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Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis
Veterinary
ELSEVIER
Veterinary immunology and immunopathology
Immunology and Immunopathology 48 (1995) 177-182
Short communication
Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis Trevor Waner aY*,Shimon Harrus b, Douglas J. Weiss ‘, Hylton Bark b, Avi Keysary d aLife Science Research Israel, P.O. Box 139, Ness Ziona 70451, Israel b Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100. Israel ’ Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, Minneapolis, MN, USA ’ Israel Institute for Biological Research. P.O. Box 19. Ness Ziona, Israel Accepted
14 December
1994
Abstract 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. Keywords: Ehrlichia canis; Antiplatelet
antibodies;
Thrombocytopenia;
Pathogenesis
1. Abbreviations
ANA, antinuclear antibodies; IFA, immunofluorescence assay; MFU, mean fluorescent channel units; MPV, mean platelet volume; PF-3, platelet factor 3.
* Corresponding
author.
0165-2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-2427(95)05420-O
178
i? Waner et al. / Veterinary Immunology and ImmunopathoIo~
48 (1995) 177-182
2. Introduction Thrombocytopenia is a relatively consistent finding in all stages of canine ehrlichiosis resulting from Ehrlichia canis infection (Breitschwerdt, 1988). Significant thrombocytopenia develops in experimentally infected dogs on approximately Day 10 postinfection, and is most severe on Day 17 (Pierce et al., 1977). Studies to determine the mechanism of reduction in platelet numbers have been inconclusive. It has been suggested that the thrombocytopenia has an immunopathological basis, supported by the hypergammaglobinaemia observed in chronic ehrlichiosis, possibly representing the development of a secondary autoimmune response to damaged host cell components (Burghen et al., 1971). Th e occurrence of antinuclear antibodies (ANA) in dogs affected with ehrlichiosis was investigated and found to be infrequent, indicating that ANA are not important in the pathogenesis of the disease (Kelly et al., 1994). Efforts to demonstrate the presence of antiplatelet antibodies by assay of platelet factor 3 (PF-3) have been used. This indirect method has failed to demonstrate antiplatelet antibodies (Pierce et al., 1977). We present here evidence for the presence of antiplatelet antibodies in beagle dogs experimentally infected with E. canis, during the acute phase of the disease.
3. Materials and Methods Six healthy adult male beagle dogs 8-12 months of age were purchased from Harlan (Indianapolis, USA) and tested seronegative for E. cunis antibodies as determined by indirect immunofluorescence assay (IFA). Haematological and clinical chemistry test results were within reference ranges. The dogs were housed individually in stainless steel cages (IFFA Credo; France). The room in which the dogs were housed was air conditioned with the temperature set to achieve a range of 21 &-2°C. An automatic timer regulated the lighting for 12 h of dark and 12 h of light. The dogs were fed 400 g per day of a commercial dog food (Bonzo; Kibbutz Ma’abarot, Israel) and watered automatically. The dogs were acclimatized for 1 week before inoculation. Each dog was inoculated intravenously with 5 ml of heparinized blood from a dog infected with E. cunis. Thereafter, blood was collected at least three times weekly from the jugular vein for haematology and serology. Serum samples were separated off after centrifugation within 1 h of blood collection. Haematological assays were performed on the same day as blood collection using a semi-automatic impedance cell counter (Cellanalyzer CA 580 A; Medonic, Sweden). The following parameters were determined: packed cell volume, haemoglobin concentration, total erythrocyte count, mean corpuscular volume, mean corpuscular haemoglobin concentration, mean corpuscular haemoglobin, total and differential white blood count, total platelet count, and mean platelet volume (MPV). Serum samples for serology were stored at - 70°C until assayed. Samples were tested for E. canis antibodies by the indirect IFA, as described previously (Ristic et al., 1972). Serum samples from each dog before inoculation with E. cunis, and 24 days after inoculation, were tested for antiplatelet antibodies at the Department of Veterinary
T. Waner et al. / Veterinary Immunology and Immunopathology
48 (1995) 177-182
119
Pathobiology of the University of Minnesota, Minneapolis, using a flow cytometric platelet IFA as previously described Wristensen et al., 1994a,b). Briefly, 20 ~1 of platelet suspension were incubated for 60 min with a 1:lO dilution of test serum. Thereafter, platelets were washed twice, incubated with a 1:20 dilution of fluorescein-labelled goat antidog IgG, washed three times and resuspended in physiological buffered saline containing 3 mM EDTA and 1% bovine serum albumin. Samples were analysed on a flow cytometer (Epics 751; Coulter Electronics, Heartland, FL). Mean fluorescent channel units (MFU) greater than 2 standard deviations (SD) than values for ten healthy control dogs were considered positive for antiplatelet antibodies. The intensity of fluorescence was graded as negative (less than 2 SD above controls), 1 + (2-5 MFU), 2 + (6-10 MFU), 3 + (11-20 MFU), and 4 + (greater than 20 MFU).
