Osnat Eyal

Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria

Background Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria. Methodology/Principal Findings Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep. Conclusions/Significance The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents.

Molecular detection of Ehrlichia canis, Anaplasma bovis, Anaplasma platys, Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

: Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools-83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)-were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the authors knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.

Babesia canis canis and Babesia canis vogeli infections in dogs from northern Portugal

Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408bp fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis and B. canis vogeli in dogs from Portugal.

Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR

The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S-23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.

Redescription of Hepatozoon felis (Apicomplexa: Hepatozoidae) based on phylogenetic analysis, tissue and blood form morphology, and possible transplacental transmission

BackgroundA Hepatozoon parasite was initially reported from a cat in India in 1908 and named Leucocytozoon felis domestici. Although domestic feline hepatozoonosis has since been recorded from Europe, Africa, Asia and America, its description, classification and pathogenesis have remained vague and the distinction between different species of Hepatozoon infecting domestic and wild carnivores has been unclear. The aim of this study was to carry out a survey on domestic feline hepatozoonosis and characterize it morphologically and genetically.MethodsHepatozoon sp. DNA was amplified by PCR from the blood of 55 of 152 (36%) surveyed cats in Israel and from all blood samples of an additional 19 cats detected as parasitemic by microscopy during routine hematologic examinations. Hepatozoon sp. forms were also characterized from tissues of naturally infected cats.ResultsDNA sequencing determined that all cats were infected with Hepatozoon felis except for two infected by Hepatozoon canis. A significant association (p = 0.00001) was found between outdoor access and H. felis infection. H. felis meronts containing merozoites were characterized morphologically from skeletal muscles, myocardium and lungs of H. felis PCR-positive cat tissues and development from early to mature meront was described. Distinctly-shaped gamonts were observed and measured from the blood of these H. felis infected cats. Two fetuses from H. felis PCR-positive queens were positive by PCR from fetal tissue including the lung and amniotic fluid, suggesting possible transplacental transmission. Genetic analysis indicated that H. felis DNA sequences from Israeli cats clustered together with the H. felis Spain 1 and Spain 2 sequences. These cat H. felis sequences clustered separately from the feline H. canis sequences, which grouped with Israeli and foreign dog H. canis sequences. H. felis clustered distinctly from Hepatozoon spp. of other mammals. Feline hepatozoonosis caused by H. felis is mostly sub-clinical as a high proportion of the population is infected with no apparent overt clinical manifestations.ConclusionsThis study aimed to integrate new histopathologic, hematologic, clinical, epidemiological and genetic findings on feline hepatozoonosis and promote the understanding of this infection. The results indicate that feline infection is primarily caused by a morphologically and genetically distinct species, H. felis, which has predilection to infecting muscular tissues, and is highly prevalent in the cat population studied. The lack of previous comprehensively integrated data merits the redescription of this parasite elucidating its parasitological characteristics.

Prevalence of Babesia microti-like infection in red foxes (Vulpes vulpes) from Portugal

The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7-78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0-6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p<0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p=0.219) or age groups (<2 years vs. ≥ 2 years) (p=1.0). This is the first report of the B. microti-like piroplasm in foxes from Portugal as well as the first report on detection by PCR and genotyping of B. canis in a red fox worldwide. A natural cycle of the B. microti-like piroplasm is suggested in red fox populations based on the high prevalence of the protozoan. Red foxes might be a reservoir of the B. microti-like piroplasm and a source of infection to dogs.

Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli.

Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/μl in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs.

First description of natural Ehrlichia canis and Anaplasma platys infections in dogs from Argentina.

Bacteria belonging to the Anaplasmataceae family are vector transmitted agents that affect a variety of vertebrate hosts including the tick-borne pathogens Ehrlichia canis and Anaplasma platys, which cause canine monocytic ehrlichiosis and cyclic thrombocytopenia, respectively. These two infections, typically reported from tropical and sub-tropical regions, have not been previously reported in dogs from Argentina. A total of 86 blood samples from dogs with suspected rickettsial disease and 28 non-suspected dogs were studied. Analysis included evaluation of hematological findings, PCR for Ehrlichia and Anaplasma species and sequencing of the positive PCR products. E. canis was detected in the blood of six dogs and A. platys in eighteen. All the dogs categorized as non-suspected were negative by PCR. Co-infection with Hepatozoon canis and Babesia vogeli was documented. This first report of E. canis and A. platys infections in dogs from Argentina indicates that these tick-borne infections have a considerably broader range than previously recognized in South America.

Prevalence and molecular characterization of Hepatozoon canis in dogs from urban and rural areas in Southeast Brazil

The objective of this survey was to investigate the prevalence of Hepatozoon infection in dogs in the rural and urban areas of Uberlândia, Brazil by PCR and molecular characterization. DNA was obtained from blood samples collected from 346 local dogs from both genders and various ages. Seventeen PCR products from positive blood samples of urban dogs and 13 from the rural dogs were sequenced. Partial sequences of the 18S rRNA gene indicated that all 30 dogs were infected with Hepatozoon canis similar in sequence to H. canis from southern Europe. Four local dog sequences were submitted to GenBank (accessions JN835188; KF692038; KF692039; KF692040). This study indicates that H. canis is the cause of canine hepatozoonosis in Uberlândia and that infection is similarly widespread in rural and urban dogs.

Failure of imidocarb dipropionate to eliminate Hepatozoon canis in naturally infected dogs based on parasitological and molecular evaluation methods.

The efficacy of imidocarb dipropionate for the treatment of Hepatozoon canis infection was studied in three naturally infected asymptomatic dogs followed longitudinally over 8 months. Response to treatment was followed by monitoring blood counts, parasitemia levels in blood, parasite in concentrated buffy-coat smears and by PCR. The dogs were initially treated with a low dose of 3 mg/kg imidocarb dipropionate twice a month and when parasitemia persisted after five treatments, with the regular dose of 6 mg/kg. In one dog, H. canis gamonts were no longer detectable by blood and buffy-coat microscopy after 2 months of therapy with 6 mg/kg while in the two other dogs gamonts were intermittently found in blood but persistently detectable in buffy-coat smears during the whole study period. Furthermore, combined therapy with doxycycline monohydrate administered at 10 mg/kg/day PO for 4 weeks also failed to eliminate H. canis. PCR revealed that parasite DNA was present in the blood of all dogs at all sampling dates regardless of treatment refuting the effectiveness of treatment suggested by negative blood microscopy. Detection of H. canis in buffy coat was found to be twice as sensitive than by blood smear and detection by PCR was even more sensitive revealing infection in eight samples (16% of total samples) negative by blood and buffy-coat microscopy. In conclusion, imidocarb dipropionate was not effective in eliminating H. canis from dogs treated repeatedly over 8 months. Microscopical detection is not sufficient for the evaluation of treatment response in H. canis infection and follow up by molecular techniques is recommended.