A serosurvey of Hepatozoon canis and Ehrlichia canis antibodies in wild red foxes (Vulpes vulpes) from Israel

A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with Ehrlichia canis and Hepatozoon canis antigens in free-ranging red foxes (Vulpes vulpes) in Israel. Of 84 fox sera assayed, 36% were seropositive for E. canis by the indirect fluorescent antibody (IFA) test and 24% were positive for H. canis using an enzyme-linked immunosrbent assay (ELISA). Canine ehrlichiosis and hepatozoonosis appear to be endemic in the wild red fox populations in Israel, and foxes may serve as a reservoir for infection of domestic dogs and other wild canine species.     

Serological survey for antibodies reactive with Ehrlichia canis and E. chaffeensis in dogs from the Bloemfontein area, South Africa

Sera from 161 dogs in the Bloemfontein area in South Africa were tested for the presence of antibodies reactive with Ehrlichia canis and E. chaffeensis by indirect fluorescent antibody testing. Overall, 68 (42%) of the dogs had significant antibody titres (> or = 1/64) against E. canis and 61 (38%) had significant titres (> or = 1/64) against E. chaffeensis. Seven (11%) dogs had higher titres to E. chaffeensis than E. canis (1/2048 and 1/1024 (2 dogs); 1/1024 and 1/512 (2 dogs); 1/2048 and 1/512; 1/512 and 1/256 and 1/512 and < 1/64, respectively). The remaining seropositive dogs had equal (n = 26; 42%) or 2-(n = 17; 25%), 3-(n = 13; 2%) or 4-fold (n = 5; 7%) higher titres against E. canis. Dogs from economically depressed, high-density suburbs (60/112; 48%) had significantly higher prevalences of antibodies against E. canis than those from more affluent, low-density suburbs (8/49; 14%) (chi 2 = 19.38, p < 0.001). Higher titres to E. chaffeensis than E. canis were found in dogs from affluent, low-density suburbs (3/49) and in dogs from economically depressed, high-density suburbs (4/112).     

A serological survey of Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi in dogs in Oklahoma

Serum samples from 259 dogs were tested for antibodies to Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi using the indirect fluorescent antibody test. The sera were obtained from submissions to the Oklahoma Animal Disease Diagnostic Laboratory during a 14-month period from June 1986 through July 1987. The rate for positive antibody titers to E. canis was 53%, to E. equi was 33%, to R. rickettsii was 38%, and to B. burgdorferi was 18%. Higher percentages of sera serologically positive to E. canis were found in the spring through the fall months, but there were no seasonal variations for E. equi, R. rickettsii, and B. burgdorferi. There was no consistent pattern of titers to the 4 antigens when age-groups of the dogs were compared. Forty-four different breeds were tested.     

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.     

Seroepidemiological study of canine ehrlichial infections in Yamaguchi prefecture and surrounding areas of Japan

Randomly selected serum samples from 150 dogs from Yamaguchi and neighbouring prefectures were subjected to the indirect immunofluorescent assay to detect antibodies against Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia muris and Ehrlichia from Ixodes ovatus. A total of 30 out of the 150 serum samples reacted with at least one of the antigens at a titer of 1:20 or more. Considerable cross-reactivity was seen and most samples reacted with at least two different antigens. Fifteen (10.0%) dogs had higher titers to E. canis than any of the other antigens. Four (2.7%) dogs had higher titers to Ehrlichia from Ixodes ovatus and one (0.6%) dog had higher titers to E. muris compared to the other antigens. The findings suggest that these five dogs may be infected with the domestic Ehrlichia of Japan. The remaining ten dogs had similar high titers to two or more of the antigens. This is the first serological evidence obtained of canine infection with the domestic Ehrlichia of Japan.     

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.     

