
Te prognosis in general is not good and it largely depends
on early diagnosis, on the location and length of the tear,
and on the degree of intestinal damage (2, 6, 8). Romero and
Rodgerson (2) reported that out of 25 horses which were op-
erated on, only 6 survived more than three months following
surgery, while 11 were euthanized during surgery. Similarly, 7
out of 44 survived to discharge in a recent retrospective study
by Hart and Brown (6). Nevertheless, long term results were
positive in the same study with 71 % of horses discharged
surviving for over a year. Recently a DH diagnosed during
abdominal exploration was successfully repaired, three weeks
later, in the standing horse, using thoracoscopy (13).
Although the thoracic approach to DH repair is described
in the literature, the conventional approach is typically ab-
dominal. Hart and Brown (6) described repairing nine DHs
via an abdominal incision, and Romero and Rodgerson (2)
reported repairing DH in 14 horses using the same approach.
In that study, fve of the horses that underwent surgery had
DH corrected by means of a polypropylene mesh applied by
direct suturing or using a hand stapling device. Four of these
horses did not survive to discharge. Tis may indicate low
chances of success in correcting DH using a mesh through
an abdominal approach. Case #4 in our series was treated by
direct suturing through the abdomen and failed immediately
after surgery. Tis defect was neither large nor dorsal but still
failed. It may further support the inferiority of the abdominal
approach for repairing DH.
Te thoracoscopy technique used by Röcken et al. (13)
agrees with our elected thoracic approach, although the
techniques used and the type of lesion was diferent. In that
report, an endoscope was used, while in case #3; in the cur-
rent report; an open approach was used performed by rib
resection. Furthermore, in that study the defect was corrected
using interrupted sutures; conversely, in our case the defect
was corrected with mesh and staples. All the above supports
our clinical impression that approaching the DH through the
thorax is preferable. Toracoscopy may prove the method of
choice for repairing DH in the future since it provides the
best access to the lesion and is minimally invasive.
By and large the surgical success rate for equine DH
repair remains low. In Romero and Rodgerson’s report
(2), 25 horses with DH underwent surgery, but only eight
survived to discharge. Similar results were reported by Hart
and Brown (6), in a population of 26 horses that underwent
surgery, seven were discharged from hospital, and two of
them died within the frst year. Te same study showed that
horses with dorsally located rents, which were 10 cm or more
in diameter, had the worst prognosis with 92% of them being
euthanized; while rents located ventrally and less than 10
cm in diameter were associated with a far better success rate
(63%). In the current study, however the only survivor had
a large and dorsal tear. No conclusions, with regards to the
efect of lesion location and size on prognosis, can be drawn
from this single case. Nevertheless, thoracic approach may
enable repair of tears that are irreparable by the traditional
abdominal approach and thus improve the overall survival
rate.
In this study, 25% of the horses with DH survived, which
is a similar survival rate to other reports (2). Two cases were
related to parturition, one in the dam and the other in the neo-
nate emphasizing that Mares and foals should be monitored
for clinical signs related to DH. Te thoracic approach proved
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Efraim, G. 44
useful in the current report as well as in a recent study. In
conclusion, earlier diagnosis and improved surgical techniques
may improve the survival rate of horses sufering from DH.
ACKNOWLEDGEMENTS
Te authors are grateful to the referring veterinarians and the
dedicated hospital staf without whom these difcult cases could
not have been managed.
REFERENCES
1. Kelmer, G., Kramer, J. and Wilson, D. A.: Diaphragmatic hernia:
etiology, clinical presentation, and diagnosis. Compend. Equine.
3:28-36, 2008.
2. Romero, A. E. and Rodgerson, D. H.: Diaphragmatic herniation
in the horse: 31 cases from 2001-2006. Can. Vet. J. 51:1247-1250,
2010.
3. Barker, I. K.: Te peritoeneum and retroperitoenum. In: Jubb, K.
V. F, Kennedy, P. C. and Palmar, N. (Eds.): Pathophysiology of
domestic animals. 4th ed. Academic Press, San Diego, pp. 425-
428, 1993.
4. Pauwels, F. F., Hawkins, J. F., McHarg, M.A., Rothenbuhler, R.
D., Baird, D. K. and Moulton, J. S.: Congenital retrosternal (Mor-
gagni) diaphragmatic hernias in three horses. J. Am. Vet. Med.
Assoc. 231:427-432, 2007
5. Schambourg, M. A., Laverty, S., Mullim, S., Fogarty, U.M. and
Halley, J.: Toracic trauma in foals: post mortem fndings. Equine
Vet. J. 35:78-81, 2003.
6. Hart, S. K. and Brown, J. A.: Diaphragmatic hernia in horses: 44
cases (1986-2006). J. Vet. Emerg. Crit. Care. 19:357-362, 2009.
7. Speirs, V. C. and Reynolds., W. T.: Successful repair of a diaphrag-
matic hernia in a foal. Equine Vet. J. 8:170-172, 1976.
8. Kelmer, G., Kramer, J. and Wilson, D. A.: Diaphragmatic hernia:
treatment, complications, and prognosis. Compend. Equine 3:37-
46, 2008.
9. Collier D.S.: Comparative aspects of diaphragmatic hernia. Equine
Vet. Edu. 31:358-359, 1999.
10. Mair T.S., Smith L.J.: Survival and complication rates in 300
horses undergoing surgical treatment of colic. Part 1: Short-term
survival following a single laparotomy. Equine Vet. J. 37:296-302,
2005.
11. Tapio, H., Hewetson, M. and Sihvo, H. K.: An unusual cause of
colic in a neonatal foal. Equine Vet. Edu. 24:334-339, 2012.
12. Goehring, L. S., Goodrich, L. R.and Murray, M.J.: Tachypnoea
associated with a diaphragmatic tear in a horse. Equine Vet. J.
31:443-445, 1999.
13. Röcken, M., Mosel, G., Barske, K. and Witte, T. S.: Toracoscopic
diaphragmatic hernia repair in a warmblood mare. Vet. Surg.
42:591-594, 2013.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 45 Enrofoxacin in Tortoises
SHORT COMMUNICATION
Fluoroquinolone antibiotics belong to a group of synthetic
antimicrobials that are widely used in veterinary medicine.
Teir spectrum of activity includes Gram positive, Gram neg-
ative and Mycoplasma species responsible for a vast array of
pulmonary, urinary and digestive infections (1). Enrofoxacin
is a prototypical fuoroquinolone, showing treatment efcacy
for the major bacterial conditions in several animal species
(2, 3). Te enhanced efcacy demonstrated by enrofoxacin
is due to the formation of an active metabolite, ciprofoxacin
which exhibits potency and spectrum of activity similar
to that of the parental drug (1). Te pharmacokinetics of
enrofoxacin following various routes of administration have
been investigated in diferent species of turtles and tortoises
with plasma concentrations of enrofoxacin and ciprofoxacin
showing wide disposition variability among the species (3-
5). Considering tortoise species are more closely related to
one another than to other orders of animals, this underlines
the importance of conducting pharmacokinetic studies for
individual species rather than extrapolating doses from data
generated in other reptile species (6).
Te treatment of bacterial infections should be based on
Pharmacokinetics of Enrofoxacin and its Metabolite Ciprofoxacin
after Intracoelomic administration in Tortoises (Testudo hermanni)
Salvadori, M.,
1
De Vito, V.,
2
Owen, H.
3
and Giorgi, M.
2
*
1
ExoticVet Veterinary Center, Via U. Dini 157, San Giuliano Terme, Pisa, Italy.
2
Department of Veterinary Sciences, University of Pisa, Via Livornese (lato monte) 1, San Piero a Grado, 56122 Pisa, Italy.
3
School of Veterinary Science, Te University of Queensland, Gatton Campus, Gatton, Queensland 4343, Australia.
*
Corresponding Author: Prof. Mario Giorgi, Chem D., Spec. Pharmacol., Department of Veterinary Sciences, University of Pisa, Via Livornese (lato monte),
San Piero a Grado (Pisa), Italy. Email: mario.giorgi@unipi.it
ABSTRACT
Enrofoxacin belongs to the fuoroquinolone class of antibiotics. It is commonly used in a variety of reptile
species due to its wide spectrum of efcacy, partly due to its formation of an active metabolite ciprofoxacin.
Enrofoxacin shows wide disposition variability among all species resulting in large diferences in the
plasma concentrations of both enrofoxacin and ciprofoxacin. Te aim of this study was to evaluate the
pharmacokinetics of enrofoxacin and ciprofoxacin after a single intracoelomic injection of 10 mg/kg of
enrofoxacin in 9 tortoises (Testudo hermanni). Blood samples were collected at 0, 0.5, 2, 4, 10, 24, 48,
72, 96, 120, 144, 168, 192, 216, 240 and 264 h and analyzed using a validated high performance liquid
chromatography (HPLC) forescence method. Plasma concentrations of enrofoxacin were quantifable
in all subjects for up to 240 h, while ciprofoxacin was detected in all subjects up to 120 h. Te C
max
(s) of
enrofoxacin and ciprofoxacin were 8614 ± 1116 ηg/mL obtained at 2.19 h and 605 ± 43 ηg/mL obtained
at 4.23 h, respectively. Te values of C
max
/MIC ratio and AUC
0-24
/MIC ratio of enrofoxacin with a MIC
value of 0.5 µg/mL were 17.23 and 132.78, respectively. In conclusion, an administration of 10 mg/kg
of enrofoxacin via the intracoelomic route in Hermann’s tortoises produced optimal pharmacodynamic
parameters.
Keywords: Enrofoxacin; Ciprofoxacin; Pharmacokinetics; Intracoelomic Administration;
Testudo hermanni Tortoises.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Salvadori, M. 46
a rational scientifc approach. Te most common pharma-
cokinetic/pharmacodynamic (PK/PD) approach for anti-
microbial agents uses plasma concentration as the PK input
value and minimum inhibitory concentration (MIC) as the
PD input value (7). Fluoroquinolones are considered to be
well-tolerated drugs in both humans and animals, however,
their intensive use has led to a signifcant increase in antimi-
crobial resistance (1). Terefore, several PK/PD indices such
as C
max
/MIC and AUC
0-24
/MIC have been included in the
present study to evaluate the clinical efcacy of enrofoxacin.
Te aim of this study was to evaluate the PK of enrofoxa-
cin and its metabolite ciprofoxacin in Testudo hermanni after
a single intracoelomic injection of 10 mg/kg enrofoxacin, and
to establish if 10 mg/kg is an optimal dosage for treatment
of diferent bacterial infections.
Nine tortoises of undetermined age, including both sexes
(fve males and four females), with a body weight range from
0.4 to 2.95 kg, were used. Te tortoises were housed indoors,
divided equally into three glass containers, with access to
indirect sun light and heat lamps (UVB 5%). Animals were
maintained at 30 to 33° C with 250-W infrared heat lamps
suspended 0.5 m above the foor to allow turtles to regulate
their own body temperature. Tortoises were conditioned
for a 2-week period prior the commencement of the study.
Tortoises were judged to be in good health based on physi-
cal examinations, normal activity, and routine acceptance of
food. Tese observations were made by specialized veterinary
personnel. All the tortoises were fed a mixture of vegetables,
and given access to fresh water ad libitum.
Animal care and handling was performed according to
the provision of the EC council Directive 86/609 EEC. Te
study protocol was approved by the University of Pisa’s ethics
committee for animal welfare (CEASA) and transmitted to
the Italian Ministry of Health.
Enrofoxacin as the commercial injectable solution
(Enrovet®
25mg/mL, Bio98, Milan Italy), was diluted with
saline to 10 mg/mL and given as a 10 mg/kg bolus by intra-
coelomic injection in the left prefemoral fossa using a sterile
22-gauge, 3.75-cm needle. Te dose used in the present study
was selected based on previous studies in turtles (3, 5, 8). Te
drug was diluted because a previous study demonstrated that
a 10 mg/kg intracoelomic injection of 10 mg/mL in yellow
bellied slider turtles did not cause local irritation and soft
tissue necrosis (5). Tese changes did occur when the same
dose was used at higher concentrations (25 mg/mL) (9-10).
