ZUHAL ÖNDER, Alparslan YILDIRIM, ABDULLAH İNCİ, Önder DÜZLÜ, ARİF ÇİLOĞLU

Molecular Prevalence, Phylogenetic Characterization and Benzimidazole Resistance of Haemonchus contortus from Sheep

Bu çalışmada koyunlarda Haemonchus contortus'un moleküler prevalansı ve karakterizasyonu ile benzimidazol (BZ) dirençliliğinin belirlenmesi amaçlanmıştır. Araştırma yöresinde toplam 300 koyundan dışkı örnekleri toplanmış ve fekal yüzdürme yöntemiyle incelenmiştir. Trichostrongylid yumurtalarıyla pozitif belirlenen örneklerde H. contortus identifikasyonu için qPCR analizleri gerçekleştirilmiştir. Filogenetik analizler H. contortus izolatlarının ribosomal ITS-2 ve mt-COI gen bölgeleri üzerinde yürütülmüştür. BZ duyarlı ve dirençli allel sıklıkları ?-tubulin izotip 1 gen bölgesindeki tek nükleotid polimorfizmi (SNPs) temelinde qPCR ile belirlenmiş, ayrıca ilgili genin sekans analizleri gerçekleştirilmiştir. Trichostrongylid yumurtalarıyla pozitif 145 örneğin 36'sında (%24.8) qPCR ile H. contortus identifiye edilmiştir. ITS-2 gen bölgesi filogenetik analizine göre H. contortus izolatlarının %100 identik oldukları ve GenBank'ta mevcut izolatlarla %0.1 genetik farklılık gösterdikleri belirlenirken mt-COI sekans analizleri izolatların ortalama %97.4±0.5 identiklik ve GenBank'ta mevcut izolatlarla da %5.5±0.8 farklılık gösterdiklerini ortaya koymuştur. H. contortus populasyonlarında BZ duyarlı ve dirençli allel sıklıkları qPCR ile sırasıyla %87.1±16,2 ve %12.9±16.2 belirlenmiştir. Dirençli allele ait izolatların sekans analizlerinde yalnızca 200. kodonda SNPs saptanmıştır. Bu çalışma ile Türkiye'de koyunlarda H. contortus'un moleküler prevalansı ve genetik karakterizasyonu üzerine ilk veriler sağlanmış ve H. contortus popülasyonlarında BZ dirençliliği ortaya konmuştur

Koyunlarda Haemonchus contortus'un Moleküler Prevalansı, Filogenetik Karakterizasyonu ve Benzimidazol Dirençliliği

This study was conducted to determine the molecular prevalence and characterization of Haemonchus contortus from sheep along with the benzimidazole (BZ) resistance in H. contortus populations. Fecal samples were collected from a total of 300 sheep in research area and analyzed by fecal flotation. qPCR assays were utilized on trichostrongylid egg positive samples in order to identify H. contortus. Phylogenetic analyses were performed on ribosomal ITS-2 and mt-COI gene regions. BZ sensitive and resistant allele frequencies were determined by qPCR along with sequence analyses of the ?-tubulin isotype 1 gene for single nucleotide polimorphizms (SNPs). H. contortus was identified in 36 (24.8%) out of 145 trichostrongylid egg positive samples. H. contortus isolates showed 100.0% identity to each other and 0.1% difference with the isolates available in the GenBank based on phylogenetic analyses of ITS-2 gene while mt-COI analyses of the isolates exhibited a mean of 97.4±0.5% identity to each other and 5.5±0.8% difference with the isolates in GenBank. BZ sensitive and resistant allele frequencies in H. contortus populations were determined as 87.1%±16.2 and 12.9%±16.2, respectively. SNPs were detected only in the codon 200 of the sequenced isolates belong to resistant allele. This study provides the first data on molecular prevalence, phylogenetic characterization and BZ resistance in H. contortus populations from sheep in Turkey

