Detection of SNPs and benzimidazole resistance in strongyle nematode eggs of horses by allele-specific PCR.

This study was conducted to determine single nucleotide polymorphisms (SNPs) and the benzimidazole (BZ) resistance in strongyle nematode egg populations in horses using molecular techniques. A total of 200 fecal samples were collected from horses in 26 farms in two provinces (Kayseri and Nevsehir) of the Central Anatolia Region of Türkiye between May and August 2022. The flotation method was used to detect strongyle nematode eggs in the fecal samples of the horses. Afterward, strongyle nematode eggs were collected, and the allele-specific polymerase chain reaction (AS-PCR) technique was used to detect the BZ resistance. BZ-susceptible and BZ-resistant PCR products were sequenced to determine single nucleotide polymorphisms (SNPs) in the ß-tubulin isotype 1 gene. The strongyle nematode eggs were determined in 85 (42.5%) out of 200 fecal samples. AS-PCR detected 50.58% (43/85) BZ-resistant (homozygous resistant) and 36.4% (31/85) BZ-susceptible (homozygous susceptible) genes in the strongyle eggs. Both BZ-resistant and BZ-susceptible genes (heterozygous) were determined in 11 samples. BZ-resistant and BZ-susceptible allele frequencies were determined as 57.0% (48.5/85) and 43.0% (36.5/85), respectively. SNPs were detected only in codon 200 of the ß-tubulin isotype 1 gene in four sequenced isolates of the two resistant and two susceptible isolates. This study is the first molecular report on BZ resistance in strongyle nematode eggs in horses in Türkiye. The widespread prevalence of BZ-resistant alleles in equine strongyle nematodes shows the requirement for the immediate usage of other anthelmintics instead of the BZ group drugs for the effective management and control of equine strongyle nematodes.

A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.

Occurrence and molecular characterization of Enterocytozoon bieneusi in water buffaloes (Bubalus bubalis) in Turkey.

Microsporidia are obligate intracellular fungus-like parasites that infect humans and animals worldwide. However, there is limited epidemiological data on the occurrence and molecular diversity of microsporidia in buffaloes worldwide. In the present study, fecal samples of 300 water buffaloes (Bubalus bubalis) in Kayseri, Sivas, and Samsun provinces of Turkey were investigated using two nested PCR assays targeting the rRNA of E. bieneusi and Encephalitozoon spp. All the fecal samples from water buffalo were found to be negative for Encephalitozoon spp. PCR positive isolates of E. bieneusi were bidirectionally sequenced for genotyping and phylogenetic analyses. Enterocytozoon bieneusi was the only microsporidian species identified in 8 water buffaloes with an overall molecular prevalence of 2.7%. Two known genotypes, YNDCEB-90 (n = 5) and J (n = 3) were identified by ITS sequence analysis. The YNDCEB-90 and J genotypes fall into zoonotic Group 1 and 2 of E. bieneusi in the phylogenetic tree, respectively. These findings suggested that water buffalo in Turkey are harbouring zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission to humans. This is the first report of detecting E. bieneusi genotypes J and YNDCEB-90 in water buffaloes. Further insight into the epidemiology of E. bieneusi in water buffaloes in different geographical areas in Turkey will be highly important to have determined the public health significance of this pathogen.

First report and genotyping of Dientamoeba fragilis in pet budgerigars (Melopsittacus undulatus), with zoonotic importance.

The protozoan Dientamoeba fragilis is one of the most common parasites in the digestive system of humans worldwide. The host range and transmission routes of D. fragilis, including the role of animals, are still ambiguous with few reports from non-human primates, sheep, rodents, pigs, a cat and a dog. In this study, we used microscopic and TaqMan qPCR analyses to investigate D. fragilisin 150 faecal samples from pet budgerigars (Melopsittacus undulatus) in the Central Anatolia Region of Turkey. Dientamoeba fragilis DNA was detected in 32 samples, resulting in a mean prevalence of 21.3%. In microscopic examination, trophozoites/cysts of D. fragilis were detected in 13 of 32 qPCR-positive samples. SSU rRNA sequence analyses of the qPCR-positive isolates identified genotype 1 of D. fragilis as predominant in budgerigars. Phylogenetic analyses of the SSU rRNA gene region clustered D. fragilis genotypes, as well as other trichomonads, in separate monophyletic clusters with bootstrap values ≥79.0. Our study provides the first evidence for the natural host status of pet budgerigars for D. fragilisand contributes to the knowledge of the epidemiology of this parasite. The high prevalence of genotype 1 of D. fragilis suggests that pet budgerigars are suitable reservoirs for zoonotic transmission. Our findings contribute to an increased awareness and knowledge of D. fragilis infections in the context of a one-health approach.