Seroprevalence of Anaplasma phagocytophilum among healthy dogs and horses in Israel

The presence of reacting antibodies to Anaplasma phagocytophilum has previously been demonstrated in Israel, both in humans and the golden jackal (Canis aureus syriacus). This study was undertaken to determine the seroprevalence of A. phagocytophilum antibodies in two additional potential hosts, domestic dogs and horses in order to investigate the possibility of exposure to the organism in Israel. Of 195 dogs tested, 9% were seroreactive with A. phagocytophilum antigen and 30% were seroreactive to Ehrlichia canis. Twenty-nine percent of the dogs seropositive for E. canis were also reactive to A. phagocytophilum. Two dogs had immunofluorescence antibody (IFA) antibody titres for A. phagocytophilum greater than E. canis. The equine serological survey (n = 300) revealed no seroreactive horses. The results presented in this study suggest that dogs in Israel could have been accidentally exposed to A. phagocytophilum, for example by ticks carried on migrating birds, however, the possibility of cross-reaction with E. canis should also be considered. In spite of the high prevalence of ticks on horses in Israel during the summer months, no evidence for exposure to A. phagocytophilum was apparent.     

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.     

Comparison of results for serologic testing and a polymerase chain reaction assay to determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis

OBJECTIVE: To determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis and examine the correlation between results for an ELISA, indirect immunofluorescent antibody (IFA) test, and polymerase chain reaction (PCR) assay. SAMPLE POPULATION: Blood samples obtained from 90 adult dogs admitted to an animal shelter in eastern Tennessee. PROCEDURE: Serum samples were analyzed for antibodies against E. canis by use of a commercially available ELISA kit, 2 IFA tests, and a PCR assay; testing was performed at the University of Tennessee (TN) and North Carolina State University (NCSU). The PCR amplification was performed by use of DNA extracted from EDTA-anticoagulated blood and primers designed to amplify DNA of Ehrlichia spp. RESULTS: Antibodies against E. canis were detected in only 1 dog by use of the ELISA. By IFA testing at TN, 10 of 90 (11%) dogs were seroreactive against E. canis antigens, all of which had medium to high titers to E. canis. Only 5 of the 10 TN seroreactors were also reactive against E. canis antigens in IFA tests conducted at NCSU, and all 5 had low to medium titers. The DNA of Ehrlichia spp was not amplified in any blood samples by use of PCR assays conducted at the TN or NCSU. CONCLUSIONS AND CLINICAL RELEVANCE: The discordant ELISA, IFA, and PCR results obtained in this study were unexpected and may have been related to exposure of dogs to an Ehrlichia species other than E. canis, such as E. ewingii.     

Detection of Ehrlichia chaffeensis in Brazilian marsh deer (Blastocerus dichotomus)

Ehrlichia chaffeensis was detected for the first time in blood samples from Brazilian marsh deers (Blastocerus dichotomus) captured in the marshes of Parana River in Southeast Brazil in 1998. Seven EDTA-blood samples from deers were analyzed by PCR and nested PCR for presence of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Neoriickettsia risticii, Anaplasma phagocytophilum and Anaplasma marginale. Three samples showed positive reactions for E. chaffeensis and Anaplasma marginale. None contained detectable A. phagocytophilum, E. ewingii, E. canis or Neorickettsia risticii DNA. In Brazil, the wild marsh deer may be a natural reservoir of the agents that cause human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis.     

Restriction and expansion of Ehrlichia strain diversity

Ehrlichia are tick-borne gram negative, obligately intracellular bacteria. The 16S rRNA gene DNA sequences are highly conserved among strains of each Ehrlichia species. The 28-kDa/Map-1 outer membrane protein genes are highly diversified among strains of Ehrlichia chaffeensis and E. ruminantium, but are highly conserved among E. canis isolates. The diversity of the immunodominant proteins of E. chaffeensis and E. ruminantium in contrast with the conservation of the immunodominant proteins of E. canis suggests that E. chaffeensis and E. ruminantium face more host immune pressure than E. canis or that E. chaffeensis and E. ruminantium evolved earlier than E. canis and have diverged.     

