Description of the pathology of a gazelle that died during a major outbreak of foot-and-mouth disease in Israel.

Naturally occurring foot-and-mouth disease (FMD) in wildlife is a relatively mild condition but occasionally it can be devastating as has been documented in impala in South Africa and in mountain gazelles in Israel. This report describes pathological changes in an adult male gazelle with FMD from an outbreak in the Nature Reserve of Ramot-Issachar region and the lower Galilee in Israel. The outbreak was characterised by the malignant form of the disease, which is uncommon among domestic animals. Lesions observed included, ulceration in the oral cavity, oesophagus and ruminal pillars, coronitis, multifocal cardiac necrosis and pancreatic necrosis and inflammation. Pneumonia, caused by Muellerius capillaries was an incidental finding.

Prognostic indicators for canine monocytic ehrlichiosis.

In order to identify prognostic factors for survival in canine monocytic ehrlichiosis (CME), clinical records of 40 cases of CME were retrospectively studied. The dogs were assigned as survivors (n=21) and non-survivors (n=19), and their signalment, anamnesis, clinical and clinicopathological signs, and treatment protocols were compared. Pale mucous membranes, bleeding tendencies and weakness were more prevalent in the non-survivors compared to the survivors. Dogs in the non-survivor group had significantly lower white blood cell (WBC), hematocrit (HCT), and platelet (PLT) counts. Pronounced pancytopenia (WBC < 4 x 10(3) microL(-1); HCT < 25%; PLT < 50 x 10(3) microL(-1)) was found as a risk factor for mortality. In this study, severe leucopenia (WBC < 0.93 x 10(3) microL(-1)), severe anemia (PCV < 11.5%), prolonged activated partial thromboplastin time (APTT>18.25s) and hypokalemia (K<3.65 mmol/L) were each found to predict mortality with a probability of 100%. In contrast, WBC counts above 5.18 x 10(3) microL(-1), platelet counts above 89.5 x 10(3) microL(-1), PCV > 33.5%, APTT < 14.5s and serum potassium concentration above 4.75 mmol/L, each provided 100% prediction for survival. These prognostic indicators can be easily obtained at presentation, are inexpensive, and may be useful aids when treatment and prognosis are being considered.

Gluconeogenesis in non-obese diabetic (NOD) mice: in vivo effects of vandadate treatment on hepatic glucose-6-phoshatase and phosphoenolpyruvate carboxykinase.

The contribution of gluconeogenesis to hyperglycemia in non-obese diabetic (NOD) mice has been investigated using oral vanadate administration. Vanadate compounds have been shown to mimic many actions of insulin; however, the exact mechanism is poorly understood. The aims of the present study were (1) to elucidate vanadate's action in vivo, and to assess the possibility that its glucose-reducing effect is dependent on the presence of a minimal concentration of insulin; and (2) to evaluate the effects of vanadate administration on the key hepatic gluconeogenesis enzymes, glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK), as well as glucose-6-phosphate dehydrogenase (G-6-PDH). Vanadate caused a significant reduction in blood glucose but failed to normalize it, despite effective serum vanadate concentrations (26.2 +/- 1.6 micromol/L). Two weeks after initiation of treatment, blood glucose levels were 26.0 +/- 1.8, 21.7 +/- 3.0, 16.0 +/- 1.6, and 14.3 +/- 2.3 mmol/L in the control (C), insulin (I), vanadate (V), and combined vanadate and insulin (V + I) groups, respectively (P < .001). G-6-Pase activity was significantly reduced by vanadate (622 +/- 134 v365 +/- 83 nmol/min/mg protein in C vV, P < .05). PEPCK activity was also significantly reduced (844 +/- 370, 623 +/- 36, 337 +/- 43, and 317 +/- 75 nmol/min/mg in the C, I, V, and V + I groups, respectively, P < .001). No significant differences in the hepatic glycogen stores and G-6-PDH activity were noted between treatment groups. Our study suggests that the inhibition of hepatic G-6-Pase and PEPCK activity by vanadate plays an important role in reducing blood glucose levels in NOD mice.