Blood samples (0.5 mL or 0.25 mL in subjects greater than
or less than 0.5 kg body weight, respectively) were collected
from the subcarapacial venipuncture site at 0, 0.5, 2, 4, 10,
24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 h after
enrofoxacin administration. Although subcarapacial blood
collection could be considered suboptimal because of po-
tential lymph contamination, enrofoxacin has been reported
to be equally distributed in blood and lymph (7), and thus
the pharmacokinetic data were not expected to be afected
by sampling method. Te blood samples were immediately
transferred to tubes containing heparin, centrifuged and
stored at -20° C until they were analyzed. Sample analysis
was completed within 30 days of collection. Te analytical
method was based on a previous method using high perfor-
mance liquid chromatography (HPLC) with a fuorescence
detector (5). Pharmacokinetic analysis of enrofoxacin and
ciprofoxacin was performed using WinNonlin 5.3.1 software
program according to a non-compartmental model.
No adverse efects at the point of injection and no be-
havioural or health alterations were observed in the animals
during or after the study. Some transient, self-resolving side
efects such as uncoordinated movements were noticed in an
earlier study (5) in yellow bellied slider turtles. Species dif-
ferences might have triggered this distinction in side efects.
Blood levels of enrofoxacin were quantifed in all
subjects up to 240 h following injection. Blood levels of
ciprofoxacin were detected in all subjects up to 120 h. Te
semi-logarithmic blood concentration vs. time average curves
for enrofoxacin and ciprofoxacin are reported in Figure 1.
Te pharmacokinetic parameters are reported in Table 1.
Te mean maximum blood concentration of enrofoxacin
(C
max
8614.64 ± 1116.36 ηg/mL) was reached at 2.19 h. Tis
value, if normalized for the dose, was within the range of peak
concentrations shown in previous studies on intramuscular
injection of 5 mg/kg enrofoxacin in Gopher tortoises (2.4 µg/
mL) (4) and Indian star tortoises (3.59 µg/mL) (11). Similar
trends were noticed also in the ciprofoxacin concentration.
Te C
max
of ciprofoxacin was 605.16 ± 43.04 ηg/mL attained
at 4.23 h. Tis value was comparable to that shown in Indian
star tortoises (0.35 µg/mL) after intramuscular injection of
5 mg/kg enrofoxacin, if normalized for the dose (11), but
higher than that shown in yellow-bellied slider turtles (0.32
µg/mL) following an intracoelomic injection of 10 mg/kg
enrofoxacin (5). Te values of apparent terminal half-life
(T
1/2
λz) of enrofoxacin and ciprofoxacin were 37.00 ± 11.97 h
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 47 Enrofoxacin in Tortoises
and 49.06 ± 5.82 h, respectively. T
1/2
λz of enrofoxacin was
longer than that reported in earlier studies: Gopher tortoises
(23.1 h) (4) and Indian star tortoises (5.1 h) (11). Tis dif-
ference might be due to the diferent routes of administra-
tion (intracoelomic vs. intramuscular) and the previously
mentioned wide variability in pharmacokinetic parameters
among tortoise species. In agreement with this speculation, a
recent study involving a 10 mg/kg intracoelomic injection of
enrofoxacin in yellow-bellied slider turtles (5) has shown that
intracoelomic administration signifcantly increased the drug
T
1/2
λz compared to the same parameter after intramuscular
and oral administration in red-eared slider turtles (8) and after
oral administration in Loggerhead sea turtles (3).
Te reported MICs of enrofoxacin for most susceptible
Gram negative, Gram positive bacteria and Mycoplasma
isolated from domestic animals were < 0.1 µg/mL, with some
additional moderately susceptible isolates having MICs of
0.125-0.5 µg/mL (4). C
max
/MIC ratio > 10 and AUC
0-24
/
MIC ratio of 100 and 125, are required for fuoroquinolones
to have antibiotic activity and to limit the development of
bacterial resistance, respectively (7, 12-13). In the present
study, considering a bacterium with a MIC value of 0.5 µg/
mL, the C
max
/MIC ratio of enrofoxacin was 17.23 and
the average AUC
0-24
/MIC ratio was higher (132.78) than
the required safety value. In contrast, C
max
/MIC ratio and
AUC
0-24
/MIC ratio of ciprofoxacin were below the target
ranges. Tese results could be due to the limited extent
to which ciprofoxacin is produced in reptiles (<15%), as
compared to mammals (35%) (14). Tis fnding is in line
with the low contribution of ciprofoxacin shown in reptiles
(5,8). It has been postulated that this minimal presence of
ciprofoxacin could be due to the slow metabolism of turtles
and tortoises. In fact, cytochrome P450 3A, the enzyme that
metabolizes enrofoxacin to ciprofoxacin, has been found to
be poorly expressed in reptiles and fsh (15-16).
In conclusion, the plasma concentrations of enrofoxacin
achieved in this study after intracoelomic administration
of 10 mg/kg enrofoxacin are adequate to reach the target
end points associated with efcacy of fuoroquinolones in
tortoises (Testudo hermanni).
CONFLI CT OF I NTEREST STATEMENT
None of the authors of this paper have a fnancial or personal
relationship with other people or organizations that could inap-
propriately infuence or bias the content of the paper.
ACKNOWLEDGMENTS
Tis work was supported by funds of University of Pisa (Athenaeum
ex 60%). Any external funding did not support the preparation of
manuscript.
Figure 1: Mean semi-logarithm plasma concentrations of enrofoxacin
(-○-) and ciprofoxacin (-●-) vs time curves following intracoelomic
injection of enrofoxacin (10 mg/kg) in tortoises (n=9). Bars represent
the standard deviations.
Table 1: Pharmacokinetic parameters of enrofoxacin and ciprofoxacin
after 10 mg/kg enrofoxacin intracoelomic injection in tortoises
(Testudo hermani) (n=9)
Enrofoxacin Ciprofoxacin
Parameter Units Mean SD Mean SD
r
2
0.99 ± 0.01 0.97 ± 0.01
λz 1/hr 0.02 ± 0.03 0.01 ± 0.01
T
1/2
λz
hr 37.00 ± 11.97 49.06 ± 5.82
T
max
hr 2.19 ± 0.58 4.23 ± 0.93
C
max
ng/mL 8614 ± 1116 605.16 ± 43.04
AUC
0-24
hr*ng/mL 66388 ± 4647 7952 ± 318
AUC
0-∞
hr*ng/mL 102123 ± 9476 12835 ± 1244
Vz/F mL/kg 5227 ± 926 55140 ± 443
CL/F mL/hr/kg 97.92 ± 19.98 779.08 ± 88.17
AUMC
0-∞
hr*hr*ng/mL 3383764 ± 42011 412688 ± 58959
MRT
0-∞
hr 33.13 ± 1.69 32.15 ± 1.28
r
2
= correlation coefcient.
λz = terminal phase rate constant.
T
1/2
λz = terminal half-life.
T
max
= time of peak.
C
max
= peak plasma
concentration.
Vz/F = apparent volume of
distribution.
CL/F = apparent clearance.
AUC
0-24
= area under the
plasma concentration-time
from 0-24 h curve.
AUC
0-∞
= area under the plasma
concentration-time from 0 h to
infnity curve.
AUMC
0-∞
= area under the frst
moment curve.
MRT
0-∞
= mean resident time.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Salvadori, M. 48
REFERENCES
1. Giguere, S. and Dowling, P.M.: Fluoroquinolones. In Giguere, S.,
Prescott, J.F. and Dowling, P.M. (Eds.). Antimicrobial therapy in
veterinary medicine. Wiley Blackwell, Oxford UK, pp. 295-314,
2013.
2. Lizodo, M., Pons, M., Gallardo, M. and Estelrich, J.: Phys-
icochemical properties of enrofoxacin. J. Pharm. Biomed. Anal.
15:1845-1849, 1997.
3. Jacobson, E., Gronwall, R., Maxwell, L., Merrit, K. and Harman,
G.: Plasma concentrations of enrofoxacin after single-dose oral
administration in loggerhead sea turtles (Caretta caretta). J. Zoo
Wildlife Med. 36:628-634, 2005.
4. Prezant, R.M., Isaza, R. and Jacobson, E.R.: Plasma concentra-
tions and disposition kinetics of enrofoxacin in Gopher tortoises
(Gopherus polyphemus). J. Zoo Wildl. Med. 25:82-87, 1994.
5. Giorgi, M., Rota, S., Giorgi, T., Capasso, M. and Briganti, A.:
Blood concentrations of enrofoxacin and the metabolite cipro-
foxacin in yellow-bellied slider turtles (Trachemys scripta scripta)
after a single intracoelomic injection of enrofoxacin. J. Exotic Pet.
Med. 22:192-199, 2013.
6. Isazar, R. and Jacobson, E.R.: Antimicrobial drug use in reptiles. In
Giguere, S., Prescott, J.F. and Dowling, P.M. (Eds.). Antimicrobial
therapy in veterinary medicine. Wiley Blackwell, Oxford UK, pp.
623-636, 2013.
7. Liu, P., Müller, M. and Derendorf, H.: Rational dosing of antibiot-
ics: the use of plasma concentrations versus tissue concentrations.
Int. J. Antimicrob. Agents19. :285-290, 2002.
8. James, S.B., Calle, P.P., Raphael, B.L., Papich, M., Breheny, J. and
Cook, R.A.: Comparison of injectable versus oral enrofoxacin
pharmacokinetics in red-eared slider turtles, Trachemys scripta
elegans. J. Herpetol. Med. Surg. 13:5-10, 2003.
9. Maxwell, L. K. and Jacobson, E. R.: Preliminary single-dose
pharmacokinetics of enrofoxacin after oral and intramuscular
administration in green iguanas (Iguana iguana). Houston, TX:
Proc. Am. Assoc. ZooVet. 25, 1997.
10. Young, L.A.,Schumacher, J., Papich, M.G. and Jacobson, E.R.:
Disposition of enrofoxacin and its metabolite ciprofoxacin after
IM injection in juvenile Burmese pythons (Python molurus bivit-
tatus). J. Zoo Wildl. Med. 28:71-79, 1997.
11. Raphael, B. L., Papich, M. and Cook, R.A.: Pharmacokinetics of
enrofoxacinafter a single intramuscular injection in Indian star
tortoises (Geochelone elegans). J. Zoo Wildl. Med. 25: 88-94, 1994.
12. Mckellar Q. A., Sanchez Bruni, S.F. and Jones D.G.: Pharmacoki-
netic/pharmacodynamic relationships of antimicrobial drugs used
in veterinary medicine. J. Vet. Pharmacol. Ter. 27:503-514, 2004.
13. Wright, D.H., Brown, G. H., Peterson, M. L. and Rotschafer, J.C.:
Application of fuoroquinolone pharmacodynamics. J. Antimicrob.
Chemother. 46:669-683, 2000.
14. Reo, G. S., Ramesh, S., and Ahmad, A.H.: Pharmacokinetics of
enrofoxacin and its metabolite ciprofoxacin after intramuscu-
lar administration of enrofoxacin in goats. Vet. Res. Commun.
25:197-204, 2001.
15. Ertl, R.P. and Winston, G.W.: Te microsomal mixed function
oxidase system of amphibians and reptiles: components, activities
and induction. Comp. Biochem. Physiol. C Pharmacol. 121:85-
105, 1998.
16. Vaccaro, E., Giorgi, M., Longo, V., Mengozzi, G. and Gervasi
P.G.: Inhibition of cytochrome P450 enzymes by enrofloxacin in
the sea bass (Dicentrarchuslabrax). Aquatic Toxicol. 62:27-33, 2003.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 49 Equine Acute Pancreatitis
INTRODUCTION
Acute pancreatitis is rarely diagnosed in horses and the true
prevalence of the disease is probably under estimated appear-
ing less commonly than in other species (1, 2, 3). It appears to
be much less commonly reported in horses than in other spe-
cies (1). Both acute pancreatitis and chronic pancreatitis have
been documented. Acute pancreatitis is usually associated
with severe acute colic, often characterized as an acute small
intestinal obstruction with signifcant refux and peritonitis.
Chronic pancreatitis tends to cause inappetance, weight loss,
lethargy and mild recurrent signs of colic (1, 2). Previous
studies have identifed horses with gastric distention and/or
rupture with acute pancreatitis (1, 3, 4, 5).
Pancreatitis has been reported to occur in adult horses
although cases of pancreatitis have been described in foals at
post mortem (4, 5, 6, 7). Ante-mortem diagnosis is difcult
on the basis of clinical and laboratory fndings. No specifc
diagnostic tests are available and although reference values
for serum and peritoneal fuid amylase and lipase activities
have been published, their diagnostic accuracy has not been
established (4, 8). Furthermore, trypsin is produced in small
amounts by the equine pancreas (4).