4. Results
All dogs developed clinical and haematological signs typical of ehrlichiosis after inoculation with E. canis. Thrombocytopenia was a consistent finding in all dogs (Fig. 1). A concurrent increase in MPV was observed (Fig. 2). The mean circulating platelet count in the dogs before inoculation with E. canis was 318 000 f 66 000 (range 228 000-369 000) platelets per ~1. Platelet counts were lowest between Days 17 and 24, with values ranging from 20 000 to 52 000 platelets per ~1 on Day 24 postinoculation. Values of MPV for the dogs during the postinoculation period approximately followed an inverse pattern when compared to circulating platelet numbers (Fig. 2). The
0
10
20
30
40
Days Fig. 1. Mean peripheral canis.
platelet count and standard error of six Beagle dogs artificially
infected with Ehrlichia
180
T. Waner et al. / Veterinary Immunology
and Immunopathology
48 (1995) 177-182
8 I 0
5
10
15
20
25
30
35
40
45
Days Fig. 2. Average canis.
mean platelet volume and standard error of six Beagle dogs artificially
infected with Ehrlichia
first increase in platelet volume was detected at Days 6 and 7 postinoculation when the MPV for the dogs rose to 9.7 and 10.5 fl, respectively. Other haematological changes observed included a mild transient leucopenia and mild non-regenerative anaemia. All dogs developed an antibody titre to E. canis on Day 15 postinoculation as determined by IFA. The initial IgG titres on Day 15 ranged from 1:160 to 1:640, and increased to 1:1280 to 1:5120 by Day 43 postinoculation. The results of the presence of serum antiplatelet antibodies in E. canis infected dogs are presented in Table 1. None of the dogs showed the presence of any serum antibody to platelets before inoculation. In five of the six dog sera, antibodies to platelets were demonstrated 24 days postinoculation with E. canis.
Table 1 Results of platelet immunofluorescence antibodies in sera of dogs after artificial
assay, in mean fluorescence channel infection with Ehrlichia canis
Dog no.
MFU at 24 days post-inoculation
51 56 98 418 83 22
1+ 2+ 0 1+ 2+ 3+
0, < 2 MFU above controls; controls.
1+ , 2-5
MFU; 2 + , 6-10
MFU; 3 + , 11-20
units (MFU),
MFU, 4 +,
for antiplatelet
> 20 MFU above
T. Waner et al. / Veterinary Immunology and Immunopathology
48 (1995) 177-182
181
5. Discussion Thrombocytopenia in dogs with E. canis infection has been attributed to several mechanisms, acting together or alone. These include increased platelet consumption or sequestration (Woody and Hoskins, 1991) increased platelet destruction in the spleen resulting in shortened platelet survival (Smith et al., 1975), suppression of platelet production in the bone marrow, mainly in the chronic phase (Woody and Hoskins, 1991) or immunologically or non-immunologically mediated platelet destruction (Breitschwerdt, 1988). This study investigated the immunological component of platelet destruction in dogs with acute ehrlichiosis and demonstrated conclusively the presence of circulating serum antiplatelet antibodies. Platelet destruction, as measured in radioactively labelled platelets, has been documented to occur at an accelerated rate during E. canis infection and was considered the primary cause of thrombocytopenia in affected dogs (Smith et al., 1975). Whole-body scans on a dog before and 7 days after infection with E. canis have shown that labelled platelets are destroyed primarily in the spleen, similar to that which occurs in immunologically mediated thrombocytopenia purpura in man (Smith et al., 1975). Smith et al. (1975) considered that the decrease in platelet survival time, 2-4 days after inoculation with E. canis, to be too rapid to be initiated by an antibody-mediated