ISRAEL JOURNAL OF

 
VETERINARY MEDICINE                        home    archive    journal

ABSTRACTS OF PAPERS PRESENTED AT THE 29th ANNUAL ISRAEL VETERINARY SYMPOSIUM FEBRUARY 2005.

Chairperson: K .Perk.
Honorary Director¨ Koret School of Veterinary Medicine.
 

Download a pdf version of the abstracts

 

 

CANINE MAST CELL TUMORS
Dank, G.

School of Veterinary Medicine The Hebrew University of Jerusalem P.o.Box 12 Rehovot

Mast cells are derived from hematopoietic precursors. They are normally found in the liver, lung, skin, GI tract, and bone marrow. These cells have an integral part in the inflammatory and allergic responses.

Mast cell tumors (MCTs) are the most common cutaneous tumor in dogs. The etiology is unknown; however, due to the increased incidence in some breeds, there may be a genetic
component. The mean age is 9 years, though this tumor has also been reported in younger dogs. There is no sex predilection.  There is a breed predilection, which includes: Boxers, Bulldogs,Terriers, Retrievers, and Chinese Sharpeis.

History and clinical signs include either a solitary or multiple nodules. In addition, systemic signs may be caused due to release of histamine and heparin, including gastrointestinal
signs due to ulcers and prolonged bleeding time and wound healing. Mast cell tumors have been reported throughout the body. Multiple cutaneous mast cell tumors have also been reported in 10-14% of the cases. Systemic mastocytosis usually is associated with cutaneous tumors. Mullins et al performed
a retrospective study to define the biological behavior, prognostic factors, and treatment outcomes in cases of multiple MCTs. They concluded that dogs with multiple mast cell tumors have a low metastatic rate and appear to have overall median disease free intervals (DFI) and survival times (ST)
similar to low or intermediate grade solitary primary MCTs.

The first step after finding a mass on physical exam is attempting to get a diagnosis. A fine needle aspirate should be performed, and is diagnostic for a mast cell tumor in most cases. The next step is an aspirate from the local lymph node, to look for evidence of metastatic spread of the disease. If the
lymph node is negative, the owners have the option of either surgery or staging. If the lymph node is positive, staging is necessary. Staging includes a complete blood count (possible
anemia, mast cells, eosinophilia), biochemistry panel, urinalysis, abdominal ultrasound, splenic / liver aspirates, thoracic radiographs (lymph nodes metastasis, pleural effusion), buffy
coat smears, and bone marrow aspirates.

Many prognostic factors have been established, including histologic grade, clinical stage, tumor location, breed, growth rate/duration, PCNA, tumor recurrence, AgNOR count, and intratumoral vessel density. Patnaik et al studied the histologic grade as a prognostic indicator in 83 dogs. The percent alive
at 1500 days was 93% in Grade I tumors, 44% in Grade II tumors, and 6% in Grade III tumors. Overall, 54% were alive at 1500 days. Northrup et al looked at the variation among pathologists in histopathologic grading in canine cutaneous  MCTs, and found that each graded the MCTs differently.

Several locations have been associated with a worse prognosis, including the perineal, vulvar, scrotal, subungual areas and the oral cavity, muzzle, and GI tract. Takahashi et al. found that 10 dogs with visceral mast cell tumors all died within 8 weeks of diagnosis regardless of therapy, indicating
that visceral MCTs have a worse prognosis. Cahalane et al. performed a retrospective study of perineal and inguinal MCTs. They concluded that when appropriately treated, survival times and TFI may be similar to MCTs in other locations. Gieger el al performed a retrospective study of MCTs of the muzzle.
They concluded that muzzle MCTs are biologically aggressive tumors with a higher regional metastatic rate.

The treatment of canine mast cell tumors is dependent on the histologic grade. The purpose in all cases is clean margins (even if an additional surgery is required); however, some cases require additional treatment modalities. If there is evidence of gross disease, supportive care may include H1
antagonists (diphenhydramine), and either H2 antagonists (famotidine) or a proton pump inhibitor (omeprazole).

The general rule until a couple of years ago was that surgery of MCTs requires 3 cm margins. However, many researchers are presently trying to understand whether 3 cm is necessary. Gieger et al evaluated the recurrence rate of incompletely or narrowly (<2 mm margins) resected MCT not treated with
adjuvant therapy in 31 dogs. Only 3/31 MCTs recurred (2 grade II, 1 grade III). They concluded that it is possible that some tumors may not recur without further therapy. Seguin B evaluated the rate of local recurrence and clinical outcome following incomplete excision in 34 dogs (36 MCTs). Only 8 tumors (22.2%) recurred locally, and the median time to local recurrence of 92 days. 13 dogs had MCTs occur at other
locations, with a median time to the other location of 546 days. The median time to death was 556 days. They concluded that a minority of the tumors recur, so ancillary local therapy may not always be necessary. Murphy et al showed similar results, and also concluded that wide surgical margins are not
a prerequisite for a successful long term outcome. Simpson et al proposed to determine whether neoplastic mast cells extended into tissue 1,2, or 3 cm laterally or deeper than 1 fascial plane
from the visible edge of MCTs. They found that a 2 cm lateral margin ad a deep margin of 1 fascial plane appear to be adequate for complete excision of grade I and grade II MCTs.

Treatment today with chemotherapy includes prednisone, vinblastine, and lomustine. There are indications that chemotherapy may be beneficial in cases of gross disease and in cases that clean surgical margins were not achieved.

Treatment with radiation therapy is efffective in incompletely resected MCTs. Sarraf et al reported a disease free interval of 100% at 1 year, and 86% at 5 years, and survival of 100% at 1 year, and 96% at 5 years in 32 incompletely resected Grade II MCTs. In addition, there have been reports of radiation
therapy for non-resectable MCTs. Dobson et al. evaluated 35 dogs with non-resectable, grade I-III MCTs on the head or limb, treated with prednisone for 10-14 days and then with radiotherapy (4 treatments, 8 Gy, once/week). The overall response rate was 88.5%.

In conclusion, these are very complex tumors, and a great deal of research is still required to understand their biologic behavior and ideal treatment required.

 

 

INTRODUCTION TO ECHOCARDIOGRAPHY

Ohad, D.

Koret School of Veterinary Medicine The Hebrew University of Jerusalem P.O.Box 12 Rehovot

 Echocardiographic technology (not technique) is introduced, emphasizing aspects that are not widely known to general practitioners, such as 2D, M-Mode, Color Doppler and Spectral Doppler imaging. Rather than teaching practitioners how to acquire echocardiographic data, the aim is to expose them to the types of information that can and should be gained from a well equipped echocardiographic examination performed by a thoroughly trained professional, to help them solve daily diagnostic and therapeutic dilemmas. At the end of the lecture they will be familiar with concepts that appear on a written echocardiographic examination report, such as received by a cardiologist in response to a cardiac referral.

Echocardiography, like all other diagnostic technologies, *cannot* and should not substitute an effective physical examination. Rather, it should help us confirm or rule out diseases we diagnose or suspect, after having prioritized their likelihood based on the 4 simplest and most important bases of the diagnostic process: signalment, history, observation, and physical examination.

Most veterinary cardiac patients (especially cats, puppies, and many adult dogs) are either too unstable or too impatient to allow a lengthy echocardiographic examination. Modern echocardiography equipment may be highly sophisticated and offers a myriad of different technologies, formulas, sensitivities, and parameters to enable a thorough diagnostic process. However, if the echocardiographic examination is conducted in a routine fashion so as to include each and every one of the available features, always in the same "systematic" order, not only it will never be effective but it will most likely end much too early, prior to providing the most crucial information. No veterinary patient is patient enough to allow that!

