ISRAEL JOURNAL OF
VETERINARY MEDICINE
EPIGENETIC CONTROL OF GENE EXPRESSION
Vol. 56 (2) 2001
A. Razin
The Hebrew University, Hadassah Medical School, Jerusalem
Epigenetics refers to modifications in gene expression that are controlled by heritable but potentially
reversible changes in DNA methylation and/or chromatin structure. DNA methylation is a postreplication process by which cytosine residues in CpG sequences are methylated, forming gene-specific
methylation patterns. Housekeeping genes possess CpG-rich islands at the promoter region that are
unmethylated in all cell types, whereas tissue-specific genes are methylated in all tissues except the
tissue where the gene is expressed. These methylation patterns obviously correlate with gene
expression. Further direct experiments proved that one of the most efficient gene-silencing mechanisms
involves DNA methylation. Methylation patterns are established in the embryo by erasure of the
gametic methylation patterns in the preimplantation embryo followed by global de novo methylation at
the pregastrula stage, leaving CpG islands unmethylated. Finally, specific demethylation shapes the
adult gene specific methylation patterns. Once a methylation pattern is established, it is clonally
inherited using a maintenance methylasse that copies the methylation pattern on the parental DNA
strand to the newly replicating strand. About 1% of the genes do not obey Mendel’s genetic rules being
expressed monoallelically in a parent-of-origin fashion. This phenomenon was called genomic
imprinting and this subset of genes is imprinted by an epigenetic mechanism. The imprint must be
established during gametogenesis, maintained during embryo development and erased in the primordial
germ cells to set the stage for establishing a new imprint according to the gender of the embryo.
DNA methylation had been suggested as a suitable candidate to serve as the imprint since it can be
established by de novo methylation, erased by demethylation and maintained by maintenence
methylation. Methylation, being involved in gene silencing, may also account for the monoallelic
expression of the imprinted genes. In fact, all imprinted genes possess differentially methylated regions
(DMRs) that may play a role in the imprinting process. To examine this hypothesis, a DMR in the
imprinted gene igf2r was studied. This DMR is established in the zygote by methylation of the
maternal allele and prevents methylation of the paternal allele. This is controlled by a 113 dp
imprinting box that includes in its 3’ end an 8 bp element that is responsible for de novo methylation of
the entire DMR and a 6 bp element at 5’ end that discriminates between the alleles by preventing
methylation of the paternal allele. The de novo methylation signal (DNS) binds a specific protein factor
(DNP) and the allele discrimination signal (ADS) binds another specific protein (ADP). Binding of
ADP to the paternal allele presumably interferes with DNP binding to this allele, resulting in an
unmethylated status of the DMR on the paternal allele. This allows synthesis of antisense RNA from a
promoter located downstream of the imprinting box. The antisense RNA abolishes transcription of the
igf2r gene mRNA. The methylated status of the DMR on the maternal allele prevents the synthesis of
antisense RNA, and as a result the igf2r mRNA is formed. It has recently been shown than in cloned
sheep, the imprinted status of igf2r fails to be reprogrammed since it has not ben passed through the
gametes. DMR is consequently unmethylated on both alleles and the gene is completely unexpressed,
resulting in large offspring syndrome (LOS). Failure to reprogram the imprinted state of imprinted
genes can cause developmental defects, neurological diseases and tumorigenesis, jeopardizing cloning
and the recent use of embryonic stem cells to grow tissues in culture for implantation.
GENE-IMMUNOTHERAPY OF CANCER: REDIRECTING GENETICALLY
ENGINEERED EFFECTOR LYMPHOCYTES TO CANCER USING
CHIMERIC RECEPTORS
Z. Eshhar
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
Gene-immunotherapy is using gene-transfer technology to induce and enhance disease-specific
immune responses. We took advantage of this new modality to develop a novel approach for adoptive
immunotherapy of cancer, combining the specificity of anti-tumor antibodies with the efficient tissue
rejection of T cells. To expand the recognition spectrum of effector lymphocytes and redirect them to
predefined targets, notably cancer cells, we endowed T and NK cells with antibody-type specificity,
using chimeric receptor genes. Several configurations of chimeric receptors have been designed,
mostly employing the anti-tumor antibody V region in the form of single chain variable fragment
(scFv) as the recognition domain. Several scFv's of antibodies recognizing human tumor associated
antigens were cloned and successfully used in the chimeric receptor context. As another recognition
unit, we have replaced the extracellular scFv with the Neuregulin/NDF ligand, which binds to human
adenocarcinoma cells over-expressing members of the erb-B onco-receptor family. We found that
optimal activity could be obtained with chimeric receptors made of the EGF-like domain of NDF. T
cells expressing the NDF based chimeric receptors responded to surface heterodimers made of HER2/3
or HER2/4. These scFv and ligand recognition units are linked through flexible spacer sequences
(usually hinge regions of the Ig gene family) and linked through transmembranal regions to the amino
termini of triggering subunits of the FcR ?-chain or TCR/CD3 complex ? chain. Recently, part of the
extracellular, transmembranal and the cytoplasmic regions of the CD28 co-stimulatory molecule was
used to bridge between the scFv and the cytoplasmic stimulatory domains of the ?/? chains. Such a
chimeric receptor combines both T cell stimulatory and co-stimulatory units and should be a preferred
configuration avoiding anergy or apoptosis due to inappropriate triggering. In an additional design, we
checked as triggering domains a few intracellular kinases inorder to bypass the TCR complex and its
proximal triggering events (which are often impaired in T cells derived from large tumor burden
bearing patients). Here we joined the scFv through a spacer and transmembranal stretch directly to
intracellular kinases and found Syk to be the preferred protein-tyrosine kinase in this configuration.
