Sezayi Ozubek

Molecular detection of tick-borne rickettsial and protozoan pathogens in domestic dogs from Turkey

BackgroundCanine tick-borne parasites have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary importance, domestic dogs play an important role in the transmission cycles of some agents by acting as reservoirs and sentinels. This study investigated Babesia, Theileria, Anaplasma, and Ehrlichia species in asymptomatic dogs in ten provinces of Turkey.MethodsDNA obtained from blood samples collected from 757 domestic dogs (243 stray, 351 shelter, 163 pet) of both sexes and various ages were evaluated using PCR and reverse line blotting (RLB) assays.ResultsOf the 757 dogs tested, 41 (5.4%) were found to be infected with one or more parasites. Ehrlichia canis (37/757, 4.9%) was the most common canine tick-borne pathogen, followed by Anaplasma platys (4/757, 0.5%). Babesia canis and Theileria annulata were each detected in 1 (0.13%) sample. Combined infection of E. canis and A. platys was detected in 2 (0.3%) samples. The prevalence of tick-borne pathogens was higher in adult dogs (6.8%) than in those under one year old (3.1%). Difference in infection rate of male and female dogs was not significant. Pet dogs had a lower prevalence of infection (1.2%) compared to stray (7.4%) and shelter dogs (6%) although the difference between stray and shelter dogs was not significant.ConclusionsBabesia canis, T. annulata, A. platys, and E. canis species were identified at the molecular level in dogs in several provinces of Turkey, with E. canis being the most common species among tick-borne pathogens. Detailed studies should be conducted regarding the existence and prevalence of B. canis and Dermacentor reticulatus in eastern Turkey.

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.

A molecular and parasitological survey of Hepatozoon canis in domestic dogs in Turkey.

In this study, asymptomatic dogs in nine provinces of Turkey were surveyed to investigate the prevalence and intensity of Hepatozoon canis infection. DNA obtained from blood samples collected from 694 domestic dogs (243 stray, 288 shelter, and 163 pets) of both genders and varying ages were evaluated by polymerase chain reaction (PCR). In addition, 285 thin blood smears prepared from these blood samples were also evaluated for microscopic examination. Direct microscopy revealed Hepatozoon gamonts in the peripheral blood of three of 285 (1.0%; 95% confidence interval (CI): 0.21-3.04) tested. Using PCR, 155 of the 694 (22.3%; 95% CI: 19.28-25.61) were found to be positive for the presence of H. canis DNA. The prevalence of infection was higher in adult dogs (26.2%; 95% CI: 22.1-30.7) than young animals (16.4%; 95% CI: 12.2-21.3). Although the prevalence determined by PCR was higher in male dogs (24.5%; 95% CI: 19.6-29.9) than in female dogs (20.8%; 95% CI: 16.9-25.1), gender differences were not significant. Pet dogs had a lower prevalence of infection (10.4%; 95% CI: 6.2-16.2) compared to stray (26.3%; 95% CI: 20.9-32.3) and shelter dogs (25.7%; 95% CI: 20.7-31.1), but no significant association between stray and shelter dogs was found for the presence of the parasite. Partial sequences of the 18S ribosomal RNA (rRNA) gene shared 99-100% similarity with the corresponding H. canis isolates. This epidemiological survey revealed a high prevalence of H. canis in dogs from several provinces in Turkey, and it suggests that the age and origin are associated with the parasite.

Bovine anaplasmosis in Turkey: First laboratory confirmed clinical cases caused by Anaplasma phagocytophilum.

Anaplasma species are obligate intracellular rickettsial pathogens that affect the health of humans and other animals. Clinical cases of anaplasmosis caused by Anaplasma phagocytophilum were evaluated, and the frequency of bovine Anaplasma species was determined in cattle. Blood samples and thin blood smears were collected from 10 cattle exhibiting clinical signs of tick-borne fever. In addition, blood samples were collected from 123 apparently healthy cattle from the same area. DNA was screened by reverse line blot assay for the presence of the hypervariable V1 region of the 16S rRNA gene of Anaplasma/Ehrlichia species. Intracytoplasmic inclusion bodies of A. phagocytophilum were observed in neutrophils of 6 sick animals. Parasitemia ranged from 0.2 to 1.6% in individual slides. Reverse line blot showed 45.1% (60/133) of the sampled cattle to be positive for one or more of five Anaplasma species. The frequency of single infections was 20.3% (27/133), while mixed infections were found in 24.8% (33/133) of samples with six different combinations of species and a maximum of four pathogens detected. A. phagocytophilum was the most prevalent (41/133, 30.8%) followed by Anaplasma marginale (25/133, 18.8%), Anaplasma centrale (24/133, 18%), Ehrlichia sp. strain Omatjenne (18/133, 13.5%) and Anaplasma bovis (1/133, 0.7%). This is the first report of A. bovis in a cow from Turkey. This is also the first report of clinical cases caused by A. phagocytophilum in cattle from the country. Therefore, A. phagocytophilum should be taken into account as differential diagnosis in cases of high fever and anorexia in pastured animals.