For this reason, every high-quality echocardiographic examination absolutely and necessarily has to begin with a thorough and high-quality physical examination that is highly attentive to the cardiovascular system. This is the only way the cardiologist can develop at least a primary diagnostic impression, if not a complete diagnosis, to help plan the echocardiographic examination per se. This way, all that is left to do is to *quantify the severity* of the diagnosed or suspected disease, after having ruled it in or out. This not only shortens the actual echocardiographic examination but also increases the chances for an effective and a complete diagnostic process without having to waste time on seeking the problem. Instead, it focuses on the most important aspects of the already diagnosed or suspected problem right from the start, while the patient is still cooperative enough.

Two-dimensional echocardiography (cardiac ultrasound) enables direct, on-line visualization and quantification of cardiac anatomy (including presence of masses or tumors and their complications, chamber dimensions, wall thickness, intra-or extra-cardiac shunts, and wall continuity), chamber condition (volume overload, pressure overload), function (myocardial force of contraction and efficiency of relaxation,  valve thickness, valve motion, valve competence), and hemodynamics (pressure gradients across stenotic or leaking valves or septal defects, peak-flow velocities, etc.). These tools enable the diagnosis of congenital and acquired heart disease and the assessment of their severity and rate of progression in a highly quantitative (although sometimes subjective) fashion. Spontaneous or artificial echo contrast can be used to assess the risk for the presence of intra-cardiac thrombus formation or shunts.

Doppler technology provides an opportunity to quantify the direction, laminarity (versus turbulence), and velocity of blood flow as well as motion of specific tissues within the heart, and identify both systolic and diastolic dysfunction.

Data are documented as reference for future follow up examinations and provide a powerful diagnostic and prognostic tool that can always be retrieved for serial monitoring of the response to therapy.

 

CLINICAL ASPECTS OF NEUTROPHIL CYTOPLASMIC TOXICITY IN DOGS AND CATS

Aroch, I.

 School of Veterinary Medicine, The Hebrew University of Jerusalem P.O. Box 12 Rehovot, 76100

Definition

The term ‘neutrophil toxicity’ refers to certain nuclear and cytoplasmic morphological abnormalities observed in circulating neutrophils on examination of Romanowsky-stained blood smears. Such changes occur during the maturation process of the neutrophils in the bone marrow, and do not result in cell death. For convenience, dogs and cats presenting neutrophil cytoplasmic toxicity will be referred to as toxic dogs and toxic cats.

 

Neutrophil toxicity versus neutrophil degeneration

Degeneration is another term to describe certain morphological abnormalities observed in neutrophils. There is some inconsistency and even controversy in the veterinary clinical pathology texts concerning the use of term degeneration versus the term toxicity. Some clinical pathologists use both these terms interchangeably, while others prefer to reserve the term ‘neutrophil toxicity’ to those abnormalities occurring in the bone marrow during the maturation process of the neutrophil, and that are observed in the blood. In contrast, ‘neutrophil degeneration,’ as the name implies, refers to those morphological changes that occur in neutrophils after their maturation process has been completed, are associated with cell death, and are observed mainly in cytological samples. Such changes may be due to programmed cell death and apoptosis, which occur in noninfectious nontoxic environments and body fluids or sometimes in the blood. These include nuclear changes, such as hypersegmentation, polyploidity, fragmentation and pyknosis, and cytoplasmic and cellular changes, such as pyknosis, observed often in old samples. Other degenerative changes in neutrophils are associated with unprogrammed cell death usually due to the presence of toxins, bacteria and other leukocidic environments and result in karyorhexis, kariolysis, degranulation and loss of cell membrane integrity. This presentation will not include a discussion on neutrophil degenerative changes, but rather will focus on neutrophil toxic changes as defined above.

 

Types and origin of neutrophil toxic changes

Neutrophil toxic changes may present as nuclear and/or cytoplasmic abnormalities. The nuclear changes include hyposegmentation (i.e. left shift, or presence of immature neutrophils such as bands and metamyelocytes) and ring-forms. The toxic cytoplasmic changes are the most commonly noted and important, and include Dצhle body formation, basophilia, vacuolation (i.e. cytoplasmic “foaminess”), granulation and giant neutrophils. Dצhle bodies are grayish to blue cytoplasmic inclusions, which are the result of lamellar retention and aggregation of rough endoplasmatic reticulum, and their number, size, and location varies. Cytoplasmic basophilia appears as a bluish-gray

to dark blue cytoplasm as opposed to the normal neutral cytoplasm of the cell, and results from RNA and ribosome retention in the neutrophil cytoplasm during the maturation process. Vacuolation, which appears as a mildly reticulated cytoplasm with loss of granule uniformity up to an intensively foamy cytoplasm, is thought to be the result of autodigestion, or alternatively, disruption of bone marrow production leading to integrity loss of granules and membranes. Toxic granulation refers to the presence of azurophilic granules in the neutrophil cytoplasm, and is presumed to result from retention of acid mucopolysaccharides and increased permeability of primary granule membranes to Romanowsky stains.  Giant neutrophils are larger compared to normal neutrophils, and their cytoplasm stains uniformly dark gray to blue, and may contain in many cases a hyposegmented nucleus. They may reflect further manifestation of toxicity, due to skipped cellular divisions during the maturation process. This presentation will focus on neutrophil cytoplasmic toxic changes.

 

Neutrophil counts versus neutrophil morphology      

The evaluation of neutrophil responses in disease is based on the interpretation of neutrophil counts (i.e. neutrophilia, neutropenia) ,as well as their morphology in Romanowsky-stained peripheral blood smears. Neutrophil and band neutrophil (i.e. left shift) counts and their associations with different disease conditions and other clinicopathological parameters have been extensively studied and reported in the human and veterinary medicine literature. In contrast, studies on the association of neutrophil morphological abnormalities on examination of blood smears with diseases and other parameters have been limited to case reports or series, and no extensive studies on this phenomenon have ever been published.  Neutrophilia or neutropenia, along with a left shift are often associated with inflammation of different causes, and have commonly been associated with differences in other hematological variables in the hemogram. In contrast, when mature neutrophilia or neutropenia occurs, or when the leukogram reveals no cell counts abnormalities, the interpretation of the leukogram is challenging and the evaluation of neutrophil morphology may serve as an additional aid in the diagnostic procedure. Neutrophil cytoplasmic toxicity may precede changes in neutrophil numbers and appearance of neutrophilic bands, and thus can be a sensitive early diagnostic and prognostic aid.

 

Assessment of blood smears for neutrophil cytoplasmic toxicity

 Although modern automated hematology analyzers perform 3-way or 5-way differential white cell counts, the reliability of such automated counts is in many cases questionable in veterinary

species, and manual counts, although limited to 100-200 leukocytes, are recommended in most instances, especially when abnormalities in cell counts are present and when the analyzer flags certain parameters. Automated blood analyzers are also quite limited in their ability to measure nucleated blood cells commonly observed in feline and canine samples, and most will count these as leukocytes (usually, lymphocytes). Blood smear evaluation is useful for the assessment of blood cell morphological abnormalities, as well as presence of hemoparasites, commonly encountered in small animal practice in Israel. In addition, the automated platelet counts in feline blood are inaccurate at best, necessitating a manual evaluation of the platelet count and size in the blood smear. Platelet clumps are very common in veterinary blood samples, and also result in inaccuracy of the automated platelet count, necessitating blood smear evaluation. For all these reasons, an evaluation of the blood smear through light microscopy should probably be performed in every veterinary patient. Once this evaluation is accepted as a routine and a valuable diagnostic measure, the assessment of neutrophil morphology should be included in the procedure.