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Brasil BS, Nunes RL, Bastianetto E, Drummond MG, Carvalho DC, Leite RC, Molento MB, Oliveira DA: Genetic diversity patterns of Haemonchus placei and Haemonchus contortus populations isolated from domestic ruminants in Brazil. Int J Parasitol, 42, 469-479, 2012. DOI: 1016/j.ijpara.2012.03.003
von Samson-Himmelstjerna G, Harder A, Schnieder T: Quantitative analysis of ITS2 sequences in trichostrongyle parasites. Int J Parasitol, 32, 1535, 2002. DOI: 10.1016/S0020-7519(02)00163-7
O'Connor LJ, Walkden-Brown SW, Kahn LP: Ecology of the free-living stages of major trichostrongylid parasites of sheep. Vet Parasitol, 42, 1-15, DOI: 10.1016/j.vetpar.2006.08.035
Akkari H, Jebali J, Gharbi M, Mhadhbi M, Awadi S, Darghouth MA: Epidemiological study of sympatric Haemonchus species and genetic characterization of Haemonchus contortus in domestic ruminants in Tunisia. Vet Parasitol, 193, 118-125, 2013. DOI: 10.1016/j. vetpar.2012.12.014
Hussain T, Periasamy K, Nadeem A, Babar ME, Pichler R, Diallo A: Sympatric species distribution, genetic diversity and population structure of Haemonchus isolates from domestic ruminants in Pakistan. Vet Parasitol, 206, 188-199, 2014. DOI: 10.1016/j.vetpar.2014.10.026
Molento MB, Fortes FS, Pondelek DA, Borges Fde A, Chagas AC, Torres-Acosta JF, Geldhof P: Challenges of nematode control in ruminants: Focus on Latin America. Vet Parasitol, 180, 126-132, 2011. DOI: 1016/j.vetpar.2011.05.033
Von Samson-Himmelstjerna G, Blackhall WJ, McCarthy JS, Skuce PJ: Single nucleotide polymorphism (SNP) markers for benzimidazole resistance in veterinary nematodes.Parasitology, 134, 1077-1086,2007. DOI: 10.1017/S0031182007000054
von Samson-Himmelstjerna G, Walsh TK, Donnan AA, Carriere S, Jackson F, Skuce PJ, Rohn K, Wolstenholme AJ: Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing. Parasitology, 136, 349-358, 2009. DOI: 10.1017/ S003118200800543X
Hodgkinson JE, Clark HJ, Kaplan RM, Lake SL, Matthews JB: The role of polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int J Parasitol, 38, 1149-1160, DOI: 10.1016/j.ijpara.2008.02.001
Beech RN, Skuce P, Bartley DJ, Martin RJ, Prichard RK, Gilleard JS: Anthelmintic resistance: Markers for resistance, or susceptibility? Parasitology, 138, 160-174, 2011. DOI: 10.1017/S0031182010001198
Barrere V, Alvarez L, Suarez G, Ceballos L, Moreno L, Lanusse C, Prichard RK: Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the beta- tubulin isotype 1 encoding gene in Haemonchus contortus. Vet Parasitol, , 344-349, 2012. DOI: 10.1016/j.vetpar.2011.11.068
Barrere V, Falzon LC, Shakya KP, Menzies PI, Peregrine AS, Prichard RK: Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: Comparison of detection methods for drug resistance. Vet Parasitol, 198, 159-165, 2013. DOI: 1016/j.vetpar.2013.07.040
Kwa MS, Veenstra JG, Roos MH: Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1. Mol Biochem Parasitol, 63, 299-303, DOI: 10.1016/0166-6851(94)90066-3
Rinaldi L, Coles GC, Maurelli MP, Musella V, Cringoli G: Calibration and diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts in sheep. Vet Parasitol, 177, 345-352, 2011. DOI: 1016/j.vetpar.2010.12.010
Bott NJ, Campbell BE, Beveridge I, Chilton NB, Rees D, Hunt PW, Gasser RB: A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep. Int J Parasitol, 39, 1277-1287, 2009. DOI: 1016/j.ijpara.2009.03.002