Presence of immune-complexes, and absence of antinuclear antibodies, in sera of dogs naturally and experimentally infected with Ehrlichia canis.

Antinuclear antibodies (ANA), immunoglobulin G (IgG) concentrations and circulating immune-complexes (CIC) were measured, over a period of 3 years, in 6 dogs experimentally infected with Ehrlichia canis, and in 10 dogs naturally infected with the rickettsia. No ANA were detected in any of the samples tested. The IgG concentrations were shown to be higher in the infected dogs when compared to the control dogs. CIC were detected in 2 of 10 naturally and 2 of 6 experimentally infected dogs, during both the acute and the subclinical phases of the disease. The results of this study suggest that ANA do not play a role in the pathogenesis of CME. It is however suggested that some manifestations in canine ehrlichiosis are immune-complex mediated.

Kinetics of serologic cross-reactions between Ehrlichia canis and the Ehrlichia phagocytophila genogroups in experimental E. canis infection in dogs.

The serological cross-reactions between the Ehrlichia canis and Ehrlichia phagocytophila genogroups, and the kinetics of development of antibodies in dogs to the E. phagocytophila group after artificial infection with the Israeli strain of E. canis was investigated. Results of this study indicate that the development of antibodies to the E. phagocytophila genogroup in dogs after infection with E. canis is a time dependent event probably conditional on the continued propagation of the rickettsia in the host. After spontaneous clinical recovery, and at the beginning of the subclinical phase, no antibodies to E. phagocytophila were yet detectable. The first evidence of IgG antibodies to E. phagocytophila were found in two of the six dogs, 55 days after artificial infection with E. canis, while another two dogs became seropositive to E. phagocytophila 22 days later. All surviving dogs were seropositive to E. phagocytophila by 150 days PI. It is suggested that the appearance of E. phagocytophila antibodies following treatment of acute E. canis infection may be used to judge treatment failure and/or persistence of infection.

Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis.

This report presents evidence for the presence of antiplatelet antibodies in sera of dogs experimentally infected with Ehrlichia canis, during the acute phase of the disease. Six healthy adult male beagle dogs were inoculated intravenously with blood from a longstanding infected dog with the Israel strain 611 of E. canis. Thrombocytopenia and concurrent increase in mean platelet volume were the most consistent haematological signs of the disease. The dogs developed an antibody titre to E. canis from Day 15 postinoculation. All dogs were antiplatelet antibody negative before inoculation. Twenty-four days postinoculation with E. canis, when the platelet count was at its lowest, antibodies to platelets were demonstrated, in the sera of five of the six dogs.

Kinetics of serum antiplatelet antibodies in experimental acute canine ehrlichiosis.

The pattern of appearance of serum antiplatelet antibodies during the acute phase of experimental canine ehrlichiosis (Ehrlichia canis) was investigated in six beagles and correlated with the development of thrombocytopenia. The earliest detection of serum antiplatelet antibodies was made on Day 7 post-inoculation in one dog, on Day 13 in three out of six dogs, and on Day 17 post-inoculation in the remaining two dogs. Thrombocytopenia developed in all infected dogs. The results of this study suggest that antiplatelet antibodies play a role in the destruction of platelets in the acute phase of the disease. It is proposed that E. canis infection in dogs alters the immune system resulting in the overproduction of natural antiplatelet antibodies.

Investigation of splenic functions in canine monocytic ehrlichiosis.

In order to determine the role of the spleen in the pathogenesis of canine monocytic ehrlichiosis (CME), the effect of splenectomy on the course of the acute phase of experimental was investigated. Intact and splenectomized dogs, sero-negative for Ehrlichia canis antibodies, were infected with the Israeli strain of E. canis. Serology, clinical signs and haematological parameters were recorded prior to infection, and over a period of 60 days post infection, and were compared between the intact and the splenectomized dogs. All dogs seroconverted for IFA E. canis antibodies by days 10 to 17 post infection. There did not appear to be any difference in the day of appearance or in the titer of anti-E. canis IgG antibodies, between the splenectomized and intact groups throughout the course of the study. During the acute stage, food consumption (percentage change) was significantly lower in the intact group compared to the splenectomized group (-66.3% and -25.3%, respectively, p < 0.0001). During this period, significant higher body temperatures were measured in the intact group (average of 39.76 degrees C vs. 38.96 degrees C, p < 0.0001). The haematocrit, red blood cell counts, haemoglobin concentrations and platelet counts were significantly lower (p < 0.05) in the intact group when compared to the splenectomized group during the whole course of the study. The clinical and the haematological findings in our study suggest that the disease process was milder in the splenectomized dogs compared to the intact dogs. The results of this study suggest that the spleen plays an important role in the pathogenesis of CME. Splenic inflammatory mediators and/or other splenic substances, are proposed to play a key role in the pathogenesis of the disease. Our results further substantiate the involvement of immune mechanisms in the pathogenesis of CME.