Assessment of neutrophil morphology requires minimal equipment; a light microscope with a x50 to x100 immersion oil magnification and a Romanowsky staining solution, such as Giemsa or Wright stains. Rapid staining solutions can also be used, however, these sometimes result in loss of cytoplasmic detail, and thus are less sensitive and especially when neutrophil cytoplasmic changes are mild, may yield a false negative result. This evaluation can be performed in any veterinary practice, and the results may be sometimes available before those of the complete blood count. It is also a cost-effective diagnostic measure.

The severity of neutrophil cytoplasmic toxicity is assessed subjectively and semiquantitatively and is based on the presence of cytoplasmic toxic changes (i.e. foaminess, basophilia, Dצhle bodies, toxic granulation and giant toxic neutrophils) on examination of a single Romanowsky-stained blood smear. Maintenance of a routine and acquiring experience will lead to quicker and repeatable assessments. The following is a proposed method protocol for the assessment of neutrophil cytoplasmic toxicity that has been used in our previous recent canine and feline studies, although other systems were also used. Each individual type of toxic change is assigned to one of three final grade scores of morphologic abnormality; mild, moderate or marked (toxicity scores 1, 2 and 3, respectively). The final grade of each type of toxic change is a combination of a quantitative assessment of the percentage of affected neutrophils (<10% - mild, 10-30% - moderate, >30% - marked) and a qualitative grade of the intensity of each of the individual toxic scores for each morphologic change. Mild cytoplasmic basophilia is defined as a presence of a non-uniform grayish to light blue cytoplasm in the neutrophils, while a uniformly light blue cytoplasm is judged as a moderately abnormal change. The presence of a uniformly blue to dark blue cytoplasm is considered as a marked abnormality. The presence of one to two small Dצhle bodies per cell is judged as a mild abnormality, while presence of three to four Dצhle

bodies is ranked as moderate toxicity, and a large prominent Dצhle body and/or more than four Dצhle bodies per cell is judged as a marked abnormality. The presence of cytoplasmic vacuolation is classified at three levels. Mild vacuolation is defined as loss in cytoplasm clarity and neutral stained granules, while a moderate change was defined when small cytoplasmic vacuoles are observed. Appearance of intense vacuolation with grayish reticulation is classified as a severe cytoplasmic vacuolation. The presence of toxic granulation and giant toxic neutrophils is always considered a marked abnormality. For the assessment of the overall neutrophil toxicity in a smear, the sum of all scores of the individual neutrophil morphologic abnormalities is calculated.

 

Interpretation of neutrophil cytoplasmic toxic changes

1. Etiology

Neutrophil cytoplasmic toxicity has been traditionally linked with systemic rather than local conditions and with inflammation of a wide range of etiologies. This has been reported in several cases reports and case series, in pancreatitis, immune-mediated hemolytic anemia (IMHA), pyometra, septic arthritis, bacteremia (dogs), drug toxicity, poisonings, myeloproliferative disorders, toxemia and in experimental inflammations (dogs and cats). Our previous retrospective studies have shown that neutrophil cytoplasmic toxicity was associated with systemic bacterial infections (dogs and cats), viral infections (cats), neoplasia (dogs), metabolic and inflammatory, non-infectious conditions (dogs). Thus, presence of neutrophil cytoplasmic toxicity, although non specific, can direct the clinician to the nature of the disease process. Nutritional, developmental, degenerative, allergic and idiopathic conditions are usually not associated with neutrophil toxicity, and are more common in animals exhibiting normal neutrophil morphology compared to dogs and cats with neutrophil cytoplasmic toxicity. Systemic inflammatory conditions of different etiologies, such as pyometra, IMHA (dogs), pneumonia, complicated upper respiratory disease (cats), pancreatitis, sepsis, parvovirus infection and peritonitis (dogs and cats) were found to be significantly more common when neutrophil toxicity was present. There is a positive association of neutrophil cytoplasmic toxicity with other quantitative markers of inflammation such as leukocytosis, leukopenia, neutrophilia, neutropenia, monocytosis and left shift. All these markers, as well as toxic changes, are non-specific. Although inflammation is in many cases a result of infection, any inflammation of any other etiology may lead to manifestation of neutrophil cytoplasmic toxicity. Thus, it is reasonable to assume that bacterial components and/or toxins cannot be the sole factors that induce neutrophil toxicity, and that other components, such as cytokines and inflammatory mediators are involved in the induction of this phenomenon.

2.  Association with clinical signs

Dogs and cats with cytoplasmic neutrophil toxicity are more ill compared to controls. They present higher body temperature and heart rate. The also have a higher prevalence of hyperthermia,

most probably of a pyrogenic origin, and they present a wider range of clinical signs. There are some differences in the clinical manifestations between dogs and cats with neutrophil toxicity compared to their respective negative controls. While toxic cats present a wider range of clinical manifestations compared to toxic dogs when compared to their controls, most of these are general and non-specific, including depression, dehydration, weakness, cachexia, vomiting and diarrhea. In both species the prevalence of icterus in toxic animals was significantly higher compared to controls. Toxic dogs also presented a higher prevalence of abdominal organomegally and vaginal discharge, the latter probably due to a higher prevalence of pyometra. Neutrophil toxicity should be regarded as an additional marker for the severity of disease, and should serve as an additional aid for the interpretation of the clinical signs.

 

3. Association with hematological variables

Dogs in particular, but also cats, with neutrophil toxicity present a wider range of additional hematological abnormalities, most of which are associated with inflammation, including leukopenia, leukocytosis, neutropenia, neutrophilia, left shift and monocytosis, and they were found to have a higher leukocyte and neutrophil counts compared to negative controls. Although toxic cats had a significantly higher prevalence of neutrophilia and neutropenia compared to controls, 53% and 47% of these cats in our study presented normal leukocyte and neutrophil counts, respectively. A combination of normal neutrophil and WBC count and no left shift was present in 43% of toxic cats, while in 45% of toxic cats, leukocytosis, a left shift or neutrophilia and a left shift were both absent.  Furthermore, no difference was found in mean band neutrophils count between toxic cats and their negative controls. These findings well demonstrate that neutrophil toxicity may not necessarily correlate with abnormalities in the absolute numbers of leukocytes, neutrophils and bands. Thus, in certain cases, presence of neutrophil toxicity may serve as the only hematological marker of inflammation and infection. This exemplifies the benefit of assessing neutrophil morphology in stained blood smears.

Toxic dogs and cats were also found to be more anemic compared to controls, and in toxic dogs, there was a higher prevalence of macrocytosis and hypochromia. The mechanisms of anemia in toxic animals are not determined, but inflammation, IMHA and blood loss are all possible causes.

4. Association with serum biochemistry variables

The same trend observed in the hematological variables is true for serum biochemistry parameters. Toxic dogs and cats have a wide range of serum biochemistry abnormalities, and were found to present significant differences in mean parameters compared to their respective negative controls. In toxic dogs, there was a significantly higher prevalence of hyperbilirubinemia, hypokalemia, hypocalcemia, hyopalbuminemia, hypoproteinemia, hypertriglyceridemia, azotemia (both urea and creatinine) and increased serum activities of LDH, ALP and GGT. Toxic cats had a higher prevalence of hyperbilirubinemia, hypokalemia, hypocalcemia and increased serum activities of LDH, and a lower prevalence of hypekalemia compared to negative control. No doubt, that these findings exemplify the fact that toxic animals are more ill compared to negative controls, and that neutrophil cytoplasmic toxicity is a negative clinicopathologic sign. It is probable that these abnormalities reflect the specific disease entities in our studies, however, when the data were analyzed for each specific disease in cats, there was no obvious pattern. The prevalence of secondary complications such as disseminated intravascular coagulation (DIC) and acute renal failure (ARF) was found to be significantly more prevalent in toxic dogs, while shock was more prevalent in toxic cats compared to their respective negative controls. These complications are usually associated with many clinicopathological abnormalities.

