Molecular prevalence, phylogenetic characterization, and epidemiological risk factors of pulmonary nematodes in domestic cats in Türkiye.

Aelurostrongylus abstrusus, Oslerus rostratus, and Troglostrongylus brevior are nematodes found in the respiratory system of domestic cats and cause a variety of symptoms. All three parasites use the same hosts and niches, and the morphological measurements of their L1s excreted in faeces overlap with each other. In this study, 300 cats brought to Ondokuz Mayis University Veterinary Teaching Animal Hospital were screened for lungworms by morphological measurements and molecular analyses. The prevalence of the lungworms was found as 1.33% (4/300) for A. abstrusus, 0.66% (2/300) for O. rostratus, and 0.33% (1/300) for T. brevior. Molecular identification of A. abstrusus, T. brevior, and O. rostratus in domestic cats was carried out for the first time in Türkiye within the present study. This study also reveals the risk factors of lungworm positivity in domestic cats in Türkiye.

Morphological and molecular characterization of Hysterothylacium larval morphotypes (Nematoda: Raphidascarididae) infecting edible marine fish in the Black Sea.

The morphological and molecular identification of Hysterothylacium larval morphotypes in the Black Sea remains unresolved and incomplete. The aim of current study was to provide a detailed morphological identification with rDNA whole ITS (ITS‒1, 5.8S subunit, ITS‒2) and mtDNA cox2 sequences data of Hysterothylacium larval morphotypes infecting four commonly edible marine fish species, including European anchovy, Engraulis encrasicolus (L.), horse mackerel, Trachurus trachurus (L.), whiting, Merlangius merlangus (L.), and red mullet, Mullus barbatus ponticus (E.) in the Black Sea (FAO fishing area 37.4.2). Hysterothylacium larval morphotypes were morphologically classified, followed by whole ITS and cox2 sequencing. Four Hysterothylacium larval morphotypes, III, IV, VIII, and IX, are described based on morphological and molecular data. The present study provides the first study reporting whole ITS and cox2 sequences for Hysterothylacium larval morphotypes III, IV, VIII and III, IV, VIII, IX, respectively, in the Black Sea. Here, we offer a foundation for future research on the distribution, morphologic and molecular identification of Hysterothylacium larval morphotypes infecting edible some marine fish in the Black Sea.

Molecular identification and infection levels of Anisakis species (Nematoda: Anisakidae) in the red scorpionfish Scorpaena scrofa (Scorpaenidae) from the Aegean Sea.

The red scorpionfish Scorpaena scrofa (Scorpaenidae) is a high commercial value marine fish species along the Mediterranean coasts. Anisakiasis is a fish-borne parasitic zoonoses caused by Anisakis larvae in consumers. To date, there are only a few epidemiological studies on the presence and molecular identification of Anisakis larvae infecting S. scrofa. A total of 272 S. scrofa captured from the Gulf of Izmir in the Turkish Aegean coasts (FAO 37.3.1) were examined for Anisakis larvae between March 2019 and March 2020. The prevalence, mean intensity and mean abundance of Anisakis larvae were 9.6% (95% CI 6.5-13.7%), 2.8 (95% CI 1.88-5.19), and 0.27 (95% CI 0.15-0.56), respectively. All Anisakis larvae were collected from the viscera and body cavity of S. scrofa. Anisakis pegreffii, A. typica, and A. ziphidarum were genetically identified by RFLP analysis of the ITS region. These species were also confirmed by cox2 sequence analysis. A weak positive and statistically significant correlation between the total length (ρS 0.204; p = 0.001) and total weight (ρS 0.200; p = 0.001) of S. scrofa and the number of Anisakis larvae was observed. This survey presents the first molecular detection of A. typica and A. ziphidarum in S. scrofa. Thus, this fish species is a new host for A. typica and A. ziphidarum. This is also the first report of the presence of A. ziphidarum in the Aegean Sea.

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.

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.

Morphological and molecular characterization of Eustrongylides excisus larvae (Nematoda: Dioctophymatidae) in Sander lucioperca (L.) from Northern Turkey.

