Babesia canis vogeli,Ehrlichia canis,and Anaplasma platys infection in a dog

A 12‐month‐old male neutered mixed breed dog was presented with a history of diarrhea, lethargy, emaciation, polydypsia, and sniffling. Physical examination findings included pale mucous membranes, increased heart and respiratory rates, and normal rectal temperature (38°C). Hematologic abnormalities included anemia and thrombocytopenia. Biochemical abnormalities included hypoalbuminemia, hyperbilirubinemia, and elevated ALP and ALT activities. A SNAP 4Dx test result was positive for Ehrlichia canis. Babesia canis vogeli organisms were found in the peripheral blood films, while morulae of E canis were not seen. Real‐time polymerase chain reaction testing confirmed the presence of both B c vogeli and E canis organisms, and also was positive for Anaplasma platys infection. The dog recovered following treatment with doxycycline and imidocarb dipropionate, with normal hematology and biochemical profiles.     

Platelet aggregation studies in dogs with acute Ehrlichia platys infection

Ten adult dogs (5 Beagles and 5 mixed-breed dogs) were inoculated IV with canine platelets containing Ehrlichia platys. Inclusions and morulae of E platys developed in platelets of infected dogs at 10 to 14 days after inoculation, followed by marked thrombocytopenia at 14 to 21 days. Parasitemia and marked thrombocytopenia recurred at 24 to 28 days after inoculation. Increased numbers of megakaryocytes were observed in marrow aspirate smears from infected dogs, indicative of regenerative thrombocytopenia. Prior to infection, platelet-rich plasma from these dogs was determined to have similar aggregatory response to arachidonate. After infection with E platys, the aggregatory response of platelet-rich plasma to collagen or 3 dilutions of adenosine diphosphate was evaluated. A statistically significant (P less than 0.05) inhibition of platelet aggregatory response to the lowest dilution of adenosine diphosphate was detected for mixed-breed dogs, whereas aggregation responses were unchanged in Beagles. Results indicate that platelet activation may occur in dogs with acute ehrlichial infection.     

Detection of Ehrlichia platys in dogs in Australia

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.     

Demonstration of Anaplasma (Ehrlichia) platys inclusions in peripheral blood platelets of a dog in Japan

A free-roaming dog in Okinawa island showed Anaplasma (Ehrlichia) platys-like inclusions within the platelets of peripheral blood samples. The inclusions were positive in indirect fluorescence test with anti-A. phagocytophila serum. The platelet count of the dog was 170,000 microl(-1). The sequence analysis of the 16S rRNA, citrate synthase and heat shock protein genes of DNA from the infected platelets confirmed that the inclusions were A. platys. This is the first detection of A. platys inclusions in dogs in Japan.     

Anemia of inflammation in dogs infected with Ehrlichia platys

Ten adult male dogs were inoculated with Ehrlichia platys, and blood samples were collected throughout the infection to evaluate the hematologic changes with respect to serum biochemical analytes. All dogs developed a mild, normocytic, normochromic anemia by postinoculation day 7, with significantly (P less than 0.05) decreased serum iron concentration and total iron-binding capacity. Stainable bone marrow iron appeared normal or increased throughout the infection. By postinoculation day 31, the PCV was not significantly different from the pretreatment value. All dogs became hypergammaglobulinemic, leukopenic, hypoalbuminemic, and hypocalcemic during the infection. These findings were compatible with the syndrome of anemia of inflammation.     

Immunocytochemical detection of Ehrlichia platys antigens in canine blood platelets

An avidin-biotin immunoperoxidase complex (ABC) immunocytochemical (ICC) stain procedure was optimized for detection of Ehrlichia platys antigens. Positive immunoreactivity was detected with dilutions of canine immune serum on acetone-fixed smears of platelet-rich plasma from E. platys-infected dogs. No E. platys antigens were detected when this ICC stain was applied to frozen or paraffin-embedded formalin- or acetone-fixed tissue sections from dogs with acute E. platys infection. Acetone fixation and freezing preserved ICC staining of ehrlichial antigens in infected blood platelets, whereas formalin treatment of similarly preserved E. platys-infected platelets nullified positive immunoreactivity. Significant E. platys infection of cells and tissues other than platelets may not occur.     

