First Molecular Evidence of Leishmania Infantum in Domestic Cats and Associated Risk Factors from the Black Sea Region of Türkiye.

PURPOSE: The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. METHODS: Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020-2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. RESULTS: All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L-) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. CONCLUSION: This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone.

Identification and distribution of some medico-veterinary important pathogens in muscid flies in two geographical regions of Türkiye.

Some dipteran flies play an important role in the transmission of pathogens such as viruses, bacteria, fungi, protozoan and metazoan parasites in humans and other animals. Despite this importance, knowledge of the prevalence and molecular characteristics of some pathogens in flies is limited, and no data are available for Türkiye. In this study, we investigated the possible vector role of muscid fly species for the transmission of Enterocytozoon bieneusi Desportes (Chytridiopsida: Enterocytozoonidae), Encephalitozoon spp., Coxiella burnetii Derrick (Legionellales: Coxiellaceae) and Thelazia spp. using polymerase chain reaction (PCR) and sequence analysis. The flies were trapped in different animal-related places and surroundings from two different geographical regions of Türkiye including Central Anatolia and Middle Black Sea. According to the morphological keys, 850 (85%), 141 (14.1%) and 6 (0.6%) of the total of 1000 fly specimens identified as Musca domestica Linnaeus (Diptera: Muscidae), Stomoxys calcitrans Linnaeus (Diptera: Muscidae) and Musca autumnalis De Geer (Diptera: Muscidae), respectively. The other species including Haematobia irritans Linnaeus (Diptera: Muscidae), Muscina stabulans Fallén (Diptera: Muscidae) and Hydrotaea ignava Harris (Diptera: Muscidae) were each represented by a single specimen. Screening of the pathogens identified E. bieneusi only in M. domestica with a prevalence of 2.4%. Sequence analyses identified three known genotypes, Type IV, BEB6 and BEB8, and one novel genotype named AEUEb of E. bieneusi in M. domestica. Coxiella burnetii was detected in M. domestica and S. calcitrans with prevalences of 2.9% and 2.8%, respectively. The one specimen of H. ignava was also positive for C. burnetii. Encephalitozoon spp. and Thelazia spp. were not found in the examined specimens. Our results contribute to the current knowledge on the vector potential of muscid flies and their possible role in the transmission dynamics of certain pathogens, especially in regions where diseases are prevalent and affect public and animal health.

Molecular detection and genotyping of Dientamoeba fragilis and Blastocystis sp. in housefly Musca domestica (Diptera: Muscidae): first report for Dientamoeba fragilis.

Dientamoeba fragilis and Blastocystis sp. are single-celled protozoan parasites of humans and animals. Although they are found in the intestines of healthy hosts, the pathogenicity of them is still unclear. To date, there is no report on D. fragilis and only two studies (without subtyping) on the occurrence of Blastocystis sp. in Musca domestica. In this study, fly samples were collected from livestock farms and their surroundings in the Kirsehir province (Central Anatolia Region) of Türkiye from May to August 2023. A total of 150 microscopically identified M. domestica samples were analyzed for the detection of D. fragilis and Blastocystis sp. molecularly. The overall prevalence of Blastocystis sp. and D. fragilis in M. domestica was determined to be 3.3% (5/150) and 8.0% (12/150), respectively. The SSU rRNA gene sequences of the isolates indicated genotype 1 of D. fragilis. Eleven isolates were identical and represented a single isolate (KAU-Dfrag1). BLAST analysis of KAU-Dfrag1 indicated identity with the isolates reported from humans, cattle, sheep, and budgerigars. The other isolate (KAU-Dfrag2) was polymorphic at two nucleotides from KAU-Dfrag1 and three nucleotides from known genotypes from GenBank and represented a variant of genotype 1. The Blastocystis sp. isolates were found to be identical and represent a single genotype (KAU-Blast1). BLAST analysis revealed that the KAU-Blast1 genotype belonged to the potentially zoonotic subtype 5 (ST5) and exhibited the highest genetic identity (ranging from 99.4 to 99.6%) with pigs, cattle, and sheep from different countries. Our study provides the first data on the molecular prevalence, epidemiology, and genotypic characterization of D. fragilis and Blastocystis sp. in M. domestica.