Detection of the new Ehrlichia species closely related to Ehrlichia ewingii from Haemaphysalis longicornis in Yonaguni Island, Okinawa, Japan

We collected a total of 206 Haemaphysalis longicornis ticks by flagging in pastures in Yonaguni Island, Okinawa, Japan, in April 2008. Four of the 206 tick DNA samples tested were positive in a polymerase chain reaction (PCR) screening for the 16SrRNA gene of Anaplasmataceae. Partial sequences of 4 PCR products were identical to each other. Longer sequences of the 16SrRNA gene were successfully determined in 2 of the 4 tick samples, and the obtained 1,392 bp and 1,300 bp sequences revealed high similarity to the 16SrRNA gene sequences of the validated Ehrlichia species, including Ehrlichia ewingii, E. chaffeensis, and E. canis (98.3-98.6%). We also sequenced 1,304 bp of the groEL gene from the 2 tick samples, and found that these had the highest similarity to sequences of E. ewingii (94.0-94.4%) in the validated ehrlichial species. Based on the 16SrRNA and groEL gene sequences, the ehrlichial agents detected in this study were similar to the Ehrlichia species detected in Asia and may compose a new Ehrlichia species with other Ehrlichia species detected in Asia.     

Serological evidence of Ehrlichia spp. exposure in cats from northeastern Spain

There is little information about Ehrlichia canis as an infectious agent in cats. In order to estimate the prevalence of antibodies to E. canis in the feline population, 235 cat sera were analysed by indirect fluorescent-antibody test. With the objective to determine some risk factors associated with seropositivity, serum samples were divided into two groups: urban stray cats and pet cats. The seroprevalence detected was 17.9%. Most positive sera (83.3%) showed low antibody titres (<1:80). Seropositivity was very similar when comparing the two groups of animals: 17.4% in urban stray cats and 18.4% in pet cats. Results revealed that cats are exposed to Ehrlichia spp. infection, as the low antibody titres detected and the serological cross-reactivity between Ehrlichia species do not allow us to confirm E. canis exposure.     

Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand

Forty-nine dogs from Thailand were evaluated for serologic evidence of exposure or polymerase chain reaction (PCR) evidence of infection with vectorborne pathogens, including Ehrlichia sp. (Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, and Ehrlichia risticii), Bartonella vinsonii subsp. berkhoffi (Bvb), spotted fever group (SFG) rickettsiae (Rickettsia rickettsii), Typhus group (TG) rickettsiae (Rickettsia canada, Rickettsia prowazekii, and Rickettsia typhi), and Babesia sp. (Babesia canis and Babesia gibsonii). All study dogs had at least 1 of 3 entry criteria: fever, anemia, or thrombocytopenia. By immunofluorescence antibody (IFA) testing, seroreactivity was most prevalent to E chaffeensis (74%) and E canis (71%) antigens, followed by E equi (58%), Bvb (38%), E risticii (38%), R prowazekii (24%), B canis (20%), R rickettsii (12%), R canada (4%), and B gibsonii (4%) antigens. There was 100% concordance between E canis IFA and Western blot immunoassay (WI) for 35 of 35 samples; 2 samples were IFA and WI reactive only to E equi antigens. By PCR amplification, 10 dogs were found to be infected with E canis, 5 with Ehrlichia platys, and 3 with B canis. Sequencing of PCR products was undertaken to compare Ehrlichia strains from Thailand to strains originating from the United States. Partial DNA sequence analysis confirmed infection with E canis and E platys, with identical 16S rRNA sequence alignment to E canis (U26740) and to E platys (M83801), as reported in GenBank. Partial E canis P28.1 and P28.2 amino acid sequences from Thai dogs were divergent from analogous sequences derived from North American E canis (AF082744) strains, suggesting that the Thai dogs were infected with a geographically distinct strain of E canis compared to North American strains. The results of this study indicate that dogs in Thailand have substantial exposure to vectorborne diseases and that coinfection with these pathogens may be common.     

Susceptibility of dogs to infection with Ehrlichia chaffeensis, causative agent of human ehrlichiosis.

