First confirmed canine case of Ehrlichia canis infection in Japan

An 11-year-old castrated Pekinese dog that had been moved from Indonesia to Japan eight years previously was diagnosed with an Ehrlichia canis infection by haematological characteristics (normocytic anaemia, mild thrombocytopenia and hypergammaglobulinaemia) and serological findings (antibody titre to E canis 1:3,200 or more). The dog did not respond to treatment with tetracycline and died from renal failure. The diagnosis was confirmed postmortem by pathological evaluation and polymerase chain reaction (PCR) followed by sequencing of the 16S rRNA gene. Typical morulae of Ehrlichia were detected in the cytoplasm of macrophages in spleen tissue by immunohistological staining. Ehrlichia-like organisms were also detected in the spleen by electron microscopy. E canis-specific PCR analysis of DNA extracted from the spleen gave a positive signal, and sequence analysis of the fragment revealed that it was identical to part of the 16s rRNA gene of E canis. The dog was the first confirmed clinical case of E canis infection in Japan.     

Molecular detection and characterization of Ehrlichia canis from dogs in Turkey

Seroprevalence of Ehrlichia canis antibodies among dogs in Turkey were previously reported, however, the ehrlichial organism has never been characterized in this region. The current study examined dogs from Ankara with febrile illness for E. canis infection with E. canis-specific PCR. Three of the 12 blood specimens from dogs showing clinical signs compatible with canine ehrlichiosis were found to be positive by PCR using E. canis-specific primers. E. canis detected in one of the blood specimens was designated as Kutahya strain. The representative E. canis strain was characterized by 16S rRNA gene sequencing and Western blot analysis of the plasma sample from the dog infected with E. canis. The 16S rRNA sequence (1,388 bp) of the E. canis Kutahya was identical to that of Ehrlichia ovina from a sheep in Turkey and Venezuelan Dog Ehrlichia (VDE) and was closely related (99.9%) to that of type strain of E. canis, Oklahoma. The plasma of the dog infected with E. canis Kutahya was analyzed by Western blotting using the purified E. canis Oklahoma strain as antigen. The reactive antibody profiles of the dog infected with E. canis Kutahya was found to be similar to those of dogs infected with E. canis Oklahoma and VDE, suggesting the antigenic similarities among these strains. The findings in this study would help for a better understanding of epidemiology of canine ehrlichiosis. This is the first report of molecular detection and characterization of an ehrlichial agent in Turkey.     

Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela

Background: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. Objective: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. Methods: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. Results: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. Conclusions: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.     

First record of autochthonous canine ehrlichiosis caused by Ehrlichia canis in Romania

This case study describes the first genetically confirmed and clinically manifested autochthonous Ehrlichia canis infection in a 9‐year‐old female mixed‐breed dog from Romania. Health screening of the dog included clinical examination, evaluation of stained peripheral blood smear and hematologic variables, as well as serologic testing and molecular analysis. Clinical signs included fever, apathy, dehydration, pale mucous membranes, and weakness. The microscopic examination of the blood smear and immunologic assays for Borrelia burgdorferi, Anaplasma phagocytophilum, and E canis antibodies, and for Dirofilaria immitis antigen yielded negative results. Hematologic abnormalities included moderate nonregenerative anemia, leucopenia with neutropenia, and moderate thrombocytopenia. The biochemical abnormalities identified were hypoalbuminemia, and mildly increased serum enzyme activities of AST and ALT. In addition, increased urea and creatinine levels associated with low urine specific gravity and proteinuria were also present. Nested PCR amplification of the partial E canis 16S rRNA gene demonstrated the presence of this rickettsial pathogen in the dog's blood, which subsequently was confirmed through sequencing based on the 100% homology with GenBank deposited E canis isolates. After specific treatment with doxycycline (10 mg/kg, orally, SID) for one month, the proteinuria, and hematologic and serum biochemical abnormalities with the exception of mild azotemia resolved. This report supports the geographical expansion of canine ehrlichiosis caused by E canis in nonendemic regions of Europe.     