Following transfection into murine and human effector lymphocytes the chimeric genes have been
expressed as functional receptors and conferred non-MHC restricted, antibody (or ligand) specificity on
the recipient cells. Specific killing, cytokine secretion and elimination of tumor cells have been
demonstrated in vitro and in vivo models. A very efficient procedure, using human PBL activation and
transduction by retrovectors expressing GaLV envelopes and RetronectinTM, is being practiced to
transduce and express the chimeric receptors in 60-80%% of T cells.
To determine and optimize the clinical applicability of the chimeric receptor approach we have
established a few human prostate cancer xenografts in SCID mice. We believe that prostate cancer is an
excellent candidate for the chimeric receptor therapy not only because direct, intratumoral application
of the genetically engineered lymphocytes is possible and because the metastatic pattern of prostate
tumor (bones, lymph nodes) is readily accessible to T cells, but also because 'biological prostatectomy'
is acceptable. Experiments in which we tested the ability of the genetically engineered human T cells to
reject the prostate cancer xenografts are encouraging and demonstrate the potential of the chimeric
receptor or “T-Body” approach.
ALLOPURINOL TREATMENT DIMINISHES THE INFECTIVITY OF DOGS
WITH CANINE LEISHMANIASIS TO LUTZOMYIA LONGIPALPIS SAND
FLIES
G. Baneth1, O. Hoffman2, C.L. Jaffe2, D. Strauss1, L.F. Schnur2, B. Sandler1, E. Sekeles3,
C.L. Eisenberger2, R.L. Jacobson2 and A. Warburg2
1. School of Veterinary Medicine, Hebrew University
2. Department of Parasitology, The Hebrew University Hadassah Medical School
3. Merhav Veterinary Clinic, Nataf
Allopurinol is used for the therapy of canine leishmaniasis in Europe and it is currently the main
treatment for this disease in Israel. Allopurinol treatment is oral, inexpensive and can be prescribed by
the attending veterinarian and administered by the owners of the dog at home. This study was designed
to assess the clinical and parasitological efficacy of allopurinol in dogs with visceral leishmaniasis
(VL). Five dogs with naturally-occurring VL were treated with 20 mg/kg of allopurinol once daily for 8
months. The dogs were physically examined and tested pre- and during treatment. Testing included: the
determination of serum anti-leishmanial antibody titers by ELISA, recording of globulin levels and
albumin/globulin ratio, evaluation of the parasitological status by culture of splene and lymph node
aspirates, xenodiagnosis and the determination of the infectivity to Lutzomyia longipalpis sand flies.
RESULTS: Clinical improvement manifested by the return to physical activity, improvement of skin
lesions and decrease in the size of peripheral lymph nodes were noted in all the dogs. Although the
anti-leishmanial antibody titers decreased in the sera of all dogs, they remained seropositive. The drop
in antibody titers was accompanied by the decrease in globulin levels and increase in the
albumin/globulin ratio. Four of the 5 dogs remained parasitologically positive at the end of the study.
Three dogs were tested by xenodiagnosis pre-treatment and four times during treatment. Initially these
3 dogs were infective to L. longipalpis with 89, 59 and 41 percent of the sand flies dissected positive
for promastigotes, respectively. The % of infected sand flies decreased during the course of treatment
and diminished by 4 to 5 months. The two additional dogs included in the study were parasitologically
positive before the initiation of allopurinol treatment, and although they were not tested by
xenodiagnosis pre-treatment, they too were not infectious to sand flies at the end of the study. In
conclusion, daily allopurinol treatment of canine leishmaniasis at 20 mg/kgs can induce clinical
improvement. Like other anti-leishmanial drugs used for treatment of canine leishmaniasis, it does not
bring about parasitological cure in most cases. The sharp decrease in infectivity of treated dogs to L.
longipalpis sand flies could be important in areas where disease transmission takes place.