Genetic diversity of Ichthyophthirius multifiliis (Fouquet, 1876) infecting farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) in Turkey.

We assessed genetic diversities among Ichthyophthirius multifiliis (Ich) field isolates collected from farmed rainbow trout (Oncorhynchus mykiss) in Turkey. The overall prevalence of Ich was 35.3% (634/1798). Five novel Ich genotypes (ImulTR1 and ImulTR3-ImulTR6) were described based on mitochondrial cox-1 and nad1_b genes. The remaining genotype ImulTR2 was identical to the previously reported NY3 (or Ark9 and TW7) genotype from the United States and South Asia. Phylogenetic analysis indicated Turkish Ich isolates separated genetically into at least four distinct groups. Our study presents the first data on the genotypes of Ich in Turkey. We also provide evidence for the wide distribution of the NY3 genotype (or Ark9 and TW7) from the United States and South Asia to Turkey. Genetic diversities within the mitochondrial genes provided adequate resolution for describing novel genotypes and identifying the known genotype within Turkish Ich isolates. Description of the Ich genotypes allows for tracking of pathogen genotypes worldwide. Thus, we can better understand the connections between Ich outbreaks in the fisheries aquaculture.

Investigation of Zoonotic Cryptosporidium and Giardia intestinalis Species and Genotypes in Cats (Felis catus)

Objective: Giardia intestinalis and Cryptosporidium spp. are important zoonotic protozoan parasites that infect humans and various animals. We investigated the occurrence of G. intestinalis and Cryptosporidium spp. infection in cats. To provide data on the zoonotic transmission dynamics of these parasites, genotypes of the detected isolates were investigated through DNA sequence characterization. Methods: A total of 100 fecal samples were collected from cats between June and October 2020 in Kayseri and Samsun provinces. Fecal samples were examined by nested polymerase chain reaction (PCR), targeting the ß-giardin gene of G. intestinalis and small subunit (SSU) rRNA gene of Cryptosporidium spp. All PCR products were sequenced for genotyping. Results: Of the samples examined, Giardia intestinalis was determined in 8 samples (8.0%), whereas none of the samples were found positive for Cryptosporidium spp. Sequence analyses of the ß-giardin PCR products indicated that all G. intestinalis isolates were classed into the zoonotic assemblage B. Conclusion: This study adds to the current data on the molecular epidemiology of cryptosporidiosis and giardiasis in cats. The findings also highlight the potential risk of cats for public health concerning the zoonotic transmission dynamics of G. intestinalis.

Complete mitochondrial genome characterization and phylogenetic analyses of the main vector of Crimean-Congo haemorrhagic fever virus: Hyalomma marginatum Koch, 1844.

The Mediterranean tick, Hyalomma marginatum, is the most important vector of Crimean-Congo haemorrhagic fever virus and several pathogens that cause animal and human diseases and economic losses to livestock production. Given the medical and veterinary importance of this tick species, we sequenced and characterized its mitochondrial genome (mitogenome) for the first time. We designed two new primer sets and combined long-range PCR with next generation sequencing to generate complete mitogenomes with deep coverage from 10 H. marginatum adults. The mitogenomes contained 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal subunits, two control regions, and three tick-box motifs. The nucleotide composition of the H. marginatum mitogenomes were A+T biased (79.76%) and exhibited negative AT- and GC- skews across most PCGs. All PCGs were initiated by ATK codons and two truncated termination codons were seen in the COX2 and COX3 genes. All tRNAs exhibited typical cloverleaf structures, except for tRNACys and tRNASer1. A total of 62 polymorphic sites defined ten unique haplotypes. Phylogenetic analyses based on the 13 PCGs of 56 tick species revealed that four Hyalomma species (H. marginatum, H. asiaticum, H. rufipes, and H. truncatum) formed a monophyletic clade with strong support. The results of this study provide a comprehensive resource for further studies on the systematics, population genetics, molecular epidemiology, and evolution of ticks.

Molecular identification and subtype distribution of Blastocystis sp. in farm and pet animals in Turkey.