5. Prognosis

Our studies have shown that toxic dogs and cats have longer hospitalization periods and higher treatment costs compared to their negative controls. Toxic dogs, but not cats, had also a significantly lower survival compared with their control. Therefore, presence of neutrophil cytoplasmic toxicity is a negative prognostic marker, and detection of such changes may suggest to the attending clinician that the animal is seriously ill, may require a longer intensive and expensive therapy. In toxic dogs, those with leukopenia (WBC<5x103/mm3) were found to have a significantly (p<0.0001, RR=2.2, CI 95%=1.3-3.6) higher case fatality compared to dogs with normal or high leukocyte counts (47.8% vs. 21.9% respectively). Therefore, presence of leukopenia, most commonly as a result of neutropenia along with neutrophil cytoplasmic toxicity should be an especially alarming sign. There was also a significant trend for increasing case fatality with increase of neutrophil toxicity; case fatalities of 22.2%, 28.9% and 40.9% were observed among low, moderate and severe toxicity dog groups respectively. In the neutrophil toxicity group, dogs that presented severe neutrophil toxicity had a significantly ( p<0.02) higher fatality compared to those presented with mild and moderate toxicity. These trends were not observed in toxic cats.

6. Species differences

Toxic dogs were considerably more ill than toxic cats, compared to their respective controls, while toxic cats were found to present a wider range of clinical signs. However, some of these were general and non-specific. This trend was also found in the clinicopathological findings, and toxic dogs presented many more abnormalities than toxic cats when compared to their respective controls, thus reflecting more severe and serious disease conditions. It seems that ill cats present toxicity “more readily’ and in milder and earlier disease stages compared to dogs, and this may result from different marrow characteristics and/or a different profile cytokines and inflammatory mediators. While in toxic dogs the survival is significantly lower compared to negative controls, in cats this trend was not observed, nor was the trend of decreased survival with an increase in neutrophil cytoplasmic toxicity

observed in toxic cats, in contrast to the findings recorded in toxic dogs. In 40-50% of the toxic cats, neutrophil cytoplasmic toxicity was the only significant marker of inflammation in the hemogram. This was not assessed in dogs.

7. Conclusions

In conclusion, evaluation of blood smears for neutrophil cytoplasmic toxic changes is a quick, simple, cost-effective and readily available process. Neutrophil cytoplasmic toxicity was found to be a non-specific marker of infectious, inflammatory, neoplastic and metabolic disease processes in dogs and cats. In some animals, such changes were present when other clinicopathological abnormalities in the CBC and serum biochemistry profile were minimal or absent. In some animals, the clinical signs along with the neutrophil toxicity were the only

indicators of disease. The presence of neutrophil toxicity was also found to be associated with a longer hospitalization period and higher treatment cost. In dogs, it was a found as a risk factor fro mortality. Neutrophil cytoplasmic toxicity was found to be an important diagnostic sign, and an aid in the assessment of the patient, the disease course, the hospitalization period length and the therapeutic plan. Neutrophil toxicity in cats, unlike in dogs, was not associated with higher mortality, and therefore may not necessarily represent the same severity of illness as in dogs. Further research is warranted to strengthen the associations found in our studies, to shed light on the molecular mechanisms that induce neutrophil toxicity, and to assess the sensitivity and specificity of such changes for the diagnosis of various diseases and conditions.

References are available on request  

 

 

ONTHOPHAGUS SELLATUS, THE INTERMEDIATE HOST OF SPIROCERCA LUPI IN ISRAEL: BIOLOGY, LIFE CYCLE, PREVALENCE AND INFECTION RATE

Shemesh, O.1, Aroch I.1, Makovics, A.2, Samish. M.2 and Lavy, E1

1. School of Veterinary Medicine, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76-100, Israel

2. Kimron Veterinary Institute, P.O. Box 12 Bet Dagan

 

The esophageal worm of the dog Spirocerca lupi, causes great morbidity in Israel. The life cycle of S. lupi is indirect. The definite hosts are carnivores (especially a Canidae), and the intermediate hosts are dung beetles (the Scarabaeidae family).

Birds, reptiles, amphibians and small mammals might serve as paratenic hosts.

 The Scarabaeidae family includes 3 subfamilies - Scarabaeinae (warm climate), Geotrupinae and Aphodiinae (cold climate). All the 3 subfamilies are represented in Israel. Onthophagus sellatus (the Onthophagini tribe, Scarabaeinae subfamily) is the most important and efficient intermediate host for S. lupi in Israel. O. sellatus is rather small, measuring 4-5 mm in length. It feeds on different sources of dung including dog feces. The dung beetle flies to the dung pad, locating it with its numerous olfactory organs in its antennae. It makes dung balls for feeding and brood balls, and buries them in tunnels below the dung pad. Tunnels are 20-25 cm deep.

O. sellatus is part of the tropical fauna, and is found in Egypt, Syria, Israel, Cyprus, Libya, Arabic Peninsula and tropical Africa.  In Israel it is recorded in the coastal plain, the Galilee, Mount Hermon, Golan Heights, Mount Karmel, Samaria, Judean hills and the northern Negev.

Our current ongoing study describes the prevalence and infection rate of O. sellatus with S. lupi larvae in 2 natural habitats in the Tel Aviv area around the year (Ramat Hen and Kikar Hamedina). Infected fecal samples are collected, and the beetles are separated from the feces, and dissected under the microscope. We record the number of beetles collected, average number of beetles per dung pad, % beetles infected with S. lupi (infection rate) and average number of larvae per beetle.

Between July and January, the infection rate (% infected beetles) was 0-9.6%, declining as we approach the colder months (as was the beetle prevalence). The prevalence and infection rate in Ramat Hen were higher then in Kikar Hemedina. Comparing our results with a survey taken 10 years ago in Ramat Hen shows that there is a decline in the infection rate of the beetles.

The full results of our current survey will allow us a better understanding of S. lupi epidemiology in Israel and optimizing preventive measures.

 

 

SUCCESSFUL TREATMENT OF KERATOCONJUNCTIVITIS SICCA (KCS) IN DOGS WITH PIMECROLIMUS EYE DROPS: A COMPARISON WITH CYCLOSPORIN A (CyA) OPHTHALMIC OINTMENT.

Ofri, R.1, Allgoewer, I.2, Graenitz, U.3, Pena, T.4, Spiess, B.M.5, Lambrou, G.N.6  and Latour, E.6 

 

1. Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Israel;

2. Veterinary Ophthalmology Clinic, Berlin, Germany;

3. Veterinary Ophthalmology Clinic, Chemnitz, Germany;

4. Autonomous University of Barcelona, Spain; University of Zurich, Switzerland;

5. Novartis Institutes for BioMedical Research, Basel, Switzerland

Purpose: The aim of this study was to conduct a clinical trial, testing the efficacy of topical administration of pimecrolimus in alleviating the clinical signs of KCS in dogs and to compare it with the veterinary commercial form of CyA (Optimmune®).