A SEROEPIDEMIOLOGICAL STUDY OF CANINE VISCERAL
LEISHMANIASIS IN ISRAEL
B. Sandler1, D. Strauss1, S. Harrus1, R. King2, C.L. Jaffe3, B. Yakobson4 and G. Baneth1.
1. School of Veterinary Medicine, Hebrew University.
2. Nature Reserve Authorities.
3. Hadassah School of Medicine, Hebrew University
4. Kimron Veterinary Institute
Zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum has been reported recently in
central Israel and in the Galilee region of northern Israel. A seroepidemilogical study to examine the
prevalence of VL among domestic and wild canids in Israel was carried out during 1999-2000 to assess
the spread of the disease and the possible association between infection in dogs, wild canids and
people. Anti-leishmanial antibodies were detected by enzyme-linked immunosorbent assay (ELISA).
Serum samples (n=790) were collected from domestic dogs in municipal pounds, society for prevention
of cruelty to animals (SPCA) kennels, military camps, and veterinary hospitals.
Surveys were also performed among dog populations in three villages that are known foci of VL
(n=146). Wild canid sera were supplied by the Natural Reserves Authorities and included samples from
jackals (Canis aureus n=158), foxes (Vulpes vulpes n=93) and wolves (Canis lupus n=16).
Out of a total of 1,203 animals sampled, 43 (3.6%) were positive. Only 0.76% (6/790) of the domestic
dogs sampled randomly from locations that were not previously recognized as disease foci were
seropositive, while 15% of the dogs from endemic villages were infected. The seroprevalence among
wild canids was 5.6% with most of the positive sera originating from jackals (7.6% positive). Passive
surveillance, which included testing of samples sent by referring veterinary practitioners to our
laboratory during 1999-2000, identified 18 new locations in villages and cities throughout central and
northern Israel where infected dogs (n=22) were diagnosed.
We conclude that the transmission of canine VL in Israel appears to be multifocal. Although the
disease is in general restricted to specific foci and less likely to be found among dogs living elsewhere.
The passive detection of infected dogs indicates that these transmission foci are numerous and are
spread throughout different geographical regions and habitats. Visceral leishmaniasis appears to be
prevalent in wild canid populations that may constitute an important natural reservoir for L. infantum in
our region.
TICK TOXICOSIS CAUSED BY IXODES REDIKORZEVI IN A DOG FROM
CENTRAL ISRAEL
Z ֶFishman, S. Harrus, I. Yeruham, I. Aroch, Y. Alekseev and G. Baneth
Koret School of Veterinary Medicine, The Hebrew University of Jerusalem
A 3-year-old female Weimaraner dog, from moshav G. was presented to the Veterinary Teaching
Hospital with an acute lameness of the front left leg. A week prior to admission, acute swelling and
edema appeared on the right eyelid and was accompanied by local pain and systemic depression. A tick
was discovered at the center of the swollen area and was detached by the owners. The swelling
resolved within 18 hours, however depression persisted for another 14 hours. On admission, the dog
was depressed and did not use the left front leg. On physical examination the following clinical signs
were observed: depression, lethargy, swelling and edema of the carpus area (predominantly of the
palmar side) accompanied by sharp pain during flexion of the carpus. A tick was discoverd on the
palmar side of the leg, above the carpus. Complete blood count revealed leukocytosis
(WBC=20.9X103/?l). A soft tissue swelling without bone involvement was noticed on radiography.
The tick was classified as an adult female Ixodes redikorzevi and the dog was diagnosed as suffering
from tick toxicosis. Three hours after detachment of the tick the dog regained use of the leg and the
swelling started to regress. The owners reported a progressive resolution of the swelling and normal
behavior by the next day.
Ixodes redikorzevi is a three-host tick. It is prevalent in Europe, eastern Mediterranean basin (Iran,
Pakistan and Afghanistan) and Nepal. The tick is transferred by migrating birds and its prevalence in
Israel is sporadic. To date, twenty-seven cases of tick toxicosis in humans and five cases in dogs were
documented in Israel. Most cases occurred in northern Israel and the coastal area. Predominant clinical
signs included restriction of movement, lameness accompanied by local edema, swelling and pain in
the affected limb, regional lymphadenomegaly and hyperthermia. Clinical signs resolved in all cases
within a day or two following tick detachment.
TRAINING GUIDE DOGS FOR THE BLIND
Y. Peres
Israel Guide Dog Center for the Blind, Bet Oved
The Center’s Goals and Activities:
The IGDCB was established in 1991, to provide safe mobility and independence for the blind in Israel
and help them become an integral part of society and labor circles.
The Center graduates 20 partnerships each year. The current number of partnerships is over 140. The
entire process takes place at the Center: Dog breeding and care; Dog training; Instruction of the blind.
The Guide Dog: instruction and follow-up aftercare are provided to the blind person at no cost.
The creation of a partnership:
Dog breeding
Puppy-raising
Dog training
Instruction course
Follow-up aftercare
Breeding:
Common breeds: Labrador Retriever, Golden Retriever, Hybrid ( Labrador-Golden cross), German
Shepherd, others
Studs and Brood Bitches - At volunteer homes, or at a breeding center.
Breeding program - For behavior and anatomical patterns, elimination of genetic disorders, high
fertility.
