ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 60 (1) 2005
THE 3RD ANNUAL SYMPOSIUM OF VETERINARY
MICROBIOLOGY AND IMMUNOLOGY
BET-DAGAN, ISRAEL. DECEMBER 2004.
Chairman: D. Elad
DYNAMICS OF ORGAN SPECIFIC INTERACTIONS OF WEST NILE VIRUS IN MICE
S. Kleiman1, S. Landes1, T. Dvorkin, D. Ben-Nathan2, A. Progador , B. Rager-Zisman1.
1. Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion
University of the Negev, Beer-Sheva,
2. Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona.
West Nile fever (WNF) is a mosquito-borne disease found in Africa, West Asia, the Middle
East, and since 1999 has emerged in Central and North America. It is known that WNV
circulates in a natural transmission cycle involving mainly Culex mosquitoes and wild birds.
Lately an exceptionally broad range of mosquito species and more incidental hosts such as
geese, crows, dogs, cats, alligators, primates and humans have been identified. The
conventional mode of transmission to humans and other incidental hosts is by mosquito bite.
Surprisingly, it was recently shown that WNV can also be transmitted to humans via blood
transfusions, lactation (breastfeeding), organ transplants and laboratory accidents. Humans
infected with WNV develop a febrile illness that may progress to meningitis or encephalitis. In
laboratory mice, WNV causes a central nervous system infection, paralysis, encephalitis
leading to death between 1-2 weeks depending on the age and strain of the mouse and the
size of the infecting dose. In view of these recent findings the goal of the present study was to
characterize the dynamics of virus dissemination and organ specific interactions of the virus
during the acute phase of infection in mice. We have tested the effects of WNV brain infection
on acetylcholine esterase (AchE) activity. BALB/c mice were injected with wild-type WNV.
Virus levels in several organs (liver, lungs, spleen, heart and brain) were determined in
individual mice by RT- PCR and plaque assay on days 1 to 7 after infection. Although WNV
was detected in all these organs, the dynamics of organ-virus interactions varied in each
organ. Viral RNA was detected as early as on day 1 after infection in most of the organs,
whereas different levels of infectious virus were found in various tissues from day 3. The
dynamics of viral replication and clearance were organ dependant. We also demonstrated
that virus infection of the brain down-regulated AchE activity. Our results demonstrated that
WNV infection is not restricted to the CNS but also affects peripheral organs.
COMPARISON OF MILK WITH SERUM ELISA FOR THE DETECTION OF
PARATUBERCULOSIS IN DAIRY COWS
M. Chaffer1, O. Koren2, S. Friedman3, M. Freed3, Z. Beider1, D. Elad1.
1. Department of Bacteriology, Kimron Veterinary Institute. Bet Dagan 50250.
2. Israel Dairy Board, Rishon Le’Zion, 75054.
3. National Service for Udder Health and Milk Quality, Cesarea, 38900.
Available tests for diagnosing paratuberculosis are based on detection of either the
pathogen or the host’s immune response. Detection by bacteriological culture of
faecal samples requires a minimum of 8 weeks of incubation; thus detection of
antibody response by ELISA is advantageous for the diagnosis of the disease. In the
last years, milk ELISA has been developed to check the disease.
Milk recording is performed monthly in Israel at the Central Milk Laboratory. More
than 100.000 cows from 819 herds, or about 88% of the cows in the country, are
checked monthly though the milk to which a preservative (Bromopol) was added.
In this study, a milk ELISA for detection of antibodies against Mycobacterium avium
subsp. paratuberculosis was evaluated.
Blood was collected from 1520 milking cows at seven farms. All serum samples were
tested by a commercial IDEXX ELISA. In addition, 737 raw milk or 783 preserved
milk samples from these cows were checked for presence of Myocbacterium avium
subsp. paratuberculosis antibodies with a commercial Pourquier ELISA.
The proportion of agreement between milk ELISA and serum ELISA was 98% when
using raw milk and 97% with preserved milk. The agreement between the 2 tests
when assessed using Kappa value was excellent (0.82) on comparing raw milk with
serum, and good (0.80) when milk with preservative was used.
Raw milk or preserved milk from the National Milk Recording seems to be a good
alternative for serodiagnosis of paratuberculosis in cows.