Clinical signs are non-specifc; the most important are
signs of colic, gastrointestinal refux and shock (2-5). Te
abdominal pain originates from gastric distention, peritonitis
and hemoperitoneum (9). Tus the lack of specifc clinical
pathological parameters and clinical signs makes the clinical
diagnosis of equine pancreatitis a challenge in the live horse.
In dogs, cats and humans pancreatitis is associated with
nutritional imbalance, abdominal trauma, hypercalcemia,
hyperlipidemia, drug induced, bacterial and viral infection,
vascular impairment, cholecystitis, small intestinal obstruc-
tion and duodenal refux (9, 10). Etiology in horses includes
partial or complete destruction of the pancreatic duct (chol-
angiohepatitis and cholethiasis), migration of Strongylus
equinus and Parascaris equorum, duodenitis, duodenal ulcers
and possible vasculitis in foals and associated with other
disorders, commonly those involving the gastrointestinal
tract or liver (4, 10, 11). Migration of Strongylus equinus and
Parascaris equorum to the pancreas can produce pancreatic tis-
Acute Pancreatitis in a Horse – a Case Report
Edery, N.,
1
Rosenbaum, A.,
2
Busnach, A.,
1
Steinman, A.,
2
Tirosh Levy, S.
2
and Perl, S.
1
*
1
Department of Pathology, Kimron Veterinary Institute, Beit Dagan, Israel.
2
Koret School of Veterinary Medicine - Veterinary Teaching Hospital, Te Robert H. Smith Faculty of Agriculture, Food and
Environment, Te Hebrew University of Jerusalem, Rehovot, Israel.
*
Corresponding Author: Prof. S. Perl, Department of Pathology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel. Email: perls@moag.gov.il
ABSTRACT
Tis report presents a case of acute pancreatitis in a 30 year old local breed horse. Te horse was diagnosed
clinically with severe acute abdominal pain, distended small intestine, a left dorsal large colon displacement
and large colon impaction. On post mortem examination pathological changes in the pancreas were observed
without intestinal impaction. Histopathologically, the pancreatic lesions were diagnosed as acute pancreatitis
with peripancreatic fat necrosis. In addition to these fndings, multifocal necrotizing hepatitis was identifed
as well as a mild interstitial nephritis and tubular nephrosis. Tis case demonstrates the difculty in making
a clinical diagnosis of pancreatitis in a horse and the importance of a thorough macroscopic and histological
evaluation of the pancreas in horses with a history of abdominal pain.
Keywords: Horse; Colic; Abdomen; Impaction; Pancreas; Pancreatitis; Hepatitis.
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Edery, N. 50
sue destruction and extensive fbrosis (12). Histopathology of
acute pancreatitis includes large numbers of neutrophils that
infltrate the pancreatic parenchyma and percolate between
the intralobular septae and acini, the peripancreatic fat shows
evidence of necrosis. Histopathology of chronic pancreatitis
is indicated by marked fbrosis between acinar lobuli and
surrounding the pancreatic ducts. Interstitial mononuclear
cell infltrate may be present (4).
Tis case study describes an adult horse with pancre-
atitis diagnosed at post mortem examination while clini-
cally as a case of severe acute colic that did not respond to
analgesic medication and required abdominal emergency
surgery. Te case illustrates the difculty in the diagnosis
of equine pancreatitis in the clinical situation and proposes
the consideration of pancreatitis as a diferential diagnosis
under conditions of colic of unknown origin.
CASE HISTORY
Clinical history
A 30-year-old local breed horse with an acute abdomen
was admitted to the Koret School of Veterinary Medicine -
Veterinary Teaching Hospital (KSVM-VTH). On physical
examination before referral, the horses demonstrated severe
abdominal pain even after NSAID (fumixin meglumine)
administration and had elevated heart rate (60 beats/minute).
Nasogastric intubation was performed by the referring vet-
erinarian and resulted in spontaneous refux of approximately
25 L of fuid.
Rectal examination revealed swollen small intestines,
large colon displacement and large colon impaction. Prior
to referral the horse received butorphanol, medetomidine
and xylazine. On arrival the horse was dehydrated (dry mucus
membranes and decreased skin turgor). Heart rate was 72/
minute. Passage of a nasogastric tube resulted in 4 liter refux.
Rectal examination at the hospital diagnosed a left dorsal
displacement of the colon and large colon impaction.
Te packed cell volume was 53% (Reference interval (RI)
32-52%), total solids were 8.6 g/dL (RI 5.3-7.9 g/dL). creati-
nine 4.54 mg/dL (RI 0.9-2.0mg/dL) and lactate 7.6 mmol/L
(RI up to 2mmol/L). Te horse was prepared for exploratory
laparotomy, while showing continuous and unrelenting pain.
It was therefore treated with xylazine (Vetmarket, Shoham,
Israel) and butorphanol (Alvegesic, Dachra Veterianary
ptroducts, Shropshire, UK). While preparing the horse, signs
of shock were evident: mean arterial pressure of 20 mm Hg,
weak pulse, disrupted ECG and cyanotic mucous mem-
branes. Prior to induction after consulting with the owners,
the horse was euthanized at the request of the owners due
to the poor prognosis.
Post mortem examination
Te horse was sent for a full post mortem examination. Post
mortem examination revealed slightly collapsed lungs and
fbrous tags on the abdominal surface of the diaphragm. Te
stomach contained sparse fuid content. Te pancreas was
edematous and with multiple white yellowish foci (Figure
1). Te small intestine showed multifocal serosal hemorrhage
for almost its entire length. Te large intestine contained dry
content without pathological lesions in the mucosa. Te liver
and kidney showed no macroscopic pathological changes.
Tere was no evidence of an impaction.
Histopathology
Histopathological examination of the intestines showed en-
gorgement of blood vessels and multifocal serosal hemorrhage
of the small intestine with difuse submucosal edema. In the
pancreatic tissue, multifocal to difuse infltrations by infam-
matory cell mainly neutrophils and histiocytes were present,
as well areas of peripancreatic fat necrosis (Figure 2 and 3). In
some areas intralesinal bacterial colonies could be seen (Figure
4). A few of the kidney’s tubules were distended and contained
an amorphus eosinophilic material. Numerous interstitial
Figure 1: Pancreas: Edema and multiple white yellowish foci of necrosis.
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 51 Equine Acute Pancreatitis
foci of infammatory mononuclear cells were evident in the
parenchyma. Difuse congestion was present mostly in cortex.
Te liver showed multifocal areas of necrosis and infltrations
with mainly neutrophils. Difuse congestion was also present.
DISCUSSION
Pancreatitis in horses can be a primary condition or may be
associated with other disorders, commonly those involving the
gastrointestinal tract or liver (4). Pancreatitis may be under
diagnosed owing to the presence of nonspecifc abdominal pain.
In this case report, it is unclear and difcult to determine
whether the horse sufered from primary pancreatitis or sec-
ondary pancreatitis associated with hepatitis. Te case history
(pain duration, other symptoms, appetite, etc.) is lacking and
in addition, blood tests including complete blood count and
chemistry are also absent. Laboratory abnormalities associ-
ated with acute pancreatitis include increased activities of
serum (and sometimes peritoneal) amylase and lipase (6).
Te histopathological fnding of multifocal necrotizing
hepatitis has many etiologies: Causes of hepatitis in horses
include serum hepatitis, cholangiohepatitis and chronic active
hepatitis with occasional cases of hematogenous bacterial
hepatitis, abscesses, viral hepatitis, parasitism and chronic
infltrative infammatory disease (7).
It has previously been hypothesized that horses with
enteritis or strangulated small intestinal lesions may develop
acute pancreatitis and hepatitis as a result of ascending infux
of intestinal fuid through the pancreatic and bile duct, with
subsequent activation of pancreatic enzymes (4, 6, 10, 11).
Once activated, these enzymes are responsible for autodiges-
tion of pancreatic tissue, resulting in necrosis of the acini and
pancreatic islets with interstitial fat necrosis and necrotizing
vasculitis. Te release of pancreatic enzymes stimulates the
production of infammatory cytokines, which, in turn triggers
an infammatory cascade which leads to a systemic infamma-
tory response syndrome (SIRS), multiple organ dysfunction
syndrome (MODS), shock and death (4, 8).
Large colon disorders such as volvulus and displace-
Figure 4: Pancreatic necrosis, infltration with infammatory cells and
bacterial colonies. x10
Figure 2: Pancreas: Interlobular edema and infltration with mainly
neutrophils and histiocytes. x20
Figure 3: Pancreas: Peripancreatic fat necrosis and multifocal
infltration with infammatory cells. x20
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Edery, N. 52
ments could cause reduced blood fow to the pancreas and
induce pancreatitis (4). In this case report, the rectal exami-
nation diagnosed large colon displacement and obstruction,
but this was not verifed in post mortem examination (5).
Similarly to previous cases, this horse had distended small
intestines and large amount of gastric refux before refer-
ral, signs that are sometimes encountered in acute cases of
pancreatitis (1, 4, 5).
In conclusion, pancreatitis should be considered in horses
with unexplained moderate to severe abdominal pain with or
without gastric refux. Although the ante-mortem diagnosis
of acute pancreatitis in the horse is difcult, it should be
included as a diferential diagnosis. Results from this case
study emphasize the importance of a pathological and his-
topathological evaluation of the pancreas in horses with a
history of abdominal pain.
REFERENCES
1. Johnson, P.J., Weidemyer, C.E. and Messer, N.T.: Conditions of
the equine pancreas. Equine Vet. Educ. 21: 26-29, 2009.
2. Bakos, Z., Krajcsovics, L. and Toth, J.: Successful medical treat-
ment of acute pancreatitis in a horse. Vet. Rec. 162: 95-6, 2008.
3. Rose, R. J.and Hodgson, D.R.: Pancreatic Diseases, in Manual of
Equine Practice. pp. 585-586, W.B. Saunders, 2000.
4. Dacre, K.J., Pirie, R.S., Scudamore, C. and Prince, D.R.: Hyperli-
paemia and pancreatitis in a pony with cushing’s disease. Equine
Vet. Educ. 15: 175-178, 2003.
5. Yamout, S.Z., Nieto, J.E., Anderson, J., De Cock, H.E., Vapniarsky,
N. and Aleman, M.: Pathological evidence of pancreatitis in 43
horses (1986-2011). Equine Vet. J. Suppl.: 45-50, 2012.
6. Lilley, C. and Beeman, G.: Gastric dilatation associated with
necrotizing pancreatitis. Equine Prac. 3: 8-15, 1983.
7. Schmidt A., W.O., Kaufmann S.H.E.: . Comparative hepatitis.
Birkauser advances in infection diseases: pp. 245-264, 2008.
8. Buote, M.: Cholangiohepatitis and pancreatitis secondary to severe
gastroduodenal ulceration in a foal. Can. Vet. J. 44: 746-8, 2003.
9. Savage, C.J.: Diseases of the Pancreas, in Equine Medicine and
Surgery. pp. 833-938, 1999.
10. Taintor, J., Sartin, E.A., Waldridge, B.M. and Schumacher, J.:
Acute pancreatitis in a 3-day-old foal. J. Vet. Intern. Med. 20:
210-202, 2006.
11. Hardy, R.: Infammatory pancreatic disease, in Veterinary Gastro-
enterology. Anderson, N. Editor. pp. 275-283, 1980.
12. Hamir, A.N.: Verminous pancreatitis in a horse. Vet. Rec. 121:
301-302, 1987.
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 53 Neosporosis and Hepatozoonosis in Pups
INTRODUCTION
Neospora caninum, an apicomplexa protozoan from the family
Sarcocystidae is the causative agent of abortion in cattle and
neuromuscular diseases in dogs (1). Te defnitive hosts of
N. caninum are domestic dogs and wild canids. Dogs may be
infected by N. caninum horizontally by consumption or verti-
cally by in utero transmission of tachyzoites during pregnancy
(1, 2). In naturally infected dogs and cattle the predominant
route of infection is considered to be transplacental (1, 2).
