The association between haemosporidian infection and non-breeding moult location in great reed warblers revisited by combining feather stable isotope profiles and geolocator data.

Stable isotope analysis provides valuable insights into the ecology of long-distance migratory birds during periods spent away from a specific study site. In a previous study, Swedish great reed warblers (Acrocephalus arundinaceus) infected with haemosporidian parasites differed in feather isotope ratios compared to non-infected birds, suggesting that infected and non-infected birds spent the non-breeding season in different locations or habitats. Here, we use a novel dataset comprising geolocator data, isotopes, and haemosporidian infection status of 92 individuals from four Eurasian populations to investigate whether parasite transmission varies with geography or habitats. We found that the probability of harbouring Plasmodium and Leucocytozoon parasites was higher in birds moulting in the eastern region of the non-breeding grounds. However, no geographic pattern occurred for Haemoproteus infections or overall infection status. In contrast to the previous study, we did not find any relationship between feather isotope ratios and overall haemosporidian infection for the entire current dataset. Plasmodium-infected birds had lower feather δ15N values indicating that they occupied more mesic habitats. Leucocytozoon-infected birds had higher feather δ34S values suggesting more coastal sites or wetlands with anoxic sulphate reduction. As the composition and prevalence of haemosporidian parasites differed between the old and the current dataset, we suggest that the differences might be a consequence of temporal dynamics of haemosporidian parasites. Our results emphasize the importance of replicating studies conducted on a single population over a restricted time period, as the patterns can become more complex for data from wider geographical areas and different time periods.

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.

Host shift and natural long-distance dispersal to an oceanic island of a host-specific parasite.

Parasite dispersal and host-switching may be better understood by knowing when they occurred. We estimated when the ancestor of a parasite of great reed warblers (Acrocephalus arundinaceus) dispersed to the Seychelles and began infecting the endemic Seychelles warbler (A. sechellensis). We used mitochondrial genomes and published molecular divergence rates to estimate the date of divergence between mitochondrial haplotypes of the parasite Haemoproteus nucleocondensis (lineage GRW01) in the great reed warbler and the Seychelles warbler. We also constructed a time-calibrated phylogeny of the hosts and their relatives to determine when the ancestor of the Seychelles warbler dispersed to the Seychelles. The two GRW01 lineages diverged ca 20-451 kya, long after the ancestor of the Seychelles warbler colonized the Seychelles ca 1.76-4.36 Mya. GRW01 rarely infects other species despite apparent opportunity. Humans were likely not involved in the dispersal of this parasite because humans settled the Seychelles long after the parasite diverged from its mainland relative. Furthermore, introduced birds are unlikely hosts of GRW01. Instead, the ancestor of GRW01 may have dispersed to the Seychelles with an errant migrating great reed warbler. Our results indicate that even specialized parasites can naturally disperse long distances to become emerging infectious diseases.

Prevalence of Borrelia burgdorferi and diversity of its outer surface protein C (ospC) alleles in blacklegged ticks (Ixodes scapularis) in Delaware.

Characterizing the diversity of genes associated with virulence and transmission of a pathogen across the pathogen's distribution can inform our understanding of host infection risk. Borrelia burgdorferi is a vector-borne bacterium that causes Lyme disease in humans and is common in the United States. The outer surface protein C (ospC) gene of B. burgdorferi exhibits substantial genetic variation across the pathogen's distribution and plays a critical role in virulence and transmission in vertebrate hosts. In fact, B. burgdorferi infections that disseminate across host tissues in humans are associated with only a subset of ospC alleles. Delaware has a high incidence of Lyme disease, but the diversity of ospC in B. burgdorferi in the state has not been evaluated. We used PCR to amplify ospC in B. burgdorferi-infected blacklegged ticks (Ixodes scapularis) in sites statewide and used short-read sequencing to identify ospC alleles. B. burgdorferi prevalence in blacklegged ticks varied across sites, but not significantly so. We identified 15 previously characterized ospC alleles accounting for nearly all of the expected diversity of alleles across the sites as estimated using the Chao1 index. Nearly 40% of sequenced infections (23/58) had more than one ospC allele present suggesting mixed strain infections and the relative frequencies of alleles in single infections were positively correlated with their relative frequencies in mixed infections. Turnover of ospC alleles was positively related to distance between sites with closer sites having more similar allele compositions than more distant sites. This suggests a degree of B. burgdorferi dispersal limitation or habitat specialization. OspC alleles known to cause disseminated infections in humans were found at the highest frequencies across sites, corresponding to Delaware's high incidence of Lyme disease.