response. In order to elucidate the mechanism of platelet destruction, Pierce et al. (1977) assayed infected dog sera for PF-3. They found that the release of PF-3 from normal canine platelets by serum from infected dogs was delayed, and concluded that thrombocytopenia was not due to the action of antiplatelet antibodies (Pierce et al., 1977). In contrast, an increased release of PF-3 (two out of three dogs) and the detection of ANA (two out of two dogs) have been reported in association with suspected chronic canine ehrlichiosis (Codner et al., 1985). The results were suggestive that autoantibodies may be important in the pathogenesis of chronic ehrlichiosis. On the other hand, Abeygunawardena et al. (1990) have shown that E. canis induced high levels of platelet migration inhibition factor which induced prominent platelet surface changes leading to increased platelet vulnerability. The finding of antiplatelet antibodies in five out of six beagle dogs, 24 days after inoculation of dogs with E. canis infected blood, is indicative that immune-mediated platelet destruction may take place in the acute phase of canine ehrlichiosis. These results do not rule out the possibility that a number of mechanisms may still be involved in the pathogenesis of ehrlichial thrombocytopenia (Breitschwerdt, 1988) of which the immune-mediated mechanism is only one. The initial appearance and persistence of antiplatelet antibodies in ehrlichiosis was not investigated in this study as antiplatelet antibodies were only examined 24 days postinoculation, the time at which thrombocytopenia was most severe. The kinetics of antiplatelet antibodies in ehrlichiosis requires further investigation.
References Abeygunawardena, I., Kakoma, I. and Smith, R.D., 1990. Pathophysiology of canine ehrlichiosis. Williams and I. Kakoma (Editors), Ehrlichiosis. A vector-borne disease of animals and humans. Dordrecht, pp. 78-92.
In: J.C. Khwer,
182
T. Waner et al. / Veterinary Immunology and Immunopathology 48 (1995) 177-182
Breitschwerdt, E.B., 1988. Infectious thrombocytopenia in dogs. Compend. Contin. Educ. Prac. Vet., 10: 1177-1186. Burghen, G.A., Beisel, W.R., Walker, J.S., Nims, R.M., Huxsoll, D.L. and Hildebrandt, P.K.,. 1971. Development of hyperglobulinemia in tropical canine pancytopenia. Am. J. Vet. Res., 32: 749-756. Codner, E.C., Roberts, R.E. and Ainsworth, A.G., 1985. Atypical findings in 16 cases of canine ehrlichiosis. J. Am. Vet. Med. Assoc., 186: 166-169. Kelly, P.J., Carter, S.D., Bobade, P.A., Matthewman, L.A. and Bell, S.C., 1994. Absence of antinuclear antibodies in dogs infected with Ehrlichia canis. Vet. Rec., 134: 382. Kristensen, A.T., Weiss, D.J. Klausner, J.S. Laber, J. and Christie, D.J., 1994a. Detection of antiplatelet antibody with a platelet immunofluorescence assay. J. Vet. Intern. Med., 8: 36-39. Kristensen, A.T., Weiss, D.J., Klausner, J.S., Laber, J. and Christie, D.J., 1994b. Comparison of microscopic and flow cytometric detection of platelet antibody in dogs suspected of immune-mediated thrombocytopenia. Am. J. Vet. Res., 55: 1111-1114. Pierce, K.R., Marrs, G.E. and Hightower, D., 1977. Acute canine ehrlichiosis: platelet survival and factor 3 assay. Am. J. Vet. Res., 38: 1821-1825. Ristic, M., Huxsoll, D.L., Weisiger, R.M., Hildebrandt, P.K. and Nyindo, M.B.A., 1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect. Immun., 6: 226-231. Smith, R.D., Ristic, M., Huxsoll, D.L. and Baylor, R.A., 1975. Platelet kinetics in canine ehrlichiosis: evidence for increased platelet destruction as the cause of tbrombocytopenia. Infect. Immun., 11: 1216-1221. Woody, B.J. and Hoskins, J.D., 1991. Ehrlichial diseases in dogs. Vet. Clin. North Am., 21: 75-98.