Hepatozoon canis is an arthropod-borne infectious agent
of the apicomplexan protozoa from the family Hepatozoidae
in the suborder Adeleorin. H. canis transmission takes place by
ingestion by the intermediate host, the dog, of the brown dog
tick Rhipicephalus sanguineus, the defnitive host that contains
mature oocysts (3). Most domestic dogs probably become
infected with H. canis through grooming ticks from their
hair coat. Also like other apicomplexan parasites horizontal
transmission through the uterus from dam to its ofspring
has been demonstrated to take place (3).
Concurrent Neosporosis and Hepatozoonosis in a Litter of Pups
Mazuz, L.M.,
1, 2
Wolkomirsky, R.,
1
Sherman A.,
3
Savitzsky, I.,
1
Waner, T.,
4
Golenser,
J.
2
and Shkap, V.
1
1
Division of Parasitology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel.
2
Department of Microbiology and Molecular Genetics, Te Kuvin Centre for the Study of Infectious and Tropical Diseases,
the Hebrew University of Jerusalem, Jerusalem, Israel.
3
HaMercaz HaVeterinary BaNegev, Rehov HaShomron 62, Beer Sheva, Israel.
4
Veterinary Clinic, 9 Meginay Hagalil Street, 76200 Rehovot, Israel.
*
Corresponding Author: Dr. Monica Mazuz, Division of Parasitology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel. Email: monica@int.gov.il
ABSTRACT
Tis case report describes a concomitant infection in a litter of puppies with two apicomplexan protozoa
Neospora caninum and Hepatazoon canis. Te diferent potential routes of infection are discussed along with a
description of the course of the disease. A private practitioner in the South of Israel (Beer Sheva) submitted
blood samples for serological testing for toxoplasmosis and neosporosis from a pup of 6 weeks of age (from a
liter of 6 pups) showing neurological symptoms. Te results were seropostive for N. caninum and seronegative
for toxoplasmosis. After 3 weeks another pup also started to develop neurological signs. Samples including
blood smears, serum samples for serology for Neospora and Toxoplasma and fecal samples from all the pups.
Out of the 6 pups tested four were found seropositive for Neospora while all pups were seronegative for
Toxoplasma. Of the six pups, four were found to be infected with Heptaozoon canis, three of which were co-
infected with N. caninum. One pup was infected with only H. canis and another pup with only N. caninum and
a third pup was found to be negative for both H. canis and N. caninum. Treatment consisted of trimethoprim
sulfadiazine and clindamycin which were administered for six weeks. Te pups recovered completely, with
the exception of paresis of the left hind limb in the frst diagnosed puppy. An important conclusion from
this case report is the need to test all pups in a litter for N. caninum where even only one pup shows clinical
signs. Furthermore treatment at an early age and for a prolonged period of time appeared to be successful in
preventing the progression of the clinical signs. To the best knowledge of the authors this is the frst natural
concomitant infection case of N. caninum and H. canis in a litter of puppies.
Keywords: Neospora caninum; Hepatazoon canis; Concurrent Infection; Dog; Pups.
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Mazuz, L.M. 54
Concurrent infections in dogs are reported to occur with
a variety of pathogens (3). Often they may be transmitted
by a common vector such as the tick. However co-infecting
pathogens seemingly unrelated may be involved, resulting in
clinical manifestations which may make the clinical diagno-
sis more complex. Te relationship between the concurrent
infections in this case involves two diseases of puppies, neo-
sporosis and hepatozoonosis, both of which can be transmit-
ted in utero. On the other hand, infection by one agent may
infuence the susceptibility to a new infection which may
infuence the progression of the existing condition.
Tis case report describes a concomitant infection in a
litter of puppies with two apicomplexan protozoa N. cani-
num and H. canis. Te diferent potential routes of infection
are discussed along with a description of the course of the
disease. To the best knowledge of the authors this is the frst
natural concomitant infection case of N. caninum and H. canis
in a litter of puppies.
CASE REPORT
A sample of serum was received from a private practitioner
from a pup named “Fistuk” of 6 weeks of age from a liter of
6 pups born in the city of Beer Sheva in the South of Israel.
Te pup showed neurological symptoms which in the opinion
of the practitioner resembled either that of toxoplasmosis or
neosporosis. Te clinical signs in the pup were that of paresis
of the left hind limb which appeared to be progressing to
the right hind limb. In addition the pup appeared to be both
fecally and urinarily incontinent.
Te serum sample was tested by the immunofuorescence
antibody test (IFAT) for both Toxoplasma gondii and Neospora
caninum. A pronounced high titer for were found for N. cani-
num (1:12,800) while no reaction to T. gondii were observed.
Te pup was treated orally with trimethoprim sulfadiazine
(Resprim, Teva Medical, Petah Tikva, Israel) and clindamycin
(Dalacin C, Pfzer Pharmaceutical, Israel). Two weeks later
the pup showed an improvement in the right hind limb but
not in the left hind limb.
When the pups reached 9 weeks of age, another pup,
named Puf, also started to develop neurological signs of
paresis in the left hind limb and furthermore had difculty
in rising. Samples including blood smears, serum samples for
serology for Neospora and Toxoplasma and fecal samples were
collected from all the pups. Out of the 6 pups tested four
were found seropositive for Neospora with high antibody titers
(Table 1) while all pups were seronegative for Toxoplasma. Of
the six pups, four were found to be infected with Heptaozoon
canis, diagnosed from examination of their blood smears and
three of which were co-infected with N. caninum. One pup
was infected with only H. canis and another pup with only N.
caninum and a third pup was found to be negative for both
H. canis and N. caninum. No oocysts, or other parasites were
found in fecal samples.
Clinical signs resembling those of neosporosis were only
seen in the two pups described above. Te other two pups
with hepatozoonosis and/or neosporosis did not appear to
exhibit any detectable clinical signs of disease.
Positive pups for either N. caninum or H. canis parasites
were treated per os with trimethoprim sulfadiazine (15mg/kg
q12h) (Resprim, Teva, Petach Tikva, Israel) and clindamycin
(10mg/kg q12h) (Dalacin, Pfzer) for six weeks (2). All pups
were found to be healthy after 6 weeks after treatment. Te
incontinent pup Fistuk improved although the left hind limb
remained paralytic. Te pup named Puf recovered completely.
No H. canis parasites were observed in the blood smears
Table 1: Antibody titer for Neospora caninum and the detection of Hepatozoon canis gamonts in a litter of 6 pups
Date 10-Oct-12 5-Nov-12 11-June-13
Name
Ab titers
N. caninum
Ab titers
N. caninum
Blood smear
Ab titers
N. caninum
Blood smear
Fanta n.d. 1:12800 Hepatozoon 1:12800 Negative
Pupa n.d. 1:12800 Negative 1:12800 Negative
Lipstick n.d. Negative Hepatozoon Not done Negative
Popcorn n.d. Negative Negative Not done Negative
Puf n.d. 1:12800 Hepatozoon 1:12800 Negative
Fistuk 1:12800 1:12800 Hepatozoon 1:12800 Negative
n.d. = Not done
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 55 Neosporosis and Hepatozoonosis in Pups
examined at the end of the six week treatment period. Te
four Neospora seropositive puppies continued with high
antibody titers.
DISCUSSION
Te predominant route of natural infection in dogs by
Neospora caninum is considered to be transplacental (1, 2). Te
bitch from this report from the south of Israel was adopted
by the current owner as a stray dog. It is suggested that free
roaming dogs in urban areas have a greater seroprevalence
for N. caninum than companion pets (4).
It has been reported that a variable number, but not all
pups in a litter from a dam infected with N. caninum may
have clinical manifestations (4, 5, 11). Indeed, in this litter
four of the six pups presented as infected, judged by sero-
logical investigation (IFAT) and of them only two showed
clinical signs at diferent intervals after birth. An important
conclusion from this study is the need to test all puppies in a
litter for N. caninum where even only one pup shows clinical
signs. From the frequency of the disease among the pups in
this litter this conclusion appears to be applicable to both N.
caninum and H. canis.
Te clinical signs presented by the pups were typical of
neosporosis for their age: Congenitally infected pups tends
to exhibit a more severe form of the disease (2). Pups of less
than 6 months of age exhibit a severe disseminated form
showing ascending paralysis of the limbs with the pelvic
limbs afected more severely than the thoracic limbs (1, 6).
Muscle atrophy, contracture and fbrosis occur in many pups
(2). Gradual, progressive, ascending paralysis with hyperex-
tension of the hind limbs in congenitally infected puppies
is the most common clinical manifestation of the disease in
dogs less than 6 months of age (2).
H. canis infection causes disease ranging in severity from
an incidental hematologic fnding in an apparently healthy
dog to a debilitating and life threatening illness (3). Even so,
when high parasitemia is present H. canis may also induce
severe clinical manifestations (e.g. lethargy, fever, anorexia,
weight loss, lymphadenomegaly and anemia) associated with
a high parasite load (3, 7). Te possibility that the clinical
signs were exacerbated due to the concurrent infection with
H. canis cannot be completely discounted, however H. canis
is found primarily in the hemolymphatic tissues and not
directly related to the neurological system (3).
Concurrent infections with H. canis in dogs have been
described with Ehrlichia canis and Babesia canis where all
three infections are transmitted by the same tick vector,
R. sanguineus. Other pathogens reported to be involved in
concurrent infections include parvovirus, canine distemper,
Anaplasma phagocytophilum, Anaplasma platys, Toxoplasma
gondii (8) and Leishmania infantum. To the best knowledge
of the authors’ coinfection of Heptozoon canis with Neospora
caninum has not been previously described.
Te seroprevalence of H. canis in dogs in Israel has been
reported to be 33% (9). Te incidence of clinical disease for
H. canis is considered to be much lower than the exposure
rate with most dogs probably undergoing a subclinical form
of the disease. In fact in the litter described in this study one
pup was infected with H. canis only and was not reported to
show clinical signs. Although the route of infection cannot
be defnitely ascertained, it seems likely that the pups were
infected in utero. Horizontal transmission through in the
uterus from the dam to its ofspring has been demonstrated
in H. canis. In a study of naturally infected pregnant bitches
meronts were found in the spleen of a pup that died 16 days
after birth and blood gamonts were detectable as early as 21
days in other pups. Te possibility of these pups being natu-
rally infected by ingestion of the Rhipicepahalus sanguineus
ticks cannot be completely discounted, however taking into
account the young age of the pups and the relatively high rate
of infection within the litter this seems unlikely. Furthermore
it would have been necessary for the young pups to ingest the
ticks and this makes the possibility of infection after birth as
even more unlikely.
Symptoms seen in the two pups were strongly sug-
gestive of infection with N. caninum (1). Treatment with
trimethoprim sulfadiazine and clindamycin for six weeks
was successful in treating the two afects pups, one pup had
recovered completely, and the other remained with paresis of
the left hind limb whereas the other signs including urinary
and fecal incontinenence were alleviated. Although the cur-
rent treatment protocol for H. canis is imidocarb dipropionate
alone or in combination with doxycycline, a similar treatment
used in this study has been recommended for several years
in the treatment of H. canis (10). Tis treatment regime was
not applied in the treatment of two puppies in this study
indicating that H. canis may not have been involved in the
symptomology of this concurrent infectious condition.
Te exact interaction of N. caninum and H. canis in the
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 Mazuz, L.M. 56
pathogenesis of the symptoms is unknown but from the
treatment outcome it does appear that N. caninum did play
a prominent or maybe even a singular role.
In conclusion, this case study is to the best knowledge
of the authors the frst report of a concomitant infection
of neosporosis and hepatozoononosis in a litter of puppies.
Treatment at an early age and for a prolonged period of time
appeared to be successful in preventing the progression of the
clinical signs and possibly preventing clinical signs in other
litter mates which were infected. Our study is in agreement
with others who shown that in naturally infected dogs, treat-
ment can improve clinical signs but may not eliminate the
infection, as the puppies remained seropositive (11).
Te clinical signs were strongly indicative of those caused
by N. caninum although the role of H. canis in the develop-
ment of the symptomology could not be established.
ACKNOWLEDGMENT:
Appreciation to the owner of the puppies Ronit, for her cooperation
and dedication.
REFERENCES
1. Dubey., J.P. and Lappin, M.R.: Toxoplasmosis and Neosporosis,
in Infectious Diseases of the Dog and Cat, C.E. Greene, Editor.