Harmon AF, Williams ZB, Zarlenga DS, Hildreth MB: Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors. Parasitol Res, 101, 71-76, 2007. DOI: 10.1007/s00436-006-0428-0
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28, 2731-2739, 2011. DOI: 10.1093/molbev/msr121
Walsh TK, Donnan AA, Jackson F, Skuce P, Wolstenholme AJ: Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes. Vet Parasitol, 144, 304-312, 2007. DOI: 10.1016/j. vetpar.2006.10.014
Germer S, Holland MJ, Higuchi R: High-throughput SNP allele- frequency determination in pooled DNA samples by kinetic PCR. Genome Res, 10, 258-266, 2000. DOI: 10.1101/gr.10.2.258
Ghisi M, Kaminsky R, Maser P: Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes. Vet Parasitol, 144, 320, 2007. DOI: 10.1016/j.vetpar.2006.10.003
Mederos A, Fernandez S, VanLeeuwen J, Peregrine AS, Kelton D, Menzies P, LeBoeuf A, Martin R: Prevalence and distribution of gastrointestinal nematodes on 32 organic and conventional commercial sheep farms in Ontario and Quebec, Canada (2006-2008). Vet Parasitol, , 244-252, 2010. DOI: 10.1016/j.vetpar.2010.02.018
Ng'ang'a CJ, Munyua WK, Maingi N, Kanyari PW: Occurrence of peri-parturient rise in trichostrongylid nematode egg output in Dorper ewes in a semi-arid area of Kajiado District of Kenya. Acta Trop, 92, 213- , 2004. DOI: 10.1016/j.actatropica.2004.05.016
Yıldırım A, İça A, Düzlü Ö, Yavuz A, İnci A: Gebelik döneminde mide-bağırsak nematodları ile doğal enfekte koyunlarda parazit yükünün takibi. ERU Vet Fak Derg, 4, 43-77, 2007.
Prichard R: Genetic variability following selection of Haemonchus contortus with anthelmintics. Trends Parasitol, 17, 445-453, 2001. DOI: 1016/S1471-4922(01)01983-3
Garg R, Yadav CL: Genotyping of benzimidazole susceptible and resistant alleles in different populations of Haemonchus contortus from Himalayan and sub-Himalayan regions of North-West India. Trop Anim Health Prod, 41, 1127-1131, 2009. DOI: 10.1007/s11250-008-9292-5
Chaudhry U, Redman EM, Raman M, Gilleard JS: Genetic evidence for the spread of a benzimidazole resistance mutation across southern India from a single origin in the parasitic nematode Haemonchus contortus. Int J Parasitol, 2015 (In Press, Corrected Proof). DOI: 10.1016/j. ijpara.2015.04.007
Redman E, Whitelaw F, Tait A, Burgess C, Bartley Y, Skuce PJ, Jackson F, Gilleard JS: The emergence of resistance to the benzimidazole anthlemintics in parasitic nematodes of livestock is characterised by multiple independent hard and soft selective sweeps. PLoS Negl Trop Dis, , e0003494, 2015. DOI: 10.1371/journal.pntd.0003494
Taylor MA, Hunt KR, Goodyear KL: The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep. Vet Parasitol, 109, 29-43, 2002. DOI: 10.1016/S0304- (02)00269-8
dos Santos JM, Monteiro JP, Ribeiro WL, Macedo IT, Camurca Vasconcelos AL, Vieira Lda S, Bevilaqua CM: Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil. Vet Parasitol, 199, 164, 2014. DOI: 10.1016/j.vetpar.2013.11.006
Silvestre A, Cabaret J: Mutation in position 167 of isotype 1 beta- tubulin gene of Trichostrongylid nematodes: Role in benzimidazole resistance? Mol Biochem Parasitol, 120, 297-300, 2002. DOI: 10.1016/ S0166-6851(01)00455-8
Kotze AC, Cowling K, Bagnall NH, Hines BM, Ruffell AP, Hunt PW, Coleman GT: Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus. Int J Parasitol Drugs Drug Resist, 2, 92-97, 2012. DOI: 10.1016/j.ijpddr.2012.02.003