ELSEVIER
Veterinary immunology and immunopathology
Immunology and Immunopathology 48 (1995) 177-182
Short communication
Demonstration of serum antiplatelet antibodies in experimental acute canine ehrlichiosis Trevor Waner aY*,Shimon Harrus b, Douglas J. Weiss ‘, Hylton Bark b, Avi Keysary d aLife Science Research Israel, P.O. Box 139, Ness Ziona 70451, Israel b Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100. Israel ’ Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, Minneapolis, MN, USA ’ Israel Institute for Biological Research. P.O. Box 19. Ness Ziona, Israel Accepted
14 December
1994
Abstract 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. Keywords: Ehrlichia canis; Antiplatelet
antibodies;
Thrombocytopenia;
Pathogenesis
1. Abbreviations
ANA, antinuclear antibodies; IFA, immunofluorescence assay; MFU, mean fluorescent channel units; MPV, mean platelet volume; PF-3, platelet factor 3.
* Corresponding
author.
0165-2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-2427(95)05420-O
178
i? Waner et al. / Veterinary Immunology and ImmunopathoIo~
48 (1995) 177-182
2. Introduction Thrombocytopenia is a relatively consistent finding in all stages of canine ehrlichiosis resulting from Ehrlichia canis infection (Breitschwerdt, 1988). Significant thrombocytopenia develops in experimentally infected dogs on approximately Day 10 postinfection, and is most severe on Day 17 (Pierce et al., 1977). Studies to determine the mechanism of reduction in platelet numbers have been inconclusive. It has been suggested that the thrombocytopenia has an immunopathological basis, supported by the hypergammaglobinaemia observed in chronic ehrlichiosis, possibly representing the development of a secondary autoimmune response to damaged host cell components (Burghen et al., 1971). Th e occurrence of antinuclear antibodies (ANA) in dogs affected with ehrlichiosis was investigated and found to be infrequent, indicating that ANA are not important in the pathogenesis of the disease (Kelly et al., 1994). Efforts to demonstrate the presence of antiplatelet antibodies by assay of platelet factor 3 (PF-3) have been used. This indirect method has failed to demonstrate antiplatelet antibodies (Pierce et al., 1977). We present here evidence for the presence of antiplatelet antibodies in beagle dogs experimentally infected with E. canis, during the acute phase of the disease.
3. Materials and Methods Six healthy adult male beagle dogs 8-12 months of age were purchased from Harlan (Indianapolis, USA) and tested seronegative for E. cunis antibodies as determined by indirect immunofluorescence assay (IFA). Haematological and clinical chemistry test results were within reference ranges. The dogs were housed individually in stainless steel cages (IFFA Credo; France). The room in which the dogs were housed was air conditioned with the temperature set to achieve a range of 21 &-2°C. An automatic timer regulated the lighting for 12 h of dark and 12 h of light. The dogs were fed 400 g per day of a commercial dog food (Bonzo; Kibbutz Ma’abarot, Israel) and watered automatically. The dogs were acclimatized for 1 week before inoculation. Each dog was inoculated intravenously with 5 ml of heparinized blood from a dog infected with E. cunis. Thereafter, blood was collected at least three times weekly from the jugular vein for haematology and serology. Serum samples were separated off after centrifugation within 1 h of blood collection. Haematological assays were performed on the same day as blood collection using a semi-automatic impedance cell counter (Cellanalyzer CA 580 A; Medonic, Sweden). The following parameters were determined: packed cell volume, haemoglobin concentration, total erythrocyte count, mean corpuscular volume, mean corpuscular haemoglobin concentration, mean corpuscular haemoglobin, total and differential white blood count, total platelet count, and mean platelet volume (MPV). Serum samples for serology were stored at - 70°C until assayed. Samples were tested for E. canis antibodies by the indirect IFA, as described previously (Ristic et al., 1972). Serum samples from each dog before inoculation with E. cunis, and 24 days after inoculation, were tested for antiplatelet antibodies at the Department of Veterinary
T. Waner et al. / Veterinary Immunology and Immunopathology
48 (1995) 177-182
119
Pathobiology of the University of Minnesota, Minneapolis, using a flow cytometric platelet IFA as previously described Wristensen et al., 1994a,b). Briefly, 20 ~1 of platelet suspension were incubated for 60 min with a 1:lO dilution of test serum. Thereafter, platelets were washed twice, incubated with a 1:20 dilution of fluorescein-labelled goat antidog IgG, washed three times and resuspended in physiological buffered saline containing 3 mM EDTA and 1% bovine serum albumin. Samples were analysed on a flow cytometer (Epics 751; Coulter Electronics, Heartland, FL). Mean fluorescent channel units (MFU) greater than 2 standard deviations (SD) than values for ten healthy control dogs were considered positive for antiplatelet antibodies. The intensity of fluorescence was graded as negative (less than 2 SD above controls), 1 + (2-5 MFU), 2 + (6-10 MFU), 3 + (11-20 MFU), and 4 + (greater than 20 MFU).