Methods: The study was an open-label, multicenter, randomized, 8-week outpatient clinical study and included 44 dogs (77 eyes) that had never been treated with CyA. KCS was diagnosed based on a Schirmer Tear Test I (STT) value ? 10 mm/min. and an aggregate score ? 4 in grading clinical signs of corneal and/or conjunctival inflammation (9 signs were evaluated and scored from 0=none to 4=severe). Dogs were randomly assigned to a treatment group, to be medicated twice daily with 1% pimecrolimus oil-based eye drops or 0.2% CyA ophthalmic ointment. Rechecks were conducted 2, 4 and 8 weeks after enrollment. The STT values and the ocular sign scores were analyzed by using ANOVA and the Wilcoxon rank sum test, respectively. As there were no substantive differences between the worst-eye (with the lowest STT value at baseline) and best-eye analyses, the results for only the worst-eye analyses are summarized.

Results: At baseline, the mean STT values in the pimecrolimus and CyA groups were 3.8±0.7 mm/min. (mean ± sem; n = 20) and 4.6 ± 0.7 mm/min. (n = 24), respectively. Statistically significant improvements from baseline were observed within both groups (P ­ 0.001) at all follow-up visits.  After 8 weeks of treatment, mean increases of 9.2 and 5.8 mm/min.were observed in the pimecrolimus and CyA groups, respectively. The difference between the 2 groups was marginally significant (P = 0.085). Both drugs also caused a significant improvement in clinical signs of corneal and conjunctival inflammation (P ­ 0.001) at all follow-up visits. At baseline, the mean total scores in the pimecrolimus and the CyA groups were 16.0 ± 1.1 and 13.9 ±1.4, respectively.  After 8 weeks of treatment, there was a significantly larger reduction in the total score in eyes treated with pimecrolimus (mean decrease of 10.3) as compared to eyes treated with CyA (mean decrease of 7.6; P = 0.024).  Two dogs assigned to the pimecrolimus group were discontinued a few days after enrollment because of severe local irritation.

Conclusions: The results of this study show that 1% pimecrolimus oily eye drops are more effective than the commercial 0.2% CyA ophthalmic ointment in controlling KCS in dogs. These findings confirm the interest to develop pimecrolimus for the treatment of dry eye in humans.

Support: Funded by Novartis Pharma AG, Basel, Switzerland.

 

MULTI-GENE MOLECULAR DETECTION OF RICKETTSIA FELIS IN CAT FLEAS (CTENOCEPHALIDES FELIS) FROM ISRAEL: A LIKELY ALL-ASIAN STRAIN OF THE HUMAN PATHOGEN

Bauer, O.1, Baneth, G.1, Eshkol, T.2, Shaw,S. E.3 and Harrus, S.1

1. Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel

2. "Let the Animals Live” Association, Moshav Talmei-Menashe, Israel

3. University of Bristol, Langford House, Langford, North Somerset BS40 5DU, UK

 

Rickettsia felis is a newly emerging bacterial pathogen that causes “flea-borne spotted fever” in humans. The bacterium is both efficiently maintained and biologically-transmitted by several flea species and has been confirmed in cat fleas (Ctenocephalides felis) globally. We investigated the presence of R. felis in 79 cat flea lots, collected from Israeli dogs and cats, by polymerase chain reaction (PCR) assay and sequencing of 5 different gene fragments: ompA, ompB, htrA (17 kDa antigen), gltA (citrate synthase) and fusA. 2 novel genotypes, closely related to a recently described Thai isolate of Rickettsia felis (Rf31), were detected in 7.59% of the flea lots. These were located to the cities of Jerusalem, Tel-Aviv, Ramla and Bat-Yam. Their genetic makeup and sequence-based taxonomic positioning were then determined. This is the first report of this flea-transmitted rickettsia within its vector in Israel and the Middle East. Further, our preliminary findings bring afore the odds for a distinct strain of R. felis endemic to Asia. Though supplementary studies are still required, we believe Israeli physicians should consider R. felis a potential agent of fever and/or rash in patients with a history of possible flea contact.

 

 

EVALUATION OF THE IMMUNIZATION STATUS TO CANINE PARVOVIRUS AND DISTEMPER VIRUS IN ADULT DOGS, USING AN IN-CLINIC ELISA TEST.

Waner, T.1, Mazar, S.2, Keren-Kornblatt, E.3

1. Veterinary Clinic, 9 Meginay Hagalil Str. Rehovot, Israel;

2. Biogal Gal’ed Laboratories, Kibbutz Gal’ed, Israel.

3. Veterinary Clinic, Hashmonaim, Israel.

 

Historically, prophylaxis against important infectious diseases of dogs has been based on annual vaccination.  Numerous studies have shown that the vast percentage of dogs is protected for a number of years after vaccination. 

In this study we used an in-clinic dot-ELISA kit (ImmunoComb®, Biogal Laboratories, Kibbutz Gal’ed, Israel) to evaluate the duration of IgG antibody titers to canine parvovirus (CPV) and canine distemper virus (CDV) in dogs in Israel vaccinated at least one year previously.  One hundred and fifty eight dogs were sampled.  An antibody titer equivalent to 1:80 was regarded as a protective for both CPV and CDV.

Overall the percentage of dogs with protective antibody titers for both CPV and CDV was 84%.  The percentage of dogs with borderline antibody titers was 11% for CPV and 10% for CDV, respectively. Four percent of the dogs had no detectable antibody to CPV and 6% had no antibody to CDV. There did not appear to be any pattern regarding the incidence of low or borderline titers to either CPV or CDV with increasing time after the last vaccination.

The results reported here are in good agreement with other laboratory IgG antibody assayed studies which indicated that a large percentage of healthy dogs have adequate serum antibody to CPV and CDV for many years after their preceding vaccination, and to the conclusion that the majority of dogs do not require annual vaccination. Use of the dot-ELISA kit in this study was able to identify dogs retaining protective antibody titers to CPV and CDV after a number of years following their last vaccination. The dot-ELISA technique allows the practicing veterinarian the opportunity of easily monitoring the vaccination status of dogs and revaccinating only those whose antibody titer to specific diseases has waned.

 

KINETICS, DIAGNOSTIC AND PROGNOSTIC POTENTIAL OF QUANTITATIVE WESTERN BLOT AND ANTIGEN- SPECIFIC ELISA IN EXPERIMENTAL CANINE LEISHMANIASIS

D. Talmi-Frank1, D. Strauss-Ayali1, C.L. Jaffe2, G. Baneth1

1. School of Veterinary Medicine, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel

2. Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem

Visceral leishmaniasis (VL) is a severe public health problem with an estimated global annual incidence of 500,000 human VL cases. It is estimated that there are more than 2.5 million dogs with VL in southern Europe alone. Current research priorities focus on developing diagnostic tools for early infection, and quantitative methods for detection of relapse.

The aim of this study was to characterize the development of anti-leishmanial antibodies in experimentally-infected dogs to specific antigenic components before, during, and after treatment with allopurinol. Antibodies against both crude Leishmania infantum antigens and recombinant proteins were examined. Quantitative western blot (WB) analysis was used to define reactivity with specific antigens as indicators of early infection and to evaluate the efficacy of therapy in experimentally infected dogs.

Six beagle dogs were infected with Leishmania infantum by intravenous inoculation, and 3 non-infected beagles served as controls. Sera of infected dogs were periodically sampled and tested by ELISA and WB during 75 weeks post-infection (PI). Seroreactivity to crude leishmanial antigen was evaluated by ELISA and WB, and reactivity to rk39 and HSP70 was evaluated by ELISA. Net WB band intensities were measured using the KODAK 1D Image Analysis Software.

