Haematology of experimental babesiosis and ehrlichiosis in steroid immunosuppressed horses.

An investigation was carried out to study the haematology of steroid immunosuppressed horses experimentally infected with Babesia equi and Ehrlichia equi, separately or simultaneously. Horses infected with both pathogens showed less marked changes in their haematology than those inoculated with either pathogen separately. This appeared to result from early elimination of the more pathogenic Babesia as Ehrlichia spread through the granulocytes. The apparent suppression of Babesia by Ehrlichia is of field clinical importance and merits further investigation for its apparent useful potentials in the control of babesiosis in endemic areas.     

Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily

A reverse line blot hybridisation (RLB) of 21 oligonucleotides with polymerase chain reaction (PCR) amplified regions of 16S rRNA (Ehrlichia/Anaplasma group) or 18S rRNA (Babesia/Theileria group) genes of haemoparasites detected Theileria annulata, T. buffeli/orientalis, Babesia bovis, B. bigemina, B. divergens, Ehrlichia bovis, Anaplasma marginale, A. centrale and unknown species within the Rickettsia tribe.A very high prevalence of mixed infections was detected, which indicated that animals infected with Babesia spp. were also infected with Theileria spp. and/or Anaplasma spp.The tick distribution appeared to be seasonal with Hyalomma marginatum as the most frequently observed tick and Boophilus annulatus and Ixodes ricinus as the least frequently observed ticks. Other species identified in the 818 ticks collected during the five sampling periods between April 1998 and November 1999 included H. lusitanicum, Rhipicephalus sanguineus group, R. bursa, Dermacentor marginatus, Haemaphysalis punctata, B. annulatus and I. ricinus.     

Ehrlichia and Babesia infections in dogs in The Netherlands

A retrospective study was performed at the Department of Clinical Sciences of Companion Animals at Utrecht University amongst 75 dogs diagnosed with a Babesia canis and/or an Ehrlichia canis infection. The majority of the dogs had visited an endemic area (most often the Mediterranean area or the Dutch Antilles), but two dogs became infected with Babesia in the Netherlands. Babesia infections were associated with a stay in an endemic area and an incubation period that are both significantly shorter (less than 3 months) than those for Ehrlichia and co-infections (more than 3 months). Reasons for the owner to seek veterinary attention (lethargy, anorexia, fever), findings from the physical examination (pale mucous membranes, hepato-/splenomegaly) and laboratory results (anemia, thrombocytopenia, hypo-albuminemia) were highly aspecific, making serology or PCR mandatory for diagnosing infections. Antigenic stimulation by the parasite sometimes resulted in immune-mediated diseases such as immune-mediated hemolytic anemia, thrombocytopenia, glomerulonefritis, and polyarthritis and in the case of ehrlichiosis in hypergammaglobulinemia. Specific therapy (imidocarb-diproprionate and/or doxycycline) was necessary, and because combined infections were common, it was considered appropriate to administer both drugs while the definitive diagnosis was being established. The prognosis was reasonably good, with almost half of all patients showing no clinical signs after treatment, although Babesia and co-infections were associated with a significantly longer survival sometimes resulted than Ehrlichia infections.     

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated intravenously with blood containing an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila, and identical to the human granulocytic ehrlichiosis agent with respect to its 16S rRNA gene sequence. The clinical signs were evaluated, and blood samples were collected for haematology, serum biochemistry and serology. Ehrlichial inclusions in the blood were monitored by microscopy, and ehrlichial DNA was detected by the polymerase chain reaction (PCR). Two of the dogs were injected with prednisolone on days 54 to 56 and days 152 to 154 after infection, and the other was injected with prednisolone on days 95 to 97 after infection. The dogs were euthanased and examined postmortem. Ehrlichial inclusions were demonstrated in the neutrophils and seroconversion occurred shortly after inoculation. Two of the dogs developed acute disease with rectal temperatures above 39.0 degrees C, after which no further clinical signs were observed. The administration of corticosteroids seemed to facilitate the detection of ehrlichial inclusions. Ehrlichial DNA was detected intermittently by PCR in blood samples from two of the dogs throughout the study. Persistent infection was demonstrated up to five-and-a-half months after inoculation.     

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.     

