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.

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.

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.

Sequence variants of CYP345a1 and CYP6a14 gene regions in Tribolium castaneum (Coleoptera: Tenebrionidae) adults treated with the novel characterized Bolanthus turcicus (Caryophyllaceae) extract.

In this study, various doses of plant extracts that obtained from Bolanthus turcicus was applied to an important storage pest Tribolium castaneum adults. Bolanthus turcicus is an endemic species and spreads on the Hasan Mountain above Karkin town (Turkey, Aksaray province). The plant species was collected from June to July with the field study to be carried out in this region. Obtained extract of plant was analyzed by gas chromatography mass spectrometry (GC-MS) method. The doses were defined during the study and the concentrations that kill 50% and 99% of the population were determined after applications. After 24 h, DNA was isolated from live and dead individuals that obtained from LC50 and LC99 concentration applications and analyzed for Cytochrome P450-mediated detoxification resistance genes, CYP345A1 and CYP6A14 gene regions, by polymerase chain reaction (PCR). CYP genes in insects are known to be rapidly regulated when exposed to insecticides. In the study, in order to screen for 206 bp and 353 bp fragments of CYP345A1 and CYP6A14 genes in T. castaneum adults were amplified using specific primers, respectively. DNA direct sequencing was performed on each template using the forward primer. When compared to the control, it is believed that mutation differences in live and dead individuals according to the sequencing results obtained from survival and dead adults, may allow these genes to play a protective role against the toxic effect of B. turcicus extract.

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.