A total of 1340 fresh fecal samples from farm and pet animals in Central Anatolia and the Middle Black Sea Region of Turkey were investigated using a PCR assay targeting the SSU rRNA of Blastocystis sp. An overall Blastocystis sp. prevalence of 19.4% (183/940) was found in farm animals, including cattle, sheep, water buffaloes, and chickens. Fecal samples of dogs, cats, and horses were negative. The highest prevalence of Blastocystis sp. was found in sheep (38.2%) among the farm animals. The SSU rRNA sequence analysis revealed two animal-specific subtypes, including ST10 in cattle and sheep and ST14 in water buffaloes. The zoonotic subtype ST7 was identified in chickens. Our results indicated a high prevalence of animal-specific subtypes in livestock and zoonotic subtype ST7 in chickens, highlighting the potential risk of chickens for zoonotic transmission of Blastocystis in the research area. This study is the first large-scale evaluation of Blastocystis in animal hosts in Turkey, and contributes to the molecular epidemiology and genetics of Blastocystis. Our results should be considered by authorities as an indication of the zoonotic importance of Blastocystis sp. and the need for surveillance in public health intervention programs.

Enterocytozoon bieneusi in raw milk of cattle, sheep and water buffalo in Turkey: Genotype distributions and zoonotic concerns.

Raw milk is a continued threat to public health due to possible contamination with zoonotic pathogens. Enterocytozoon bieneusi is one of the most prevalent pathogenic fungi in a wide range of vertebrate hosts, causing diarrheal disease. Although there has been some evidence, the role and potential risk of raw milk of dairy animals in the transmission dynamics of E. bieneusi is not clear. Therefore, we aimed to determine the occurrence and genotypes of E. bieneusi in raw milk of dairy animals in several farms of the Central Anatolia Region. We also investigated if there is a relation between the presence of E. bieneusi and mastitis. Genomic DNAs from a total of 450 raw milk including 200, 200 and 50 samples from cattle, sheep and water buffalo respectively were analyzed using nested PCR, targeting the internal transcribed spacer of E. bieneusi. Totally milk samples of 9 (4.5%) dairy cattle, 36 (18.0%) sheep, and 1 (2.0%) water buffalo were PCR-positive. A significant relationship was determined between mastitis and the presence of E. bieneusi. Sequence analysis revealed the presence of eight genotypes: two known (ERUSS1, BEB6) and six novel genotypes (named as TREb1 to TREb6). The genotype ERUSS1 and BEB6 were the most common genotypes, found in all cattle and sheep farms. Phylogenetic analysis clustered all the identified genotypes in Group 2. This study provides novel findings that contribute to the transmission dynamics and molecular epidemiology of E. bieneusi. Our study also highlighted the potential risk of raw milk for public health with respect to microsporidia infections.

Investigation of Anisakis larvae in different products of ready-to-eat fish meat and imported frozen fish in Turkey.

Globalization opens new market areas and affects food consumption habits, resulting in rapid and remarkable cultural change. Food habits such as consumption of raw fish meat have become popular, resulting in increased risk of emerging infectious diseases. Anisakis simplex sensu stricto (s.s) and A. pegreffii are the most common and important fish-borne zoonotic nematodes responsible for human anisakiasis, which occurs through the consumption of raw or undercooked fish as well as cooked fish due to their heat-stable allergens. Here, we investigated the prevalence, intensity, and abundance of Anisakis larvae in imported fish and ready-to-eat local fish products in Turkey. A total of 205 ready-to-eat fish products, 100 imported frozen Atlantic salmon (Salmo salar) fillets, and 100 imported frozen whole Atlantic mackerel (Scomber scombrus) were sampled from supermarkets, sushi restaurants, and fish markets. All samples were individually examined using a pepsin digestion technique. In total, 602 Anisakis type I larvae were recovered from 98/100 mackerel. No larvae were found in ready-to-eat products or frozen Atlantic salmon fillets. Overall, 8.8% of the larvae were found in the muscle tissue. The overall mean intensity and abundance of infection in mackerel were 6.14 and 6.02, respectively. The larvae were molecularly identified and their phylogenetic relationships with the relevant Anisakis sequences in GenBank were investigated. For this purpose, a subsample of randomly selected 100 Anisakis larvae were analyzed with PCR-RFLP of the ITS region. The larvae were identified as A. simplex (s.s.) (n = 87) and hybrids (n = 13). ITS and cox2 gene regions of all hybrids and randomly selected 50 A. simplex (s.s.) larvae were sequenced for species confirmation and phylogenetic analyses. No intraspecific nucleotide variation was found among the ITS sequences of either species. Seven and three haplotypes, respectively, were identified for A. simplex (s.s.) and hybrid species according to DNA polymorphism of the cox2 gene. Hybrids in our study clustered within the common A. simplex (s.s.) clade in the cox2 phylogenetic tree indicating the dominance of A. simplex (s.s) in the catching area of Atlantic mackerel. Consequently, our study indicates high occurrence of A. simplex (s.s.) larvae with an overall 98.0% prevalence in imported Atlantic mackerel, and highlights the importance of these fish as potential reservoirs for human allergic anisakiasis in Turkey and possibly in other countries.