Ehrlichia chaffeensis, the newly recognized agent of human ehrlichiosis, is closely related to E canis, the causative agent of canine ehrlichiosis. Eight pups were inoculated IV with E chaffeensis-, or with E canis-infected DH82 cells, or organisms released from these host cells. Two additional pups served as nonexposed controls. Marked thrombocytopenia was observed in the E canis-infected pups, but not in those infected with E chaffeensis. Homologous serologic response was observed in the E chaffeensis-exposed pups by postinoculation day (PID) 14 and in the E canis-exposed pups by PID 21. Ehrlichia chaffeensis and E canis were reisolated from the respective inoculated pups on each of 8 attempts from PID 7 to 26. One E chaffeensis-exposed pup that was challenge exposed with E canis via blood transfusion, developed fever, anorexia, and thrombocytopenia, suggesting lack of cross protection against E canis.     

Antibodies reactive with Bartonella henselae and Ehrlichia canis in dogs from the communal lands of Zimbabwe

The prevalences of antibodies against Bartonella henselae and Ehrlichia canis were determined in sera from 228 dogs in 5 communal lands of Zimbabwe, areas where traditional subsistence agro-pastoralism is practised. The sera were collected from apparently healthy dogs during routine rabies vaccination programmes and tested with indirect fluorescent antibody assays using B. henselae (Houston-I) and E. canis (Oklahoma) as antigens. We found reactive antibodies (> or =1:80) against B. henselae in 14% of the dogs tested. Seropositive animals were found in Bikita (41%; 17/42), Omay (13%; 6/48), Chinamora (5%; 2/38) and Matusadona (15%; 7/48). No seropositive dogs were found in Chiredzi (0%; 0/52). Antibodies reactive with E. canis (> or =1:80) were found in 34% of the dogs tested, from Bikita (88%; 37/42), Chiredzi (31%; 16/52), Omay (17%; 8/48), Chinamora (26%; 10/38) and Matusadona (15%; 7/48). Our survey shows dogs in the communal lands of Zimbabwe are frequently exposed to E. canis and B. henselae or closely related species. Further studies are indicated to determine the pathogenicity of the organisms infecting these dogs and their clinical significance.     

Molecular and serologic evidence of tick-borne Ehrlichiae in three species of lemurs from St. Catherines Island, Georgia, USA.

In recent years, several species of ehrlichiae have been recognized as tick-borne disease agents of veterinary and medical importance. Clinically normal free-ranging or previously free-ranging lemurs, including 46 ring-tailed lemurs (Lemur catta), six blue-eyed black lemurs (Eulemur macaco flavifrons), and four black and white ruffed lemurs (Varecia variegata variegata) from St. Catherines Island, Georgia, were tested for evidence of exposure to tick-borne ehrlichiae. All 52 adult lemurs were serologically tested for exposure to Ehrlichia chaffeensis and Anaplasma phagocytophilum. Polymerase chain reaction (PCR) assays for E. chaffeensis, A. phagocytophilum, Ehrlichia ewingii, and Ehrlichia canis were conducted on blood samples from all 56 lemurs. Blood from all lemurs was inoculated into DH82 cell cultures for E. chaffeensis isolation. Of the adult lemurs, 20 (38.5%) and 16 (30.8%) had antibodies reactive (> or =1:128) for E. chaffeensis and A. phagocytophilum, respectively. Two ring-tailed lemurs were PCR and culture positive for E. chaffeensis. Molecular characterization of the two E. chaffeensis isolates showed that both contained 5-repeat variants of the variable-length PCR target (VLPT) antigen gene and 3-repeat variants of the 120-kDa antigen gene. Sequencing of the VLPT genes revealed a novel amino acid repeat unit (type-9). One lemur infected with E. chaffeensis was slightly hypoproteinemic and had moderately elevated serum alanine aminotransferase (ALT) levels. These lemurs from St. Catherines Island have been exposed to or infected with tick-borne ehrlichiae, or both, but showed no clinical disease.     