Puppy-raising:
Volunteer foster families raise the pups from the age of 7-8 weeks, for one year. Expose the pups to
“human” environment - Family, guests, urban and country environments, stores, shopping malls, public
transport, restaurants, etc. Basic training - House training, teaching basic commands. Tender loving
care
Dog training:
Training takes 5-6 months. Initial evaluation : Health - History, physical examination, blood tests and
radiographs; Social behavior - Foster family report, observation. Reaction to stimuli - Other dogs, cats,
other animals, objects, noises
Training : Discipline - Work on a leash and response to hand and verbal commands; Guiding position Turns, ignoring distractions; Regulating pace - Speed, turns, halts; Obstacle recognition - Sidewalk,
road, various obstacles; Working in harness - Continue walks and obstacle training, in full harness. At
more advanced stage - the trainer walks blindfolded; Working in various environments - Urban and
country, public transport, shopping centers; Locating doors, stairs, cross-walks, bus stops
Course instruction:
A four-week course, four students in a class; At the Center - First three weeks: Orientation Introduction to the Center’s facilities; Frontal lectures
Dog health and care, dog behavior and
psychology, anatomy and physiology; Introduction to the harness and handle; Instruction - Walks with
the dog under the instructor’s supervision; Gradual withdrawal of the instructor’s supervision; Walks
along various routes; At the blind person’s home - Fourth week: Aquainting the dog to his new home
and environment, under the instructor’s guidance; Introduction to new routes with the instructor
Aftercare:
Tightening of the bond - To full cooperation and harmony may take several months; Home visits Routine periodic visits; Support and assistance - As needed by telephone or home visits; Guide dog
replacement - Upon retirement of the dog
THE EFFECT OF ELECTROCARDIOGRAPHIC FILTERS ON THE RAMPLITUDE OF CANINE ELECTROCARDIOGRAMS
E. Dvir, P.J. Cilliers, R. G. Lobetti,
University of Pretoria, RSA.
The goal of this study is to describe the influence of low-pass filtering on R-amplitude in the canine
ECG and to propose a simple formula to compensate for these changes.
Additionally the correlation between the mass of the dog and the magnitude of the reduction in the Ramplitude by the filtering was evaluated.
Lead II ECGs were recorded in 88 dogs with canine babesiosis ranging in mass from 3 to 59 kg. A
commercial direct writing electrocardiograph was used with manual notch filter at 50/60 Hz and a lowpass filter with a cutoff frequency of -3 dB at 35 Hz ON, immediately followed by recording with both
filters OFF.
The reduction in R-amplitude from filters OFF to filters ON settings was evaluated and ranged from
22-100% (mean =53 +18%, median = 15%). The R-amplitude with filters OFF was related to the Ramplitude with filters ON, providing a possible practical formula to retrospectively correct for the
effect of the filters. The R-amplitude reduction was found to be inversely correlated to body mass and
to QRS-complex duration. Other known changes induced by filters, such as the elimination of notches
and slurring of the junction between the QRS-complex and the ST-interval to a point of coving, were
also observed.
These servers R-amplitude reduction indicate that for canine practice an electrocardiograph with better
high frequency response than that obtained with filters in an ECG machine designed for humans is
needed. Electrocardiograms should preferably be recorded without filters. However, severe electrical
noise artifacts sometimes necessitate the use of filters, indicating the need for specifically designed
ECG-filters for veterinary use. The inverse relation between dog mass and the magnitude of Ramplitude reduction due to ECG-filters indicates the need for different ECG-machine characteristics
for different size animals.
PREVALENCE AND CONTROL OF COCCIDIOSIS IN DAIRY CALVES
A. Markovics
Department of Parasitology, Kimron Veterinary Institute, P.O.Box 12, 50250 Beit
Dagan, Israel.
A study conducted on calves of different ages from 31 large (kibbutz) dairy farms during 1998-19999
demonstrated that coccidiosis is prevalent in young calves. Coccidial oocysts were detected in all 16
farms previously known to be infected and in 12 out of 15 farms in which coccidial infection was not
diagnosed prior present study. Similar rates of infection were found in calves of 2-4 and 4-6 months
age groups (43.4% and 39.1%, respectively). The infection rate in 1-2 month calves was significantly
lower (10.9%) and oocysts were detected only in 5 out of 28 positive farms. In one farm, where
newborn calves were kept in proximity with older infected calves, the morbidity was detected already
in one month old calves. Seven species of Eimeria were identified in the present survey of which the
predominant ones were E. bovis and E. zuernii. Results of the survey and the records of outbreaks of
clinical coccidiosis in recent years indicated that only E. bovis and E. zuernii are pathogenic in dairy
calves.
The influence of husbandry and environmental factors (humidity and low temperatures) prevailing
during the winter on levels of coccidial infection were evaluated. Rearing newborn calves separately
from older ones prevented coccidial infection until weaning. Frequent changes of bedding and
prevention of feed and water contamination by raising the water fountains and feeding troughs above
floor level significantly reduced the levels of infection and morbidity on several farms affected with
clinical coccidiosis. Elevated levels of environmental contamination and calf morbidity were found in 3
out of 4 farms examined during the winter months. Treating calves with lasalocid at a dose of 35 mg/kg
concentrated feed did not prevent infection and morbidity in 2 —6 month old calves. However a dose
of 100 mg/kg concentrated feed reduced significantly the number of oocysts shed by treated calves and
prevented clinical coccidiosis on four affected farms.