RAPID TRANSLOCATION OF SKIN EPITHELIAL BARRIERS BY
STREPTOCOCCUS INIAE, AN INVASIVE BUT NON-INTRACELLULAR
PATHOGEN
M. Eyngor, D. Lahav and A. Eldar
Kimron Veterinary Institute, Beit Dagan.
Despite the widespread importance of the disease, mechanisms of S. iniae
pathogenesis have been poorly characterized, and the few available experimental data
have relied strongly on the use of non-fish models. Clearly, there are some
deficiencies in this approach, raising questions as to the validity of these findings in
fish. Additionally, as most previous models used to assess strain virulence and
pathogenicity by intraperitoneal or intramuscular inoculation of bacteria, data
pertaining to initial sites of adhesion, colonization and invasion were not available.
While external surfaces provide a primary defense against invading organisms,
several pathogenic bacteria possess the (in vitro) ability to translocate an epithelial or
mucosal cell barrier without causing apparent damage. Such translocation in vivo is
an important virulence feature, as it allows the invading organism access to
underlying tissues without causing an initial inflammatory reaction and may permit
the pathogen to spread through the host. By constructing a biologically relevant model
based on in vitro culture of rainbow trout (polarized) primary skin epithelial cell
monolayers, we have investigated the series of early events that precede S. iniae
infection, particularly in colonization and translocation through external barriers. In
vitro data, supported by electron microscopy microphotographs, demonstrate that S.
iniae successfully invades skin epithelial cell, exists free in the cytoplasm after release
from the endosome and translocates the rainbow trout skin barrier. Bacterial invasion
and transcytosis were not accompanied by morphological changes of host cells.
THE USE OF OZONE FOR EXTENDING SURVIVAL OF LIVE AND
PROLONGING SHELF LIFE OF CHILLED FISH
Gelman A.,1 Glatman L.,1 Sachs O.,2 Khanin Y.,2 Drabkin V.,1 Chechik K.,1 Gabay
I.1
1. Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250
2. Dor Aquaculture Experimental Station, Dor.
Live tilapia were ozone-treated at Dor Aquaculture Experimental Station, and were
transported to Fishery Products Laboratory for storage and performing sensory,
bacteriological, chemical, and physical studies.
Long-term, low-level ozonation of live fish in the tank prolonged their survival and
improved their physical condition. By the 5th day almost all the treated fish were
alive and healthy, whereas most of the control fish were dead by the 3rd day. This
could be the result of reduction of bacterial contamination, and especially the
prevention of S. putrefaciens growth in the ozonated fish. This treatment could be
used for improving commercial fish quality in fish shops.
Chilled tilapias were stored at 0oC and 5oC after short ozone (6 ppm) pretreatment of
live fish. Sensory analysis showed that ozone pretreatment prolonged their storage life
by 12 days (40%) and improved their quality characteristics through one month's
storage at 0oC. Total counts of bacteria on the surface of the pretreated fish were less
by 2-3 log CFU cm-2 than the controls, and their muscles were practically sterile by
day 30. At 5oC no differences in bacterial contamination and only a three-day
extension of the shelf-life were recorded. These effects could be the result of initial
reduction and prevention of growth of spoilage bacteria such as Pseudomonas
fluorescens, Shewanella putrefaciens, and Aeromonas sobria. The combination of
ozone pretreatment with storage at 0oC appears to be a feasible means of prolonging
the storage life of fish, and extending their marketability and exportation potential.
“TIMED TESTING PROTOCOL”- A COMBINATION OF SEROLOGY AND
MANAGEMENT CHANGES TO REDUCE HERD PREVALENCE OF
JOHNE'S DISEASE
N. Galon,1, A. Arnin,1, H. Adler,2
1. Hachaklait Vet Services, P.O.Box 3039, Caesarea Industrial Park, 38900
2. Milouda Vet Laboratory, Miluot, Western Galilee, Israel
The usual practice of testing for Johne’s disease in Israel is to test the whole herd at
the same time. This gives a more accurate evaluation of the true herd seroprevalence, but is more time and effort consuming for the farm staff, a greater burden
on the cows and on the farm routine. It also tests cows which are due to be culled and
have therefore a reduced risk of transmitting the disease.
