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.

A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.

Genomic sequence capture of Plasmodium relictum in experimentally infected birds.

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

Occurrence and molecular characterization of Enterocytozoon bieneusi in water buffaloes (Bubalus bubalis) in Turkey.

Microsporidia are obligate intracellular fungus-like parasites that infect humans and animals worldwide. However, there is limited epidemiological data on the occurrence and molecular diversity of microsporidia in buffaloes worldwide. In the present study, fecal samples of 300 water buffaloes (Bubalus bubalis) in Kayseri, Sivas, and Samsun provinces of Turkey were investigated using two nested PCR assays targeting the rRNA of E. bieneusi and Encephalitozoon spp. All the fecal samples from water buffalo were found to be negative for Encephalitozoon spp. PCR positive isolates of E. bieneusi were bidirectionally sequenced for genotyping and phylogenetic analyses. Enterocytozoon bieneusi was the only microsporidian species identified in 8 water buffaloes with an overall molecular prevalence of 2.7%. Two known genotypes, YNDCEB-90 (n = 5) and J (n = 3) were identified by ITS sequence analysis. The YNDCEB-90 and J genotypes fall into zoonotic Group 1 and 2 of E. bieneusi in the phylogenetic tree, respectively. These findings suggested that water buffalo in Turkey are harbouring zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission to humans. This is the first report of detecting E. bieneusi genotypes J and YNDCEB-90 in water buffaloes. Further insight into the epidemiology of E. bieneusi in water buffaloes in different geographical areas in Turkey will be highly important to have determined the public health significance of this pathogen.

First report and genotyping of Dientamoeba fragilis in pet budgerigars (Melopsittacus undulatus), with zoonotic importance.

The protozoan Dientamoeba fragilis is one of the most common parasites in the digestive system of humans worldwide. The host range and transmission routes of D. fragilis, including the role of animals, are still ambiguous with few reports from non-human primates, sheep, rodents, pigs, a cat and a dog. In this study, we used microscopic and TaqMan qPCR analyses to investigate D. fragilisin 150 faecal samples from pet budgerigars (Melopsittacus undulatus) in the Central Anatolia Region of Turkey. Dientamoeba fragilis DNA was detected in 32 samples, resulting in a mean prevalence of 21.3%. In microscopic examination, trophozoites/cysts of D. fragilis were detected in 13 of 32 qPCR-positive samples. SSU rRNA sequence analyses of the qPCR-positive isolates identified genotype 1 of D. fragilis as predominant in budgerigars. Phylogenetic analyses of the SSU rRNA gene region clustered D. fragilis genotypes, as well as other trichomonads, in separate monophyletic clusters with bootstrap values ≥79.0. Our study provides the first evidence for the natural host status of pet budgerigars for D. fragilisand contributes to the knowledge of the epidemiology of this parasite. The high prevalence of genotype 1 of D. fragilis suggests that pet budgerigars are suitable reservoirs for zoonotic transmission. Our findings contribute to an increased awareness and knowledge of D. fragilis infections in the context of a one-health approach.

Genetic diversity of Ichthyophthirius multifiliis (Fouquet, 1876) infecting farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) in Turkey.

We assessed genetic diversities among Ichthyophthirius multifiliis (Ich) field isolates collected from farmed rainbow trout (Oncorhynchus mykiss) in Turkey. The overall prevalence of Ich was 35.3% (634/1798). Five novel Ich genotypes (ImulTR1 and ImulTR3-ImulTR6) were described based on mitochondrial cox-1 and nad1_b genes. The remaining genotype ImulTR2 was identical to the previously reported NY3 (or Ark9 and TW7) genotype from the United States and South Asia. Phylogenetic analysis indicated Turkish Ich isolates separated genetically into at least four distinct groups. Our study presents the first data on the genotypes of Ich in Turkey. We also provide evidence for the wide distribution of the NY3 genotype (or Ark9 and TW7) from the United States and South Asia to Turkey. Genetic diversities within the mitochondrial genes provided adequate resolution for describing novel genotypes and identifying the known genotype within Turkish Ich isolates. Description of the Ich genotypes allows for tracking of pathogen genotypes worldwide. Thus, we can better understand the connections between Ich outbreaks in the fisheries aquaculture.