Molecular evidence for trans-stadial and transovarial transmission of Babesia occultans in Hyalomma marginatum and Rhipicephalus turanicus in Turkey.

A total of 1064 adult ticks identified as Hyalomma marginatum (n=965), Rhipicephalus turanicus (n=86), Haemaphysalis inermis (n=9) and Rhipicephalus bursa (n=4) along with 20 egg masses derived from H. marginatum (n=18) and R. bursa were screened for Babesia occultans. The ticks were combined into 328 pools and infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of these pools, 23 (7%) were positive for B. occultans, and the overall MLE was calculated as 2.27% (CI 1.48-3.35). Infection prevalence in tick species was 7.09% for H. marginatum (22/310, MLE 2.41, CI 1.55-3.59) and 7.14% for R. turanicus (1/14, MLE 1.11, CI 0.07-5.18). None of H. inermis and R. bursa was infected with the parasite. Host-seeking H. marginatum females displayed an infection prevalence of 1.68% (2/119, MLE 0.63, CI 0.11-2.06), while no parasite DNA was detected in the males. The prevalence of infection in feeding H. marginatum varied, ranging from 27.27% (9/33, MLE 4.61, CI 2.26-8.52) in males to 25.92% (7/27, MLE 8.22, CI 3.72-15.75) in females. Of the egg masses examined, 4 (22.22%) of H. marginatum and 1 (50%) of R. turanicus were infected with B. occultans. To the best of our knowledge, this is the first time that B. occultans has been detected in R. turanicus.

Molecular evidence of a new Babesia sp. in goats

In this study, a novel Babesia sp. infecting goats was detected and its phylogenetic relationship to related species was determined. A total of 200 blood samples collected from sheep (n=78) and goats (n=122) were examined in the study. The V4 hypervariable region of the 18S rRNA gene of the novel Babesia sp. was amplified by PCR and analysed using a reverse line blot hybridization assay adapted for small ruminants. Samples from seven goats hybridized to Theileria/Babesia catch-all and Babesia catch-all probes and did not hybridize to any species-specific probe tested, suggesting the presence of an unrecognized Babesia species or genotype. Sequencing results showed the isolate to clearly differ from ovine Babesia species and genotypes currently available in the GenBank database. The isolate showed 90.9%, 93.5%, and 93.4% identity to B. ovis, B. motasi, and B. crassa, respectively and 91-93% similarity to Babesia genotypes recently described in small ruminants. The highest homology (∼96-97%) observed was with Babesia odocoilei, Babesia sp. EU1, and Babesia divergens. The new isolate was provisionally designated Babesia sp. The study contributes to better insight into the distribution and phylogenetic diversity of piroplasms in small ruminants. The survey indicated a high prevalence of piroplasms in small ruminants (21.5%). Of those detected, T. ovis was the most prevalent (17%), followed by Babesia sp. (3.5%), and B. ovis (2%).

A survey of canine haemoprotozoan parasites from Turkey, including molecular evidence of an unnamed Babesia

Canine tick-borne apicomplexan parasites have emerged in recent years, showing a wider geographic distribution and increased global prevalence. A reverse line blot assay was performed on 219 blood samples collected from domestic dogs for simultaneous detection of all named canine piroplasm species as well as Hepatozoon canis. Ten samples hybridized to the Theileria/Babesia and Babesia catch all probes but did not hybridize to any species-specific probe tested, suggesting the presence of an unrecognized Babesia species or genotype. Sequencing results showed 91.5%, 91.9%, 92.4%, 92.4%, and 89.2% similarity to B. canis, B. vogeli, B. rossi, B. gibsoni, and B. conradae, respectively. The highest homology (98.1-98.5%) observed was with unnamed Babesia sp. isolates (Ludhiana and Malbazar) described in dogs, Babesia sp. of buffalo origin, Babesia sp. Kashi 2, and Babesia orientalis, along with Babesia occultans of cattle origin. The partial cox1 sequence indicated that this isolate was most similar to Babesia sp. 1 HG-2012, with an identity of 86.5%. The survey revealed high prevalence of haemoprotozoans in domestic dogs (57.5%, CI 50.7-64.2), with Hepatozoon canis the most prevalent (54.3%, CI 47.5-61.117%), followed by Babesia sp. (4.6%, CI 2.2-8.2), B. vogeli (1.4%; CI 0.3-3.9), and B. canis (0.4%, CI 0-2.5). Combined infection of Hepatozoon canis and Babesia sp. was detected in five (2.3%, CI 0.7-5.2) samples and of H. canis and B. vogeli in two (0.9%, CI 0.1-3.2) dogs. The study contributes insight into the distribution and phylogenetic diversity of canine piroplasms in Turkey.