BSE SURVEY IN ISRAEL AND ITS CURRENT STATUS IN WESTERN
EUROPE
S. Perl, Y. Zacharine, N. Sheichat, D. Lahav and U. Orgad
Department of Pathology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel
O. Israeli and S. Ben Said - Tel Aviv Municipality Abattoir.
Bovine spongiform encephalopathy (BSE) was first diagnosed in the United Kingdom in 1986, and
until the end of 2000 the disease was found in 180,924 cattle. Increasing numbers of BSE cases are
now being observed in certain other European countries through 1994 to 2000. In December, 2000
Germany diagnosed its first local BSE cases.
BSE belongs to the transmissible spongiform encephalopathies (TSE) group that can affect humans
(Creutzfeldt-Jacob, Gerstmann-Straussler syndrome, kuru and fatal familiar insomnia), sheep (scrapie),
mink (mink transmissible encephalopathy), felines, deer and other wild animals. The cause of the
disease is a distorted prion that affects the central nervous system being expressed histologically by the
presence of vacuolar changes in the neurons and neuropil in specific areas of the brain such as the
obex, thus giving a spongiform aspect to the brain tissue.
The origin of BSE may be explained by oral exposure to a scrapie-like agent in the ruminant-derived
protein of meat and bone meal included in concentrates or feed supplements. Experimental
transmissibility of BSE to cattle has been demonstrated following parenteral and oral challenge with
brain tissue from affected cattle. The disease became extremely important because of the link of BSE to
a new variant (vCJD) of human Creutzfeldt-Jacob disease (CJD), of which 10 cases were diagnosed in
1996. Until 31 December 2000, a total of 83 confirmed deaths from the disease were reported in the
UK. The most likely explanation for the occurrence of vCJD in humans is the exposure of the victims
to the etiological agent of BSE believed to be an abnormal form of the prion protein (PrPres).
Israel is one of the few countries that banned the importation of meat and bone meal of ruminant origin
from the U.K. in 1988. The Department of Pathology of the Kimron Veterinary Institute is conducting
an annual BSE survey according to OIE protocols to monitor the bovine population for specific
manifestations of the disease. During 1997-2000, 988 bovine brains were examined; some of the cattle
had CNS signs (but none even remotely resembled of BSE) as well from cattle randomly sampled at
abattoirs. All were found to be negative.
In 2001 the BSE survey will be extended by introducing the new Prionics Check test. This method was
approved by the OIE.
It is possible that in the future, every cow over 30 months old, will be tested for BSE before entering
the food chain if such a decision is taken by the veterinary services.
SEROLOGICAL AND IMMUNOLOGICAL RELATIONSHIP OF NEOSPORA
CANINUM AND BESNOITIA BESNOITI
Reske, Adi, V. Shkap and E. Pipano
Kimron Veterinary Institute, P.O.Box 12, Bet Dagan 50250, Israel
Neospora caninum is a tissue cyst-forming coccidian parasite, recognized as a major causative agent of
abortion in bovines. Infection with N. caninum might occur concomittantly in the same herd with
another taxonomically related parasite of bovines, Besnoitia besnoiti, which affects the reproductive
system and causes infertility in bulls. Both parasites stimulate immune responses and produce specific
antibodies. Cross-reactivity tests with the two parasites were performed to exclude the possible
interference in serodiagnosis of neosporosis and besnoitiosis. In the present study, performance of the
IFA and ELISA, were compared with that of the commercially available IFA and the competitive
ELISA (cELISA), the latter assays considered as “gold standards”. Using the commercially available
IFA and cELISA as “gold standard” on 30 and 105 randomly selected sera, respectively, our “homemade” assays were found to be both specific (90.9% + 9.1%) and sensitive (95.7% + 2.1%).
Comparison between the two assays, IFA and ELISA, showed the two tests to be both compatible as
serodiagnosis assays with a 95% agreement (k = 0.773 + 0.097), IFA being more specific and ELISA
being more sensitive.
Western immunoblotting analysis of B. besnoiti and N. caninum tachyzoites revealed proteins common
to both N. caninum and B. besnoiti. Negligible reactivity was detected when heterologous antibodies
were applied to either antigen. Immunologic relationship was studied by immunization and challenge
cross-reaction in Meriones unguiculatus. No cross-protection was observed when infected gerbils were
challenged with heterologous parasites. Neospora caninum could be differentiated from Besnoitia
besnoiti infection by both serological tests applied, IFA and ELISA. Evidently, there seems to be no
cross-reactivity between the two coccidian protozoa, hence, a possible infection with B. besnoiti does
not interfere with serodiagnosis of N. caninum.