The objective of this study was to evaluate the efficacy of a different protocol to
reduce the herd prevalence of Mycobacterium avium subspecies paratuberculosis
(MAP) by testing cows at a specific, critical time in their lactation. Serum was
sampled just before drying off. This protocol tests only cows that will calve and be
milked in their next lactation. The farm veterinarian does the sampling during his
routine weekly visit while cows are tied up for a pre-drying off pregnancy check. A
cohort of 2,264 cows from 3 commercial Holstein dairy herds (ranging 300-900 cows)
was tested in the northwestern part of Israel between March 2002 and October 2004.
All serum samples were tested at the Kimron Veterinary Institute, using a commercial
IDEXX ELISA kit with a published sensitivity of 50% and a specificity of 99%.
Cows were considered positive if their S/P was above 0.25. Cows between 0.16 and
0.24 were considered suspected positive. Both positive and suspected positive cows
were marked and were sent to calve in an isolated pen. Colostrum and waste milk of
these cows was not used to feed heifer calves. All farms kept well-managed individual
calving pens, identified non-pooled colostrum feeding practice and raised calves in
individual pens or hatches for the first few weeks of life. A drop in herd positive and
suspect rates was recorded for all three herds. In the first herd the change was from
3.9% to 3.6% and to 0.7%, and in the second herd from 6.8% to 6.2% and to 1.9% in
2002, 2003 and 2004 respectively. In the third herd, it was from 6.5% in 2003 to 2.9%
in 2004. The average age of the cows at the time of positive testing was 5.2 years.
There was no policy of immediate culling of sero-positive cows, and on average they
left the herd after 14 months in one herd and 7 months in the other two herds. On one
farm, when both testing protocols were compared, the “Timed Test” protocol reduced
both the number of cows to be tested (470 per annum) as well as the cost of sampling
compared with testing the entire herd at one time (850 cows). These preliminary
results demonstrate that testing cows before drying off combined with management
practices can achieve the same reduction in herd prevalence of MAP at a reduced cost
and effort.
RELAPSING FEVER BORRELIOSIS IN A DOG, CAT AND MONKEY IN
ISRAEL - MOLECULAR CHARACTERIZATION OF THE ISOLATE
G. Baneth,1 T. Halperin,2 M. Yavzuri,2 E. Klement,1,2 Y. Anug,3 H. Almagor,4 I
Aizenberg,1 M. Cohensius5 and N. Orr2
1. School of Veterinary Medicine, The Hebrew University, P.O. Box 12, Rehovot
76100
2. Center for Vaccine Development and Evaluation, Medical Corps, Israel Defense
Force
3. PathoVet Diagnostic Veterinary Pathology Services. Kfar Bilu
4. Haiviva Veterinary Clinic, Jerusalem
5. Kineret Veterinary Clinic, Moshava Kineret.
Spirochaetemia was detected during the second half of 2003 in a dog, domestic cat
and Marmoset monkey in 3 different locations in Israel. The main presenting clinical
signs were lethargy and anorexia. All three animals were anemic and
thrombocytopenic. A large number of organims were detected as single forms or in
clusters of several spirochetes in the blood smears from all animals. The dog and cat
improved within two days of antibiotic treatment; the monkey however, died one day
after the initiation of therapy.
PCR amplification of a fragment of the Borrelia glycerolphosphodiester
phosphodiesterase (GlpQ) gene was positive in blood samples taken from the dog and
cat and in brain tissue taken at necropsy from the monkey (no blood samples were
available from the monkey). Amplification of a 253 bp fragment of Borrelia 16S
rRNA gene was positive in blood of the dog and cat. Sequencing of the partial 16S
rRNA amplicons showed identity with the sequence of Borrelia persica available in
the GenBank.
B. persica is the causative agent of human tick-borne relapsing fever (TRBF) in Israel
and other Middle Eastern countries. TRBF is a notifiable disease that has been
reported from northern, central and some parts of southern Israel, and is often
associated with entering caves. It causes serious morbidity and is potentially fatal if
not treated appropriately. To our best knowledge, this is the first description of B.
persica infection in a dog and cat. Future research is warranted to investigate the
potential role of animals as reservoirs of this infection.
MOLECULAR CHARACTERIZATION OF ISRAELI ISOLATES OF
AKABANE VIRUS (AKAV)
AND INHIBITION OF VIRUS REPLICATION BY siRNA TARGETED TO
THE GENOME S SEGMENT
Y. Stram1,. Levin2, L. Kuznetzova1, J. Brenner1, Y. Braverman1, M. Ginni1.
1. Kimron Veterinary Institute, P.O. Box 12 Beit Dagan, 50250.
2. Molecular Virology Department, Faculty of Medicine, Hebrew University of
Jerusalem, P.O. Box 12272 Jerusalem, 91120.