Elsevier, Saunders: St. Louis, Missouri. pp. 806-827, 2012
2. Lyon, C.: Update on the diagnosis and management of Neospora
caninum infections in dogs. Top Companion Anim. Med. 25:
170-175, 2010.
3. Baneth, G.: Hepatozoon canis Infection, in Infectious Diseases of
the Dog and Cat, C.E. Greene, Editor. Elsevier, Sauders: St. Louis,
Missouri. pp. 750-757, 2012.
4. Canon-Franco, W.A., Bergamaschi, D.P., Labruna, M.B., Cama-
rgo, L.M., Souza, S.L., Silva, J.C., Pinter, A., Dubey, J.P. and Gen-
nari, S.M.: Prevalence of antibodies to Neospora caninum in dogs
from Amazon, Brazil. Vet. Parasitol. 115: 71-74, 2003.
5. Dubey, J.P., Koestner, A. and Piper, R.C.: Repeated transplacental
transmission of Neospora caninum in dogs. J. Am. Vet. Med. Assoc.
197: 857-860, 1990.
6. Reichel, M.P., Tornton, R.N., Morgan, P.L., Mills, R.J. and
Schares, G.: Neosporosis in a pup. N Z Vet. J. 46: 106-110, 1998.
7. Karagenc, T.I., Pasa, S., Kirli, G., Hosgor, M., Bilgic, H.B.,
Ozon, Y.H., Atasoy, A. and Eren, H.: A parasitological, mo-
lecular and serological survey of Hepatozoon canis infection in
dogs around the Aegean coast of Turkey. Vet. Parasitol. 135:
113-119, 2006.
8. Harmelin, A., Dubey, J.P., Yakobson, B., Nyska, A. and Orgad, U.:
Concurrent Hepatozoon canis and Toxoplasma gondii infections in
a dog. Vet. Parasitol. 43: 131-136, 1992.
9. Baneth, G., Shkap, V., Presentey, B.Z. and Pipano, E.: Hepatozoon
canis: the prevalence of antibodies and gametocytes in dogs in
Israel. Vet. Res. Commun. 20: 41-46, 1996.
10. Voyvoda, H., Pasa, S. and Uner, A.: Clinical Hepatozoon canis
infection in a dog in Turkey. J. Small Anim. Pract. 45: 613-617,
2004.
11. Dubey J.P, Vianna M.C.B., Kwok O.C.H., Hill, D.E., Miska,
K.B. , Tuo, W., Velmurugan, G.V., Conors, M. and Jenkins, M.C.:
Neosporosis in Beagle dogs: Clinical signs, diagnosis, treatment,
isolation and genetic characterization of Neospora caninum. Vet.
Parasitol. 149: 158–166, 2007.
12. McGarry, J.W., Stockton, C.M., Williams D,J. and Trees, A.J.:
Protracted shedding of oocysts of Neospora caninum by a naturally
infected foxhound. J. Parasitol. 89:628-630, 2003.
Case Reports
Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 57
Bartonellosis and Ehrlichiosis in Dogs and Cats
Harrus, S.
Koret School of Veterinary Medicine, Te Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 761001, Israel.
Bartonelloses are a group of diseases caused by Bartonella species. Te latter are small pleomorphic Gram-
negative bacilli, belonging to the α-2 subdivision of Proteobacteria. Tey are intracellular bacteria, parasitizing
RBCs and endothelial cells, transmitted by arthropod vectors. Tey cause persistent/cyclic bacteremia. More
than 30 species exist in the genus (with many more variants). Over than 15 species are associated with human
diseases. Six species have been reported in cats, including Bartonella henselae, Bartonella clarridgeiae, Bartonella
koehlerae, Bartonella quintana, Bartonella bovis and Bartonella vinsonii subsp. berkhofi. Most cats naturally or
experimentally infected with Bartonella spp., exhibit no clinical signs. Teir CBC, biochemistry and urinalysis
results are usually within normal reference intervals. In Israel, a molecular survey indicated that 25.1% of the
general cat population (31% of stray cats and 19% of indoor cats) were positive for Bartonella spp. Tree species
were detected, including B. henselae (15.6 & 15.5%), B. clarridgeiae (12.3 & 3.2%) and B. koehlerae (5.6 & 1.3%)
in stray and indoor cats, respectively. Cat scratch disease (CSD) is the best known disease caused by Bartonella
species. Te etiologic agent of CSD is B. henselae, causing several syndromes in humans. Immunocompetent
human individuals present usually more local signs, while immunocompromised individuals may present
systemic signs, which may be fatal. Veterinarians and veterinary staf are at greater risk for CSD. Transmission
of B. henselae from cats to humans probably occurs through contamination of cat scratches with fea excrement.
Transmission may also occur through cat bites (blood, saliva, fea-excrement). Dogs can be infected with several
Bartonella spp. including B. vinsonii subsp. berkhofi, B. henselae, B. clarridgeiae, B. washoensis, B. elizabethae,
and B. quintana. Domestic dogs are more likely to be accidental hosts. Tey are excellent sentinels for human
infections because a similar disease spectrum develops in dogs.
Canine monocytic ehrlichiosis (CME) is an important canine disease worldwide. It is caused by the obligate
intracellular rickettsia Ehrlichia canis and is transmitted by the brown dog-tick of the Rhipicephalus sanguineus
complex. Recent molecular studies detected E. canis-DNA in cats from Portugal and Brazil. However, to
date E. canis was not isolated and cultured from suspected cats. Diagnosis of the disease is challenging due
to its variable presentations. Molecular techniques are becoming the defnitive diagnostic methods of CME.
Polymerase chain reaction (PCR) and sequencing are sensitive methods for detecting and characterizing E.
canis-DNA, respectively. Detection of E. canis DNA can be achieved as early as 4 to 10 days post-inoculation.
Several assays are based on diferent target genes, however the 16S rRNA and the p30-based PCR assays are
commonly used. Te p30-based assay is more sensitive than the 16S rRNA-based assay, probably because the
fact that each E. canis contains one gene copy of the 16S rRNA while multiple copies of the p30 gene. Splenic
samples are considered more sensitive samples for the evaluation of ehrlichial elimination by PCR. Real time
PCR is a more sensitive assay than conventional PCR, allowing quantitative analysis of specifc DNA. It is less
prone to contaminations than conventional methods, and therefore is rapidly becoming the preferred method
for diagnosis of E. canis. Recently, loop-mediated isothermal amplifcation (LAMP) is becoming an important
assay for the in clinic diagnosis of infectious diseases. LAMP-based assays have been developed for the diagnosis
of E. canis. Tey will probably become important assays in future diagnosis of CME.
Sponsored by
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Israel Journal of Veterinary Medicine  Vol. 70 (1)  March 2015 58
MERS-CoV: A Humped Zoonosis?
Shimshony, A.
In June 2012, a 60-year-old male citizen of Bisha, southeast Saudi Arabia, presenting with pneumonia associated
with acute renal failure and was hospitalized in Jeddah. A novel beta corona virus, later coined Middle East
Respiratory Syndrome Coronavirus or MERS-CoV, was isolated from his sputum and sequenced. Te patient
died and became to be known as the “index case” of a hitherto unknown corona infection.
Since then, the epidemic has spread, mainly in Saudi Arabia and neighbouring countries with sporadic cases
elsewhere. As of 16 Feb 2015, a total of 1016 cases of MERS-CoVhave been reported by local health authorities
worldwide, including 406 deaths (case fatality rate 39.96%). Te numberof reported cases in the most afected
countries were (in brackets, fatal): Saudi Arabia 886 (372), United Arab Emirates (UAE) 71 (10), Qatar 11 (5) and
Jordan 10 (4). Retrospectively it was found that thevery frst clinical cases of MERS-CoVoccurred, in fact, in April
2012 in Jordan, involving acluster of patients in a public hospital in Zarqa, about two months before the detection of
the “index” case in Saudi Arabia. Clinical cases have been reported, so far, in 10 countries in the Middle East (Saudia,
UAE, Jordan, Qatar, Oman, Kuwait, Egypt, Yemen, Lebanon and Iran),and in 13 countries elsewhere. In Europe
(Austria, UK, France, Germany, Italy, Netherlands, Greece), Asia (Turkey, Malaysia, Philippines), Africa (Algeria,
Tunisia) and America (USA). Most of the infections, in the Middle East have been shown to occur in healthcare
settings. All cases which have been reported outside the Middle East, frst developed infection within the region
and were then exported. Tese travel-related cases have, so far, rarely infected others in the destination countries.
Several international research centers have invested eforts to unravel theepidemiology of MERS-CoV with
partial success. In particular, they tried to identify the animal reservoir of the virus and its mode of transmission.
Te SARS (“Severe acute respiratory syndrome”) experience in 2003, namely a novel, zoonotic coronavirus
disease casing seriousdisease in humans (but with lower fatality rates and higher inter-human infectivity),
became the benchmark. Bats were found to be the primary reservoir of SARS virus. Hence, bats became main
suspects as the primary reservoir of MERS-CoV. So far, there is just one single report on the detection of MERS
coronavirus genetic material in a bat (Taphozousperforatus, the Egyptian tomb bat, Saudia,October 2012). No
viable, whole virus could be obtained from this or anyother bat-derived sample to present (February 2015).
Te initial suspicion of dromedary (single humped) camels as possibleintermediate hosts of MERS-CoV
was raised in March 2013, when it became apparent that a MERS patientfrom the UAE who died in a hospital
in Munich, Germany, had kept 4 camels as pets in his yard and had beenexposed to a sick camel shortly before
falling ill. Tat animal was not tested. Tese observations encouraged researchersto focus attention to camels.
Between October 2013 and December 2014, 4 countries reported to the World Organisation of Animal Health
(OIE) the irrespective detections of MERS-CoV or its genetic material in sub-clinically infected camels. Tey
were Kuwait and Oman (5 each), Qatar (9), and Iran (14 camels, reportedly illegally imported from Pakistan).
In Qatar, the virus was detected in raw camel milk samples. Infections in Saudi camels have been described in
scientifc papers, but – so far – not reported to the OIE.
Evidence from MERS-CoV infections in camels suggests virus shedding for a limited period. During an
experimental infection trial in Colorado, USA, 3 adult dromedary camels were inoculated with a human isolate of
MERS-CoV. Transient, primarily upper respiratory tract infection developed in each of the 3 animals. Clinical signs
were benign. Each camel shed large virus quantities from the upper respiratory tract. Te infectious virus was detected
in nasal secretions through 7 days postinoculation, and viral RNA was detectable up to 35 days postinoculation.
Te possibility for reinfection of camels cannot at this stage be excluded since immunity to infection is
poorly understood. MERS-CoV has been identifed in camels which have antibodies against the virus. Tere
are still many MERS-CoV cases in humans of which the source of infection remains obscure. Te implications
of these fndings for management and control recommendations need further investigation.
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Serological studies suggest that antibodies to MERS-CoV have been detected with a prevalence range of
0-100% (varying within countries and between countries) in populations of camels in Middle East and African
countries. Tis range of prevalence indicates the need to assess risk factors for infection between and within
herds. Te results are suggestive of MERS-CoV circulating since decades; the earliest positive camels in Saudi
Arabia date back to 1993, corresponding with the start of increased camel imports, in particular from Somalia and
Sudan. International trade may have contributed to the virus’ diversity and persistence, but its epidemiology, and
particularly its late emergence as cause of human disease, is still in need of considerable research efort. Tere does
not seem to be at present room for animal culling nor practicality of trade restrictions within the afected regions.
According to published literature, other species of animals, including sheep, goats, cattle, water bufalo and
wild birds, have tested negative for the presence of antibodies to MERS-CoV. However owing to the relatively
small sample sizes the results of these studies cannot exclude infection in other animal species. Based on receptor
studies other animal species have been identifed as potential hosts.
Although genetically related viruses have already been detected in bat species around the world, and a
fragment of viral genetic material matching the MERS-CoV was found in one bat from Saudi Arabia, current
evidence does not (yet) indicate a direct link between bats and MERS-CoV in humans. More evidence is
needed to directly link the MERS-CoV to bats or other animal species.
In Jordan, 10 human cases were reported in 2012. A serological investigation in domesticanimals was
applied there between June and September 2013; neutralising MERS-CoV antibodies were found in all (11)
camel sera tested, while sera from goats and cattle tested negative. In view of the fact that Israel borders Jordan,
Israeli investigators are encouraged to take notice of these data and include MERS-CoV within their scientifc
objectives. Tis may include serology in camels (Israel’s population estimated at 3000-4000, most of them in
the south). Te scarce information on MERS-CoV in bats calls for enhanced research.