Clinical and hematologic effects of experimental infection of dogs with recently identified Babesia gibsoni-like isolates from Oklahoma

OBJECTIVE: To characterize clinical and hematologic responses in dogs following experimental inoculation with Babesia gibsoni-like isolates from infected dogs in Oklahoma. DESIGN: Prospective study. ANIMALS: 6 mixed-breed dogs. PROCEDURE: 2 dogs were inoculated with organisms from a naturally infected dog, and 3 were inoculated with organisms from a second naturally infected dog (1 of these 3 dogs was splenectomized 1 week prior to inoculation). One dog was not inoculated. Complete blood counts were performed weekly. RESULTS: In the 5 dogs inoculated with organisms, parasites were initially detected 1 to 5 weeks after inoculation, and severity of parasitemia peaked with 1.9 to 6.0% of RBC infected by 4 to 6 weeks after inoculation. Parasitemia was easily detectable (> 0.1% of RBC infected) for 3 to 4 weeks. Clinical abnormalities included lethargy, fever, and pale mucous membranes but were mild to nearly inapparent in 2 dogs. All dogs developed regenerative anemia and marked thrombocytopenia. Thrombocytopenia developed before and lasted longer than the parasitemia. Profound but transient neutropenia was detected in some dogs. The splenectomized dog developed more severe parasitemia and anemia and more pronounced clinical abnormalities. Three dogs with intact spleens recovered without treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 2 or more genotypically distinct, but morphologically identical, small Babesia parasites can infect dogs in the United States. Compared with infection with small Babesia parasites from California, infection with these isolates resulted in less severe parasitemia and clinical abnormalities. Parasitemia was transient, indicating that identification of organisms in blood smears may be difficult in some dogs.     

Resistance to tick-borne fever in young lambs.

Lambs were inoculated with Ehrlichia phagocytophila infected blood at the age of either two or six weeks. The temperature response and degree of parasitaemia were both most pronounced in the lambs inoculated at six weeks old. The half-life of antibodies to E phagocytophila was estimated to be 17.5 days. Lambs inoculated with tick-borne fever (TBF) had lower liveweights in the autumn than TBF-free controls.     

Experimental inoculation of beagle dogs with Ehrlichia species detected from Ixodes ovatus

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.     

A survey of ixodid ticks feeding on cattle and prevalence of tick-borne pathogens in the Black Sea region of Turkey

The study reports the frequency of infestation and the prevalence of tick-borne pathogens in feeding adult ticks detached from cattle in two climatic zones of the Black Sea region of Turkey. A total of 2160 adult ticks were collected during 2007-2008. Of these, 1062 were randomly selected, divided into 224 pools, and tested for the presence of bovine Theileria, Babesia, and Anaplasma species. Eleven tick species were recognized on cattle in the study. Hyalomma marginatum was widely disrubuted in the semi-arid bioclimatic zone, but few specimens were collected in the humid bioclimatic zone. The most prevalent tick species in the humid climatic zone was Ixodes ricinus. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). Overall, 4% (CI 2.87-5.44) of 224 tick pools were found to be positive for the pathoges by Reverse line blot. Maximum likelihood estimation of the infection rate varied among tick species, ranging from 2.68% (CI 0.16-12.68) in Haemaphysalis sulcata to 10.49% (CI 4.07-23.66) in Rhipicephalus bursa. The most prevalent tick-borne pathogen was Anaplasma phagocytophilum at 6.78% (CI 3.41-12.18) followed by A. centrale (6.56%, CI 0.42-31.47), Anaplasma/Ehrlichia spp. (3.61%, CI 1.99-6.06), Babesia spp. (3.33%, CI 1.65-6.03), and T. buffeli/orientalis (2.71%, CI 0.73-7.18). Sequencing results indicated that Babesia spp. shared 99% to 100% similarity with the unnamed Babesia sp. Kashi 1 and 2, Babesia sp. Kayseri 1 and Babesia sp.CS58. Anaplasma/Ehrlichia spp. were 98% and 100% identical to Ehrlichia canis and Ehrlichia sp. Omatjenne strain, respectively.     

