Trevor Waner

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.

Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): an overview.

Canine monocytotropic ehrlichiosis (CME), caused by the rickettsia Ehrlichia canis, an important canine disease with a worldwide distribution. Diagnosis of the disease can be challenging due to its different phases and multiple clinical manifestations. CME should be suspected when a compatible history (living in or traveling to an endemic region, previous tick exposure), typical clinical signs and characteristic hematological and biochemical abnormalities are present. Traditional diagnostic techniques including hematology, cytology, serology and isolation are valuable diagnostic tools for CME, however a definitive diagnosis of E. canis infection requires molecular techniques. This article reviews the current literature covering the diagnosis of infection caused by E. canis.

Canine monocytic ehrlichiosis: a retrospective study of 100 cases, and an epidemiological investigation of prognostic indicators for the disease

One hundred cases of monocytic ehrlichiosis diagnosed in Israeli dogs were confirmed by the presence of anti-Ehrlichia canis indirect immunofluorescent antibody titres greater than 1:40. The disease occurred in all age groups and there was no sex predilection. German shepherd dogs were significantly over-represented whereas crossbreed dogs were significantly under-represented (P>0.0005). The most common clinical signs were depression, lethargy, lymphadenomegaly, fever, anorexia, panting, pale mucous membranes and bleeding, of which epistaxis was most common. Thrombocytopenia, anaemia (mainly normocytic normochromic) and lymphopenia were the predominant haematological findings. Forty-nine of the 100 cases were followed up for a year. Thirty-two dogs survived and 17 died. A Cox proportional hazards regression model was used to examine the effect of host, environmental, and haematological prognostic factors on survival. It was concluded that severe anaemia, severe leucopenia, pancytopenia, a tendency to bleed (especially epistaxis) and being a German shepherd dog were important indicators of poor survival in cases of monocytic ehrlichiosis in dogs.

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.

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.

Comparison of Simultaneous Splenic Sample PCR with Blood Sample PCR for Diagnosis and Treatment of Experimental Ehrlichia canis Infection

ABSTRACT This report presents evidence that dogs recover from acute canine monocytic ehrlichiosis (CME) after 16 days of doxycycline treatment (10 mg/kg of body weight every 24 h). Blood PCR was as valuable as splenic aspirate PCR for early diagnosis of acute CME. Splenic aspirate PCR was, however, superior to blood PCR for the evaluation of ehrlichial elimination.

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.

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.

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.

Comparison of three enzyme-linked immunosorbant assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis

The aim of this study was to compare three different enzyme-linked immunosorbant assays (recombinant major antigenic protein 2 (rMAP2)-ELISA, the Immunocomb (Biogal, Israel) and the Snap 3Dx assay (IDEXX Laboratories Inc., USA)) with the indirect immunofluorescent antibody test in detecting anti-Ehrlichia canis immunoglobulin-G (IgG) antibodies. Samples tested were collected from dogs suspected to be naturally infected with E. canis and from experimentally infected dogs. When qualitative results (positive/negative) were compared, there was an overall agreement of 81% (54/67) between the indirect immunofluorescence antibody (IFA) test and the rMAP2-ELISA. An overall agreement of 94% (63/67) was found between the IFA test and the Immunocomb, and an overall agreement of 91% (61/67) was found between the IFA test and the Snap 3Dx assay. In 50 of 67 (74.6%) samples tested, complete agreement in the qualitative results was found in all four tests. Sixteen of 17 samples with disagreement in the qualitative results were found to have IFA titers of 1:320 or less. The sensitivities and specificities of the tests were found to be 0.71 and 0.85 for the rMAP2-ELISA, 0.86 and 0.98 for the Immunocomb, and 0.71 and 1.00 for the Snap 3Dx assay. The tests performed in this study were found to be highly specific in detecting E. canis antibodies. Their sensitivity was found to be low with sera having IFA titers of < or =1:320, while high with sera having titers greater than 1:320. Repeating the serological tests 1-2 weeks after the first antibody assay may overcome the sensitivity problem with titers of < or =1:320.