Experimental Ehrlichia canis infection in the dog does not cause immunosuppression

A carrier state develops in some Ehrlichia canis-infected dogs due to ineffective host defenses. The subsequent development of immune-mediated diseases or opportunistic infections in chronic ehrlichiosis suggests dysregulation of immunity; however, the immunobiology of this infection has not been well characterized. In this study, eight dogs were infected with E. canis, and changes in seroreactivity, serum immunoglobulin (Ig) concentrations, peripheral blood T cell subsets, lymphocyte blastogenesis (LBT), and lymphokine-activated killer (LAK) activity were evaluated over 4 months. Infection, which was documented by seroconversion, polymerase chain reaction, and blood culture, caused self-limiting fever and thrombocytopenia. Infected dogs developed an anti-E. canis antibody response but were not immune to re-infection. Serum IgM, IgG, and IgA concentrations were unaffected by E. canis. The percentage of circulating CD4(+) T cells was similar in uninfected and infected dogs at all points. Infected dogs developed a CD8(+) lymphocytosis 6 weeks after inoculation that subsequently subsided, despite organism persistence. Functional defects of cell-mediated immunity, measured as suppression of LAK activity or mitogen-driven LBT, were not observed. These results suggest that immune responses are not grossly impaired in young dogs during the first several months following experimental E. canis infection.     

Antibodies to Ehrlichia canis, Ehrlichia platys, and Spotted Fever Group Rickettsiae in Louisiana Dogs

Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group (SFG) rickettsiae were detected by indirect immunofluorescence in sera from 27 ill individually owned thrombocytopenic dogs (platelet concentrations less than 200,000 platelets/microliters) and 59 healthy kenneled dogs located in southern Louisiana. Platelet concentrations less than 100,000 platelets/microliters were detected in 63% of ill thrombocytopenic dogs and 6.8% of healthy kennel dogs. One ill thrombocytopenic dog had intracytoplasmic E platys morulae detected within platelets. The prevalence of increased serum antibody titers to E canis and E platys was 25.9% and 40.7% for the ill thrombocytopenic dogs and 20.3% and 54.2% for the healthy kennel dogs, respectively. All dogs with seropositivity to E canis had increased antibody titers of greater than or equal to 1:100 to E platys. Simultaneous examination of increased serum antibody titers (greater than or equal to 1:64) to four SFG rickettsiae indicate that Rickettsia rhipicephali and Rickettsia montana accounted for the majority of the antibodies detected in these dogs. Of 86 dogs tested, 44.2% were seronegative to E canis, E platys, and SFG rickettsiae.     

Serum hyperviscosity syndrome associated with Ehrlichia canis infection in a dog

A 10-year-old male Lhasa Apso was examined because of pyrexia, vomiting, weight loss, lethargy, and disorientation. The clinical and laboratory findings were supportive of a diagnosis of serum hyperviscosity syndrome associated with an Ehrlichia canis infection. After tetracycline therapy the serum hyperviscosity syndrome and E. canis infection were successfully resolved.     

Genetic characterization of Anaplasma (Ehrlichia) platys in dogs in Spain

This paper reports the first genetic characterization of Anaplasma (Ehrlichia) platys in Spain from a naturally infected dog. The dog presented clinical signs compatible with canine ehrlichiosis. After DNA extraction and PCR amplification, 16S rRNA gene and citrate synthase gene ( gltA) of this agent were amplified. The GenBank accession number for the nucleotide sequence of the 16S rRNA gene of this strain is AY530806. The A. platys strains registered in France and Japan showed the highest similarity to the 16S rRNA gene sequence obtained from the Spanish strain. In the amplification of the gltA gene, a 1443 bp fragment was obtained, and three nucleotide differences were detected in comparison with other strains sequences. These data confirm the presence of A. platys in a dog showing clinical signs compatible with ehrlichiosis in Spain.     

Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from the North of Portugal

Tick-transmitted rickettsial pathogens belonging to the Ehrlichia and Anaplasma genera can infect dogs and humans. In this study, four dogs from the North of Portugal, in which an ehrlichial disease was suspected clinically, were tested by molecular methods. After DNA extraction from blood on filter paper, a 345 bp fragment of the Ehrlichia/Anaplasma 16S rRNA gene was amplified by the polymerase chain reaction (PCR). Sequence analysis of PCR products revealed one dog infected with Ehrlichia canis and three with Anaplasma platys. One of these latter animals was co-infected with Babesia canis subspecies vogeli. This is the first report of the genetic characterisation of both A. platys and E. canis in naturally infected dogs from the North of Portugal.     