A Molecular Survey of Rickettsias in Shelter Dogs and Distribution of Rhipicephalus sanguineus (Acari: Ixodidae) sensu lato in Southeast Turkey

Abstract Canine tick-borne pathogens are the source of emerging diseases and have important zoonotic relevance. Dogs play a major role in the transmission of several zoonotic tick-borne pathogens, as reservoirs and/or sentinels. To simultaneously detect Anaplasma and Ehrlichia species, a reverse line blot assay was conducted on 219 blood samples collected from autochthonous asymptomatic shelter dogs. One hundred and three (47.0%, CI 40.3–53.9) dogs were positive for one or both rickettsial pathogens. Seventy-one (32.4%, CI 26.3–39.0) dogs were infected with Anaplasma platys and 23 (10.5%, CI 6.8–15.3) with Ehrlichia canis. Concurrent infection with A. platys and E. canis was detected in nine (4.1%, CI 1.9–7.6) dogs. Partial sequences of the 16S ribosomal RNA gene shared 100% identity with the corresponding published sequences for A. platys and E. canis. Infection with Anaplasma phagocytophilum was not detected in the examined dogs. In total, 1018 (range 1–70, mean intensity 13.1, mean abundance 4.6) Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) sensu lato ticks (45.7% nymphs, 54.3% adults) were collected from the dogs.There was no significant association between Anaplasma/Ehrlichia infection and dog sex or age, but a significant correlation was found between rickettsia infection and presence of R. sanguineus. Improved tick control strategies to reduce the risk of these pathogens spreading among dogs and humans are needed in the region.

A molecular survey of small ruminant hemotropic mycoplasmosis in Turkey, including first laboratory confirmed clinical cases caused by Mycoplasma ovis

Hemotropic mycoplasmas are emerging zoonotic pathogens that cause mild to severe hemolytic anemia. Blood samples and thin blood smears were collected from six sheep exhibiting clinical symptoms typical of hemolytic anemia. Coccoid and ring-shaped M. ovis were found on the surface of erythrocytes of infected sheep. Parasitemia ranged from 0.7% to 3.7% in individual smears. Clinical cases caused by Mycoplasma ovis were evaluated, and prevalence, based on DNA screening for the 16S rRNA gene of hemoplasmas in apparently healthy sheep (n=328) and goats (n=272) from the same area, was 11.3% (CI 8.1-15.2) and 6.2% (CI 3.7-9.8), respectively, a significant difference between species (p<0.05). Sequences of the nearly complete 16S rRNA gene shared 99% identity with the corresponding published sequences for M. ovis and Mycoplasma wenyonii. The overall M. ovis prevalence in healthy animals was 9% (CI 6.8-11.6) with no significant differences in infection rate between young and adults (p>0.05). The proportion of hemoplasma-positive small ruminants was higher with tick infestation (p<0.05). The presence of tick-borne Babesia spp. and Theileria spp. was also significantly associated with ovine hemoplasma infection (p<0.05) in the healthy sample. This is the first report of M. wenyonii in sheep. M. ovis should be considered in differential diagnosis in cases of high fever and anorexia in pastured sheep and goats.

Outbreak of anaplasmosis associated with novel genetic variants of Anaplasma marginale in a dairy cattle

During early lactation, dairy cows may present a transient immunosuppressive state and develop anaplasmosis caused by Anaplasma marginale. In this study, clinical anaplasmosis in dairy cattle in the Thrace region of Turkey was investigated with respect to within-herd prevalence, vertical transmission, and genetic diversity. In March and September 2015, thirty lactating cows showed primary clinical signs of anaplasmosis, including fever, anaemia, decreased milk yield, anorexia, and laboured breathing. Symptoms disappeared in most cows after administration of long-acting oxytetracycline, but nine of them (30%) died. Following diagnosis based on clinical signs, microscopy and molecular findings, blood samples were collected from apparently healthy lactating cows (n=184), pregnant heifers (n=39) and newborn calves (n=24). DNA was extracted from each sample and analyzed for the presence of major surface proteins (MSPs) of A. marginale, followed by sequencing to assess diversity of isolates. Microscopic examination of erythrocytes revealed A. marginale inclusion bodies in symptomatic cows. Examination of thin blood smears showed 3.8% of the lactating, clinically asymptomatic, cows to be infected with A. marginale, while nPCR detected 31.0% positive. A. marginale infection was not detected in pregnant heifers by either method. Congenital infection was found in one calf by nPCR. This is the first report of transplacental transmission of A. marginale in Turkey. The MSP4 sequence analyses showed high genetic diversity among the isolates, presenting 97.6-99.6% homology at the amino acid level. The sequences of MSP1a amplicons revealed genetic diversity providing three new tandem repeats.