Three bands of 24 kDa, 48 kDa and 68 kDa showed a significantly increased intensity (p<0.05) as early as 4-6 weeks PI whereas crude antigen ELISA reached the cut-off (0.6 OD) at 8 weeks PI. These 3 bands reached peak intensity at 8-11 weeks PI, significantly earlier than the ELISA values, peaking at 19-21 weeks PI (P<0.05). A second reaction pattern was observed with 6 bands, 12, 14, 19, 29, 71, and 102 kDa. Reaction with these bands was first observed at 4 weeks PI and steadily increased in intensity until 25 weeks PI. Shortly after treatment was begun on week 32 there was a decrease in intensity of these bands. Reactivity subsequently returned to previous intensity after the treatment had been stopped on week 45 PI following 3 months of treatment. Antibody reactivity to HSP70 and rk39 was stronger than to crude antigen (OD values peaking at 2.27, 2.05 and 1.5 for HSP70, rk39 and crude antigen, respectively).

In conclusion, the 24, 48 and 68 kDa protein bands appear to be good markers of early infection, whereas the 12, 14, 19, 29, 71 and 102 kDa bands re-intensified following cessation of therapy and may be useful as markers of disease relapse. HSP70 and rk39 were superior to crude antigen ELISA in evaluating the initial serological response and response to treatment.

 

SEVERE TONGUE NECROSIS ASSOCIATED WITH PINE PROCESSIONARY MOTH (THAUMETOPOEA WiLKINSONI) INGESTION IN THREE DOGS

Bruchim,Y., Ranen, E., Saragusty, J., Aroch I,

School of Veterinary Medicine, The Hebrew University of Jerusalem, Israel

The pine processionary moth (Thaumetopoea wilkinsoni) is among approximately 200 species of Lepidoptera that have been described as causing inflammatory dermatologic reactions in man, and occurs in pine stands, as well as planted pine trees in urban areas in Mediterranean countries, including Israel. Several caterpillar species are covered with poisonous hair containing chitinous spines capable of penetrating the human epidermis and leading to dermatitis, known as erucism. This report describes three exposure cases in dogs due to ingestion of caterpillars in Israel. All three dogs were observed in direct contact with caterpillars or pinecones in infested gardens. The disease course and progression of signs were acute in all three cases, and included vomiting and severe tongue swelling. Physical examination findings included hyperthermia, tachypnoea, respiratory distress, cyanosis and tongue oedema, labial angioedema, ptyalism, bilateral submandibular lymphadenomegaly and conjunctivitis.  Severe tongue necrosis and sloughing of its distal portion occurred two to five days after admission to the hospital.

All dogs recovered and were discharged within 2-7 days of admission. Two staff members, attending one of the dogs, experienced an itchy rash and wheals on their arms, wrists and necks. Exposure to PPM caterpillars should probably be included in the differential diagnoses of unexplained tongue necrosis in dogs in endemic areas such as Israel, when the history is obscure. To the best of our knowledge, this should be the first report of severe oral lesions and tongue necrosis due to contact with T. wilkinsoni caterpillars.

 

RETROSPECTIVE STUDY AND PHYLOGENETIC ANALYSIS OF CANINE BABESIOSIS IN ISRAEL

Baneth, G.,1 Shkedy, N.,1 Helps, C.R.2 Shaw, S.E. 2

1. School of Veterinary Medicine, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel

2. School of Clinical Veterinary Science, University of Bristol, Langford House, Langford BS40 5DU , United Kingdom

Babesia species are tick-borne apicomplexan parasites of erythrocytes that infect a variety of domestic and wild animals. Babesia infections in dogs are caused by several species including B. canis and B. gibsoni. Three subspecies of B. canis have been recognized in dogs based on differences in the pathological and clinical syndromes caused by each subspecies, antigenic properties, genetic characterization and transmission by different vector ticks.  A retrospective study of canine babesiosis included all dogs diagnosed with Babesia infection during 1997-2003 at the Hebrew University Veterinary Teaching Hospital (HUVTH). In addition, blood was collected from dogs that were admitted to the HUVTH with babesiosis. Following DNA extraction,  PCR was performed to amplify a 450 bp fragment of the 18S rRNA gene of Babesia species.

Thirty seven cases of babesiosis were detected. Fifteen dogs (40%) were younger than 1 year old. No breed or gender predilections were found. The majority of the cases were detected during the warm months from April to November. On physical examination, 51% had fever, 44% had pale mucous membranes and 18% had visible icterus. Ninetysix % were thrombocytopenic and 77 % were anemic, mostly with normocytic normochromic anemia. Babesiosis was the primary diagnosis in only 10 cases (27%) and concurrent ehrlichiosis was diagnosed in an additional 18 dogs (49%). Other concurrent diagnoses included: neoplasia, parvoviral enteritis, hepatozoonosis, dermatologic abnormalities and an incidental finding in dogs before spay. Sequencing of the amplicons and phylogenetic analysis of nine isolates indicated that the sub-species infecting all these dogs was B. canis susbsp. vogeli, that was most closely related to an isolate from Egypt (GenBank accession no. AY371197). In conclusion, canine babesiosis in Israel is usually a mild to moderate disease frequently complicated by concurrent illness and mostly associated with B. canis subsp. vogeli infection.

 

AORTIC THROMBOEMBOLISM ASSOCIATED WITH SPIROCERCA LUPI INFECTION

Gal, A.,  Kleinbart, S.,  Aizenberg Z. and Baneth G.

School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Spirocerca lupi is a nematode pathogen of canines, distributed world wide, mostly in warm climates. Affected dogs usually show signs of gastrointestinal and respiratory involvement, although, clinical signs related to aberrant larval migration have been documented. A 2 year old castrated male Cavalier King Charles Spaniel was presented with paraplegia, cold caudal extremities and lack of femoral pulse. A thrombus of 2 cm length occluding the aortic trifurcation and an abdominal aortic aneurysm of 3 cm length with a thrombus were detected by ultrasonographic examination. The clinical and ultrasonographic findings were consistent with aortic thromboembolism. Anti-thrombotic andvasodilative therapy was not helpful and the dog was euthanized 3 days after the onset of paraplegia. A thrombus in the aortic trifurcation, multiple thoracic and abdominal aneurysms and a distal mediastinal esophageal granuloma with S. lupi worms were found on necropsy. The abdominal aortic aneurysms formed by S. lupi larval migration are believed to be responsible for the formation of the thrombus that occluded the aortic trifurcation.  To the best of our knowledge, this is the first report of aortic thromboembolism associated with S. lupi infection.

 

IS ERADICATION OF AIDS VIRUS IN MAN  AND ANIMALS POSSIBLE?

Perk, K.

Koret School of Veterinary Medicine, P.O.B 12 Rehovot.

The dissemination of lentiviruses ( a subfamily of retroviruses)  maedi-visna (MVV) in sheep¬ caprine arthritis encephalitis(CAEV) in goats and human immunodeficiency virus (HIV) in man, are the most catastrophic examples of emergence, transmission and propagation of a viral genome.

Morphologically the retrovirus family are classified as A, B, C and D virus particles.  The intracytoplasmic A particles are a stage in the morphogenesis of the lentiviruses.

In an epidemiological study of CAEV incidence in goats and MVV in sheep ¬blood examinations of heavily afflicted herds revealed a small number of goats and sheep, that on previous blood examinations (6 month earlier) had shown high levels of infection, had become negative (i.e had undetectable virus in the blood).  In post-mortem examinations, the main findings were only typical intracytoplasmic A particles in ganglial cells. This concealed location (of the long living cells) may be in some analogy with HIV infection. Despite having undetectable blood levels of HIV in patients after potent combination therapies, eradication is still a daunting task¬ since viruses were detected in a certain population of T cells. This latent reservoir in long living cells appears sufficient to ensure lifelong persistence of the virus. Furthermore, A particles were also present in glial cells of one patient where blood tests were negative for the virus. These concealed locations of lentivirus infection will be discussed.

