Ibrahim Balkaya

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.

Prevalence and molecular characterization of bovine coenurosis from Eastern Anatolian region of Turkey

This study was conducted to determine the prevalence and molecular characteristics of Coenurus cerebralis, the metacestode of Taenia multiceps in cattle from Erzurum province located in eastern region of Turkey between November 2009 and April 2010. Five of 1045 brains of cattle (0.47%) were found to be infected with Coenurus cysts. The characteristics and morphology of C. cerebralis were seen in all the cysts. The cyts from three infected cattle were genetically analyzed and confirmed to be T. multiceps metacestodes by NAD1 and COX1 mitochondrial gene sequence analysis. Pairwise comparison between the NAD1 sequences of the T. multiceps isolates from Erzurum and other T. multiceps isolates available in GenBank showed differences ranging from 0.6 to 2.9%, while COX1 sequences showed differences ranging from 0.2 to 2.6%. Considering the two genes, it was seen that all of the three isolates from Erzurum province were in the same group according to phylogenetic analyses. The present findings could provide a stimulus for future studies on the systematic relationships and epidemiology of lesser-known taeniid cestodes in the region, employing mitochondrial sequence data sets.

The Relationship of Public Park Accessibility to Dogs to the Presence of Toxocara Species Ova in the Soil

This survey was conducted to determine prevalence of Toxocara spp. ova in public parks in Erzurum, Turkey. A total of 214 soil samples were collected from July 2007 to June 2008 in 36 public parks, of which 28 were unfenced and 8 were fenced. Prevalence of Toxocara spp. was 64.28% in unfenced public parks, while no contamination was observed fenced public parks (p<0.001). Average number of Toxocara spp. ova was 1.43 per 50 g sand ranging from 1 to 7. Moreover, soils from unfenced public parks were contaminated with Taeniid spp. ova (3.12%). In conclusion, public parks and/or playgrounds should be fenced to prevent fecal contamination, suggesting that a more frequent surveillance should be performed and preventive measures should be taken and enforced by local governments to reduce likelihood of zoonoses in children.

Epidemiological survey and molecular characterization of Echinococcus granulosus in cattle in an endemic area of eastern Turkey.

Cystic echinococcosis (CE) is a zoonotic disease affecting mainly various species of livestock and humans. A survey of cystic echinococcosis in cattle was conducted from December 2008 to April 2009 in an endemic area of eastern Turkey. A total of 1758 cattle were examined and hydatid cysts were found in 33.9% of the cattle (595/1758). Most of the cattle (75.8%) had hydatid cysts only in the lungs, 10.9% only in the liver, 12.4% in the both liver and lungs, 0.7% in the spleen and 0.2% in the heart. 220 of these cysts were examined by PCR of 12S rRNA gene and sequencing of mt-CO1 gene. 147 of 220 cattle isolates showed the same band pattern with 12S rRNA analyses and were identified as G1-G3 complex (Echinococcus granulosus sensu stricto) and also 28 of these were confirmed by mitochondrial CO1 sequencing as G1 genotype. The other 73 samples that did not amplified with 12S rRNA gene specific primers were analyzed for mt-CO1 gene and only 7 samples yielded 446bp product and after the CO1 sequencing these were identified as G3 genotype. This study confirms the predominance of the sheep strain (G1 genotype) in Turkey and this is the most comprehensive genetic survey of cattle CE in Turkey.

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PCR in Turkey.

Cystic echinococcosis (CE), caused by hydatid cysts, is a widespread and hazardous disease in humans and animals worldwide. The aim of the current study was to investigate the genetic characteristics of sheep and cattle isolates of Echinococcus granulosus obtained from eastern Turkey using Single Stranded Conformation Polymorphism (SSCP) analysis and conventional PCR method. A total of 54 isolates collected from Erzurum and Elazig provinces of east-Turkey were examined. The 31 of these were obtained from liver of sheep while 23 cattle isolates (12 of liver and 11 of lung) were tested. After the total genomic DNA isolation 12S rRNA gene of all isolates were examined by PCR for the aim of genetic characterization by conventional PCR and mitochondrial CO1 gene for SSCP analysis. The 12S rRNA-PCR yielded 254 bp of amplification product with all samples analyzed. Thus, these samples were identified as G1-G3 cluster (E. granulosus sensu stricto). At least two major single stranded bands were resolved for G1-G3 cluster and G5 in SSCP analysis. While the resolution of more than two additional single stranded bands in SSCP indicated the existence of G7 genotype. The SSCP analysis was identified the G5 and G7 while failed to G1 and G3. The present SSCP analysis classified all 54 cyst isolates from sheep and cattle as E. granulosus sensu stricto (G1-G3 cluster). However, some sequenced samples for G1 and G3 showed the same band patterns by SSCP.