The genus Eustrongylides Jägerskiöld, 1909 includes parasitic nematodes (Dioctophymatidae) affecting various fish species and piscivorous birds of freshwater ecosystems. Currently, there is little information on the molecular characterization of E. excisus based on nuclear ribosomal internal transcribed spacer (ITS) rDNA regions. However, before the present study, there had been no reports of characterizing the E. excisus using nuclear small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) genes sequences. In the present study, Eustrongylides spp. larvae were collected from pike-perch Sander lucioperca (L.) in Northern Turkey, and characterized by sequencing of ITS regions, SSU rRNA, and COI markers. Larvae herein morphologically identified as the fourth stage of Eustrongylides spp. were genetically identified as E. excisus based on the ITS sequence analysis. This study is the first record of SSU rRNA and COI sequences for E. excisus in GenBank. This is also a molecular characterization of E. excisus for the first time in Turkey. The ITS, SSU rRNA, and COI sequences of E. excisus can be used to establish the phylogenetic relationships of Eustrongylides species from Turkey and worldwide for further studies.

Novel and known myxobolids (Cnidaria, Myxozoa) infecting Chondrostoma angorense (Cypriniformes: Leuciscidae) in Turkey.

Turkey has more than 200 endemic freshwater fish species, one of which is the Ankara nase, Chondrostoma angorense Elvira, 1987 (Cypriniformes: Leuciscidae), a food fish in northern Turkey. Like most endemic fish species in Turkey, its myxosporean parasite fauna (Cnidaria: Myxosporea) are not yet described. We surveyed twenty C. angorense from Lâdik Lake in northern Turkey, and identified two myxosporean parasites from gills of these fish: Myxobolus arrabonensis Cech, Borzák, Molnár, Székely, 2015, and a co-infection of a novel species, Myxobolus polati sp. nov. We characterized both infections based on myxospore morphology, morphometry, tissue tropism, small subunit ribosomal DNA sequence and phylogenetic analysis. Plasmodia of both species were observed in gills, but had distinct tropism: M. arrabonensis is an intrafilamental vascular type, and M. polati sp. nov. is an intralamellar vascular type. We identified M. arrabonensis on the basis of myxospore characters and 100% similarity to the type DNA sequence from the closely-related host C. nasus. The small subunit ribosomal DNA sequence of M. polati sp. nov. (1946 base pairs; GenBank Accession number MH392318) had a maximum similarity of 98% with any Myxobolus sp. from other Eurasian cypriniforms. Phylogenetic analysis revealed that M. polati sp. nov. is most closely related to gill-infecting Myxobolus diversicapsularis from Rutilus rutilus (L.). The present study is the first record of myxosporean species infecting C. angorense comprising a novel species, M. polati sp. nov. and a known species M. arrabonensis.

First report and molecular prevalence of potential zoonotic Enterocytozoon bieneusi in Turkish tumbler pigeons (Columba livia domestica).

A total of 250 droppings of tumbler pigeons (Columba livia domestica, Columbidae) were collected individually from different breeders in Turkey, to investigate the presence and genotyping of microsporidian species by nested PCR and to reveal their zoonotic potential. In the present study, Enterocytozoon bieneusi was the only microsporidian species identified in 35 pigeons with an overall molecular prevalence of 14.0%. Only one known genotype zoonotic Peru6 was identified in all positive samples according to the sequence analyses of the internal transcribed spacer region of ribosomal DNA of E. bieneusi. This study represents the first report of E. bieneusi in pigeons in Turkey. Our study also confirms the competence of breeding pigeons as hosts for the zoonotic Peru6 genotype, corroborating its potential role as a source of human infection and environmental contamination. LAY SUMMARY: Microsporidia are spore-producing fungi defined as emerging opportunistic pathogens of humans. The occurrence of microsporidia in animals could be risky for human public health. Home kept breeding pigeons pose a high risk for transmission of the microsporidians to humans.

Occurrence and molecular identification of zoonotic microsporidia in pet budgerigars (Melopsittacus undulatus) in Turkey.