Te “Egyptian tomb bat” is known to be present in the Jordan valley, which separates Jordan and Israel and
is shared by both. According toa recent report of Israel’s nature protection agencies, there are at least 4 active
colonies of T. perforatus in Israel. Te largest cave, found to include hundreds of breeding bats, is situated on the
northern shore of the Dead Sea, very close to the Jordanian border. Sampling and testing these bats deserves to
become a prioritised “One Health” project, encouraged and supported by local and international public health,
animal health, andnature protection agencies, preferably combining regional cooperative eforts.
Severe Subcutaneous Mast Cell and Eosinophilic Infltration Associated with Presence
of Multiple Diroflaria repens Microflariae in Tree Dogs
Mazaki-Tovi, M.,
1
Reich, M.,
2
Karnieli, A.,
3
Kuzi, S.
1
and Aroch, I.
1
1
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine. Te Hebrew University of
Jerusalem, P.O. Box 12, Rehovot, 761001, Israel.
2
Ramat Hasharon Veterinary Center, 21 Yaakov Cohen, Ramat Hasharon, 47213, Israel.
3
Karnieli Vet Ltd, Molecular Veterinary Diagnosis, Kiryat Tivon, Israel.
Diroflaria repens is a parasitic nematode in the subcutaneous tissue of carnivores, including dogs and cats,
transmitted by mosquitos. It is a zoonosis, since humans might serve as accidental hosts. Infection of a dog with
D. repens was frst reported in the Palestine in 1934, and two additional cases were reported in dogs in Israel to
date. Tis report describes D. repens infection in 3 dogs from Ramat Hasharon, Israel that presented subcutane-
ous masses, which were cytologically characterized by severe mast cell and eosinophil infltration, with numerous
microflariae in 2/3 cases; rare microflariae were present in the third case. In all three dogs, polymerase chain
reaction of fne needle aspirates was positive for D. repens. Te mast cells observed in all lesions were uniform
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and highly granular, and with the presence of the microflariae, mast cell tumor was considered unlikely. Tis
report suggests that D. repens infection-associated subcutaneous lesions, characterized cytologically by massive
mast cell and eosinophil infltration, should be considered a diferential diagnosis for mast cell tumor, especially
in geographic locations endemic for this nematode. Tese dogs were infected with D. repens despite a routine
preventative doramectin therapy against Spirocerca lupi administered every three months, probably due to the
relatively long time-interval between treatments. It seems that Ramat HaSsharon is endemic for the nematode.
Hemostatic Abnormalities in Dogs with Naturally-Occurring Heatstroke
Kelmer, E., Bruchim, Y., Cohen, A., Codner, C., Segev, G. and Aroch, I.
Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 761001, Israel.
Heatstroke in dogs induces both procoagulant and anticoagulant states, characterized by several hemostatic dis-
turbances, often leading to DIC and death. Tis prospective observational study aimed to characterize hemostatic
analytes and their association with mortality in dogs with naturally-occurring heatstroke. Citrated and ETDA
blood samples were collected at presentation and at 4, 12, 24, 36 and 48 hours post-presentation (PP) from 30
client-owned dogs with naturally-occurring heatstroke. Hemostatic tests performed included platelet count,
prothrombin and activated partial thromboplastin times (PT and aPTT, respectively), antithrombin activity
(ATA), total protein C activity (tPCA) and fbrinogen and D-dimer concentrations. Te survival rate was 60%
(18/30 dogs). Older age, higher heart-rate and rectal temperature at presentation and time from onset of clinical
signs to presentation were signifcantly associated with mortality. Hemostatic analyte results at presentation
were not associated with mortality; however prolonged PT and aPTT at 12-24 hours PP, lower tPCA at 12
hr PP and hypofbrinogenemia at 24 hours PP were signifcantly (P≤0.05) associated with mortality. Increased
D-dimer concentration and low ATA were common at all time-points, but were not associated with mortality.
Te frequency of DIC increased in non-survivors throughout hospitalization, but DIC was not associated with
mortality. Te number of abnormal hemostatic abnormalities during the frst 24 hours was signifcantly (P=0.04)
higher in non-survivors. All but one dog received fresh frozen plasma (FFP), with no diference in FFP dose
between survivors and non-survivors (4.4 vs. 4.5 units per dog, respectively, P=0.45). In conclusion, hemostatic
derangements are very common in dogs with naturally-occurring heatstroke. PT, aPTT, tPCA and fbrinogen
concentrations became associated with the outcome at 12-24 hours PP, exemplifying the need for serial sequential
measurement of multiple laboratory hemostatic tests during hospitalization, even if results are within reference
interval at presentation. In this cohort, DIC was not associated with outcome, possibly because FFP was used
extensively compared to previous studies of heatstroke in dogs.
Canine Protothecosis in Israel: A Case-Series of Four Dogs
Ben-Oz, J.,
1
Nivi, R.,
1
Yas, E.,
1
Merbl, Y.,
1
Sarfati, H.,
2
Gelman, I.
3
and Zaretzky, G.
3
1
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem,
Rehovot, P.O. Box 12, Rehovot, 761001, Israel.
2
Eyecare Clinic, Yehud-Monoson, Israel.
3
Veterinary Center for Pets, Pardes-Hana, Israel.
Protothecosis is caused by Prototheca spp., a saprophytic achlorophyllous algae. Te disease has been identifed
in people and animals in Europe, Asia, Africa, Australia, Pacifc Ocean islands and North America. Te most
frequently reported clinical sign in dogs is intermittent, protracted bloody diarrhea. Dogs generally develop
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a disseminated disease, and the type and severity of the clinical signs vary, depending on the tissue involved.
Between 2013 and 2014, four dogs were diagnosed with protothecosis in Israel, based on identifying Prototheca
organisms in cytological smears or histopathology of biopsies. Tree of the four dogs had a history of protracted
bloody diarrhea. Two dogs had disseminated disease, while two had localized infection, one of which with
recurrent subcutaneous lesions, while in the other, clinical signs were limited to the gastrointestinal tract. Shortly
after the diagnosis, one dog with systemic disease died, while the other was euthanized. Te two remaining
dogs were both treated orally with itraconazol, while the dog with gastrointestinal infection was treated ad-
ditionally with oral nystatin. Tis dog was euthanized six months after the diagnosis. Te other dog, treated
with itraconazol, showed no signs of disseminated infection 14 months after diagnosis, although reoccurrence
of the subcutaneous infection occurred.To the best of our knowledge, this is the frst report of protothecosis in
dogs in Israel. Although an uncommon disease, care should be taken to diagnose such infected dogs early, to
facilitate early specifc treatment. However, the prognosis is poor and although treatment might prolong life,
it usually will not result in disease resolution.
Sleeping and Resting Respiratory Rates in Dogs with Subclinical Left-Sided Heart Disease
Ohad, D.,
1
Rishniw, M.,
2
Ljungvall, I.,
3
Porciello, F.
4
and Häggström, J.
3
1
Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 761001, Israel.
2
Veterinary Information Network, 777 W, Covell Blvd, Davis, CA, 95616, USA.
3
Department of Clinical Sciences, University Animal Hospital, Swedish University of Agricultural Science,
SE-750 07 Uppsala, Sweden.
4
Department of Clinical Sciences, School of Veterinary Medicine, University of Perugia, Perugia, 06100, Italy.
Te objective of this study was to characterize sleeping respiratory rates (SRRs) and resting respiratory rates
(RRRs) measured in the home environment, of dogs with subclinical heart disease that did result in left-sided
congestive heart failure. Te study was a prospective cross-sectional study and included 190 adult dogs with
subclinical left-sided heart disease. Most (n=174) dogs had mitral valve disease of various severities. Clients
collected ten 1-minute SRRs or RRRs during a period ranging from 5 to 178 days (median 15 days). Clinicians
provided echocardiographic and medical data on each patient.Te within-dog mean SRR (SRRmean; 16
breaths/min, range: 9-33) was signifcantly (P<0.001) lower than the within-dog mean RRR (RRRmean; 21
breaths/min, range 10-43). Seven dogs had SRRmean and 33 dogs had RRRmean > 25 breaths/min; 1 dog
had SRRmean and 12 dogs had RRRmean > 30 breaths/min; these dogs mostly had a left atrial (LA)-to-
aortic ratio > 1.8. Dogs with moderate LA enlargement had a signifcantly higher SRRmean than did other
dogs (P = 0.003). However, median SRRmean for each of 4 levels of LA enlargement was < 20 breaths/min;
median RRRmean for each of 4 levels of LA enlargement was < 25 breaths/min. Both within-dog SRR and
RRR remained stable for 10 consecutive measurements (P = 0.08-0.4). Treatment with cardiac medications
and presence of pulmonary hypertension were not associated with SRRmean or RRRmean. It was concluded
that dogs with confrmed subclinical left-sided heart disease of various severities generally had SRRmean <
25 breaths/min, which was infrequently exceeded at any time. SRR and RRR remained stable, regardless of
individual within-dog SRRmean or RRRmean.
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Septic Lens Implantation Syndrome in a Dog Following a Lens’ Cat Claw Injury
with Positive Medical Outcome
Sarfati, H. and Segev, Y.
Eyecare Clinic, Yehud-Monoson, Israel.
Septic implantation syndrome (SIS) refers to traumatic inoculation of bacteria into the lens cortex and development
of lenticular abscess following capsule rupture. Tis results in a delayed-onset severe endopthalmitis. A squela
of glaucoma frequently leads to enucleation. A 3-year old male, Labrador retriever dog was referred to Eyecare
Clinic two days after a cat claw injury to the right eye. On presentation, examination revealed corneal perforation,
a 3-mm lens capsule rupture and mild anterior uveitis. Due to good response to medical treatment, the owner
declined lens removal and pursuit medical treatment. During the following month, the corneal ulcer healed and
the anterior uveitis resolved, and on bimonthly rechecks the eye remained quiet. Eight months post presentation,
the dog showed acute severe blepharospasm. Examination revealed whitish swollen lesion in the lens and severe
uveitis. Cytology from aqueous humor aspirate reveled neutrophils, and a tentative diagnosis of SIS was made.
Following aggressive medical treatment for two days, the uveitis has improved but the lens lesion remained
unchanged. Since lenticular abscess was suspected, the dog underwent cataract surgery. Lens capsule histopatology
revealed severe supportive infammation and Gram stain was positive for bacteria, confrming the diagnosis of
SIS. Following surgery, the eye improved dramatically. Six months post-surgery, the dog was visual, pain free,
with no clinical sign of uveitis. Te few SIS published cases were all diagnosed based on globe’s histopathology
following enucleation. To our knowledge, this is the frst documented clinical case of SIS with a positive outcome.
Evaluation of Intratestiscular and Insicional Line Infltration with Ropivacaine for Castration
in Dogs under Sedation with Medetomidine-Butorphanol-Midazolam
Kushnir, Y., Toledano, N., Cohen, L. and Shilo-Benjamini, Y.
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Te Robert H. Smith Faculty of
Agriculture, Food and Environment, Te Hebrew University of Jerusalem, Rehovot, 761001, Israel.
Administration of local anesthetics can decrease general anesthesia requirements and reduce post-operative pain.
Te goal of this study was to evaluate whether intratesticular and incisional ropivacaine infltration produces
sufcient intra- and post-operative analgesia in castration of dogs under deep sedation.Twenty-three healthy
male dogs presented for castration were sedated with medetomidine 0.01 mg/kg, butorphanol 0.2 mg/kg and
midazolam 0.2 mg/kg IM, and were randomly assigned to receive 0.2-0.4 mL/kg 0.5% ropivacaine (group R) or
an equivalent volume of saline (group S) injected intratesticularly and along the incision line. Gross movement
during the procedure was treated with diazepam 0.5 mg kg
-1
and if it recurred this was considered a failure, and
general anesthesia was induced. Post-operatively, all dogs received 2.2 mg/kg carprofen SC and atipamezole
IM to reverse sedation. Pain was evaluated before sedation and 1, 2, 4, 8 and 24 hours post-operatively using a
visual analog scale (VAS; 0-100), the Glasgow composite pain scale (GCPS; 0-24) and a mechanical algometer.
