Ehrlichia

Background and Aim: Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs.

Materials and Methods: Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids.

Results: Both dsb and gltA were amplified with a high degree of linearity (R2≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10–7 and 10–6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR.

Conclusion: RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.

Eight ixodid tick species, associated with 59 free-ranging mammals belonging to 10 species, were collected at five different localities in the Free State Province, South Africa. Four of the study areas were nature reserves (Willem Pretorius, Sandveld, Tussen-die-Riviere, and Soetdoring), and one site was a private farm located in Senekal district. The collection was performed from March 2006 until June 2006. Ticks (n5569) and tissues from animals (n552) were analyzed by polymerase chain reaction, reverse line blot, and sequencing for various tickborne pathogens belonging to the genera Babesia, Theileria, Anaplasma, and Ehrlichia. Rhipicephalus (Boophilus) microplus, the known vector of Babesia bovis responsible for Asiatic redwater in South Africa, was found for the first time in the Free State Province. Rhipicephalus appendiculatus also was collected in areas in the Free State where it has not been previously described. Anaplasma marginale was detected for the first time in a gemsbok (Oryx gazella gazella). Gene sequences recovered in this study were 98-100% homologous with GenBank sequences for Anaplasma bovis, Theileria separata, and Theileria sp. Malelane sable antelope.

Dogs are susceptible to a number of ehrlichial diseases. Among them, canine monocytic ehrlichiosis is an important and potentially fatal disease of dogs caused by the rickettsia Ehrlichia canis. Diagnosis of the disease relies heavily on the detection of antibodies and is usually carried out using the indirect immunofluoresence antibody (IFA) test. The IFA test may be confounded by cross-reactivities between a number of the canine ehrlichial pathogens. This article presents a review of the ehrlichial diseases affecting dogs with reference to their immune responses, host specificities, cross-reactivites and diagnosis. Diagnostic means such as Western immunblot, dot-blot and PCR are discussed. The use of the IFA test as a diagnostic means for E. canis is presented along with its potential pitfalls. The review emphasizes that the disease process, cross-reactivites with other ehrlichial species, multiple tick-borne infections and persistent IFA antibody titers post-treatment, should all be considered when interpreting E. canis serological results.

Background: In recent years, several clinical cases and epidemiological studies of feline vector-borne diseases (FVBD) have been reported worldwide. Nonetheless, information on FVBD agents and their prevalence in Portugal is scarce. Methods: Three-hundred and twenty domestic cats presented to 30 veterinary medical centres in the north and centre regions of Portugal were randomly sampled. Blood was assayed by real-time polymerase chain reaction (PCR) for genera Anaplasma/Ehrlichia, genus Babesia, Hepatozoon canis, Hepatozoon felis, Leishmania infantum and the genus Rickettsia. Babesia-positive samples were further tested for Babesia canis and Babesia vogeli. Results: Eighty (25.0%) out of the 320 cats were positive to at least one vector-borne agent, including seven (2.2%) cats co-infected with two agents. Two cats (0.6%) were infected with Anaplasma/Ehrlichia spp., four (1.3%) with B. canis, 26 (8.1%) with B. vogeli, 50 (15.6%) with H. felis, one (0.3%) with L. infantum and four (1.3%) with Rickettsia spp. No cat tested positive for H. canis. One cat (0.3%) was co-infected with B. canis and B. vogeli, three (0.9%) with B. vogeli and H. felis, one (0.3%) with H. felis and L. infantum, and two (0.6%) with H. felis and Rickettsia spp. Conclusions: A considerable prevalence of infection with vector-borne pathogens among the domestic feline population of the north and centre of Portugal has been revealed by the present study. Additionally, this is the first detection of B. vogeli in cats from Europe and of H. felis in cats from Portugal.

OBJECTIVES: This study aimed to characterise tick species responsible for avian tick infestations in the UK, to analyse various risk factors for tick-related syndrome in tick-infested birds and to test samples for the presence of certain tick-transmitted pathogens. METHODS: Ticks, blood, splenic tissue and tick attachment site tissue from birds with attached ticks were requested from veterinarians and wildlife sanctuaries around the UK. Ticks were identified according to standard keys, and samples were analysed via DNA PCR test for Borrelia burgdorferi sensu lato, Babesia species, Bartonella species and Ehrlichia species. RESULTS: Ixodes frontalis was the most commonly identified tick, and an association of adult female I frontalis with tick-related syndrome in birds was demonstrated. Tick infestation was markedly seasonal. I frontalis was found on 32 species of birds. DNA PCR testing was uniformly negative. Of the birds known to have been treated, 75 per cent (nine of 12) survived. CLINICAL SIGNIFICANCE: Tick-related syndrome is a poorly understood syndrome, with sporadic distribution, both geographically and seasonally. This study confirms I frontalis as the most common cause of this syndrome in the UK and identifies some features of the tick life cycle in this country. The benefit of treatment in affected birds is highlighted. Risk factors for tick-related syndrome are examined and preventive strategies discussed.