In the last few decades the ruminant population of Middle Eastern countries including
Israel was considered to be exposed endemically to Akabane virus (AKAV).
More recently, outbreaks of newborn calf sydromes with teratogenic malformations in
Israel have appeared. Surviving calves were found to have high titers of AKAV, and
in some cases Aino virus (AINV) neutralizing antibodies, indicating exposure to these
viruses.
AKAV and AINV belong to the Simbu serogroup of the arthropod-borne
Bunyaviridae which consists of 24 antigenically different viruses. They can cause
severe teratogenic malformations when susceptible pregnant ruminants are infected.
Infection of susceptible cows usually causes subclinical viremia of short duration and
the virus is cleared rapidly from the blood. In pregnant cows, the virus can invade the
central nervous system and/or the skeletal tissues of the fetus and may cause
arthrogryposis (AG) or hydranencephaly/hydrocephaly/microencephaly (HE/ME)
encephalomyelitis. Blood-sucking insects such as biting midges and mosquitoes serve
as vectors and transmit the viruses to vertebrates. AKAV and AINV have been
identified serologically or by virus isolation in Japan, Korea, Taiwan, Israel, Turkey,
Saudi Arabia and Australia.
The virion is enveloped and the genome consists of three segments of ss (-) RNA. The
Lsegment RNA carrying the polymerase gene, the M segment RNA encodes for the
two G1, G2 glycoproteins, and S segment RNA is 858 bases long and encodes for the
nucleocapsid (N) and nonstructural (NSs) proteins.
In order to enable the detection of AKAV and AINV genomes in affected calves, a
multiplex quantitative reverse-transcriptase real-time PCR, using MGB TaqMan
chemistry was developed.
Each specific probe was labeled with a different fluorescent dye - VICR for detecting
AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV.
Using the developed real-time RT PCR, AKAV was identified in Culicoidies imicola
trapped at the Volcani Center. It was calculated that the insect extract contains
1.5x105 copies of the genome segment S. Following amplification of the entire S
genome segment, its nucleotide sequence was determined and found to have over
93.4% identity with the S segment of other AKAV isolates. The deduced amino acid
(aa) sequence of the combined nucleocapsid and non-structural proteins showed more
than 96.6% identity. Phylogenetic trees constructed using the combined deduced
nucleocapsid and the nonstructural protein aa sequences and the nucleotide sequence
showed that the Israeli isolate forms a fourth cluster of AKAV indicating a separate
virus lineage.
AKAV genome was also identified in the brain of a calf with typical Simbu-related
teratogenic malformations. When trying to amplify the entire viral S segment the viral
genome was shown to be cleaved between nucleotides 430-431.
To explore the ability to inhibit AKAV in tissue cell culture, 3 different siRNA
expression cassettes targeted to the viral S segment were prepared. Following
transfection of siRNA cassettes and virus inoculation the amounts of viral RNA as
well as virus titers were examined at various time points post- inoculation. By real
time RT-qPCR and virus titration experiments, it could be shown that virus
replication can be inhibited in cells transfected with the mixture of all 3 cassettes as
well as with each one separately.
THE ROLE OF CD44 IN ADHESION AND INVASION OF
MYCOBACTERIUM AVIUM PARATUBERCULOSIS TO MACROPHAGES
AND ITS EFFECT ON INTRACELLULAR SURVIVAL
I. Peleg1, E. Gonen1,2, D. Naor2 and N. Shpigel1
1. The Koret School of Veterinary Medicine, Faculty of Agriculture
2. Faculty of Medicine, The Hebrew University of Jerusalem
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of a
severe gastroenteritis in ruminants, known as Johne’s disease. Johne’s disease is
prevalent in domestic animals worldwide, and has a significant impact on the global
economy. Johne’s disease is considered to be one of the most serious diseases of dairy
cattle. Isolation of MAP organisms from intestinal tissues and blood of patients with
Crohn’s disease has led to concern that it may also be pathogenic in humans. MAP
organisms have been found in dairy product, drinking water and meat products, and
infected animals are a constant source of environmental contamination.