An Overview of One Health Concept Focusing on Toxoplasmosis.

The "One Health" concept is a universal approach to sustainably balancing and optimizing the health of humans, animals, and ecosystems. This approach is based on the health of humans, domestic and wild animals, and plants in a wider environment in which self-renewable ecosystems exist, with essential characteristics of integration, unifying and holistic perspective. Toxoplasmosis, one of the most common zoonotic infections in both terrestrial and oceanic ecosystems in the world, is an ideal model disease for the "One Health" approach. Toxoplasmosis is a zoonotic disease caused by the obligate intracellular pathogen protozoan Toxoplasma gondii. In the life cycle of T. gondii, the definitive host is domestic cats and felines, and the intermediate hosts are all mammals (including humans), birds and reptiles. The infected cats have primary importance and play a crucial role in the contamination of habitats in the ecosystems with T. gondii oocysts. Thus, ecosystems with domestic cats and stray cats are contaminated with cat feces infected with T. gondii oocytes. T. gondii positivity has been scientifically demonstrated in all warm-blooded animals in terrestrial and aquatic habitats. The disease causes deaths and abortions in farm animals, resulting in great economic losses. However, the disease causes great problems in humans, especially pregnant women. During pregnancy, it may have effects such as congenital infections, lesions in the eye and brain of the fetus, premature birth, intrauterine growth retardation, fever, pneumonia, thrombocytopenia, ocular lesions, encephalitis, and abortion. The mechanism of death and abortion of the fetus in a pregnant woman infected with T. gondii occurs as a result of complete disruption of the maternal immune mechanism. The struggle against toxoplasmosis requires the universal collaboration and coordination of the World Organization for Animal Health, the World Health Organization and the World Food Organization in the "One Health" concept and integrative approaches of all responsible disciplines. Establishing universal environmental safety with the prevention and control of toxoplasmosis requires the annihilation of the feces of the infected cats using suitable techniques firstly. Then routinely, the monitoring and treatment of T. gondii positivity in cats, avoiding contact with contaminated foods and materials, and development of modern treatment and vaccine options. Particularly, mandatory monitoring or screening of T. gondii positivity during the pregnancy period in humans should be done. It would be beneficial to replace the French model, especially in the monitoring of disease in humans. In this article, the ecology of toxoplasmosis was reviewed at the base of the "One Health" concept.

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.

Molecular survey of Anaplasma phagocytophilum and related variants in water buffaloes: The first detection of Anaplasma phagocytophilum-like 1.

Anaplasma phagocytophilum infects various hosts and lead to mild to severe infection. Currently, two A.phagocytophilum-related variants have been documented in different countries. Although limited, there are studies revealing the presence of A.phagocytophilum in water buffaloes, but no study investigating A.phagocytophilum-like 1 and -like 2. A.phagocytophilum and related variants were investigated using PCR, PCR-RFLP, and DNA sequence analysis in water buffaloes in Türkiye. 364 buffalo blood samples were examined for A.phagocytophilum and related strains. Seven buffaloes were determined to be positive with PCR and PCR-RFLP revealed that all samples were A.phagocytophilum-like 1. According to the partial sequence of 16 S rRNA gene, A.phagocytophilum like-1 may split into two different variants. This work supplies the first molecular report of A.phagocytophilum-like 1 in water buffaloes. However, a lack of information is present on the pathogen's clinical manifestations and vector species. There is still a need to investigate vectors and clinical signs of the pathogen.

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.