Prevalence and genetic diversity of avian haemosporidian parasites at an intersection point of bird migration routes: Sultan Marshes National Park, Turkey.

Avian haemosporidians (Haemosporida) represent a globally distributed, species-rich multiparasite-multihost host-parasite system. Each year, many of these parasite lineages are carried between temperate and tropical regions by migratory birds. While several factors can limit the transmission of avian haemosporidians to new areas, recent studies have shown that some abundant parasites can sometimes disperse and be transmitted in new areas to become emerging infectious diseases. In this study, we investigated the prevalence and diversity of avian haemosporidian parasites in Sultan Marshes National Park (SMNP), a major stopover site in the eastern Mediterranean flyway, and we evaluated the potential for avian haemosporidians in SMNP to be transmitted to areas outside of their known distributions. We sampled a total of 565 migratory and resident birds belonging to 39 species and 23 families. We applied both molecular and microscopic methods to detect and identify avian haemosporidian infections and also quantified the frequency of potential abortive infections. We identified a total of 52 different mitochondrial cytochrome b (cyt b) parasite lineages belonging to the genera Plasmodium (N = 12), Haemoproteus (N = 31), and Leucocytozoon (N = 9) in 193 (34.2%) infected birds. Ten of the lineages were reported for the first time. Our findings show that numerous parasite lineages are actively transmitted among resident bird species of SMNP. Our findings also revealed new parasite-host interactions while considering the role of possible abortive infections. The relatively high frequency of presumed abortive infections suggests that analyses of datasets generated only by PCR-based methods should be interpreted with caution. We also compared the prevalence and distribution of avian haemosporidian infections in both resident and migratory bird species and showed that haemosporidian prevalence was related to bird migratory behavior. The results of this study contribute to a better understanding of the ecological and genetic adaptations associated with changes in transmission areas of avian haemosporidian parasites.

Molecular prevalence and genotyping of Giardia duodenalis in cattle in Central Anatolia Region of Turkey.

The molecular prevalence and genotypes of Giardia duodenalis in cattle were investigated. A total of 450 fecal samples were collected from cattle in three provinces of Central Anatolia from August 2017 to July 2019. Genomic DNA was extracted from the fecal samples and used in molecular analysis carried out by nested PCR analyses of the ß-giardin (bg) gene of G. duodenalis. Positive samples were further analyzed by nested PCR at two gene loci (triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh)) for genotyping of G. duodenalis isolates. PCR analyses of the bg gene indicated that the overall prevalence of G. duodenalis was 30.2%. However, lower rates were determined with PCR analyses for gdh and tpi loci. The sequence analyses of the bg, gdh, and tpi genes revealed the presence of zoonotic assemblage A and livestock-specific assemblage E. Combined-sequence analyses revealed that assemblage E was the most common in the study area. Our study provides the first data on the wide prevalence of livestock-specific assemblages E in cattle in Turkey. The prevalence of assemblage A in cattle also reveals the importance of cattle for zoonotic transmission of giardiasis in Turkey.

First report on the molecular prevalence of Enterocytozoon bieneusi in horses in Turkey: genotype distributions and zoonotic potential.

Horses might play an important role as reservoir hosts in the epidemiology of Enterocytozoon bieneusi, which is one of the most important zoonotic microsporidian pathogens, with a wide range of hosts. Nevertheless, limited information is available on the infection rates and genotypes of E. bieneusi in horses, and no data are available on the occurrence and molecular characteristics of E. bieneusi in horses in Turkey. We determined the prevalence of E. bieneusi among horses raised on farms from two provinces of Central Anatolia Region, by amplification of the partial small subunit ribosomal RNA gene using nested PCR. We identified the genotypes of E. bieneusi isolates by analyzing the ribosomal internal transcribed spacer (ITS) sequences. The overall prevalence of E. bieneusi was 18.7% (56/300), with no significant differences in infection rates among age groups or between genders of horses. Sequence analysis revealed eight genotypes: two known genotypes (ERUSS1, BEB6) and six novel genotypes (named ERUH2 to ERUH7). The genotype ERUSS1 was the most common and was found on all farms, age groups, and genders. Phylogenetic analysis clustered all the identified genotypes in ruminant-specific group 2. Our findings contribute to the molecular epidemiology of E. bieneusi.

