Molecular Characterization of Trichophyton Verrucosum Strains Isolated From Cattle by PCR-RFLP

Öz

Dermatophytes infect tissues containing keratin in humans and animals, causing dermatophytosis infection. Trichophyton verrucosum is the most common agent of bovine dermatophytosis cases. Trichophytosis causes big economic lossess throughout the world and also threatens human health by being a zoonosis. T. verrucosum is usually isolated from cattle. Cattle are the natural reservoirs of this agent. The aim of this study is to isolate disease-causing dermatophytes in cattle and to carry out molecular separation of Internal Transcribed Spacer (ITS) regions of the isolated T. verrucosum strains by PCR- Restriction Fragment Lenght Polymorphism (PCR-RFLP). For this purpose, 90 samples were taken from the cattle with dermatophytosis for cultural examination. As a result of the culture of these samples, 35 (38.8%) T. verrucosum were isolated and identified. DNA isolation of these strains was made and amplification of ITS regions was performed. It was only one RFLP profile was found according to the results of RFLP analysis of T. verrucosum strains using MvaI and HinfI enzymes. At the end of study, it was founded that the isolated T. verrucosum strains showed a single profile by PCR-RFLP analysis and PCR-RFLP was a useful tool for the molecular characterization of the strains. İt was also concluded that PCR-RFLPs of strains from different regions or even from different countries might be necessary in order to detect different profiles of the tested samples.

Anahtar Kelimeler

• Cattle
• Dermatophytosis
• PCR-RFLP
• Trichophyton verrucosum

Sığırlardan İzole Edilen Trichophyton verrucosum Suşlarının PCR –RFLP ile Moleküler Karakterizasyonu.

Öz

Dermatofitler, insan ve hayvanlarda keratin içeren dokuları infekte ederek dermatofit infeksiyonuna neden olmaktadırlar. Trichophyton verrucosum sığır dermatofitozis olgularının en yaygın etkenidir. Trichophytosis, bütün dünyada hayvancılık sektöründe önemli ekonomik kayıplara neden olması yanında zoonoz olmasıyla da insan sağlığını tehdit etmektedir. Sığırlardan genellikle T. verrucosum izole edilmektedir. Sığırlar bu etkenin doğal rezervuarıdırlar. Bu çalışmanın amacı, sığırlarda hastalığa neden olan dermatofitlerinin izolasyonu ve izole edilen T. verrucosum suşlarının Internal Transcribed Spacer (ITS) bölgelerinin PCR-RFLP ile moleküler ayrımının yapılmasıdır. Bu amaçla dermatofitozisli sığırlardan 90 adet örnek alınarak kültürleri yapıldı. Bu örneklerin kültürü sonucunda 35 (%38,8) adet T. verrucosum izole ve identifiye edildi. Bu suşların DNA izolasyonu gerçekleştirilerek ITS bölgelerin amplifikasyonu gerçekleştirildi. T. verrucosum suşlarının MvaI ve HinfI enzimleri kullanılarak yapılan Restriction Fragment Lenght Polymorphism (RFLP) analizleri sonucunda bir adet RFLP profiline rastlandı. Sonuç olarak, izole edilen T. verrucosum suşlarının PCR-RFLP sonucunda tek bir profile sahip olduğu, farklı profil örneklerinin saptanması için farklı bölgelerden hatta farklı ülkelerden suşların PCR-RFLP’lerinin yapılması gerektiği kanısına varıldı.

Anahtar Kelimeler

• Dermatofitosis
• PCR-RFLP
• Sığır
• Trichophyton verrucosum

Kaynakça

1. Abd-Elmegeed M, El-Mekkawi MF, El-Diasty EM, Fawzi EM. Dermatophytosis among Ruminants in Egypt: The Infection Rate, Identification and Comparison between Microscopic, Cultural and Molecular Methods. Zagazig Veterinary Journal. 2020; 48(2):116-127.
2. Abou-Eisha A, Sobih M, Fadel H, Elmahallawy H. Dermatophytes in animals and their zoonotic importance in Suez canal area. Suez Canal Vet Med J. 2008; 13(2):625-642.
3. Aghamirian MR, Ghiasian SAJM. Dermatophytes as a cause of epizoonoses in dairy cattle and humans in Iran. Epidemiological and clinical aspects. 2011; 54(4): e52-e6.
4. Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series 1999; 41:95-98.
5. Hsiao CR, Huang L, Bouchara JP, Barton R, Li HC, Chang TC. Identification of medically important molds by an oligonucleotide array. J Clin Microbiol. 2005; 43:3760–3768.
6. Jackson CJ, Barton, RC, Evans EG. Species identification and strain differantiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions. J. Clin. Microbiol. 1999; 31: 931-936.
7. Jha BK, Murthy SM, Devi NL. Molecular identification of dermatophytosis by polymerase chain reaction (PCR) and detection of source of infection by restricted fragment length polymorphism (RFLP). Journal of College of Medical Sciences. Nepal. 2012; 8(4):7-15.
8. Kac G. Molecular approaches to the study of dermatophytes. Med. Mycol. 2000; 38:329-336.

9. Khosravi AR, Mahmoudi M. Dermatophytes isolated from domestic animals in Iran. Mycoses. 2003; 46:222-225.
10. Larone, D.H. Medically Important Fungi, In: A Guide to Identification, 4nd Ed., Washington, ASM press. 2002.
11. Liu D, Coloe S, Baird R, Pedersen J. Rapid mini-preparation of fungal DNA for PCR. Journal of clinical microbiology. 2000; 38(1):471-471.
12. Mirzahoseini H, Omidinia E, Shams-Ghahfarokhi M, Sadeghi G, Razzaghi-Abyaneh M. Application of PCR-RFLP to rapid identification of the main pathogenic dermatophytes from clinical specimens. Iranian Journal of Public Health. 2009; 18-24.
13. Mitra SK, Sikdar A, Das P. Dermatophytes isolated from selected ruminants in India. Mycopathologia. 1998; 142:13–16.
14. Mochizuki T, Kawasaki M, Ishizaki H, Makimura K. Identification of several clinical isolates of dermatophytes based on the nucleotide sequence of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA. J. Derm. 1999; 26:276-281.
15. Neji S, Trabelsi H, Hadrich I, Cheikhrouhou F, Sellami H, Makni F, Ayadi A. Molecular characterization of strains of the Trichophyton verrucosum complex from Tunisia. Medical Mycology. 2016; 1-7.
16. Papini R, Nardoni S, Fanelli A, Mancianti F. High Infection Rate of Trichophyton verrucosum in Calves from Central Italy. Zoonoses Public Health. 2009; 56:59–64.