Population genetics and detection of anthelmintic resistance of gastro-intestinal nematode species in small ruminants in Turkey

This study was carried out to determine the population genetics and anthelmintic resistance of gastrointestinal nematode species in small ruminants in Turkey. Fecal samples were collected from 1108 sheep from 19 provinces in 7 geographically distinct regions of Turkey, namely Afyonkarahisar, Konya, Antalya, Aydın, Balıkesir, Batman, Bitlis, Çanakkale, Denizli, Erzurum, Kars, Kütahya, Maraş, Ordu, Rize, Sakarya, Tekirdağ, Urfa, Uşak and 200 goats from 4 provinces in 4 geographically distinct regions, namely Antalya, Afyon, Konya and Kars in accordance with the technique between September-December 2019. The collected feces were examined microscopically in terms of parasitology with Fulleborn saturated salt flotation method. 813 (81,3%) of the sheep and 159 (79,50%) of the goats were found positive for various parasite eggs and/or oocysts. In sheep, Trichostrongylidae spp. (73,20%), Eimeria spp. (27,7%), Nematodirus spp. (26,80%), Moniezia spp. (20,70%), Marshallagia spp. (11,10%), Trichuris ovis (%) 5,60), while in goats Trichostrongylidae spp. (77%), Eimeria spp. (40%), Nematodirus spp. (24,50%), Marshallagia spp. (15,50%), Moniezia spp. (8,5%), Trichuris ovis (4%) was the most common parasites. Fecal egg count reduction test (FECRT) was performed for albendazole, ivermectin and control group with 12 sheep in each of 3 different sheep farms in Şuhut, İhsaniye and Çayırbağ provinces. Accordingly, susceptibility to albendazole was 85,9% in Şuhut, 72,6% in İhsaniye, 77,6% in Çayırbağ, susceptibility to ivermectin was 80,1% in Şuhut, 69,8% in İhsaniye, and 82,4% in Çayırbağ. In this study, species identification was carried out with PCR using DNA obtained from third stage trichostrongylid type larvae (L3) with Proteinase K enzyme in sheep for the first time in Turkey. Sequencing was used for larvae that could not be identified. H. contortus was detected at the highest prevalance in Antalya and Sakarya (100%) and the lowest in Rize (2,12%) in sheep, and, the highest prevalance in Afyonkarahisar, 92,55%, and the lowest prevalance 1,06% in Kars in goats. Teladorsagia circumcincta was detected at the highest prevalence in Erzurum (89,36%) and the lowest in Kars (1,06%) in sheep, and the highest prevalance in Konya (54,25%) and the lowest in Kars (4,25%) in goats. Ostertagia leptospicularis was detected as 1,06% in Çanakkale in sheep and was the first report in Turkey. Trichostrongylus axei was detected at the highest prevalance in Bitlis (55,31%), and lowest in Afyonkarahisar, Kütahya and Çanakkale (1,06%) in sheep, and highest prevalence in Kars (63,82%), and the lowest in Konya ( 13,82%) in goats, T. vitrinus was highest in Uşak (7,63%), lowest in Rize (1,06%) in sheep, in goats only in Afyonkarahisar (1,06%), T. colubriformis was the highest in Kars (27,65%), and the lowest in Balıkesir and Erzurum (1,06%) in sheep, and the highest in Kars (30,85%), and lowest in Antalya (1,06%) in goats. Chabertia ovina was found highest in Rize (23,40%) and lowest in Denizli and Çanakkale (1,06%) in sheep. Oesophagostomum venulosum was detected at highest prevalance in Çanakkale (25,53%), and the lowest in Denizli (1,06%) in sheep, and the highest in Konya (29,75%) and lowest in Antalya (5,31) in goats. Bunostomum trigonocephalum was found only in Kars (1,06%), Marshallagia marshalli in Denizli (1,06%), Cooperia curticei in Konya (1,06%). Benzimidazole resistance was determined in Teladorsagia circumcincta populations of sheep in Afyonkarahisar, Rize, Uşak and Erzurum with allele-specific PCR method. It was determined that benzimidazole resistance developed in H. contortus populations in Batman, Sakarya, Afyonkarahisar, Antalya, Tekirdağ, Şanlıurfa, Ordu, Balıkesir in sheep and in Afyonkarahisar and Antalya in goats. In this study, benzimidazole resistance was detected for the first time in Turkey by allele-specific PCR method on H. contortus and T. circumcincta populations. For population genetics study, Mtg15, Mtg68, Tc13604, Tc7989, Tc2066 microsatellite primers were used for third stage larvae of T. circumcincta, and Hc22193, Hc2884, Hc3086, Hc53265, Hc22c03 microsatellite primers were used for third stage larvae of H. contortus. As a result of the analysis, high within-population variation, low population genetic differentiation and high gene flow were detected in H. contortus and T. circumcincta populations in Turkey. In this study, population genetic analysis was carried out on H. contortus and T. circumcincta populations using microsatellite markers for the first time in Turkey. As a result, with this study, the molecular prevalence of T. circumcincta was carried out first time in Turkey and the molecular prevalence of H. contortus in the study provinces were determined for the first time in Turkey, and genetic characterization was carried out. Benzimidazole resistance was detected molecularly in T. circumcincta and H. contortus populations in the study provinces and homozygous susceptibility, heterozygous susceptibility and homozygous resistant alleles were determined. It has been concluded that it is necessary to increase awareness of anthelmentic resistance in veterinarians and breeders in terms of resistant H. contortus and T. circumcincta populations, and review current control ve treatment methods, and creating alternative strategies in Turkey.