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis.

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.

Comparison of a clinic-based ELISA test kit with the immunofluorescence test for the assay of Ehrlichia canis antibodies in dogs.

The "gold standard" for the detection of antibodies to Ehrlichia canis, the cause of canine monocytic ehrlichiosis (CME), is the indirect immunofluorescence antibody (IFA) test. The IFA test however is generally available only in selected laboratories and requires extensive equipment and trained personnel. A double-blind study was conducted to compare the ability of an in-clinic standardized enzyme-linked immunosorbent assay (ELISA) test kit to measure E. canis IgG antibodies in dogs compared with the standard IFA technique. A good correlation was found between the 2 techniques (r2 = 0.8793; P < 0.0001). Evidence for the sensitivity of the ELISA technique for the early detection of E. canis IgG antibodies was demonstrated by comparing the appearance of E. canis antibody titers by the IFA and ELISA techniques after artificial infection of 2 sets of dogs. In both experimental infections, both tests were equally sensitive for the early detection of IgG antibodies against E. canis, and the results correlated well with the appearance of fever and clinical signs. Proposed application of the in-clinic ELISA test is to aid in the diagnosis of CME.

Cultivation of Ehrlichia canis in a continuous BALB/C mouse macrophage cell culture line.

This report describes the successful adaptation of the Israeli isolate of Ehrlichia canis on a continuous mouse macrophage cell line (J774.A1). Successful infection of the J774.AI cells was first judged by the direct immunofluorescence antibody test using an anti-E. canis-IgG:FITC conjugate. A particular property of infected J774.A1 cells was the ability to reestablish after harvesting of the monolayer by scaping. Infected cells were used as antigen for immunofluorescence antibody tests (IFA), and the results compared well with those of DH82 cells. It was concluded that the J774.A1 continuous cell line could serve as an alternate propagation cell line for E. canis organisms.

Assessment of maternal antibody decay and response to canine parvovirus vaccination using a clinic-based enzyme-linked immunosorbent assay.

Interference caused by maternal antibodies is considered a major cause of canine parvovirus (CPV) vaccination failure. In this study, an immunoblot clinic-based enzyme-linked immunosorbent assay (ELISA) method was used to detect CPV antibodies in sera of pregnant bitches and their offspring to study the response of pups to vaccination. With a easily accessible procedure for CPV antibody determination, the veterinarian should be able to gauge the response of pups after vaccination. The validity of the technique was tested in parallel against the standard hemagglutination inhibition (HI) test. Results of the ELISA were correlated with those of the standard HI method for quantification of CPV antibodies. With the ELISA, successfully immunized pups were identified, allowing for a more reliable and cost-effective program of vaccination. This simple clinic-based test could be used for the assessment of vaccination status of pups during the critical phase of 6 to about 16 weeks of age. This study is the first in which vaccination response to CPV in pups was followed, using a clinic-based ELISA for CPV antibody monitoring.

Acute blindness associated with monoclonal gammopathy induced by Ehrlichia canis infection.

Ehrlichia canis infection was diagnosed in a Labrador retriever presented with a primary complaint of acute blindness. Ocular signs on admission included bilateral hyphema, retinal haemorrhage and retinal detachment. Serum protein electrophoresis results revealed monoclonal gammopathy. This report discusses and suggests the pathogenesis of ocular bleeding in canine monocytic ehrlichiosis. Blood hyperviscosity, elevation in oncotic pressure, vasculitis, thrombocytopenia and platelet dysfunction are all proposed to be important factors in the pathogenesis of acute blindness in canine monocytic ehrlichiosis.