Encephalitozoon spp. and Enterocytozoon bieneusi are well-known microsporidian pathogens, recently classified as fungi, infecting humans and reptiles, mammals, and birds. Budgerigars (Melopsittacus undulates) are the most preferred captive pet birds in the households. Prevalence and molecular data on microsporidian species in budgerigars are scarce worldwide. The aim of the present study was to investigate the occurrence and genotypes of Encephalitozoon spp. and E. bieneusi in budgerigars, and to reveal their zoonotic potential. A total of 143 fecal samples were collected from owned healthy budgerigars in Turkey. Encephalitozoon spp. and E. bieneusi were examined by nested PCR targeting the ribosomal internal transcribed spacer (ITS) region and sequenced for identifying Encephalitozoon spp. and E. bieneusi. The overall prevalence of E. hellem and E. bieneusi was 14.7% (21/143) and 3.5% (5/143), respectively. Two genotypes of E. hellem were identified, including one known 1A (n = 18) and a novel TURK1B (n = 3). In addition, we determined two E. bieneusi genotypes, including one known N (n = 2) and a novel TURKM1 (n = 3). E. hellem 1A and novel TURK1B clustered as a sister taxon, and genotype N and novel TURKM1 genotypes fall into group 2 of E. bieneusi in the phylogenetic tree. Novel genotypes of E. hellem and E. bieneusi were described for the first time in the avian host. Moreover, E. bieneusi genotype N was first detected in avian hosts in the present study. This study contributes to the current knowledge on the molecular epidemiology and transmission dynamics of E. hellem and E. bieneusi. LAY SUMMARY: Spore producing microsporidia are ubiquitous, obligate, and intracellular fungus defined as emerging opportunistic pathogens of humans, livestock, companion animals, wild mammals, birds, and water worldwide. The occurrence of microsporidia in animals could be risky for human public health.

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.

Morphological and molecular identification of Hysterothylacium larvae (Nematoda: Raphidascarididae) in marine fish from Tunisian Mediterranean coasts.

The taxonomy of Hysterothylacium genus in Mediterranean waters remains incomplete and unresolved. The aim of the current study was to investigate the morphological and molecular identification of selected species of Hysterothylacium larvae in marine fish from the Tunisian Mediterranean coasts. A total of 192 marine fish samples were examined. In total, thirty-seven third-stage larvae of Hysterothylacium were morphologically identified as Hysterothylacium type V. In the present study, representatives of this type from the Mediterranean Sea were genetically characterized for the first time by sequencing the rDNA ITS (ITS1-5.8S-ITS2) regions and mtDNA cox2 gene. This study represents the first report of Hysterothylacium type V from the Mediterranean Sea. We also report Mullus barbatus, M. surmuletus, and Pagellus erythrinus as new hosts for this larval type. Based upon molecular and phylogenetic analyses considering the rDNA ITS regions, the Hysterothylacium type V described here was classified as a new genotype, named Genotype B. The valid genetic data of the described Hysterothylacium type V in the present study can be used to establish the phylogenetic relationships among Hysterothylacium species from the Mediterranean Sea and worldwide for future research.

Occurrence of Anisakis pegreffii (Nematoda: Anisakidae) Larvae in Imported John Dory (Zeus faber) from Senegalese Coast Sold in Turkish Supermarkets.

BACKGROUND: West African goatfish Pseudupeneus prayensis, bluespotted seabream Pagrus caeruleostictus and John Dory Zeus faber are commercially marketed as fresh and frequently imported from Senegalese coast (FAO area 34.3.12) in Turkish supermarkets. PURPOSE: The aim of the current study was to collect data of occurrence and molecular identification of Anisakis species in imported P. prayensis, P. caeruleostictus and Z. faber caught in the Senegalese coast and to support epidemiological report for a risk evaluation of Anisakis species in Turkish supermarkets. METHODS: Forty imported fish from each species at a total of 120 samples were investigated for the presence of Anisakis larvae. Based on ITS region of RFLP analysis Anisakis larvae were identified and randomly selected five larvae were also sequenced for further confirmation for cox2 gene. RESULTS: No Anisakis larvae were isolated from P. prayensis, P. caeruleostictus whereas Anisakis larvae were only found in Z. faber. A total of 156 Anisakis larvae were collected from Z. faber. All larvae were molecularly identified as Anisakis pegreffii. The prevalence (%), intensity and abundance of Anisakis infection in Z. faber were detected to be 82.5%, 8.3 and 6.8, respectively. CONCLUSION: This is the first assessment of the occurrence of A. pegreffii in imported Z. faber from the Senegalese coast in Turkish supermarkets. Moreover, consuming imported P. prayensis and P. caeruleostictus present low to non-existent risk for anisakiasis in Turkish consumers. Furthermore, the presence of A. pegreffii larvae in imported Z. faber from the Senegal waters could have public health implications in Turkish consumers.