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.

Genomic sequence capture of Plasmodium relictum in experimentally infected birds.

BACKGROUND: Sequencing parasite genomes in the presence of host DNA is challenging. Sequence capture can overcome this problem by using RNA probes that hybridize with the parasite DNA and then are removed from solution, thus isolating the parasite DNA for efficient sequencing. METHODS: Here we describe a set of sequence capture probes designed to target 1035 genes (c. 2.5 Mbp) of the globally distributed avian haemosporidian parasite, Plasmodium relictum. Previous sequence capture studies of avian haemosporidians from the genus Haemoproteus have shown that sequencing success depends on parasitemia, with low-intensity, chronic infections (typical of most infected birds in the wild) often being difficult to sequence. We evaluate the relationship between parasitemia and sequencing success using birds experimentally infected with P. relictum and kept under laboratory conditions. RESULTS: We confirm the dependence of sequencing success on parasitemia. Sequencing success was low for birds with low levels of parasitemia (< 1% infected red blood cells) and high for birds with higher levels of parasitemia. Plasmodium relictum is composed of multiple lineages defined by their mitochondrial DNA haplotype including three that are widespread (SGS1, GRW11, and GRW4); the probes successfully isolated DNA from all three. Furthermore, we used data from 25 genes to describe both among- and within-lineage genetic variation. For example, two samples of SGS1 isolated from different host species differed by 11 substitutions across those 25 genes. CONCLUSIONS: The sequence capture approach we describe will allow for the generation of genomic data that will contribute to our understanding of the population genetic structure and evolutionary history of P. relictum, an extreme host generalist and widespread parasite.

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.

Low MSP-1 haplotype diversity in the West Palearctic population of the avian malaria parasite Plasmodium relictum.

BACKGROUND: Although avian Plasmodium species are widespread and common across the globe, limited data exist on how genetically variable their populations are. Here, the hypothesis that the avian blood parasite Plasmodium relictum exhibits very low genetic diversity in its Western Palearctic transmission area (from Morocco to Sweden in the north and Transcaucasia in the east) was tested. METHODS: The genetic diversity of Plasmodium relictum was investigated by sequencing a portion (block 14) of the fast-evolving merozoite surface protein 1 (MSP1) gene in 75 different P. relictum infections from 36 host species. Furthermore, the full-length MSP1 sequences representing the common block 14 allele was sequenced in order to investigate if additional variation could be found outside block 14. RESULTS: The majority (72 of 75) of the sequenced infections shared the same MSP1 allele. This common allele has previously been found to be the dominant allele transmitted in Europe. CONCLUSION: The results corroborate earlier findings derived from a limited dataset that the globally transmitted malaria parasite P. relictum exhibits very low genetic diversity in its Western Palearctic transmission area. This is likely the result of a recent introduction event or a selective sweep.

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.

Evolution of vector transmitted parasites by host switching revealed through sequencing of Haemoproteus parasite mitochondrial genomes.

Parasite species evolve by switching to new hosts, cospeciating with their current hosts, or speciating on their current hosts. Vector transmitted parasites are expected to speciate by host switching, but confirming this hypothesis has proved challenging. Parasite DNA can be difficult to sequence, thus well resolved parasite phylogenies that are needed to distinguish modes of parasite speciation are often lacking. Here, we studied speciation in vector transmitted avian haemosporidian parasites in the genus Haemoproteus and their warbler hosts (family Acrocephalidae). We overcome the difficulty of generating parasite genetic data by combining nested long-range PCR with next generation sequencing to sequence whole mitochondrial genomes from 19 parasite haplotypes confined to Acrocephalidae warblers, resulting in a well-supported parasite phylogeny. We also generated a well-supported host phylogeny using five genes from published sources. Our phylogenetic analyses confirm that these parasites have speciated by host switching. We also found that closely related host species shared parasites which themselves were not closely related. Sharing of parasites by closely related host species is not due to host geographic range overlap, but may be the result of phylogenetically conserved host immune systems.

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.