Dynamics of IgG1 and IgG2 subclass response in dogs naturally and experimentally infected with Ehrlichia canis.

Immunoglobulin (Ig) G subclasses were measured in dogs naturally and experimentally infected with Ehrlichia canis using enzyme-linked immunosorbant assay (ELISA). In this study, a higher IgG2 subclass response was noticed to natural and experimental E. canis infection in dogs. Anti-E. canis-IgG2 optic density (OD) values were found to be significantly higher than anti-E. canis-IgG1 during the different phases of the disease, and no differences in the IgG subclass responses to E. canis infection were found between symptomatic and asymptomatic dogs. Doxycycline treatment, which eliminated the rickettsia in three of four persistently infected dogs, had no noticeable influence on the E. canis-IgG subclass OD values during the treatment period. In order to facilitate the study, an ELISA for the detection of anti-E. canis IgG was developed and was shown to be sensitive and specific for E. canis-IgG, and in a significant correlation with the indirect immunofluorescence antibody test.

Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis.

Dogs are susceptible to a number of ehrlichial diseases. Among them, canine monocytic ehrlichiosis is an important and potentially fatal disease of dogs caused by the rickettsia Ehrlichia canis. Diagnosis of the disease relies heavily on the detection of antibodies and is usually carried out using the indirect immunofluoresence antibody (IFA) test. The IFA test may be confounded by cross-reactivities between a number of the canine ehrlichial pathogens. This article presents a review of the ehrlichial diseases affecting dogs with reference to their immune responses, host specificities, cross-reactivites and diagnosis. Diagnostic means such as Western immunblot, dot-blot and PCR are discussed. The use of the IFA test as a diagnostic means for E. canis is presented along with its potential pitfalls. The review emphasizes that the disease process, cross-reactivites with other ehrlichial species, multiple tick-borne infections and persistent IFA antibody titers post-treatment, should all be considered when interpreting E. canis serological results.

Characterization of the subclinical phase of canine ehrlichiosis in experimentally infected beagle dogs.

Beagle dogs were examined during the subclinical phase of canine ehrlichiosis under controlled conditions. Emphasis was placed on gathering data before artificial inoculation with Ehrlichia canis, and comparing these data with those of the subclinical phase of the disease. In this study all dogs were clinically healthy throughout the 6 month examination period. All subclinically infected dogs had IFA antibody titers to E. canis at a dilution varying from 1:2560 to 1:20480. The most prominent haematological finding was mild thrombocytopenia with a concomitant increase in platelet size, seen in eight of the nine dogs examined. Leukocyte counts were statistically significantly reduced in 78% of the dogs, compared with their preinfection values, with 71% of dogs having significantly reduced absolute neutrophil counts. None of the dogs were either leukopenic nor neutropenic. Six of the nine dogs had increased serum gamma-globulin concentrations. No dogs were overtly anemic, although declines in packed cell volume, haemoglobin concentration and total erythrocyte count were detected in an inconsistent manner among the dogs. It was concluded that, the most reliable parameters for judging possible subclinical ehrlichial infection in beagle dogs was mild thrombocytopenia, together with a persistently high antibody titer to E. canis. Hypergammaglobulinemia would increase the suspicion further. Based on the results presented, routine testing of dogs in E. canis endemic areas is recommended in order to identify and treat dogs in the subclinical phase of the disease.

Serum protein alterations in canine ehrlichiosis.