We studied coinfection with four genera of bacteria-Borrelia, Bartonella, Anaplasma, and Ehrlichia-in ticks collected between December 2005 and November 2006 from Zhejiang Province and Liaoning Province. Four hundred fifty Haemaphysalis longicornis and 86 Ixodes sinensis ticks were collected and divided into 236 groups. Polymerase chain reaction (PCR) or nested PCR was used to detect the bacteria. The amplicons were sequenced and compared to published sequences. Phylogenic analysis was performed with MEGA3.1 and the data were analyzed using SPSS 11.0. Borrelia, Bartonella, Anaplasma, and Ehrlichia positive rates were 30.1% (71/236), 28.8% (68/236), 1.3% (3/236), and 0.8% (2/236), respectively. One tick was coinfected with all four bacteria (Borrelia, Bartonella, Anaplasma, and Ehrlichia) and another group was coinfected with three bacteria, Borrelia, Bartonella, and Ehrlichia. Statistical analysis showed most coinfections were significant, indicating that once a tick was infected with one bacteria, there was a greater chance to be infected with another bacteria.

The presently known tick-borne diseases in Germany include Lyme Borreliosis, tickborne encephalitis (TBE-virus, Central European Type), Q-fever, Babesiosis and presumably Ehrlichiosis. Blood samples of 4.368 forestry workers in the State of Baden-Wuerttemberg (B-W), Southwest Germany, were tested for presence of antibodies against Borrelia burgdorferi sensu lato, TBE-virus and Ehrlichia spp.

Background and Aim: Ehrlichia canis is a well-known cause of both anemia and thrombocytopenia in dogs. There are insufficient epidemiological data on this blood parasite in Thailand and the association of infections with hematological abnormalities. This study aimed to analyze the molecular characteristics and to identify E. canis as well as the risk factors associated with E. canis infection in dogs in Khon Kaen, Thailand.

Materials and Methods: Blood samples from 126 dogs that visited animal clinics were subjected to molecular detection using nested polymerase chain reaction for E. canis 16S rRNA gene. The risk factors and hematological profiles associated with the infection were analyzed using the logistic regression test in program SPSS version 19.

Results: Forty-one dogs were infected, indicating a 32.5% molecular infection rate of E. canis. The factors significantly associated with E. canis infection include animal housing status, low packed cell volume, low red blood cell count, and low platelets (p<0.05). Ten positive samples were amplified, sequenced, and phylogenetically analyzed. Sequence and phylogenetic analysis confirmed the current ten samples as E. canis compared with reference sequences in GenBank, using the BLAST program hosted by NCBI, which showed 99.74-100% similarity.

Conclusion: This study provided the first data of infection rate of E. canis using nested PCR and molecular characteristics of E. canis in randomly selected domestic dogs in Khon Kaen, Thailand.

A rapid immuno-migration test for the serological detection of canine monocytic ehrlichiosis, Witness ® Ehrlichia (WE) (Zoetis, France), was evaluated in 528 serum samples from dogs living in endemic areas of West and East Africa: Senegal (N = 208), Ivory Coast (N = 7), Sudan (N = 27), and Djibouti (N = 286). Of these dogs, 200 were French military working dogs (MWD) temporarily residing in Africa. The WE test results were compared with those obtained by indirect immunofluorescence (IFA). The sensitivity of WE was 97% [94.2, 98.7] with a specificity of 100% [98.6, 100]. In MWD, the seroprevalence (IFA) was 7%; in native dogs, it reached 77.1%. This significant difference can be explained by prophylactic measures from which MWD benefit. The WE test represents a simple, fast and reliable test for the detection of anti-Ehrlichia canis antibodies. Its implementation for the diagnosis of clinical cases has been validated in the field, and its use allows easy detection of asymptomatic dogs that may be carriers of E. canis.