AVANOS YÖRESİ SOKAK KÖPEKLERİNDE DİROFİLARİA İMMİTİS’İN PREVALANSININ BELİRLENMESİ

Abstract

Bu çalışma, Avanos ve çevresindeki sokak köpeklerinde filarial nematod enfeksiyonlarının prevalansını tespit etmek amacıyla yürütülmüştür. Bu amaçla, Mayıs-Eylül 2013 tarihleri arasında farklı yaş, cinsiyet ve ırktan olmak üzere toplam 138 sokak köpeğinden kan örnekleri alınmıştır. Alınan kan örnekleri öncelikle natif, modifiye knott ve membran filtrasyon-asit fosfataz histokimyasal boyama yöntemleri ile perifer kandaki mikrofilerler yönünden incelenmiştir. Sonraki basamakta toplanan kanlardan genomik DNA ekstraksiyonu yapılmış ve elde edilen genomik DNA’lar Dirofilaria immitis’in 5.8S-ITS2-28S ve cytochrome oxidase subunit 1 (COI) spesifik gen bölgelerini parsiyel olarak amplifiye eden primerler ile PCR analizine tabii tutulmuşlardır. PCR analizi sonucunda, 138 köpeğin 3’ü (%2.17) D. immitis pozitif olarak belirlenmiş, diğer filarial nematod enfeksiyonlarına ise rastlanmamıştır. Moleküler olarak pozitif belirlenen 3 örneğin 2’sinde perifer kan muayene yöntemleri ile mikrofilerler saptanmıştır. Pozitif belirlenen izolatlardan birine ait PCR ürünü jelden pürifiye edilerek her iki gen bölgesi için çift yönlü sekans analizlerine tabii tutulmuştur. Sekans analizi sonucu Avanos yöresinden elde edilen D. immitis izolatının Dünya’da GenBank veri tabanına kayıtlı homolog izolatlarla 5.8S-ITS2-28S ve parsiyel COI gen bölgelerinin çoklu hizalama analizlerine göre sırasıyla %99.5-%100 ve %94.3-%100 identiklik gösterdiği belirlenmiştir. Bu çalışma ile Avanos yöresi sokak köpeklerinde D. immitis enfeksiyonlarının hem mikroskobik hem de moleküler yöntemlerle prevalansı belirlenmiş ve pozitif belirlenen bir izolatın 5.8S-ITS2-28S ve COI gen bölgelerinin moleküler karakterizasyonu ve filogenetik analizi yapılmıştır.

Keywords

• Avanos, Dirofilaria immitis, köpek, moleküler karakterizasyon, prevalans

The Prevalence of Dirofilaria Immitis in Stray Dogs in Avanos Province

Abstract

This study was carried out to determine the prevalence of filarial nematode infections in stray dogs in Avanos and vicinity. For this aim, a total of 138 blood samples were collected from stray dogs in different ages, sexes and breeds between May-September 2013. The blood samples were firstly examined by wet mount (direct microscopic examination), modified knott’s and membrane filtration-acid phosphates histochemical staining techniques in order to detect the circulating microfilaria in peripheral blood. In the next step, genomic DNA extraction was obtained from collected blood samples, and the obtained genomic DNAs were analyzed by PCR with primers amplifying specific gene regions of 5.8S-ITS2- 28S and cytochrome oxidase subunit 1 (COI) of Dirofilaria immitis. Three (2.17%) out of 138 blood samples were found to be D. immitis positive whereas no other filarial nematode species were detected. Two out of three positive samples determined as molecularly positive were also found positive by peripheral blood examination techniques. The PCR product belonging to one of positive isolates was gel purified and sequenced in both directions for two gene regions. The identity rates between the obtained D. immitis isolate obtained from Avanos region and the other homologous isolates from the world GenBank were determined as 99.5%-100% and 94.3%-100% according to the phylogenetic analyses of 5.8S-ITS2-28S and COI gene regions, respectively. Both microscopic and molecular methods the prevalence of D. immitis from stray dogs of Avanos region was revealed and the molecular characterization and phylogenetic analyses of isolate for 5.8S-ITS2-28S and COI gene regions were investigated in this study.

Keywords

• Avanos, Dirofilaria immitis, dog, molecular characterization, prevalence

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