Serum protein electrophoresis was performed in 42 dogs with naturally occurring Ehrlichia canis infection and in 15 clinically healthy dogs (control dogs). The infected dogs were found to have a significant hypoalbuminaemia, hyperglobulinaemia and hypergammaglobulinaemia compared to the control dogs (P < 0.001). A polyclonal gammopathy was found in all but one of the infected dogs which presented a monoclonal gammopathy. alpha-1 globulin was lower while alpha-2 and beta-2 globulin concentrations were significantly higher in the infected dogs (P < 0.0001, P < 0.05 and P < 0.005, respectively). The infected dogs were divided into two subgroups according to haematological parameters, defined as pancytopenic (n = 13) and non-pancytopenic (n = 29). When compared, the pancytopenic group revealed significantly lower concentrations of total protein, total globulin and gammaglobulin (P < 0.01, P < 0.05 and P < 0.005 respectively). The lower concentrations of the gammaglobulins coupled with the pancytopenia suggest that the immune state of the pancytopenic E. canis infected dogs is more compromised, and therefore secondary infections should be expected more frequently in these dogs.

Detection of ehrlichial antigen in plasma of beagle dogs with experimental acute Ehrlichia canis infection.

Six beagles were experimentally infected with Ehrlichia canis. All dogs developed typical clinical signs of ehrlichiosis and sero-converted. Ehrlichial antigenemia in the plasma of the infected dogs was detected using a sandwich enzyme-linked immunosorbent assay (ELISA). Ehrlichial antigen was present starting 15-20 days post-infection, after the development of clinical signs and antibody titre to Ehrlichia canis. The appearance of ehrlichial antigen in the plasma for a relatively short and variable period after the clinical and haematological signs, limits its potential as an early diagnostic prognosticator of canine ehrlichiosis.

Antibodies reactive with Ehrlichia canis, Ehrlichia phagocytophila genogroup antigens and the spotted fever group rickettsial antigens, in free-ranging jackals (Canis aureus syriacus) from Israel.

A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with the Ehrlichia canis and Ehrlichia phagocytophila genogroup antigens, and the spotted fever group (SFG) rickettsiae antigens in jackals in Israel (Canis aureus syriacus), to assess the possible role of the jackal in the epidemiology of these diseases. Fifty-three serum samples from jackals were assayed by the indirect immunofluorescence antibody test. Antibodies to E. canis were detected in 35.8% serum samples while 26.4% of the samples tested were positive to Ehrlichia chaffeensis. Twenty-six percent of the jackals tested were seropositive to E. phagocytophila, of which 5.7% were seropositive to E. phagocytophila alone without any seroreactivity to either E. canis or E. chaffeensis. Fifty-five percent of the jackals were seropositive to the SFG-rickettsiae antigens. The results suggest a high exposure rate of jackals in Israel to E. canis. Positive reactivity to E. chaffeensis was considered to be due to antigenic cross-reactions with E. canis. The study demonstrated for the first time the presence of E. phagocytophila antibodies in free-range jackals. The high incidence of antibodies to the SFG-rickettsiae and their relatively high antibody titers was suggestive of either recent or persistent infection. The possibility that jackals may play a role in the transmission of E. canis, E. phagocytophila and the SFG-rickettsiae for human and canine infections is discussed.

The first isolation, in vitro propagation, and genetic characterization of Ehrlichia canis in Israel.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, was isolated in Israel from a naturally infected dog with acute signs of the disease. The organism designated E. canis 611, was passaged experimentally to a beagle, from which it was propagated in primary canine monocytes. The organism was then grown in vitro in a continuous canine cell line, DH82. Nine beagles subsequently injected with whole E. canis-infected blood all developed typical symptoms of ehrlichiosis. An indirect immunofluorescence antibody test to E. canis was developed and compared with a commercial kit, revealing a good correlation between the two assays. Transmission electron microscopy of DH82 cells infected with the Israeli strain of E. canis (611), revealed organisms similar to those described in the literature: two different forms of morulae appeared, one tightly, the other loosely, packed. The 16S rRNA gene sequence obtained from the Israeli Ehrlichia isolate was compared with other isolates, E. canis Oklahoma and E. canis Florida. The Israeli strain 16S rRNA had three nucleotide differences from the Oklahoma isolate, and four nucleotide differences from the Florida isolate, in addition to one nucleotide gap in each. The Israeli isolate was found to be 0.54% different from the Oklahoma strain, and 0.61% different from the Florida strain. There are the same magnitudes of differences displayed by the other most closely related group in the phylogenetic tree, namely Ehrlichia equi, Ehrlichia phagocytophilia and the human granulocytic ehrlichia.