On the occurrence and molecular identification of Contracaecum larvae (Nematoda: Anisakidae) in Mugil cephalus from Turkish waters.

Anisakis and Contracaecum species are fish borne zoonotic nematodes. In our previous studies, other larval anisakid and raphidascarid nematodes, Anisakis and Hysterothylacium species, were genetically identified in marine fish from Turkish waters. However, there is no information on molecular identification of larval Contracaecum species in marine fish from Turkey. Therefore, the aim of this study was only to investigate the presence and molecular identification of Contracaecum species in commonly commercialized marine fish from Turkish waters. A total of 475 marine fish, which belong to 21 different species, were sampled from the Aegean (FAO 37.3.1), Mediterranean (FAO 37.3.2), and Black Sea (FAO 37.4.2). The prevalence of Contracaecum L3 larvae in the Aegean Sea was identified as 10% in Mugil cephalus. All Contracaecum L3 larvae were molecularly characterized with RFLP targeting the ITS region and rrnS gene. Moreover, all larvae were analyzed by sequencing of ITS region, rrnS and cox2 gene. All Contracaecum larvae were identified as C. overstreeti based on the cox2 sequence analysis. This is the first report of C. overstreeti larvae in M. cephalus as paratenic and intermediate hosts. Furthermore, the analysis reveals novel information on ITS region. Additionally, the rrnS gene of C. overstreeti was also achieved and deposited in Genbank for the first time. The PCR-RFLP patterns of the ITS region and rrnS gene from C. overstreeti were presented in the present study. Consequently, the presence of C. overstreeti larvae in M. cephalus from the Aegean Sea may also potentially capable of inducing allergic sensitization in humans.

Molecular Detection of Zoonotic Microsporidia in Domestic Cats in Turkey: A Preliminary Study.

OBJECTIVE: This preliminary study was conducted to reveal that the molecular identity of Encephalitozoon spp. and Enterocytozoon bieneusi in indoor domestic cats' fecal samples from Turkey was screened using the PCR. MATERIALS AND METHODS: Nested PCR was performed using MSP and EBITS primers. All of the amplification products were sequenced to identify the microsporidia species. RESULTS: Four (5.5%) and three (4.1%) genomic DNA isolates of the fecal samples from 72 indoor domestic cats showed amplification of the ITS regions of E. bieneusi and Encephalitozoon spp., respectively. Two different genotypes, D and IV, of E. bieneusi were determined in two cats each based on the ITS sequence analyses. Moreover, Encephalitozoon spp. sequence analyses revealed three isolates belonging to E. intestinalis. CONCLUSIONS: This preliminary study has provided the first molecular data on the zoonotic genotypes of E. bieneusi and E. intestinalis in cats in Turkey. Furthermore, E. bieneusi genotype IV (accession number MG727664) was submitted to GenBank for the first time in the Western Palearctic Region as hosted by a domestic cat. Additionally, E. intestinalis (accession number MG570080) was also submitted to GenBank as a valid ITS sequence for the first time as hosted by a domestic cat worldwide.

Occurrence and molecular characterization of Clinostomum complanatum (Trematoda: Clinostomidae) in freshwater fishes caught from Turkey.

The metacercariae of Clinostomum species which known as yellow grubs have zoonotic potential by infecting humans. In the present study, a total of 403 freshwater fish specimens belonging to different genera from Central Anatolia Region of Turkey were examined for yellow grub metacercariae infections. Only three specimens belonging to Squalius cephalus were found to be infected with metacercariae with a prevalence on this host species of 2.4% and an overall prevalence of 0.7%. All the metacercariae were morphologically identified as Clinostomum complanatum. Partial fragments of mitochondrial cytochrome oxidase I (mt-COI) gene and internal transcribed spacer 2 (ITS-2) were amplified for sequence and phylogenetic analyses. The sequence analyses of ITS-2 and mt-COI revealed three and nine polymorphic sites leading to detection of four and five haplotypes within the related gene regions, respectively. Moreover, the intraspecific genetic distances for C. complanatum isolates ranged from 0.0 to 0.7% for ITS-2 and 0.0 to 1.4% for mt-COI data sets. Consequently, the present study has provided first combined morphologic and molecular data on C. complanatum infecting Turkish freshwater fishes.