Methadone was used for rescue. Te anesthetist and pain assessors were blinded to the treatment.Tere was no
signifcant diference in the frequency of general anesthesia induction, however, the median (range) time to failure in
group S was signifcantly shorter than in group R (6 [3-25], and 56 [36-76] minutes, respectively).VAS at 8 hours
was signifcantly higher in group S (13.87±10.02) than in group R (5.5±4.01). In conclusion, intratesticular and
incisional ropivacaine infltration reduces anesthetic requirements, and produces analgesia after castration in dogs.
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Laboratory Findings in Cats with Naturally-Occurring Sepsis
Klainbart, S., Agi, L., Aroch, I. and Kelmer, E.
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Te Robert H. Smith Faculty of
Agriculture, Food and Environment Te Hebrew University of Jerusalem, Rehovot, 761001, Israel.
Sepsis is a severe, often fatal syndrome, resulting from systemic infammatory response to infection. Te
aims of this prospective study were to characterize the laboratory fndings in cats with naturally-occurring
sepsis. Te study group included 31 client-owned cats that met set criteria for sepsis, and 34 healthy
staf-owned control cats. Blood samples were collected within 24 hours from presentation for CBC, serum
chemistry, prothrombin time, activated partial thromboplastin time (PT and aPTT, respectively), D-dimer
and fibrinogen concentrations and activities of protein C (PCA) and antithrombin (ATA). Septic cats
were signifcantly more anemic, had a higher nucleated red blood cell count, lower serum albumin, total
protein and total calcium concentrations, and higher serum bilirubin and triglycerides concentration, as
well as aspartate transaminase and creatinekinase activities (P < 0.008 for all) compared to the controls.
Te septic cats had signifcantly higher aPTT, and D-dimer, and lower PCA and ATA (P ≤ 0.001 for all),
and had a lower platelet count compared to the controls (P = 0.037). Neutrophil cytoplasmic toxicity was
common in septic cats. Te 10-day survival rate of the septic cats was 65%. Serum urea concentration
was signifcantly higher (P = 0.04), and ATA and PCA tended to be lower (P = 0.06) in non-survivors
compared to survivors. Naturally-occurring sepsis in cats is characterized by anemia, neutrophil toxic-
ity, hypoalbuminemia, hypertriglyceridemia, increased muscle enzymes activity, hyperbilirubinemia and
hemostatic derangement, manifested by prolonged aPTT, low PCA and ATA, increased plasma D-dimer
and thrombocytopenia.
Minimally Invasive Unilateral Arytenoid Lateralization in Dogs: A Cadaveric Study
Shipov, A., Israeli, I., Weiser, M., Kelmer, E., Klainbart, S. and Milgram, J.
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem,
P.O. Box 12, Rehovot, 761001, Israel.
Te aims of this anatomic study were to develop a percutaneous thyroarytenoid lateralization (PTAL) technique,
and to evaluate its application in dog cadavers. Twenty-two unilateral thyroarytenoid lateralization procedures
were performed on 11 dog cadavers weighing 15-40 kg. Dogs were placed in ventral recumbency with the
neck extended and the mouth open. A rigid endoscope was used to visualize the rima glottides. Te larynx was
palpated, and two hypodermic needles were passed through the skin into the lumen of the larynx, penetrating
both the thyroid and arytenoid cartilages. Nylon suture material was passed through the needles to lateralize
the arytenoid cartilage. A key-hole approach to the larynx was performed, and the suture material was knotted
adjacent to the thyroid cartilage. Te procedure was performed bilaterally; however, each side was assessed as
a standalone/unilateral procedure. Te change in the rima glottidis area was recorded as were the duration of
the procedure and complications encountered. Te landmarks for needle insertion were easily palpated in all
dogs. Te time required to place the suture material and to perform a unilateral PTAL was 4:30-20:19 and
9:30-16:00 min, respectively. A signifcant increase in the area of the rima glottidis was documented after
performing unilateral PTAL. In conclusion, unilateral PTAL is a quick, minimally invasive procedure, which
increases the area of the rima glottidis in cadaveric dogs. Further evaluation is needed to assess whether this
technique can serve as an alternative for conventional surgical options in clinical cases.
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Polycystic Kidney Disease in Four British Shorthair Cats and Successful
Treatment of Bacterial Cyst Infection
Nivy, R.,
1
Lyons, L.A.,
2
Aroch, I.
1
and Segev, G.
1
1
Department of Small Animal Internal Medicine, Hebrew university Veterinary Teaching Hospital, Koret School of
Veterinary Medicine, the Hebrew University of Jerusalem, P. O. Box 12, Rehovot, 761001, Israel.
2
Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri - Columbia, E109
Vet Med Building, 1600 E. Rollins St., Columbia, MO 65211, USA.
Feline polycystic kidney disease (PKD) is the most common inherited disorder in cats. Renal cysts progres-
sively increase in size and number, resulting in gradual decrease in kidney function. An autosomal dominant
mutation in exon 29 of the polycystin-1 gene has been identifed, mostly in Persian and Persian-related breeds.
Te same gene is involved in autosomal dominant PKD in humans. Tis case-series describes PKD, for the
frst time, in four British Shorthair cats, in Israel, of which in 2/4 cats, the same mutation reported in Persian
and Persian-related cats was identifed by polymerase-chain reaction testing. Tis mutation was introduced
to the British Shorthair breeding line in past outcrossing with Persian cats. We also describe the diagnosis
and successful treatment of an Escherichia coli-infected renal cyst in one of these cats, a common complication
occurring in human PKD, unreported previously in cats with PKD. Treatment in this case included ultrasound
cyst drainage, intra-cystic enrofoxacin injection and systemic antibiotic therapy.
Intraoral Approach for Sialoliths Removal in 15 Horses
Oref, G.L.,
1
Shiraki, R.
2
and Kelmer, G.
1
1
Department of Large Animal Medicine and Surgery, Koret School of Veterinary Medicine, Te Robert H. Smith Faculty of
Agriculture, Food and Environment, the Hebrew University of Jerusalem, P. O. Box 12, Rehovot, 761001, Israel.
2
G.V. Ling Urinary Stone Analysis Laboratory School of Veterinary Medicine University of California, Davis, California.
Sialolithiasis is uncommon in horses, and mainly afects horses in arid environment, such as Israel. Te
defnitive treatment of sialolithiasis is surgical removal. Although direct and easily accessed, the transcutaneous
approach is frequently associated with signifcant complications, mainly fstula formation. Te objective of
the current study was to describe intraoral approach for sialolith removal in horses and the results of the
stones chemical analysis. Te study design was a prospective case series, which included 15 client-owned
horses diagnosed with sialolithiasis. Te diagnosis of sialolithiasis was based on clinical presentation and
palpation. Sialoliths were removed using the intraoral approach under general anesthesia (n=4) or in stand-
ing position with sedation and local analgesia (n=11). A mouth gag was placed, and an incision in the oral
mucosa along the entire length of the sialolith was performed, followed by manual extraction of the calculi.
Complications, success rate and chemical analysis of the sialoliths were recorded. Results of the study showed
that sialoliths were removed successfully, and all horses resumed their previous level of work shortly after the
surgery. Tree horses had mild complications, including seroma (n=2) and post-operative weakness (n=1), all
if which resolved with no intervention. Sialoliths were composed of 80-95% calcium-carbonate and 5-20%
apatite. Te vast majority contained a nidus of plant material in their center. Tus, sialoliths in the current
horse population had a uniform mineral composition, consisting mainly of calcium carbonate. Tis study
confrms that removal of sialoliths via the intraoral approach results in a high a success rate, with minimal
complications.
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Case Presentation: Metastatic Melanoma Causing Hind-Limb Neurogenic Paresis
in an Arabian Mare
Kelmer, G. and Qura’n, L.
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem,
P.O. Box 12, Rehovot, 761001, Israel.
Melanomas occur in over 80% of geriatric grey horses. Tese dermal tumors are usually benign, however, may
metastasize. Terefore, any melanotic tumor should be considered potentially metastatic. Tentative diagnosis
of melanoma often arrived at by assessing the signalment and the clinical presentation. A 12-year old Arabian
mare presented to the Hebrew University Teaching Hospital with a 2-month history of progressive weight
loss, poor condition, and a gradually worsening left hind-limb lameness. Historically, the mare had a mass
under the tail and a mass on the neck for unknown durations, both assumed to be discrete dermal melanomas.
On presentation to the hospital, the mare was weak and reluctant to move, with very poor body condition
and severe atrophy of the left hind-limb and pelvis musculature. Te mare sufered from severe ataxia, and
a non-weight bearing lameness on the left hind-limb. Tere was no response to cutaneous stimulation. On
rectal examination, a very large mass was palpated on the left dorsum within the pelvic area. Due to the poor
prognosis, the mare was euthanized and sent for necropsy. Post mortem examination revealed a large mass
(~40x30cm) in the pelvic area, with extensive metastasis to most internal organs, as well as invasion into the
pelvic bones and the spinal column. Tese fndings support the diagnosis of neurogenic paresis of the left
hind-limb, caused by metastatic melanoma. Benign neglect of melanotic tumors is often recommended and
practiced, but a more proactive clinical approach may be warranted to delay or prevent metastasis.
Risk Factors Associated with Mortality, and the Use of Antivenom to Treat Horses Following
Vipera palaestinae Envenomation
Tirosh Levy, S., Solomovitch, R. and Steinman, A.
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem,
P.O. Box 12, Rehovot, 761001, Israel.
Snakebites are a common problem in human and veterinary medicine. Te most common venomous snake
in Israel and the Middle East is Vipera palaestinae, which causes most of the envenomations in humans and
animals in Israel. V. palaestinae venom consists of several components, which adversely afect coagulation, the
heart and nervous system. Te clinical presentation and the hematological efects of the envenomation were
investigated in humans and dog, while little is known of its efect in horses. In human medicine, treatment
often includes administration of antivenom–hyperimmune serum produced from equine blood. Its use has
decreased the mortality rate in humans from 10% to 1%. Horses are considered susceptible to the venom,
and envenomations led to complications such as cardiotoxicity and coagulation disorders. Nevertheless, the
extent of these phenomena is unclear. Te efects of antivenom and its therapeutic dose in envenomed horses
have not been determined. Te aim of this study was to examine the risk factors associated with mortality in
horses envenomed by V. palaestinae, and to access if antivenom administration decreases mortality. Data of 45
envenomations in horses, occurring between April and November 2013 were collected from veterinarians using
questionnaires. Six horses died following the envenomation (13.3%), three (7%) presented with signs of shock,
and eight (18%) had local bleeding. Te risk factors that were signifcantly associated with mortality included
clinical signs of shock (P=0.002) and the response to treatment (P=0.005). Twenty nine horses were treated
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with antivenom (64%), of which only one died (3.5%), whereas fve horses of 16 untreated horses (31%) died.
Administration of antivenom was signifcantly associated with decreased mortality (P=0.017). In conclusion, V.
palaestinae envenomation occurs commonly in horses in Israel during the summer, and is life-threatening. Signs
of shock and response to treatment were associated with the prognosis. Administration of antivenom decreased
the mortality, and should be recommended in V. palaestinae-envenomed horses. Further research is needed to
evaluate the appropriate its timing and dosage.
Evaluation of the Pharmacokinetic Properties of a Combination of Marbofoxacin and
Imipenem Administered by Regional Limb Perfusion to Standing Horses
Dahan, R.,
1
Britzi, M.,
2
Sutton,G.A.,
1
Sorek, S.
1
and Kelmer, G.
1
1
Hebrew University Veterinary Teaching Hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem,
P.O. Box 12, Rehovot, 761001, Israel.
2
National Residue Control Laboratory, Kimron Veterinary Institute, Bet Dagan, Israel.
Combining antimicrobials of diferent classes may aid in broadening the antimicrobial spectrum, as well as
decreasing the emergence of resistant bacteria. Our goal was to evaluate the pharmacokinetics, feasibility and
safety of using a combination of marbofoxacin/imipenem in regional limb perfusion (RLP) in standing horses.