Investigation of Zoonotic Cryptosporidium and Giardia intestinalis Species and Genotypes in Cats (Felis catus)

Objective: Giardia intestinalis and Cryptosporidium spp. are important zoonotic protozoan parasites that infect humans and various animals. We investigated the occurrence of G. intestinalis and Cryptosporidium spp. infection in cats. To provide data on the zoonotic transmission dynamics of these parasites, genotypes of the detected isolates were investigated through DNA sequence characterization. Methods: A total of 100 fecal samples were collected from cats between June and October 2020 in Kayseri and Samsun provinces. Fecal samples were examined by nested polymerase chain reaction (PCR), targeting the ß-giardin gene of G. intestinalis and small subunit (SSU) rRNA gene of Cryptosporidium spp. All PCR products were sequenced for genotyping. Results: Of the samples examined, Giardia intestinalis was determined in 8 samples (8.0%), whereas none of the samples were found positive for Cryptosporidium spp. Sequence analyses of the ß-giardin PCR products indicated that all G. intestinalis isolates were classed into the zoonotic assemblage B. Conclusion: This study adds to the current data on the molecular epidemiology of cryptosporidiosis and giardiasis in cats. The findings also highlight the potential risk of cats for public health concerning the zoonotic transmission dynamics of G. intestinalis.

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

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

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.

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.

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.

Bovine Babesiosis in Turkey: Impact, Current Gaps, and Opportunities for Intervention.

Bovine babesiosis is a global tick-borne disease that causes important cattle losses and has potential zoonotic implications. The impact of bovine babesiosis in Turkey remains poorly characterized, but several Babesia spp., including B. bovis, B. bigemina, and B. divergens, among others and competent tick vectors, except Rhipicephalus microplus, have been recently identified in the country. Bovine babesiosis has been reported in all provinces but is more prevalent in central and highly humid areas in low and medium altitude regions of the country housing approximately 70% of the cattle population. Current control measures include acaricides and babesicidal drugs, but not live vaccines. Despite the perceived relevant impact of bovine babesiosis in Turkey, basic research programs focused on developing in vitro cultures of parasites, point-of-care diagnostic methods, vaccine development, "omics" analysis, and gene manipulation techniques of local Babesia strains are scarce. Additionally, no effective and coordinated control efforts managed by a central animal health authority have been established to date. Development of state-of-the-art research programs in bovine babesiosis to address current gaps in knowledge and implementation of long-term plans to control the disease will surely result in important economic, nutritional, and public health benefits for the country and the region.

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.

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

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

Investigation of Anisakis larvae in different products of ready-to-eat fish meat and imported frozen fish in Turkey.

Globalization opens new market areas and affects food consumption habits, resulting in rapid and remarkable cultural change. Food habits such as consumption of raw fish meat have become popular, resulting in increased risk of emerging infectious diseases. Anisakis simplex sensu stricto (s.s) and A. pegreffii are the most common and important fish-borne zoonotic nematodes responsible for human anisakiasis, which occurs through the consumption of raw or undercooked fish as well as cooked fish due to their heat-stable allergens. Here, we investigated the prevalence, intensity, and abundance of Anisakis larvae in imported fish and ready-to-eat local fish products in Turkey. A total of 205 ready-to-eat fish products, 100 imported frozen Atlantic salmon (Salmo salar) fillets, and 100 imported frozen whole Atlantic mackerel (Scomber scombrus) were sampled from supermarkets, sushi restaurants, and fish markets. All samples were individually examined using a pepsin digestion technique. In total, 602 Anisakis type I larvae were recovered from 98/100 mackerel. No larvae were found in ready-to-eat products or frozen Atlantic salmon fillets. Overall, 8.8% of the larvae were found in the muscle tissue. The overall mean intensity and abundance of infection in mackerel were 6.14 and 6.02, respectively. The larvae were molecularly identified and their phylogenetic relationships with the relevant Anisakis sequences in GenBank were investigated. For this purpose, a subsample of randomly selected 100 Anisakis larvae were analyzed with PCR-RFLP of the ITS region. The larvae were identified as A. simplex (s.s.) (n = 87) and hybrids (n = 13). ITS and cox2 gene regions of all hybrids and randomly selected 50 A. simplex (s.s.) larvae were sequenced for species confirmation and phylogenetic analyses. No intraspecific nucleotide variation was found among the ITS sequences of either species. Seven and three haplotypes, respectively, were identified for A. simplex (s.s.) and hybrid species according to DNA polymorphism of the cox2 gene. Hybrids in our study clustered within the common A. simplex (s.s.) clade in the cox2 phylogenetic tree indicating the dominance of A. simplex (s.s) in the catching area of Atlantic mackerel. Consequently, our study indicates high occurrence of A. simplex (s.s.) larvae with an overall 98.0% prevalence in imported Atlantic mackerel, and highlights the importance of these fish as potential reservoirs for human allergic anisakiasis in Turkey and possibly in other countries.