Serological survey for Ehrlichia canis in urban dogs from the major population centres of northern Australia

OBJECTIVE: To detect evidence of Ehrlichia canis infection of dogs from the major population centres of northern Australia, if present. DESIGN: Serological investigation for E. canis. PROCEDURE: The sera of 316 domestic dogs, collected from the northern Australian population centres of Townsville, Cairns, Darwin, Kununurra and Broome from May 1997 to August 1999, were investigated for evidence of infection with E. canis. Samples were tested for antibodies to E. canis using an indirect fluorescent antibody (IFA) test. The buffy coats from blood of dogs whose serum reacted in the IFA test were subsequently tested with a nested PCR to detect E. canis DNA. When available, blood from these dogs was injected into suckling mice, which were then examined for clinical disease and tested for the presence of E. canis antibodies. RESULTS: Of the 316 samples tested seven reacted in the IFA test for E. canis. None of the dogs from which these samples were obtained exhibited clinical signs of acute or chronic ehrlichiosis. The six positive samples available for testing were negative when tested with the nested PCR. Suckling mice inoculated with blood from three of the dogs whose serum was positive by IFA test showed no signs of clinical disease nor did their give positive reactions in the IFA test. CONCLUSIONS: No evidence of E. canis infection was confirmed in any of the dogs examined. Northern Australia would appear to remain free of this obligate parasite.     

Antibodies to selected canine pathogens and infestation with intestinal helminths in golden jackals (Canis aureus) in Israel.

Blood and fecal samples, collected from 46 healthy adult free-ranging golden jackals captured in two different locations in Israel, were examined. A serological Study was conducted to investigate the prevalence of circulating antibodies reacting with four common canine pathogens: canine distemper virus (CDV), canine parvovirus (CPV), Ehrlichia canis and Leishmania infantum. Faecal flotation and haematological tests were also performed. The seroprevalence of CPV, E. canis, CDV, and L. infantum were 72.3% (34/47), 54.3% (25/46), 52.2% (24/46), and 6.5% (3/46) respectively. Faecal flotation tests revealed a high prevalence of Ancylostoma caninum (13/17, 76%) and a low prevalence of Dipilidium caninum infestation. Examination of blood smears revealed Hepatazoon canis gamonts in one jackal. Golden jackals are among the most common free-ranging carnivores in Israel and neighboring countries. Their habitats are in proximity to densely populated areas and they bear close phylogenic relation to the domestic dog. These facts, combined with the high prevalence of the jackals' exposure to the major canine pathogens demonstrated in this study, suggest that they may serve as a reservoir for the transmission of certain diseases to domestic dogs.     

Antinuclear antibodies can be detected in dog sera reactive to Bartonella vinsonii subsp. berkhoffii, Ehrlichia canis, or Leishmania infantum antigens

The presence of antinuclear antibodies (ANAs) is used to support a clinical diagnosis of systemic lupus erythematosus (SLE) in dogs. However, clinicians must interpret the detection of ANAs with caution, particularly in light of increasing evidence that dogs with known bacterial and protozoal infections can have high ANA titers. Retrospectively, medical records were reviewed for all dogs that were concurrently tested for antinuclear antigens and Bartonella vinsonii (berkhoffii), Ehrlichia canis, or Rickettsia rickettsii antigens between 1990 and 2000. When analyzed on the basis of reactivity to a specific infectious agent, 75% of the B vinsonii (berkhoffii) seroreactors, 16.7% of the E canis seroreactors, and 0% of the R rickettsii seroreactors had concurrent ANAs. Subsequent prospective testing did not detect ANAs in convalescent sera from dogs experimentally infected with B vinsonii (berkhoffii), E canis, or R rickettsii. However, 10-20% B vinsonii (berkhoffii), E canis, or Leishmania infantum reactive sera from naturally infected dogs contained ANAs. In addition, 45% of sera from dogs that are reactive to multiple vectorborne organisms were more likely to contain ANAs when compared to sera from dogs reactive to only 1 test antigen. When interpreting the relevance of seroreactivity to nuclear antigens, clinicians should recognize that dogs with seroreactivity to B vinsonii (berkhoffii), E canis, or L infantum antigens (especially those with seroreactivity to more than one of these pathogens) may produce ANAs.