Prevalence,molecular characterization and risk factor analysis of Ehrlichia canis and Anaplasma platys in domestic dogs from Paraguay

This is the first study to investigate the prevalence and risk factors associated with Ehrlichia canis and Anaplasma platys positivity in dogs from Paraguay. Conventional PCR assays for the E. canis 16SrRNA gene and A. platys p44 gene were carried out in blood samples from 384 dogs from Asunción city, Paraguay. Sequencing and phylogenetic analysis were performed in selected positive E. canis and (16SrRNA gene) and A. platys (16S and p44 genes) samples. The overall prevalence of E. canis and A. platys in dogs in Paraguay was 10.41% (40/384) and 10.67% (41/384), respectively. Older dogs without veterinary care had higher odds for E. canis positivity and a higher number of dogs in the same household, as well as absence of anti-tick treatment were considered risk factors for A. platys. Ehrlichia canis and A. platys circulate in the dog population from Asunción, and are described for the first time in Paraguay.     

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.     

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.     

Seroprevalence of Ehrlichia canis, Ehrlichia equi, and Ehrlichia risticii in sick dogs from North Carolina and Virginia

Ehrlichia canis, E. equi, and E. risticii seroprevalence was determined by microimmunofluorescent antibody testing (IFA) in a sequential population of 1,845 sick dogs admitted during a 1-year period to the North Carolina State University Veterinary Teaching Hospital. A seroreactor was defined by a reciprocal IFA titer of > or =80 to E. canis, E. equi, or E. risticii antigens. Of the 48 IFA seroreactors, 44 dogs were seroreactive to E. canis, 21 to E. equi, and 0 to E. risticii. Seventeen dogs reacted to both E. canis and E. equi antigens. There was concordance of E. canis IFA and western immunoblot (WI) test results for 36/44 dogs. Because of cross-reactivity of E. canis sera with E. equi antigens, WI was of less utility to confirm E. equi exposure. After elimination of E. canis seroreactors, there was concordance of 2/4 E. equi IFA and WI test results. Based upon a retrospective review of medical records, ehrlichiosis was diagnosed in 10/48 (21%) IFA seroreactive dogs, 9 of which were confirmed positive by WI. Of the remaining 38 IFA seroreactors, 29 also were confirmed by E. canis or E. equi WI. These results indicate that (1) ehrlichiosis was not diagnosed in the majority of serologically confirmed cases, (2) based upon E. canis and E. equi WI analysis, IFA testing was not specific (21% false positive), (3) E. canis sera cross-react with E. equi antigens, and (4) serologic evidence of E. risticii infection was lacking in the dog population studied.     

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.     

Serosurvey of Ehrlichia canis and Hepatozoon canis infection in dogs in Yamaguchi Prefecture, Japan

Antibodies to Ehrlichia canis and Hepatozoon canis in dogs at the Animal Hospital in Yamaguchi University were surveyed and potential risk factors for both pathogens were evaluated. Among 430 dogs examined, 20 (4.7%) and 18 (4.2%) dogs showed positive findings for E. canis and H. canis, respectively. Neither, sex nor age was associated with the seropositivity of either pathogen, but the positive rate in dogs kept outside was slightly higher than that in dogs kept inside for both pathogens. A higher seropositive reaction to E. canis and H. canis was observed in dogs that lived in certain cities and towns. Beagles, golden retrievers and pointers had higher seropositivity than other breeds in E. canis, whereas shibas, akitas, beagles, pointers and mongrels had higher positive rates than other breeds in H. canis.     

犬埃立克体实验模型初步研究

将犬埃立克体标准毒株（Florida株）体外细胞纯培养物和我国犬埃立克体病病犬血白细胞分别接种于C3H小鼠，接毒后d，经PCR检测证实均感染成功。本试验为犬埃立克体病小动物模型的建立打下了基础。     

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.