THE EFFECT OF RAISING TESTIS TEMPARATURE BY UNILATERAL
CRYPTORCHILD SIMULATION ON RAM SEMEN QUALITY
R. Yehuda, S. Marcus, M. Reichart and B. Bartoov
Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
Cryptorchidism in domestic animals is hereditary. In general it is recognized as a unilateral or bilateral
undescendence of the testicles into the scrotum. In our case a system was established to simulate a right
unilateral ram cryptorchid mainly by raising the right side scrotal temperature.
The objects of this research was to examine the influence of long term insulation of the right scrotum
on semen quality, semen characteristics and its fertility potential in IVF of sheep ova.
Among other things we wanted to find whether the insulation of the right scrotum has any effect on the
performance of the collateral testicle (sympathy). The recovery potential of the breeding performance
of the ram was also examined after the removal of the insulation. The following subjects were
examined during the trial scrotum temperature, general clinical condition of the testes, semen volume
and pH, biochemistry of the seminal plasma (secondary sex gland markers and hormonal levels) and
semen quality: concentration, vitality, motility, acrosin activity, peroxidation activity, cell membranes
integrity, nuclear chromatin integrity, morphology and in vitro fertilization (IVF) capacity.
The hyperthermia effects could be divided into 5 different groups by the kinetics of the parameters: I.
Parameters that are sensitive to thermoregulatory insult of the testicles with partial sympathy between
the testicles but witout connection to IVF results. II. Parameters that serve as markers to
thermoregulatory insult of the testicles with complete sympathy between the testicles but without
connection to IVF results. II. Parameters that serve as markers to thermoregulatory insult of the
testicles with complete sympathy between the testicles but without connection to IVF results. III.
Parameters that are sensitive to thermoregulatory insult of the testicles only after the removal of the
insulation instrument from the ram without sympathy between the testicles and without connection to
IVF results. IV. P arameters that are sensitive to thermoregulatory insult of the testicles with complete
symphaty between the testicles and with connection to IVF results. V. Parameters that are insensitive to
themoregulatory insult of the testicles.
It was concluded that in the ram and perhaps in other mammals there exist a physiological
communication (sympathy) between the teste.
THE STATE OF ART OF CAMEL RESEARCH AND DEVELOPMENT
M. Plaves-Kitron,1 Z. Nevo,1 C. VanCreveld,1 U. Merin2 and R. Yagil1
1. International Camel Center, Central Arava
2 . Department of Food Technology, Volcani Research Institute, Bet Dagan
The International Camel Center in the Arava is the leading research and development center of
camelides with connection all over the world. The Camel Center has developed the milking techniques
which yields 15 liters of milk in twice a day milking, have developed camel milk products based on the
special properties of the milk and are at present carrying out a series of experiments into the medicinal
properties of the milk. The Camel Center organized the Second International Conference on Camels
”Agro-economics of Camelid Farming” which was held in September, 2000 in Almaty, Kazahstan. The
Center is training persons from Africa, Asia and South America in the development of camel
enterprises and the veterinary services of Israel and Jordan will soon begin a series of collaborative
camel programs under the guidance of the Camel Center. The European Community is supporting the
development of industrialized products for camel farms. The connection between basic scientific
research, agricultural development and industrial development induced the organizers of EXPO 2000
which was held in Hannover from June to November, to register the Israeli Camel Project as one of the
200 “Projects Around the World”.
THE USE OF ENZYME RESTRICTION FOR PRODUCTING TRANSGENIC
ANIMALS
E. Harel-Markowitz, M. Shemesh, M. Gurevich, L.S. Shore and Y. Stram
Kimron Veterinary Institute, P. O. Box 12, 50250 Bet Dagan, Israel
Integration of exogenous DNA into the vertebrate genome with subsequent transference to offspring
is the basis of producing transgenic animals. In domestic animals, transgenesis is seldom performed
due to its high cost. The present method for producing transgenic animals is by injection of DNA into
the male pronucleus of the zygote. This is an efficient system in rodents but gives poor results in
domestic animals.
The purpose of this work was to create cloned sperm using enzyme restriction and to use the resultant
sperm to produce transgenic animals. We used as a reporter gene plasmid green fluorescent protein
(pGFP). Sperm cells from bulls and roosters were lipofected with pGFP using restriction enzyme
mediated insertion (REMI). The restriction enzyme was used to open a specific area of the genomic
DNA and to insert the linearized DNA with sticky ends by cutting the plasmid DNA with the same
restriction enzyme. The cloned sperm cells were used for artificial insemination in cows and hens.
It was found that 89% (17/19) of the F-1 birds, which were randomly sampled from a group of 30
inseminated birds, expressed the inserted gene in their lymphocytes as determined by RT-PCR, PCR
and FACS. Homology between the sequence of cDNA as compared to the appropriate sequence in the
reporter gene was 99%. 8/9 of the birds were shown to be positive in Southern blot for the reporter
gene sequence. Similar results were obtained in calves: 2/2 were positive in PCR and one of the two
calves was also positive by Southern blot. REMI and lipofection are therefore a basis for an
inexpensive technology for producing transgenic farm animals.