Prevalence and genetic diversity of avian haemosporidian parasites at an intersection point of bird migration routes: Sultan Marshes National Park, Turkey.

Avian haemosporidians (Haemosporida) represent a globally distributed, species-rich multiparasite-multihost host-parasite system. Each year, many of these parasite lineages are carried between temperate and tropical regions by migratory birds. While several factors can limit the transmission of avian haemosporidians to new areas, recent studies have shown that some abundant parasites can sometimes disperse and be transmitted in new areas to become emerging infectious diseases. In this study, we investigated the prevalence and diversity of avian haemosporidian parasites in Sultan Marshes National Park (SMNP), a major stopover site in the eastern Mediterranean flyway, and we evaluated the potential for avian haemosporidians in SMNP to be transmitted to areas outside of their known distributions. We sampled a total of 565 migratory and resident birds belonging to 39 species and 23 families. We applied both molecular and microscopic methods to detect and identify avian haemosporidian infections and also quantified the frequency of potential abortive infections. We identified a total of 52 different mitochondrial cytochrome b (cyt b) parasite lineages belonging to the genera Plasmodium (N = 12), Haemoproteus (N = 31), and Leucocytozoon (N = 9) in 193 (34.2%) infected birds. Ten of the lineages were reported for the first time. Our findings show that numerous parasite lineages are actively transmitted among resident bird species of SMNP. Our findings also revealed new parasite-host interactions while considering the role of possible abortive infections. The relatively high frequency of presumed abortive infections suggests that analyses of datasets generated only by PCR-based methods should be interpreted with caution. We also compared the prevalence and distribution of avian haemosporidian infections in both resident and migratory bird species and showed that haemosporidian prevalence was related to bird migratory behavior. The results of this study contribute to a better understanding of the ecological and genetic adaptations associated with changes in transmission areas of avian haemosporidian parasites.

Molecular prevalence and genotyping of Giardia duodenalis in cattle in Central Anatolia Region of Turkey.

The molecular prevalence and genotypes of Giardia duodenalis in cattle were investigated. A total of 450 fecal samples were collected from cattle in three provinces of Central Anatolia from August 2017 to July 2019. Genomic DNA was extracted from the fecal samples and used in molecular analysis carried out by nested PCR analyses of the ß-giardin (bg) gene of G. duodenalis. Positive samples were further analyzed by nested PCR at two gene loci (triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh)) for genotyping of G. duodenalis isolates. PCR analyses of the bg gene indicated that the overall prevalence of G. duodenalis was 30.2%. However, lower rates were determined with PCR analyses for gdh and tpi loci. The sequence analyses of the bg, gdh, and tpi genes revealed the presence of zoonotic assemblage A and livestock-specific assemblage E. Combined-sequence analyses revealed that assemblage E was the most common in the study area. Our study provides the first data on the wide prevalence of livestock-specific assemblages E in cattle in Turkey. The prevalence of assemblage A in cattle also reveals the importance of cattle for zoonotic transmission of giardiasis in Turkey.