Molecular Characterization and Expression Analysis of the Sterol-carrier Protein-2 Fragment in Anopheles sacharovi Generations

Objective: It was aimed to characterize the sterol carrier protein-2 (SCP-2) gene in Anopheles sacharovi using molecular methods for the first time, and to reveal the expression levels of An. sacharovi in the developmental stages and female generation in different tissues such as salivary gland, midgut and adipose tissue. Methods: The adult female An. sacharovi collected from the Sultan Sazligi region and the development stages in the insectarium constituted the study material. cDNA isolation was performed following total RNA extraction from An. sacharovi strains. The 216 bp fragment of the SCP-2 gene was amplified with optimized primers in cDNA templates and was sequenced. Genetic characterization of the sequences was provided in silico analysis. Results: Twelve of the SCP-2 nucleotide sequences of 14 isolates included in the sequence analysis were 100% identical and the SCP-2 sequences of the other two isolates that were homologous to each other showed a single nucleotide change at base 183. The 216 bp fragment of the SCP-2 gene region was found encoding the 72 amino acid chain. SCP-2 gene sequences clustered the isolates monophyletically on the basis of mosquito species and strains, and that Anopheles sacharovi isolates formed a subcluster together with Anopheles stephensi and Anopheles funestus within the Anopheles cluster in phylogenetic analysis. Because of q-polymerase chain reaction-mediated expression analysis, SCP-2 gene was expressed highest in adult males, followed by an adult female, ss L4, L3, L2, L1, and pupal stages, respectively. In adult female tissues, the SCP-2 gene was expressed the highest in the fat body, followed by the midgut and salivary glands, respectively. Conclusion: SCP2, which is an important vaccine candidate or target drug site for Anopheles sacharovi with high vector potential, was firstly characterized in this study and the developmental stages and expression differences in the tissues of the mosquito were revealed.

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.

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.

Vector-borne Zoonotic Diseases in Turkey: Rising Threats on Public Health

Vector-borne zoonotic diseases (VBZDs) are a major problem for public health and animal welfare all over the world. In recent years, there has been an alarming increase in VBZDs, mainly caused by new or re-emerging arboviruses, bacteria and parasites. The World Health Organization enumerated 10 threats to global health for 2019, notably emphasizing climate change and emerging pathogens as growing priorities. It is important to review potential threats and develop new control programs for rising threats against human health and safety. Changes in host and vector population diversity and density may affect pathogen transmission patterns and influence VBZD emergence processes. In addition to environmental and climate-related changes, human and animal migratory patterns pose future threats. The geographic location and habitat features of Turkey support the establishment of many arthropod species as vectors of various diseases. To date, a total of 107 zoonotic infections have been reported originating from Turkey. Arthropods transmit 19 of 107 such infections, including 2 mosquito-borne, 9 tick-borne, 1 sandfly-borne, 3 flea-borne, 1 simuliid-borne, 1 mite-borne and 2 fly-borne diseases. In this review, we focus on the present status of knowledge on VBZDs as a rising threat to public health in Turkey to provide a foundation for future control efforts.

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.

Molecular detection and genotyping of microsporidia species in chickens in Turkey.

Microsporidia are obligate intracellular pathogens that infect various hosts including invertebrates and vertebrates. Despite the importance, knowledge on the prevalence and molecular characteristics of microsporidia in chickens is limited, and no data are available for Turkey. A total of 300 fecal samples from chickens in the Central Anatolia Region of Turkey were analyzed by using a nested polymerase chain reaction assay targeting the rRNA internal transcribed spacer (ITS) region for the common microsporidia species. Corresponding PCR amplicons from the positive samples were sequenced for genotyping. Enterocytozoon bieneusi was identified in 22 (7.3 %) samples, whereas Encephalitozoon spp. was not detected. The prevalence of E. bieneusi was 63.6 % in Kayseri and 36.4 % in Nevsehir provinces, and 8.8 % in soft fecal samples and 9.7 % in diarrhoeic samples. No infections were found in Kirsehir Province. Significant differences were found for the distribution of E. bieneusi among provinces and fecal conditions. Infections were found only in free-range chickens. As a result of ITS region sequencing, two genotypes were characterized. The novel genotype ERUNT1 (n = 21), belonging to zoonotic group 1, was the most common genotype throughout the study area. The other known genotype, ERUSS1 (n = 1), had a restricted distribution and was previously detected in cattle and sheep in the same region. Our study provides the first data on microsporidia species from chickens in Turkey. None of these genotypes have been reported in humans; thus, the risk potential for public health is limited but needs further investigation.