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.

A serological and molecular survey of cattle hypodermosis in east-Turkey

The aim of this study was to determine the seroprevalence of hypodermosis in cattle in East-Turkey and to identify Hypoderma species using morphological and molecular methods. For this purpose, a total of 778 serum samples of cattle were collected from Erzurum provinces of east-Turkey from December 2008 to February 2009. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. In addition, 10 Hypoderma spp. larvae were collected from slaughtered animals in the abattoir for morphological identification and molecular characterization on the basis of mitochondrial CO1 gene sequence analysis and PCR-RFLP. Two hundred and twenty three (28.6%) out of 778 cattle were seropositive for hypoderma antibodies. All positive cattle were female of a local breed. Seven out of 10 Hypoderma larvae were morphologically classified as third instar larvae (L3) of H. bovis and 3 were classified as L3 of H. lineatum. The TaqI restriction enzyme was used to differentiate the Hypoderma species on the basis of the 438 and 250 bp bands for H. bovis and the 488 and 200 bp bands for H. lineatum resulting from PCR-RFLP. According to the alignment of the mitochondrial CO1 sequences of the Hypoderma species and the PCR-RFLP results, all examined larva samples were classified as H. bovis.

First Molecular Characterization of Echinococcus multilocularis in Turkey.

This study aimed to find out the occurrence of Echinococcus multilocularis in foxes in Erzurum province, the highest endemic region for human alveolar echinococcosis in Turkey. The sedimentation and counting technique was used to reveal adult Echinococcus spp. in the intestines of foxes. One out of the 10 foxes was infected with E. multilocularis. The adult worms were analyzed morphologically and molecularly and were confirmed to be E. multilocularis by species-specific PCR. Pairwise comparisons between the 12S rRNA sequences of the E. multilocularis isolate from Erzurum and other E. multilocularis isolates showed 100% similarity of the Erzurum isolate with European isolates. With this study, the presence of E. multilocularis in a fox in Erzurum was confirmed by PCR, and molecular identification of E. multilocularis is reported for the first time in Turkey.

Prevalence and molecular characterization of Theileria equi and Babesia caballi in jereed horses in Erzurum, Turkey

Equine piroplasmosis (EP) is a hemoprotozoan tick-borne disease with worldwide distribution that is caused by Theileria equi and Babesia caballi. There are studies reporting the presence of equine piroplasmosis in Turkey but the situation in Erzurum is unknown. The aim of the current study was to determine the situation of equine piroplasmosis in jeered horses in Erzurum. Between April and August 2015, a total of 125 Arabian horse were examined and blood samples were collected. At the time of sampling, animals were also examined for tick infestations and clinical signs. Besides microscopic examination of Giemsa-stained blood smears, multiplex PCR performed with species specific primers partially amplifying the 18S rRNA gene of B. caballi and T. equi. During the microscopic examination of blood smears, T. equi piroplasms were found in 6 (4.8%) samples. In total, 11 (8.8%) T. equi DNA were detected with multiplex PCR. B. caballi piroplasms or DNA were not obtained. BLAST analysis of the sequenced T. equi samples (GenBank: KU921661–KU921667) indicated 98.8–100% identity to each other, and 100% similarity to T. equi isolates in South Africa, Iran, China, Sudan, India, Mongolia, Trinidad, Kenya, Spain, Serbia, Bosnia and Herzegovina and Turkey (Bursa). The results of our study indicate that T. equi occurs more frequently than B. caballi in the study area. To the authors’ knowledge, this is the first report of the molecular detection of equine piroplasmosis in jeered horses in Erzurum, Turkey.