Ehrlichia platys (Anaplasma platys) in dogs from Maracaibo,Venezuela: an ultrastructural study of experimental and natural infections

Since 1982 Ehrlichia platys, now emended as Anaplasma platys, has been diagnosed in dogs from Maracaibo, Venezuela, using buffy coat smears stained with Dip Quick. Three dogs were inoculated with an A. platys strain. When parasitemia reached 60-97%, blood samples obtained from the inoculated dogs and from two naturally infected dogs were centrifuged to obtain platelet-rich plasma, which was mixed with 0.1% glutaraldehyde at 37 C for 10 minutes. Platelet pellets were fixed in 3% glutaraldehyde for 72 hours and processed for conventional transmission electron microscopy. Platelets contained pleomorphic organisms with a distinct double membrane that was not observed when the bodies were in a determinate developmental stage. There were 1-15 individual bodies included in a host cell vacuole. The organisms had an electron-lucent inner area, whereas the internal surface of their inner plasma membranes exhibited an electron-dense rough substance. In naturally infected dogs, organisms with different ultrastructural features were found inside the same platelet. Some organisms contained central dense material surrounded by a pale zone, which was in turn surrounded by a moderately dense peripheral area. Other organisms contained an eccentrically electron-dense material. The intravacuolar space appeared fully electron-lucent. Each organism usually exhibited inner fine strands. Empty structures displaying junctions with the vacuolar membrane were observed. Our results indicate that distinct ultrastructural characteristics are associated with different stages of A. platys development and may differ among A. platys strains.     

Evaluation of Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys

The brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae), transmits several diseases among dogs including Ehrlichia canis infection. The role of Rhipicephalus sanguineus as a biologic vector for E platys, the rickettsial agent of infectious canine cyclic thrombocytopenia, was studied in dogs. Laboratory-cultured, pathogen-free nymph ticks were fed to repletion on dogs acutely infected with E platys. Tick engorgement coincided with the development of initial parasitemia and thrombocytopenia in the infected dogs. Following repletion, nymph ticks were allowed to molt under controlled conditions. One-month-old E platys-exposed adult ticks failed to infect naive dogs in animal transmission studies. The presence of E platys was not detected in midguts or salivary glands of similarly exposed adult ticks by use of light and transmission electron microscopy. These studies indicate that R sanguineus may not transmit E platys infection.     

Ehrlichia canis-related polyarthritis in a dog

Ehrlichia canis-related polyarthritis was diagnosed in a 7-month-old Boxer. The diagnosis was based on intraneutrophilic morulae found in synovial fluid specimens, thrombocytopenia, a positive result for indirect fluorescent antibody testing for E canis, the presence on the dog of the known vector of E canis infection (Rhipicephalus sanguineus), and a favorable response to treatment with tetracycline hydrochloride. The dog has had no recurrence of lameness for 18 months after cessation of treatment.     

Ehrlichia canis infection in a dog with no history of travel outside the United Kingdom

A two‐year‐old female neutered Tibetan terrier was referred following a one‐month history of lethargy, inappetence and pancytopenia, which had been poorly responsive to immunosuppressive and fluoroquinolone treatment. The dog was diagnosed with pure red cell aplasia and was found to be positive for Ehrlichia canis by both antibody titre measurement and polymerase chain reaction. The dog lived in London and had not travelled outside the UK. The dog was treated with doxycycline, prednisolone and ciclosporin, but died as a result of gastrointestinal tract haemorrhage. To the authors’ knowledge, this represents the first reported case of Ehrlichia canis in a dog in the UK with no previous travel history.     

Neospora caninum and Ehrlichia canis co‐infection in a dog with meningoencephalitis

An 8‐year‐old mixed‐breed dog was presented for acute, progressive weakness and ataxia, inappetence, and weight loss. The patient was mentally normal, but nonambulatory, with a right head tilt, right positional ventral strabismus, and slight head tremors. A neurologic lesion was localized to the cerebellum and right brainstem. Cerebrospinal fluid (CSF) analysis showed a markedly increased protein concentration and mixed pleocytosis, with eosinophil predominance (44%), intracytoplasmic inclusions within eosinophils, consistent with Ehrlichia canis (E canis) morulae, and Toxoplasma gondii (T gondii) or Neospora caninum (N caninum) tachyzoites within eosinophils and monocytes. A serum indirect immunofluorescent antibody test was positive for N caninum (titer 1:12 800) and negative for T gondii. Both blood and CSF PCR results were N caninum‐ and E canis‐positive and T gondii‐ and Anaplasma phagocytophilum‐negative, and blood PCR, but not CSF PCR, was Hepatozoon canis‐positive. The dog was treated for 30 days with clindamycin, sulfamethoxazole‐trimethoprim, doxycycline, prednisone, and cephalosporin, but did not improve neurologically, and was euthanized. Brain histopathology showed moderate multifocal, subacute meningoencephalitis with necrosis and gliosis. The neurologic disease was mostly attributed to central nervous system (CNS) neosporosis, with the possible contribution of ehrlichiosis, which was likely a manifestation of blood‐brain barrier disruption. Hepatozoonosis was probably a result or cause of underlying immunosuppression. To our knowledge, this is the first report of CNSN caninum and E canis co‐infection detected by both CSF PCR and cytology and E canis morulae identified within CSF eosinophils.