Six healthy adult horses participated in the study. After being sedated, a tourniquet was applied on randomly
selected front leg, approximately 10 cm proximal to the site of injection. A perfusate of 1 g marbofoxacin
and 500 mg imipenem, diluted to 100 mL was injected, using 22g butterfy catheter. Synovial samples were
collected from the metacarpophalengeal (MCP) joint, and blood samples were collected from the jugular vein
at times: 0, 0.5, 2, 6, 12, 24 and 36 hours after injection. All samples were analyzed for marbofoxacin and
imipenem concentration using liquid chromatography tandem mass spectrometry. Maximum concentration
(Cmax) of imipenem and marbofoxacin in the MCP joint was 71 and 73 µg/mL, respectively. Te area under
the concentration curve (AUC) of imipenem and marbofoxacin in the MCP joint was 148 and 186 µg-hr/
mL, respectively. Te results indicate that using RLP with the combination of marbofoxacin and imipenem
is safe, ofers a broad spectrum of antimicrobial coverage, and has no negative pharmacokinetic efect on each
single antibiotic efect.
Immobilization of Captive Nubian Ibexes (Capra Nubiana) with Butorphanol-Midazolam-
Medetomidine or Butorphanol-Azaperone-Medetomidine and Atipamezole Reversal
Lapid, R.
1
and Shilo-Benjamini, Y.
2
1
‘Hai-Park’ Zoo, Kiryat-Motzkin, Israel.
2
Koret School of Veterinary Medicine, Te Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew
University of Jerusalem, P.O. Box 12, Rehovot, 761001, Israel.
Seventeen captive Nubian ibex (Capra nubiana) were immobilized for transportation and/or hoof trimming,
deworming, and vaccinations. Of these, 11 were immobilized with a mean ± SD combination of butorphanol
(0.13 ± 0.03 mg/kg), midazolam (0.13 ± 0.03 mg/kg), and medetomidine (0.13 ± 0.03 mg/kg) (BMM), and 6
were immobilized with a combination of butorphanol (0.11 ± 0.03 mg/kg), azaperone (0.22 ± 0.06 mg/kg), and
medetomidine (0.11 ± 0.03 mg/kg) (BAM) by IM injection. Induction and recovery times were recorded. Heart
rate, respiratory rate, rectal temperature, blood pressure, and oxygen saturation were measured. Te quality of
induction, immobilization, and recovery were scored (scale 1-5; 1 = poor, 5 = excellent). Mean induction time was
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signifcantly shorter in the BMM group compared to the BAM group (8.8 ± 2.7 vs. 20.1 ± 7.8 min, respectively).
Median induction score and median immobilization score were signifcantly higher (i.e., better) in the BMM
group versus the BAM group (5 vs. 2.5, and 4 vs. 3, respectively). Te mean and diastolic blood pressures were
signifcantly higher in the BMM group at the 25 minutes time point. Atipamezole was administered at the end
of procedure, and all ibexes recovered smoothly. Mean recovery time was signifcantly longer in the BMM group
versus the BAM group (9.5 ± 4.3 and 3.3 ± 2.2, respectively). In conclusion, at the doses used, the combination
of BMM was superior to BAM for short-term immobilization of captive Nubian ibexes.
Bilateral Corneal Squamous Cell Carcinoma in a 9-Year Old Hafinger Mare
Shnaiderman-Torban, A., Kahane, N., Kelmer, G. and Ofri, R.
Hebrew University Veterinary Teaching hospital, Koret School of Veterinary Medicine, Hebrew University of Jerusalem,
P.O. Box 12, Rehovot, 761001, Israel.
A 9-year old Hafinger mare was referred to the Hebrew University Veterinary Teaching Hospital due to
bilateral recurrent keratoconjuctivis. Over the past year, the mare had left eye corneal edema and opacity, and
was treated with topical antibiotics and non-steroid anti-infammatory drugs. Two weeks prior to referral,
a right eye lesion appeared. Ophthalmic examination at the hospital revealed bilateral blepharospasm and
corneal opacity, composed of infltration, edema, vascularization and white-yellowish granules in the limbus.
Corneal cytology was unremarkable. Diferential diagnoses included eosinophilic keratitis, squamous cell
carcinoma (SCC), equine herpes virus infection and corneal lipid dystrophy. Initial treatment included oph-
thalmic cyclosporine and dexamethasone.Bacterial culture was positive for Mycoplasma bovis, and therefore,
dexamethasone was discontinued, and ophthalmic tetracycline was added. With no improvement, the mare
was rechecked two months later. Corneal cytology revealed a few hyperbasophilic keratinocytes with moder-
ate anisocytosis. Cyclosporine and dexamethasone were prescribed. Two months later, due to deterioration,
bilateral keratectomy and cryotherapy were performed. Corneal cytology revealed keratinocytes in diferent
maturation stages. Some of the highly cornifed cells demonstrated lacy nuclei. Histopathological examination
of corneal biopsies showed bilateral SCC. SCC is the most common equine corneal neoplasm. However, the
bilateral clinical presentation was atypical. To the best of our knowledge, this is the frst report of bilateral
corneal SCC in the horse. Corneal tumors may be misdiagnosed, and a deep corneal biopsy is necessary for a
defnitive diagnosis. Interestingly, a recent study in a Hafinger breeding farm suggested a possible inherited
basis for SCC in the breed.
Bovine Ephemeral Fever: Past, Present and Future
Kedmi, M.
Hachaklait and Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Israel.
Bovine ephemeral fever (BEF) is a unique disease afecting cattle and bufalo, caused by a virus of the
family Rhabdoviridea, probably transmitted by Mosquitos (Culicidae) or by biting midges (Ceratopogonidae).
Terefore, BEF is most prevalent in tropical and subtropical Australia, Africa and Asia. BEF outbreaks
occur mainly in the warm seasons or during seasonal transitions. BEF is characterized by a short acute
phase, manifested by 2-3 fever cycles, lameness, stif gait, and commonly by sternal or lateral recumbency.
Herd morbidity rates may be very high (up to 80%), but the mortality rate is low (up to 1-2%). In Israel,
the disease was described already as early as 1931. Since then, several outbreaks were documented, of which
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the last ones occurred in 2004, 2008-2010, and 2014. In addition, BEF occurs sporadically in diferent
parts of Israel, but its extent is unclear. In this lecture, the epidemiological characteristics of BEF and its
outbreaks were reviewed, including the risk factors, its damage, changes in morbidity characteristics, and
methods of control and prevention of BEF in Israel. New research and supplementary information were
presented to deepen the understanding of BEF.
Chlamydia Psittaci: A Primary Pathogen Not Just of Parrots
Lublin, A., Mechani, S. and Farnoushi, Y.
Division of Avian and Fish Diseases, Kimron Veterinary Institute, Bet Dagan 50250, Israel.
Chlamydia psittaci is an intracellular obligatory bacteria that may cause endemic avian chlamydiosis, epizootic
outbreaks in mammals, and psittacosis in humans. Chlamydiosis in birds is considered an ubiquitous subclinical
pathogen, however, latent infections may transform the infection to an overt disease with shedding of bacteria
and infection of other organisms. In commercial poultry establishments, chlamydiosis appears to occur mostly
in turkeys and ducks, but commercial focks having clinical chlamydiosis as a primary or unique pathogen are
considered uncommon. Tere is a question concerning the signifcance of C. psittaci as a primary pathogen of the
domesticated avian host, or, the importance of these species only as reservoir of C. psittaci infections in mammals.
Samples of domesticated birds, pet birds, zoo and wild birds (cloacal, tracheal, eye swabs and blood) or carcasses
of dead birds, were submitted routinely or as part of surveillance studies to the Laboratory of Avian Diseases in
Kimron Veterinary Institute, Bet Dagan, Israel, to be tested for C. psittaci (n=395 submissions annually; average
over the last 10 years).Te laboratory methods in determination of C. psittaci included direct immunofuorescence
with anti-LPS-fuorescent antibodies, regular and real-time PCR, and serological determination of Chlamydial
antibodies by ELISA. Genotypic classifcation of C. psittaci was made by using the gene encoding the major
outer membrane protein of the bacterial envelope (ompA), and further nucleotide sequencing. In some cases the
bacterium could be diagnosed only following isolation in embryonated chicken eggs, by yolk-sac inoculation of
5-7 days-old specifc pathogen-free eggs. Te prevalence of C. psittaci positive cases over the last 10 years was
22.0 ± 3.7% (mean ± standard error of the mean). Genotype E was the predominant (69% of positive cases), then
genotypes B (21%) and A (10%). Typical respiratory and gastrointestinal signs of C. psittaci such as sinusitis or
conjunctivitis, and diarrhea, were noted in all groups of birds. Clinical signs that are considered atypical for this
pathogen, such as neurological signs, were interestingly found in most groups (excluding psittacines). However,
locomotor signs, such as difculties in walking and limb paresis associated with leg joint infammation, were noted
only in chickens and turkeys. Additionally, the reperoductive system signs (embryonic mortality, non-hatching)
were seen only in commercial poultry, including chickens and turkeys. A syndrome of ischemia and necrosis of
the non-feathered head and neck skin, was seen only in turkeys. In some of these, we could reproduce the signs in
artifcial inoculation studies. To summarize, C. psittaci is a multi-systemic pathogen that may injure many organ
systems in the bird, including domesticated birds, chickens and turkeys, is involved in pathologies unknown so far.
Diferences between bird groups exist in clinico-pathological manifestations of C. psittaci, and most important:
the interaction between C. psittaci. Most importantly, the avian host is not only as an avian reservoir but it can
be defned also as host-parasite interaction with clinico-pathological manifestations.
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A Case Report of Bacillus cereus Poisoning from Minced Beef
Zagron, G.,
1
Uly, S.
2
and Stein-Zamir, C.
1
1
Jerusalem District Health Ofce, Ministry of Health, Israel.
2
Jerusalem District Food Laboratory (Central Laboratories), Ministry of Health, Israel.
On March 2013, Jerusalem District Health Ofce team investigated a notifcation of gastrointestinal symptoms
among fve family members after eating in a local restaurant. Symptoms, which included vomiting and, in one
case, fainting, occurred within several hours of consuming food at the restaurant, and were consistent with toxin
poisoning. Environmental and epidemiological investigations were initiated, aiming to determine the cause of
this food poisoning. Tis report summarizes the results of the investigation. After environmental inspection
and food sampling in the restaurant, the suspected dish causing the food poisoning included minced meat. Te
cooked dish was not kept under temperature control, and reached the danger zone for over 2 hours (5-60 °C).
Te suspected pathogen was identifed as Bacillus cereus. B. cereus is a well-known food poisoning organism. It
can causes two types of food poisonings, known as the emetic and the diarrheal types. Te emetic type is caused
by a heat-stable toxin, named cereulide, produced in the food. Te symptoms are usually mild, but can be severe,
up to fatal sometimes. In this case, all fve ill family members recovered. Control measures were recommended
in order to prevent such future events.
Sustained Release Injection of Doxycycline in Chicks Based on Termo-Sensible Polymer
Frank Kuhnreich, R.,
1
Lublin, A.,
2
Britzi, M.,
3
Soback, S.,
3
Rosenbluth, E.,
2
Friedman, M.
4
and Lavi, E.
1
1
Koret School of Veterinary Medicine. Te Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 761001, Israel.
2
Division of Avian and Fish Diseases, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
3
National Residue Control Laboratory, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
4
School of Pharmacy, Te Faculty of Medicine, Te Hebrew University of Jerusalem, P.O. Box 2065, Jerusalem 91120, Israel.
Avian chlamydiosis, is caused by the bacterium Chlamydia psittaci, afecting birds in general, and particularly
psittaciformes that are most susceptible. Te disease is a zoonotic, posing a public threat, and leads to major
economic loss. Te drug of choice to treat the disease is doxycycline, administered intramuscularly every 5 days or
orally every 24 hours, for 45 days. Tis treatment induces prolonged stress for the bird, and frustrates the caretak-
ers, therefore resulting in low level of owners’ compliance. In this study, we evaluated in chicks a sustained-release
formulation of doxycycline dissolved in-situ gelation system, based on Poloxamer, a thermo-sensible polymer.
Te aim was to maintain a serum minimal inhibitory concentration (MIC) of 1µg/mL against Chlamydia, for
at least 21 days, following a single dose. Tirty-four light-breed chicks were injected subcutaneously once, and
serum doxycycline levels were measured for 42 days at predetermined time-points. Te average doxycycline
concentration was maintained > MIC for 21 days. An erosive reaction developed at the injection site, however,
it resolved with no medical intervention. Future studies are needed in order to improve the formulation, while
keeping the duration > MIC for at least 21 days, with minimizing the local reaction at the injection site.
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