Prevalence and genotyping of bovine Cryptosporidium species in the Mediterranean and Central Anatolia Region of Turkey.

The prevalence of Cryptosporidium species in calves and heifers with relation to diarrhea from several herds was investigated in this study. Fecal samples were collected from 135 and 120 pre-weaned calves and 79 and 130 heifers raised in the Central Anatolia (CAR) and Mediterranean Regions (MR) of Turkey, respectively. A total of 86 post-weaned calves in CAR were also included in the study. For diagnostic comparison, all samples were examined by microscopic examination, SSU rRNA nested PCR and TaqMan real-time PCR for the presence of oocyst and Cryptosporidium DNA. In total, 102 (34.0 %) and 93 (37.2 %) of the examined samples from CAR and MR were found positive for Cryptosporidium DNA with both nested PCR and real-time PCR analyses, respectively with an overall prevalence of 35.5 %. The diagnostic sensitivity and specificity of microscopic examination were determined as 68.7 % and 100.0 % compared to molecular tools, respectively. RFLP and sequence analyses of the SSU rRNA from the PCR products revealed that 138 (70.8 %) out of 195 positive isolates were C. parvum further confirming the species-specific real-time PCR results. Among the remaining 57 (29.2 %) positive isolates, 30 (15.4 %) and 27 (13.8 %) were characterized as C. ryanae and C. bovis, respectively. C. parvum was the dominant species in pre-weaned calves especially with diarrhea while C. bovis and C. ryanae were mostly found in post-weaned calves and heifers. The sequence analyses of the gp60 gene of C. parvum isolates revealed two subtypes (IIaA13G2R1, IIaA14G1R1) belonging to zoonotic family IIa, with IIaA13G2R1 being the most common in diarrheic calves.

Development and field evaluation of a species-specific mt-COI targeted SYBR-Green Real Time PCR for detection and quantification of Haemonchus contortus in cattle in Turkey.

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per µl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite.

First Molecular Detection and Phylogenetic Analyses of Zoonotic Giardia intestinalis in Horses in Turkey.

The goal of our study was to investigate the molecular prevalence of Giardia intestinalis in naturally infected horses in Kayseri, Central Anatolia Region in Turkey, to determine the molecular characterization of the obtained isolates and to exhibit the potential role of horses in zoonotic transmission of G. intestinalis. Fecal samples were randomly collected from totally 150 horses with clinically healthy between March and June of 2018. After the genomic DNA extractions, 25 (16.6%) of the 150 fecal samples, were found positive for G. intestinalis by nested PCR analyses of ß-giardin gene. Phylogenetic analysis of the ß-giardin gene sequences of G. intestinalis showed that the sequences detected in this study belonged to assemblage A that is regarded as zoonotic. Our study is the first report on the presence of G. intestinalis in horses in Turkey. The findings of the present study indicate that future research studies are required to determine molecular epidemiology and geographical distribution of G. intestinalis infections in horses nationwide. In addition, this study also may be helpful to assess the zoonotic potential for public health of G. intestinalis infections.

Molecular characterization of black flies (Diptera: Simuliidae) in areas with pest outbreaks and simuliotoxicosis in Northeast Anatolia Region, Turkey.

Accurate species identification provides the foundation for successful pest management and vector control of black flies. Accordingly, we examined the mitochondrial DNA cytochrome oxidase I (COI) gene sequences of four morphologically and chromosomally identified species of black flies (Simulium vernumgroup sp., S. bergi Rubtsov, S. bezzii (Corti), and S. kiritshenkoi Rubtsov) in Northeast Anatolia Region of Turkey where simuliid pest problems and simuliotoxicosis cases have been reported among cattle. COI gene sequences of these species and closely related species available in GenBank were used to provide species-level diagnoses and infer relationships. Both subgenera (Nevermannia and Simulium) were monophyletic, and subclades generally corresponded with species groups. Intraspecific genetic divergence was 0.2-1.6%, whereas the mean interspecific genetic divergence among the four species was 11.2-14.5%. The COI analysis produced results congruent with morphological concepts of the nominal species S. bergi and S. bezzii. Probable misidentifications in GenBank were revealed, especially for species in the S. ornatum and S. vernum groups, complicating identification capability. Sequence variation in the COI barcode region also might not be adequate for species delineation and identification among some species in these two species groups.