NEW RABIES VARIANT IN ISRAEL
D. David, N. Devers, D. Rotenberg and B. Yakobson
Rabies Laboratory, Kimron Veterinary Institute, Bet Dagan 50250, Israelֶ
Rabies in Israel is endemic throughout Israel. Previous work using RT-PCR and direct sequencing of
the N gene fragment of Israeli rabies isolates revealed the existence of five sequence groups in four
geographic regions. During a period of 4 months between June to October 2000, four rabid foxes from
the Arava valley were positive for rabies. A molecular and antigenic study of these rabies isolates and
of others isolates collected from all over the country was conducted. The molecular analysis of the 469bp fragment from the pseudogene showed that the sequences of the four rabies isolates were identical,
but they were different from the sequences of other Israeli isolates. The antigenic characterization of
these isolates using a panel of 19 monoclonal antibodies showed the existence of a new antigenic
variant. The molecular and antigenic comparison of the Israeli and Jordanian rabies isolates revealed
close a relationship between the four foxes and a Jordanian isolate obtained from Amman, Irbid and
Mafrak. In conclusion based on the molecular and the antigenic study, the four foxes might penetrate
from Jordan. The control of rabies in the Middle East is benefited by cooperation between Israel and
Jordan.
PHARMACOKINETICS OF GENTAMICIN COMPONENTS IN HORSES
A. Steinman 1, N. Isoherranen3, O. Ashoach1 and S. Sobac2
1. Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, P.O.
Box 12, 76100 Rehovot, Israel
2. National Residue Control Laboratory, Ministry of Agriculture, Kimron Veterinary
Institute, P.O. Box 12, 50250 Beit Dagan, Israel
3. Laboratory of Analytical Chemistry, Department of Chemistry, University of
Helsinki, P.O. Box 55, 00014 Helsinki, Finland
The objective of this study was to investigate the pharmacokinetic behavior of gentamicin C 1, C1a and
C2 following intravenous administration at 6.6 mg/kg body weight to 6 healthy adult horses.
Significant difference in clearance, half-life, and mean residence time between the gentamicin C1a and
the two other components was found. The mean ± standard deviation clearance of
gentamicin C1a (1.62±0.50 ml/min*kg) was similar to the glomerular filtration rate (GFR) reported
for horses, indicating lack of tubular secretion and tubular reabsorption. The mean ± standard deviation
clearance of gentamicin C1 (1.03?0.08 ml/min*kg) and C2 (1.10±0.15 ml/min*kg), which were
smaller than the GFR in the horse, indicated reabsorption of these components by the renal tubules.
Consequently contributing to the gentamicin nephrotoxic potential, which is attributable to uptake of
the drug into proximal convoluted tubular cells. The mean ? standard deviation mean residence time
and half-life of gentamicin C1a (2.7±0.31 h and 2.4±0.46 h, respectively) were about 30%
shorter than gentamicin C1 and C2 values. The mean ± standard deviation clearance of
gentamicin (1.25±0.21 ml/min*kg), volume of distribution at steady state (0.24±0.06 l/kg), and halflife (2.9±0.73 h), were similar to previously reported values. A wide variation in the component ratio
between different pharmaceutical gentamicin preparations as well as between different batches of the
same product, and this difference in pharmacokinetics between the gentamicin components, may affect
the gentamicin mixture pharmacokinetic profile, thus, supporting the need for individual
pharmacokinetic monitoring and dosage adjustment.
In the last few years new approaches for silencing gene expression on the RNA level have been
developed. By using these techniques, one can specifically cleave designated mRNA which will lead to
inhibition of gene activity. One approach for silencing specific genes is the use of ribozymes that can
cleave target RNA at a specific sequence and thus inhibit its activity. A second system termed RNA
interference (RNA) can completely degrade target mRNA which will also lead to gene silencing. In the
present project, we apply these systems to inhibit viral replication Foot-and-mouth disease virus
(FMDV) was chsen as a model, FMDV a ssRNA virus, infects cloven-hoofed animals is major threat to
the milk and meat industry.
Ribozymes are RNA molecules with catalytic activity able to cleave RNA molecules. The recognition
and cleavage site on the target RNA molecule comprisse a sequence of 3 nucleotides which statistically
can be found in almost any mRNA molecules, thus making it a target for ribozyme activity.
In our laboratory ant-FMDV RNA ribozyme genes targeted to cleave several sites on the viral genome
were designed and prepared. We could show specific cleavage of Asial VP3 and VP4 seqences. We
also designed and prepared several ribozymes targeted to clrave the viral polymerase (3D) gene. Since
the viral polymerase gene sequence is conserved among all 7 viral serotypes we were able to
demonstrate that one of these RNA enzymes can cleave the polymerase sequences originated from A,
Asial, C, and O1 serotypes and probably the other 3 serotypes.