4. Results
All dogs developed clinical and haematological signs typical of ehrlichiosis after inoculation with E. canis. Thrombocytopenia was a consistent finding in all dogs (Fig. 1). A concurrent increase in MPV was observed (Fig. 2). The mean circulating platelet count in the dogs before inoculation with E. canis was 318 000 f 66 000 (range 228 000-369 000) platelets per ~1. Platelet counts were lowest between Days 17 and 24, with values ranging from 20 000 to 52 000 platelets per ~1 on Day 24 postinoculation. Values of MPV for the dogs during the postinoculation period approximately followed an inverse pattern when compared to circulating platelet numbers (Fig. 2). The
0
10
20
30
40
Days Fig. 1. Mean peripheral canis.
platelet count and standard error of six Beagle dogs artificially
infected with Ehrlichia
180
T. Waner et al. / Veterinary Immunology
and Immunopathology
48 (1995) 177-182
8 I 0
5
10
15
20
25
30
35
40
45
Days Fig. 2. Average canis.
mean platelet volume and standard error of six Beagle dogs artificially
infected with Ehrlichia
first increase in platelet volume was detected at Days 6 and 7 postinoculation when the MPV for the dogs rose to 9.7 and 10.5 fl, respectively. Other haematological changes observed included a mild transient leucopenia and mild non-regenerative anaemia. All dogs developed an antibody titre to E. canis on Day 15 postinoculation as determined by IFA. The initial IgG titres on Day 15 ranged from 1:160 to 1:640, and increased to 1:1280 to 1:5120 by Day 43 postinoculation. The results of the presence of serum antiplatelet antibodies in E. canis infected dogs are presented in Table 1. None of the dogs showed the presence of any serum antibody to platelets before inoculation. In five of the six dog sera, antibodies to platelets were demonstrated 24 days postinoculation with E. canis.
Table 1 Results of platelet immunofluorescence antibodies in sera of dogs after artificial
assay, in mean fluorescence channel infection with Ehrlichia canis
Dog no.
MFU at 24 days post-inoculation
51 56 98 418 83 22
1+ 2+ 0 1+ 2+ 3+
0, < 2 MFU above controls; controls.
1+ , 2-5
MFU; 2 + , 6-10
MFU; 3 + , 11-20
units (MFU),
MFU, 4 +,
for antiplatelet
> 20 MFU above
T. Waner et al. / Veterinary Immunology and Immunopathology
48 (1995) 177-182
181
5. Discussion Thrombocytopenia in dogs with E. canis infection has been attributed to several mechanisms, acting together or alone. These include increased platelet consumption or sequestration (Woody and Hoskins, 1991) increased platelet destruction in the spleen resulting in shortened platelet survival (Smith et al., 1975), suppression of platelet production in the bone marrow, mainly in the chronic phase (Woody and Hoskins, 1991) or immunologically or non-immunologically mediated platelet destruction (Breitschwerdt, 1988). This study investigated the immunological component of platelet destruction in dogs with acute ehrlichiosis and demonstrated conclusively the presence of circulating serum antiplatelet antibodies. Platelet destruction, as measured in radioactively labelled platelets, has been documented to occur at an accelerated rate during E. canis infection and was considered the primary cause of thrombocytopenia in affected dogs (Smith et al., 1975). Whole-body scans on a dog before and 7 days after infection with E. canis have shown that labelled platelets are destroyed primarily in the spleen, similar to that which occurs in immunologically mediated thrombocytopenia purpura in man (Smith et al., 1975). Smith et al. (1975) considered that the decrease in platelet survival time, 2-4 days after inoculation with E. canis, to be too rapid to be initiated by an antibody-mediated