 

HYPERADRENOCORTICISM IN DOGS WITH SKIN LESIONS AS THE ONLY CLINICAL MANIFESTATION- A CASE SERIES

Zur, G.

Veterinary Teaching Hospital Koret School of Veterinary Medicine

Abstract

This report presents 11 dogs that had skin lesions as the only clinical sign of hyperadrenocorticism (HAC).

Skin lesions were variable. All dogs exhibited alopecia. Nine dogs had signs of pyoderma, six had hyperpigmentation, and four had thin skin. One dog had alopecia and very thin skin on the dorsal part of both pinnae as the only sign. Another dog had severe pedal pruritus with secondary dermatitis as the only presenting sign. Two dogs had skin infections with dermatophytes and one was presented with adult onset general demodicosis. Two dogs were diagnosed with “castration responsive dermatosis" a few years earlier. Skin lesions resolved after treatment of HAC in 8 dogs. One dog was not treated, one did not continue treatment and one dog was lost to follow-up.

This study demonstrates that skin lesions may be the only manifestation of hyperadrenocorticism in dogs.

 

LARGE MAGNITUDE LAMENESS IN FEEDLOT FARM IN TURKEY

  Bargai,U.

Koret school of Veterinary Medicine, The Hebrew University of Jerusalem

Much was published about the economical losses caused by  dairy cows lameness. Little however was published about such losses in feedlot  farms.                           

The following is a description of heavy losses inflicted on large feedlot farm in Turkey due to wide spread lameness among the young and maturing fattening bulls in the farm.                          

In a feedlot farm that sated in Mesopotamia in south east Turkey which houses 7000 baby and maturing bull calves there were about 1300 new cases of lameness in the year 2004. This indicates about 18% incidence of lameness annually. The prevalence of the lameness reached about 50 cases of lameness at any given day most year around.

The baby bulls were purchased at the age of 2 months and were put in quarantine  cubicles  for 21 days and were moved later to feedlots.

The medical staff contained 3 veterinarians and several agronomists all graduates of Turkish schools. 

During 2004 the average daily gain in the bulls was 1.120 gms/day  as against 1.280  During 2004.  In a 7000 bull farm that was a loss of  400.000 dollars.

The investigation was requested to establish the etiology of the lameness.

The investigation on the farm Included 6 stages;

1) Careful analysis of the medical and marketing data in the office.

2) Examination of about 10 % of the bulls present in the hospital of the farm on the day of arrival.

3).Examination of about 10%  of the lame bulls in the feedlots of every age group by careful treaming.

4) Treaming of  5% of apparently normal bulls  in several of the age groups.

5)  Survey  of the entire farm   for nutritional and housing  risk factors  for  lameness.

6) Integration of the findings and discussion of the conclusions with the staff.  

 

 

Results

1) The only lesions found in the lame animals up to the age of 4 months were swollen fetlock joints.

2) There was not a single case of"footrot" (paronychia) among the lame animals.

3) There were no cases of digital or interdigital dermatitis cases among the lame animals in the farm.

4) The lesions found in the lame amimals from the age of 5-6 months of age to marketing time were all complications of sequelea of subclinical laminitis such as separation of the white line and double sole

5) Examination of the housing risk factors for subclinical laminitis revealed that there were many sharp edges stones in the dust floors of the yards.

    Further inquiry revealed that the floorings of the yards are made of quarry surplus which is pressed  by manual roller into the dirt  until the surface looks smooth.

6) Examination of the ration provided to the various age groups proved to be normally accepted levels of carbohydrates and protein as recommanded by  the NRC. Further inquiry into the preparation of the ration revealed that changes of the components are often made on a day to day basis when the supply of the specific component is short in the market.

 

Analysis of the results of the examinations pointed to two causes  to the widespread subclinical laminitis on the farm: The first is the  sharp edge stones embedded in the floors which come out to the surface by the constant  movement of the bulls because of their sexual activities. These stones  cause solar hemorrhages  which in turn become soft and abscessed.  The second cause of the wide spread subclinical laminitis is the sudden changes in the components of the ration with no transitional period for the ruminal flora to accommodate.

The farm management was advised ways and means to solve these problems.

 

 

RAPID DETECTION OF INFECTIOUS BRONCHITIS VIRUSES IN CHICKENS BY REAL-TIME REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION

Meir, R. and Maharat O.

Division of Avian and Aquatic Animal Diseases - The Kimron Veterinary Institute, Bet Dagan

The feasibility of applying the real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) method as a rapid and reliable tool for detection of infectious bronchitis viruses was examined. The aim of this study was to develop a more sensitive method, and to test the presence of RT-PCR inhibitory factors in negative samples. In the RRT-PCR method, a fluorescent dye is added to the PCR reaction mix, which emits fluorescent light when bound to the amplified  DNA and indicates the amount of DNA produced in each cycle of the reaction.

In this study we made use of a commercial kit containing SYBR Green dye. Two primers amplifying a 101 base-pair section of the viral M gene were designed and used to amplify the Israeli variants  I,  II, IS/720, and IS/885, and the reference strains Massachusetts 41, and H120. The primers were specific and did not amplify Newcastle disease virus, avian influenza virus, avian pneumovirus, reovirus, turkey encephalomyelitis virus (ITME), and infectious bursal disease virus.

Sensitivity of the test was compared to that of conventional reverse transcriptase-polymerase chain reaction (RT-PCR) based on amplification of a fragment of the viral M, and S1 genes. RT-PCR for the amplification a fragment of the M gene was one-fold more sensitive than that for the S1 gene. RRT-PCR for the M gene was two-fold more sensitive than RT-PCR with the same primers.  In addition, negative samples, as tested by RT-PCR with primers for the S1 viral gene, were tested by the RRT-PCR method. Out of 85 samples, 30 tested positive. Samples that tested negative by both methods were further examined for the presence of RT-PCR inhibitory factors. That was accomplished by adding viral RNA to an amplification reaction for the ITME virus. Out of 55 negative samples 15 had an inhibitory effects on the amplification of TIME.

The RRT-PCR method proved more sensitive than RT-PCR, and both methods were more sensitive when primers for the M gene were used rather than primers for the S1 gene. Thus, false negative results can be due to low sensitivity of the method, or as a result of inhibitory factors present in the specimen.

 

LOW PATHOGENIC H9N2 AVIAN INFUENZA AS A THREAT TO THE POULTRY INDUSTRY

Perk, S.1, Shihmanter E.1,Gissin I.1, Rosenbluth E.1, Pokamunsky, S.2, Pirak M.2 and Panshin, A 1

1. Department of Avian and Aquatic Animals Diseases, Kimron Veterinary Institute, Beit  Dagan, Israel.

2. Veterinary Services, Israel

The present study aimed to characterize the recent avian influenza viruses (AIV) isolated from Israeli commercial poultry and to determine the vaccine compatibility. About 250 AI isolates were obtained from chicken, turkey and domesticated ostrich farms during the last five years.  The viruses were identified as H9N2 and classified as apathogenic on the basis of intravenous pathogencity index (IVPI). However, under field conditions the behavior of the H9N2 isolates seemed to be different. The average mortality rates in turkey chicks ranged from 3 to 30%, although in several cases it reached up to 100%, probably due to mixed infections with other pathogens.