If this ribozyme can be demonstrated to act whole cell, it will be able to inhibit viral replication
regardless of the serotype. By using the restriction enzyme mediated insertion (REMI) technique we
prepared transgenic bovine carrying and expressing the ribozyme gene. Currently we are testing viral
inhibition activity in cells prepared from these transgenic animals.
A second system for silencing gene expression is RNAi. In the last 3 years it was discovered that
dsRNA prepared according to a specific mRNA sequence can completely digested the target mRNA
thus silencing its function and this activity was termed RNAi. RNAi activity have been shown in
plants, nematodes, insects and single cell parasites. Recently it was also shown that RNAi activity can
take place in cells of the mous in the early embryoinic development stages. In our laboratory we are
planning to extend this observation to bovine embryonic cells and in cells from advanced embryonic
stages and to live offspring.
GENE SILENCING AS AN OPTION FOR PROTECTING LIVE STOCK
ANIMALS AGAINST VIRAL INFECTION
Y. Stram, A. Shore, L. Kuznetsova, T. Molad, M. Gurevitch, H. Yadin, D. Hai, B.
Gelman.and M. Shemesh
In the last few years new approaches for silencing gene expression on the RNA level have been
developed. By using these techniques, one can specifically cleave designated mRNA which will lead to
inhibition of gene activity. One approach for silencing specific genes is the use of ribozymes that can
cleave target RNA at a specific sequence and thus inhibit its activity. A second system termed RNA
interference (RNA) can completely degrade target mRNA which will also lead to gene silencing. In the
present project, we apply these systems to inhibit viral replication Foot-and-mouth disease virus
(FMDV) was chsen as a model, FMDV a ssRNA virus, infects cloven-hoofed animals is major threat to
the milk and meat industry.
Ribozymes are RNA molecules with catalytic activity able to cleave RNA molecules. The recognition
and cleavage site on the target RNA molecule comprisse a sequence of 3 nucleotides which statistically
can be found in almost any mRNA molecules, thus making it a target for ribozyme activity.
In our laboratory ant-FMDV RNA ribozyme genes targeted to cleave several sites on the viral genome
were designed and prepared. We could show specific cleavage of Asial VP3 and VP4 seqences. We
also designed and prepared several ribozymes targeted to clrave the viral polymerase (3D) gene. Since
the viral polymerase gene sequence is conserved among all 7 viral serotypes we were able to
demonstrate that one of these RNA enzymes can cleave the polymerase sequences originated from A,
Asial, C, and O1 serotypes and probably the other 3 serotypes.
If this ribozyme can be demonstrated to act whole cell, it will be able to inhibit viral replication
regardless of the serotype. By using the restriction enzyme mediated insertion (REMI) technique we
prepared transgenic bovine carrying and expressing the ribozyme gene. Currently we are testing viral
inhibition activity in cells prepared from these transgenic animals.
A second system for silencing gene expression is RNAi. In the last 3 years it was discovered that
dsRNA prepared according to a specific mRNA sequence can completely digested the target mRNA
thus silencing its function and this activity was termed RNAi. RNAi activity have been shown in
plants, nematodes, insects and single cell parasites. Recently it was also shown that RNAi activity can
take place in cells of the mous in the early embryoinic development stages. In our laboratory we are
planning to extend this observation to bovine embryonic cells and in cells from advanced embryonic
stages and to live offspring.
OVINE AND CAPRINE NEOPLASMS - AS ANALOGUES TO HUMAN
LUNG CANCER
K. Perk
Koret School of Veterinary Medicine Rehovot, Israel
In contrast to the prevalence of primary neoplasms of the lungs in humans, primary neoplasms of the
lungs in the animal kingdom are rare. Sheep and goats are exception in having two proliferative lung
diseases distributed world wide. These diseases are
1. Sheep pulmonary carcinoma (S.P.C) whice exhibits all the criteria included in the definition of
bronchoalveolar cell carcinoma. The infection and diseas are induced by a retrovirus in nature or in the
laboratory. The S.P.C- virus do not contain a typical oncogene but carries a unique region in the
regulatory signal region, of which a product has been demonstrated to activate the arthifisically
trascription of various cellular genes as well as the viral genes. Some of the cellular genes activated
contribute to cell proliferation and transformation. These will be further discussed. Epidemiological
evidence indicates furthermore that this neoplasm is horizontally transmitted. Comparative aspects and
its relevance to human bruonche carcinoma will be discussed.
2. Maedi/Visna (M.V) of sheep and caprine arthritis-encephalitis (CAV) are manifested by typical
interstitial proliferation of the lymphoid aggregates confined to the lungs. Alveolar epithelial
hyperplasia in the lungs is generally also seen, especially in older goats and sheep. Proliferation of
these epithelial cells may form acine and papillary structures and in some cases are histopathologically
indistinguishable form tumor nodules seen in sheep pulminary carcinoma. Since maedi/visna and
caprine arthritis-encephalitis diseases are caused by lentiviruses closely related to the human
immunodeficiency virus (HIV) Their significana to the human disease which will be discussed.