Neonates and juvenile animals are at the highest risk for acquiring an infection of
MAP. Young animals are most commonly infected through the fecal-oral route. This
occurs either by ingesting the organism in contaminated milk or food products, or by
accidental ingestion from contaminated surfaces. MAP, like other pathogenic
mycobacteria, targets the mucosa-associated lymphoid tissues (MALT) of the host.
MAP preferentially targets the MALT of the upper gastrointestinal tract, where it is
endocytosed by the M cells of the ileal Peyer’s patches and is subsequently
phagocytosed by subepithelial and intraepithelial macrophages. MAP bacilli probably
remain in the phagosome, where they multiply intracellularly. The persistency of the
organism in macrophages leads to a celluar immune response culminating in a chronic
granulomatous enteritis. Thus, the ability of MAP to invade and persist in intestinal
macrophages is the hallmark of the disease.
The adhesion mechanism of intracellular pathogens is known to affect invasion and
survival of the organisms. Recent studies demonstrated the role of the transmembranous glycoprotein receptor, CD44, in the adhesion and invasion of various
microbial pathogens (including Mycobacterium tuberculosis) of macrophages. The
objective of our study was to investigate the role of CD44 in the adhesion, invasion
and survival of MAP organisms in macrophages.
Thus, we have used knockout (KO), CD44(-/-) and wild type (WT) CD44(+/+) mice,
as sources of peritoneal macrophages. Fluorescent techniques were used to detect and
quantify adhesion, phagocytosis and survival of MAP organisms in macrophages.
Binding of MAP organisms to CD44 was evaluated using in-house developed ELISA
systems.
Our results indicate that the presence of membranous CD44 significantly enhances
adhesion and phagocytosis of MAP by macrophages. However, intracellular survival
of MAP did not differ between KO and WT macrophages. Furthermore, we were
unable to demonstrate direct binding of MAP to CD44 as previously reported for
Mycobacterium tuberculosis. We plan to elucidate the mechanism by which CD44
affects MAP adhesion and phagocytosis in macrophages.
We believe that this research will improve our understanding of the pathogenesis of
paratuberculosis in ruminants.
AN EPIDEMIC OF TRANSMISSIBLE GASTRO-ENTERITIS (TGE) IN PIGS
IN ISRAEL
1Brenner, J., 1Yadin, H., 2Lavi, J., 1Perl, S., 1Edery, N., 1Elad, D., 2Bargut, A.,
2Pozzi, S., 3Lavazza A., and 3Cordioli P.
1. Kimron Veterinary Institute, 50250, Bet Dagan
2. Freelancer Veterinary Surgeon, Israel
3.Istituto Zooprofilatico Sperimentale della Lombardia e dell’Emilia-Romagna
“Bruno Ubertini” Via Bianchi 9, Brescia (BS) Italy.
This communication reports the first epidemic of transmissible gastro-enteritis (TGE)
in pigs in Israel.
TGE virus is a common cause of diarrhea in pigs affecting all ages, but significant
death only occurs in suckling pigs where its severity is related to the age of the
infected animals. Almost all susceptible piglets under 10 days of age die within a few
days of exposure, but mortality decreases with increasing age. Only mild signs such
as vomiting, regurgitation and agalactia are seen in the lactating sows. As a member
of the coronavirus group, TGEV is primarily an enteric virus, destroying enterocytes
of the small intestine, and causing villous atrophy.
When the TGEV spreads within a fully susceptible herd with no previous history of
infection, up to 100% mortality is reached among newborn pigs, and weaned pigs
show marked diarrhea and dehydration, while inappetence, vomiting and diarrhea are
typical signs in adult animals.
The first cases of diarrhea followed by dehydration and death of piglets aged 1 to 7
days, were noted in one piggery in northern Israel in May 2004. The episode gained
epidemic proportions with mortality as high as 70 to 80% during the first week of life
and proportionally less in convalescent groups of piglets. The area where this
outbreak occurred is known as “pig hill” (Evlin). Thirteen pig herds are concentrated
in this demarcated zone, and one to two days after the first outbreak, other herds
reported the same clinical signs.
PCR, electron microscopy, immuno-electron microscopy, immunofluorescence
performed by investigators from IZS, together with our findings of typical findings of
villous atrophy, lymphopenia, and the epidemiological features lead to the conclusion
that TGEV was responsible for the outbreak.
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References
1.