Experimental Babesia equi infection in mature horses

Nine 4-year-old Arabian geldings were experimentally infected with Babesia equi of European origin. All horses developed detectable parasitemia an average of 30 days after they were inoculated, which was accompanied by a decrease in PCV. The infections were generally mild with no animal deaths. All horses became serologically positive by the indirect fluorescent antibody test within an average of 23 days after they were inoculated and by the complement-fixation test 30 days after they were inoculated.     

比格犬埃立克体病实验模型的建立

为犬埃立克体病的病理学、临床学、病原学的深入研究，我们用Beagle(比格犬）复制犬埃立克体病以建立犬埃立克体病实验模型。比格犬静脉接种E.platy和E.canis后，利用PCR和电镜技术确证感染成功。进一步的观察发现，接种后比格犬与最最感的德国牧羊犬自然感染埃立克体病的临床症关放病理学变化基本吻合。结果说明通过静脉接种病原成功建立了比格犬埃立克体病实验模型。     

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.     

Serum cardiac troponin I concentration in dogs with ehrlichiosis

Background: Ehrlichiosis is a multisystemic disease with the potential to cause cardiomyocyte injury in naturally infected dogs.
Hypothesis: Myocardial injury occurs in dogs infected with Ehrlichia canis .
Animals: One-hundred and ninety-four dogs from Brazil with clinical and laboratory abnormalities indicative of ehrlichiosis. Sixteen healthy dogs served as controls.
Methods: Electrocardiogram, echocardiogram, noninvasive blood pressure measurement, and serum cardiac troponin I (cTnI) concentrations were evaluated. Serologic assays and PCR determined the exposure and infection status for E. canis, Anaplasma spp., Babesia canis vogeli, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia chaffeensis, Ehrlichia ewingii, Leishmania chagasi , and spotted-fever group Rickettsia . Dogs were assigned to groups according to PCR status: E. canis infected, infected with other vector-borne organisms, sick dogs lacking PCR evidence for infection, and healthy controls.
Results: E. canis -infected dogs had higher serum cTnI concentrations than controls (median: 0.04 ng/dL; range 0.04–9.12 ng/dL; control median: 0.04 ng/dL; range: 0.04–0.10 ng/dL; P = .012), and acute E. canis infection was associated with myocardial injury (odds ratio [OR]: 2.67, confidence interval [CI] 95%: 1.12–6.40, P = .027). Severity of anemia was correlated with increased risk of cardiomyocyte damage ( r = 0.84, P < .001). Dogs with clinical signs of systemic inflammatory response syndrome (SIRS) were at higher risk for myocardial injury than were other sick dogs (OR: 2.55, CI 95%: 1.31–4.95, P = .005).
Conclusions and Clinical Importance: Acute infection with E. canis is a risk factor for myocardial injury in naturally infected Brazilian dogs. Severity of anemia and SIRS might contribute to the pathophysiology of myocardial damage.     

Tick-Borne Fever in Lambs of Different Ages

Four groups of lambs aged 1 week, 4 weeks, 1/2 year and 1 year old respectively were inoculated with Ehrlichia phagocytophila infected blood. Clinical signs, temperature reactions, haematological changes and parasitaemia were more moderate in lambs inoculated with E. phagocytophila at the age of 1 week than those recorded in the older animals. The clinical response to tick-borne fever (TBF) appears to be more severe with increasing age of the lambs. The lymphocyte reactivity to mitogens was reduced in the TBF infected lambs, and was most pronounced in lambs in the 3 older age groups.     

Experiences with the use of Imizol in treating canine blood parasites in Nigeria

Imizol* (imidocarb diproprionate 12 per cent w/v) at 5 mg/kg body weight was found to be highly effective in the treatment of canine babesiosis, mixed infections of Babesia canis, Hepatozoon canis and Ehrlichia canis, but slightly less effective against uncomplicated Ehrlichia canis infections.     

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.     