Molecular detection and identification of Wolbachia endosymbiont in fleas (Insecta: Siphonaptera).

The aim of this study was to determine the presence and prevalence of Wolbachia bacteria in natural population of fleas (Insecta: Siphonaptera) in Turkey, and to exhibit the molecular characterization and the phylogenetic reconstruction at the positive isolates with other species in GenBank, based on 16S rDNA sequences. One hundred twenty-four flea samples belonging to the species Ctenocephalides canis, C. felis, and Pulex irritans were collected from animal shelters in Kayseri between January and August 2017. All flea species were individually screened for the presence of Wolbachia spp. by polymerase chain reaction (PCR) targeting the 16S ribosomal RNA gene. According to PCR analyses, Wolbachia spp. were found prevalent in C. canis and P. irritans fleas, while it was not detected in the C. felis species. Totally, 20 isolates were purified from agarose gel and sequenced with the same primers for molecular characterization and phylogenetic analyses. The sequence analyses revealed 17 polymorphic sites and 2 genetically different Wolbachia isolates, representing two different haplotypes in two flea species. The distribution patterns, molecular characterization, and phylogenetic status of Wolbachia spp. of fleas in Turkey are presented for the first time with this study. Understanding of the role of Wolbachia in vector biology may provide information for developing Wolbachia-based biological control tools.

Prevalence and Genotyping of Microsporidian Parasites in Dogs in Turkey: Zoonotic Concerns.

Microsporidia are opportunistic pathogens that infect a wide range of invertebrates and vertebrates. To assess the potential role of dogs in the transmission of these zoonotic pathogens, a total of 282 fecal samples from dogs in the Central Anatolia Region of Turkey were analyzed by utilizing species specific polymerase chain reaction for the four most frequent human microsporidia. Two microsporidia species were recognized in 41 samples (14.5%). Encephalitozoon intestinalis was detected in 35 samples (12.4%) and it was the most common microsporidium. The second microsporidium, E. cuniculi, was identified in six (2.1%) of the samples. Sequence analysis of the intergenic spacer of the ribosomal ribonucleic acid (RNA) internal transcribed spacer (ITS) gene revealed the presence of three E. intestinalis haplotypes closely associated with each other. No polymorphic region was found among the ITS sequences of E. cuniculi isolates and they were characterized as genotype III. This study provides the first data on the zoonotic microsporidia species from dogs in Turkey.

Genetic diversity of Culicoides species within the Pulicaris complex (Diptera: Ceratopogonidae) in Turkey inferred from mitochondrial COI gene sequences.

Identification of Culicoides (Diptera: Ceratopogonidae) biting midges to species has become important due to their potential role in the transmission of arboviruses such as bluetongue virus, bovine ephemeral fever virus, Akabane virus, African horse sickness virus, epizootic haemorrhagic disease virus and Schmallenberg virus. In several studies, molecular tools, used for the identification of biting midges, revealed the presence of cryptic and undescribed species especially within Pulicaris complex. The presence of cryptic species within species complexes raise questions about their role in viral disease transmission as there are apparent differences in the vectorial capacity between closely related species. In this study, we analyzed the mitochondrial DNA cytochrome oxidase I (COI) gene sequences of species within the Pulicaris complex present in Turkey and determined their phylogenetic relationships. Twenty-one haplotypes within the already described species C. pulicaris P1, C. lupicaris, C. lupicaris L2, C. newsteadi, C. newsteadi N1, C. punctatus, C. fagineus F2 and C. flavipulicaris were determined from the study areas. The molecular analysis revealed further two haplotypes belonging to new non-described cryptic species named as C. lupicaris L3 and Culicoides WBS corresponding to C. lupicaris and Fagineus complex which diverged by 17.9% to 25.7% and 18.7% to 31.8%, respectively from other species in the subgenus Culicoides. Genetic divergence within species was <2.0% and phylogenetic analyses of the COI dataset revealed 22 different monophyletic separate clades within two major cluster. The results of this study emphasize the applicability of COI sequences as a diagnostic marker for differentiating Culicoides species and revealing cryptic species.