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.

Xenorhabdus and Photorhabdus Bacteria as Potential Candidates for the Control of Culex pipiens L. (Diptera: Culicidae), the Principal Vector of West Nile Virus and Lymphatic Filariasis.

Vector-borne diseases pose a severe threat to human and animal health. Culex pipiens L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress Cx. pipiens populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [Xenorhabdus and Photorhabdus spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) (Sterinernema spp. and Heterorhabditis spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 107 cells mL-1) of four different symbiotic bacteria (Xenorhabdus nematophila, X. bovienii, X. budapestensis, and P. luminescens subsp. kayaii) was assessed against different development stages of Cx. pipiens (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the X. bovienii KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides.

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 Prevalence and Phylogenetic Characterization of Enterocytozoon bieneusi in Sheep in the Van Region

OBJECTIVE: In this study, we aimed to determine the prevalence of Enterocytozoon bieneusi in healthy sheep in Van province using molecular techniques and to reveal genotypes of the detected isolates. METHODS: A total of 200 healthy appearance sheep comprise 38 male and 162 female, 32 preweaned, 38 postweaned lamb and 130 adult sheep from several farms in the Van region were included in the study between May and September 2021. Genomic DNA (gDNA) extractions were utilized on fecal samples collected from sheep by commercial kits, and E. bieneusi DNA was investigated by Nested polymerase chain reaction (PCR) amplifying ITS rRNA in the gDNA isolates. PCR products of the positive isolates were subjected to sequence analyze for genotyping and phylogenetic analyses of E. bieneusi. RESULTS: E. bieneusi DNA was determined in 16 out of 200 examined sheep fecal gDNA samples (8.0%) by Nested PCR. The highest E. bieneusi prevalence was determined in preweaned lambs with a rate of 18.8%. This was followed by postweaned lambs and adult sheep with a prevalence of 10.5% and 4.6%, respectively. The prevalence of the infection in males and females was 7.9% and 9.3%, respectively. All the ITS rRNA amplicons from 16 positive isolates were subjected to sequence analyses for genotyping and phylogenetic analyses. Sequence analyses revealed that all the isolates determined in sheep belonged to the BEB6 genotype and clustered in genogroup 2 of E. bieneusi with the BEB6 isolates from different hosts in several countries. CONCLUSION: Molecular epidemiological data on the prevalence of E. bieneusi in sheep in Turkey were obtained with this study and the common genotype was determined as BEB6 in the research area. The obtained data contribute to the molecular epidemiology and diversity of E. bieneusi in sheep.

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.

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.

Seroprevalence of Neospora caninum in Goats from Korkuteli District of Antalya

Objective: This study aimed to determine the seroprevalence of Neospora caninum in goats from the Korkuteli district of Antalya. Methods: During the study, sera samples were obtained from 184 female goats and analyzed for the presence of antibodies against N. caninum using a commercial ELISA kit. Results: Seroprevalence of N. caninum was determined as 4.89%. Seropositivity of N. caninum in goats was not statistically significant (p>0.05) in terms of study centers, age groups, and abort situation. Conclusion: This study reports the first data on the presence and seroprevalence of N. caninum in the goats in the region.

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 molecular characterization of hypodermin genes of Hypoderma bovis and serodiagnosis of bovine hypodermosis with recombinant hypodermin C antigen and a synthetic peptide containing its linear B-cell epitope.

Hypodermins A (HA), B (HB), and C (HC) of warble flies are modulatory antigens involved in host inflammation and immune responses during migration of the warble fly larvae through host connective tissues. In the current study, molecular characteristics of the genes encoding HA, HB, and HC were revealed from cDNA constructs of third-instar larvae of Hypoderma bovis. The open reading frame (ORF) of each hypodermin gene was amplified with modified gene-specific primers, and the resulting PCR products were cloned into pGEM-T Easy Vector to produce recombinant plasmids (rHA, rHB, and rHC). The ORF sequences of rHA, rHB, and rHC genes are 705 bp, 771 bp, and 783 bp long and encode proteins of 234, 256, and 263 amino acids with predicted sizes of 25.74 kDa, 27.79 kDa, and 28.51 kDa, respectively. The rHC gene was subcloned into the pET 100/D-TOPO Expression Vector, and the recombinant HC was purified using affinity chromatography. Western blotting indicated that rHC was recognized by the sera of cattle naturally infested with H. bovis. The rHC and a synthetic peptide (sHC) containing its linear B cell-specific epitope were evaluated as serological markers in indirect ELISA (iELISA) for the diagnosis of bovine hypodermosis. Both sHC and rHC iELISAs had sensitivity values equal to or higher than 90 % and specificity values of 100 %. A total of 200 serum samples from cattle in the Central Anatolia Region of Turkey were also analyzed by rHC and sHC-iELISAs to reveal the seroprevalence of bovine hypodermosis. The results of both iELISAs were consistent with one another and revealed a hypodermosis prevalence of 62 %. Our study provides the first data on molecular characterization of hypodermin genes of H. bovis and indicates the efficacy of recombinant antigen and peptide-based iELISA for serodiagnosis of bovine hypodermosis.

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.

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.