The effect of two different oxytetracycline treatments in experimental Ehrlichia phagocytophila infected lambs

The effect of 2 different oxytetracycline treatments in acute E. phagocytophila infected lambs was investigated. Twenty 5-month-old lambs of the Dala and Rygja breeds were used. Ten lambs were inoculated intravenously with a stabilate of an ovine E. phagocytophila strain. On the third day of fever, 4 lambs were given long-acting oxytetracycline (Terramycin prolongatum vet, Pfizer) (20 mg/kg) intramuscularly and another 4 lambs were given short-acting oxytetracycline (Terramycin vet, Pfizer) (10 mg/kg) intravenously for 5 consecutive days. The lambs were examined for the presence of Ehrlichia infection by blood smear evaluation, polymerase chain reaction (PCR) and antibody titre against E. equi. One month after the last antibiotic treatment, 250 ml citrate blood from each of these lambs were inoculated into each of 10 susceptible lambs, which were observed during the following 6 weeks. The results indicate that oxytetracycline given in the acute stage of the infection may effectively terminate the development of fever, rickettsemia and weight reduction in E. phagocytophila infected lambs. No difference was observed between the 2 treatment groups. However, at least 3 of 8 antibiotic treated lambs (37.5%) were still infected with granulocytic Ehrlichia 3 months after treatment.     

Antigenic analysis of four species of the genus Ehrlichia by use of protein immunoblot

The antigenic profile of Ehrlichia canis, E risticii, E sennetsu, and E equi was investigated by the use of protein (western) immunoblot technique. Results of analysis of serum from acutely and chronically infected animals indicated that the 4 Ehrlichia species share a unique 25-kD polypeptide in addition to other peptides. Immune sera from dogs inoculated with E canis recognized a wide range of E canis polypeptide antigens, as determined by western blot analysis. A larger number of E sennetsu polypeptides were detected when homologous antiserum and antiserum to E equi were used. The latter antiserum did not recognize antigens of E canis or E risticii. Antisera to E canis, E risticii, and E sennetsu detected E equi antigens. Data indicate that a 25-kD protein is a common antigen among the species of the genus Ehrlichia and that the ascending order of abundance of immunodominant determinants in the 4 species of Ehrlichia studied would be: E risticii----E equi----E sennetsu----E canis. Implications of these findings for diagnosis of ehrlichial infections and prophylaxis are evident.     

Susceptibility of cats to infection with Ehrlichia risticii, causative agent of equine monocytic ehrlichiosis

Eight adult cats were inoculated IV (n = 6) or SC (n = 2) with Ehrlichia risticii-infected P388D1 (continuous murine macrophage) cells or with E risticii released from P388D1 cells. Three additional cats were inoculated with organism-free P388D1 cultured monocytes, and 1 cat, which served as a medium control was inoculated with balanced salt solution. Clinical signs of illness were observed in the IV inoculated cats from which E risticii was isolated. One cat developed intermittent diarrhea between postinoculation days (PID) 8 and 18, and the other cat developed lymphadenopathy, acute depression, and anorexia between PID 20 and 24. Ehrlichia risticii was isolated in cultures from 2 of 6 IV inoculated cats on PID 6, 10, and 17. Both cats were inoculated with E risticii released from the P388D1 cells. Ehrlichia risticii was not isolated from SC inoculated cats or from control cats. All 8 cats inoculated with E risticii seroconverted between PID 10 and 23. A pony inoculated with E risticii isolated from 1 of the inoculated cats developed clinical signs of equine monocytic ehrlichiosis including fever, anorexia, depression, and mild colic. Ehrlichia risticii was isolated from the blood of this pony on PID 7, 9, 11, and 16.     

Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings

Clinical signs, humoral and cellular immune responses, and microscopic and gross tissue alterations resulting from acute experimental Ehrlichia canis infection in dogs were studied. Four dogs were inoculated with E. canis and four were used as uninfected controls. After a 10-14-day incubation period, infected dogs developed pyrexia up to 41 degrees C for 6-8 days. Antibody titers to E. canis antigen were demonstrable in all inoculated dogs at 30 days post-infection. Necropsy of infected animals revealed pale mucous membranes, generalized lymphadenopathy, splenomegaly, edema and ascites. Microcopically, the main lesions were: lymphoreticular hyperplasia in cortical areas of lymph nodes and spleenic white pulp, periportal accumulation of mononuclear cells and centrolobular fatty degeneration of the liver. Kidneys presented with glomerulonephritis characterized by interstitial mononuclear infiltration. Immunophenotyping of lymphocytes from lymph nodes and spleen sections displayed alterations in IgG, IgM, CD3+ and CD8+ cells population in infected dogs.