 The hemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) genes of the H9N2 AIV isolated from recent outbreaks in commercial flocks were characterized. Phylogenetic analysis of the HA, NA and NP genes demonstrated their relation to the viruses circulating in the Middle East and Asia. Since 2001, distinguished genetic feature appeared in the AIV lineage. We detected the presence of new amino acids motif RSKR in the HA protein cleavage site.

To determine the mutations that may have occurred after the vaccine introduction, we compared the sequence of the vaccine strain with sequences from field isolates obtained before and after beginning the vaccination program. Furthermore, we also attempted to elucidate the detailed structure of antigenic sites on H9 HA. We mapped the amino acid changes on the three-dimensional structure of H9 HA field viruses that were obtained before and after the beginning of the vaccination. A total of 4 amino acid substitution sites were identified, which indicated that mutations were introduced in a progressive manner from earlier isolates to the recent isolates. The four H9 AI substitutions (at positions 135, 179, 188, and 216) occurred in regions previously mapped as discontinuous antigenic sites of the HA. 

In summary, we found that recently isolated H9 AIs  showed some genetic differences compared to the vaccine strains. Circumstantial evidence supports the idea that vaccine pressure drove this phenomenon, however, other possibilities for explaining the genetic drift cannot be ruled out.

 

SCREENING FOR FMD VIRUS IN VACCINATED HERDS* AFFECTED BY FIELD INFECTION.

Yadin H.,1*, Chai D.,1, Brenner J.,1, Oved Z.,2, Hadany Y.,2, Kusak A.2.  

 1. Kimron Veterinary Institute, Bet Dagan 50250, Israel.

2. Regional Veterinary Services, Bet Dagan 50250, Israel.

Introduction:

Israel is located in the Middle East in a FMD endemic area and since August 1999 has been free of FMD outbreaks. In January 2004 Israel was facing again different FMD outbreaks, eight farms we re affected; two dairy and fattening herds, four calf feedlots, one sheep flock and one game animals farm. The morbidity of clinically sick animals was up to 30% in the feedlots and 0% at the dairy operations.

The scope of this study was to detect the prevalence of FMD subclinical sick animals in vaccinated herds by searching for NSP positive animals in the vaccinated population.

 

Materials and Methods:

Five FMD affected farms were blood sampled 30 - 80 days after outbreak; two of them dairy farms with annex feedlot, two feedlot operations and one sheep herd. As a control three unaffected dairy farms were blood sampled. Certified commercial ELISA kits were used for evaluation of the presence of NSP antibodies.

 

Results:

The two dairy farms were free of clinical FMD although in the feedlots calves showed typical signs. On those two dairy farms 27% and 37 % of the animals were sampled (a total of 236 animals) 14 of them were NSP positive. Of these12 were cows (66/260 sampled) aged >5 years, having 5 - 8 vaccinations and 2 were calves (45/290 sampled) just after one vaccination.

 At the four feedlots, part of those dairy farms 35% and 89% of calves were sampled a total of 194 and 79 were NSP positive. Of these 90% were calves 6 - 10 months old, 4 - 7 months after a single vaccination. At the two dairy - associated feedlots, 64 were sampled, 20 were NSP positive, 5-6 months after a single vaccination. The unvaccinated sheep flock, 4 of the 80 sheep were sick and 60 of the 69 tested were NSP positive. The prevalence of NSP positive in multi vaccinated unaffected 3 dairy herds was 1/398 and in a dairy sheep flock 1/131.

 

Discussion:

The vaccinated dairy farms were well protected against clinical as well as subclinical infection with very low NSP - positive animals. It seems that even the young calves before first vaccination are well protected by colostral antibodies.

Feedlots with imported calves from FMD free countries are susceptible to infection four months after a single vaccination. Unvaccinated lambs form a vector of high risk for spreading of the disease even without showing clinical signs.

 

The support of this work by the EUFMD commission is gratefully acknowledged.

 

QUICK DETECTION OF SHEEP POX VIRUS BY IMMUNOHISTHOCHMICAL METHODS

Eligulashvili R., Bombarov V. and Yadin H.

Virology Division, Kimron Veterinary Institute, Beit Dagan.

Peste des Petits Ruminants (PPR) is a contagious viral disease, which form world wide threatening for the small ruminants livestock. The causative agent belongs to the family Paramyxoviridae, genus, Morbilli virus. PPR was recognized in Israel for the first in 1992 caused remarkable losses in the effected herds. The main tool in the control policy is general vaccination of all small ruminants livestock. Beside this a rapid and specific diagnosis is an important support in the control strategy. The diagnosis method consisting on the Immunohistochemistry (IHC) of fresh organs on cryostat sections is used as a quick and specific laboratory diagnosis method for different diseases.  The present study compares two IHC techniques for rapid diagnosis of PPR: avidin-biotin complex and Polymer conjugate system. Freshly frozen tissue sections from organs (intestine, lung, tongue and lymph node) from suspected dead animals or epithelial cells from ocular swabs from sick animals were tested with the IHC method. The detection system consisted on monoclonal anti PPR antibodies (France, CIRAD/EMVT), that can differentiate PPR from Rinder Pest. With both IHC techniques PPR virus antigen was apparently demonstrated in the frozen tissue sections of tongue, intestine, lymph node and in epithelial cells from ocular swabs, without any background staining.

 

The duration of the complete diagnostic procedure was 2-2,5 hours for the tissue sections and 3,5-4 hours for the ocular swabs epithelial swabs.

 

MECHANISM OF MSP2 ANTIGENIC VARIATION IN A. CENTRALE PERSISTENTLY  INFECTED CATTLE

  Molad T., Mazuz M., Fleiderovitz L., Fish L., Krigel Y., Leibovitz B., Shkap V.

Kimron Veterinary Institute,, Beit Dagan

Anaplasma, a member of the Ehrlichial genogroup II, is an intra-erythrocytic pathogen, containing a circular genome  with estimated size of 1.2 to 1.6 Mb. Two  Anaplasma spp. infect cattle: A. marginale and A. centrale. A live strain of A. centrale that caused only  a mild form of anaplasmosis  is  used  for vaccine production in Israel.  Major surface protein 2 (MSP2) of A. centrale is encoded by a polymorphic multigene family. Antigenic variation of Anaplasma MSP2 occurs during persistent infection and allows the rickettsiae to evade the pre-existing immune response. MSP2 is composed of conserved amino- and carboxy-terminal regions flanking  a central hypervariable region, characterized by substitutions, insertions and deletions. A. centrale MSP2  was found to be expressed from the operon, which has four open reading frames (ORFs) containing msp2 at the 3’ terminus. The operon  appeared to be only expression site  of the full-length msp2 transcripts. The sequences of the  three upstream orfs are conserved; polymorphism was found within the msp2 coding region in the hypervariable region. Seven msp2 pseudogenes were identified in the A. centrale genome. The pseudogene copies of msp2 in the genome are truncated, they contain a central hypervariable region flanked  by short portions of the 5’ and  3’ conserved regions. We have shown that in A. centrale MSP2 variants are generated by two mechanisms: recombination of the whole pseudogene into the single msp2 expression site, or  recombination of small segments of pseudogenes into the expression site by segmental gene conversion. Antigenic variation of A.centrale msp2 occurs by the same mechanism as antigenic variation in A.marginale msp2.

It appears that A. centrale must  infect vaccinates in order to induce immunity; presumably by allowing generation of A. centrale MSP2 variants during persistence.The shared structure of the MSP2 in all Anaplasma strains may be responsible , in part, for the cross -protection inducted by A.centrale  vaccination  against  A.marginale challenge. The efficacy of the A. centrale vaccine suggests that strategies that mimic  A.centrale vaccine responses are the key to development of an effective, non-blood-based vaccine.