response. In order to elucidate the mechanism of platelet destruction, Pierce et al. (1977) assayed infected dog sera for PF-3. They found that the release of PF-3 from normal canine platelets by serum from infected dogs was delayed, and concluded that thrombocytopenia was not due to the action of antiplatelet antibodies (Pierce et al., 1977). In contrast, an increased release of PF-3 (two out of three dogs) and the detection of ANA (two out of two dogs) have been reported in association with suspected chronic canine ehrlichiosis (Codner et al., 1985). The results were suggestive that autoantibodies may be important in the pathogenesis of chronic ehrlichiosis. On the other hand, Abeygunawardena et al. (1990) have shown that E. canis induced high levels of platelet migration inhibition factor which induced prominent platelet surface changes leading to increased platelet vulnerability. The finding of antiplatelet antibodies in five out of six beagle dogs, 24 days after inoculation of dogs with E. canis infected blood, is indicative that immune-mediated platelet destruction may take place in the acute phase of canine ehrlichiosis. These results do not rule out the possibility that a number of mechanisms may still be involved in the pathogenesis of ehrlichial thrombocytopenia (Breitschwerdt, 1988) of which the immune-mediated mechanism is only one. The initial appearance and persistence of antiplatelet antibodies in ehrlichiosis was not investigated in this study as antiplatelet antibodies were only examined 24 days postinoculation, the time at which thrombocytopenia was most severe. The kinetics of antiplatelet antibodies in ehrlichiosis requires further investigation.
References Abeygunawardena, I., Kakoma, I. and Smith, R.D., 1990. Pathophysiology of canine ehrlichiosis. Williams and I. Kakoma (Editors), Ehrlichiosis. A vector-borne disease of animals and humans. Dordrecht, pp. 78-92.
In: J.C. Khwer,
182
T. Waner et al. / Veterinary Immunology and Immunopathology 48 (1995) 177-182
Breitschwerdt, E.B., 1988. Infectious thrombocytopenia in dogs. Compend. Contin. Educ. Prac. Vet., 10: 1177-1186. Burghen, G.A., Beisel, W.R., Walker, J.S., Nims, R.M., Huxsoll, D.L. and Hildebrandt, P.K.,. 1971. Development of hyperglobulinemia in tropical canine pancytopenia. Am. J. Vet. Res., 32: 749-756. Codner, E.C., Roberts, R.E. and Ainsworth, A.G., 1985. Atypical findings in 16 cases of canine ehrlichiosis. J. Am. Vet. Med. Assoc., 186: 166-169. Kelly, P.J., Carter, S.D., Bobade, P.A., Matthewman, L.A. and Bell, S.C., 1994. Absence of antinuclear antibodies in dogs infected with Ehrlichia canis. Vet. Rec., 134: 382. Kristensen, A.T., Weiss, D.J. Klausner, J.S. Laber, J. and Christie, D.J., 1994a. Detection of antiplatelet antibody with a platelet immunofluorescence assay. J. Vet. Intern. Med., 8: 36-39. Kristensen, A.T., Weiss, D.J., Klausner, J.S., Laber, J. and Christie, D.J., 1994b. Comparison of microscopic and flow cytometric detection of platelet antibody in dogs suspected of immune-mediated thrombocytopenia. Am. J. Vet. Res., 55: 1111-1114. Pierce, K.R., Marrs, G.E. and Hightower, D., 1977. Acute canine ehrlichiosis: platelet survival and factor 3 assay. Am. J. Vet. Res., 38: 1821-1825. Ristic, M., Huxsoll, D.L., Weisiger, R.M., Hildebrandt, P.K. and Nyindo, M.B.A., 1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect. Immun., 6: 226-231. Smith, R.D., Ristic, M., Huxsoll, D.L. and Baylor, R.A., 1975. Platelet kinetics in canine ehrlichiosis: evidence for increased platelet destruction as the cause of tbrombocytopenia. Infect. Immun., 11: 1216-1221. Woody, B.J. and Hoskins, J.D., 1991. Ehrlichial diseases in dogs. Vet. Clin. North Am., 21: 75-98.