Türkiye, Sivas'taki İshalli Barınak Köpeklerinde Köpek Koronavirüsünün Moleküler Tespiti ve Kısmi Membran Gen Dizisine Dayalı Karakterizasyonu

Öz

Köpek koronavirüsü (CCoV) enfeksiyonu dünya çapında köpekler arasında yaygındır ve gastroenteritin bilinen bir nedenidir. Özellikle yavru köpek popülasyonlarında öldürücüdür. Virüs, hayvan barınakları gibi köpek yoğunluğunun yüksek olduğu ortamlarda kolayca yayılır ve dışkı yoluyla yayılır. Bu çalışma, Sivas Belediye Hayvan Barınağı'ndaki ishalli barınak köpeklerinde köpek koronavirüsünün (CCoV) varlığını ve moleküler özelliklerini tespit etmeyi amaçlamıştır. İshal semptomları gösteren 150 barınak köpeğinden (127 yetişkin ve 23 yavru) dışkı örnekleri toplandı. CCoV M genini (CCV1/CCV2 ve CCV1a/CCV2) hedef alan iki primer seti kullanılarak RT-PCR analizi gerçekleştirildi. CCV1/CCV2 primer çifti, ishalli köpeklerin %38'inin (57/150) CCoV için pozitif olduğunu gösterdi. Pozitiflik oranı yetişkin köpeklerde %31,50 (40/127) ve yavru köpeklerde %73,91 (17/23) idi. Tiplendirme, M genine dayalı ikinci primer seti (CCV1a/CCV2) kullanılarak gerçekleştirildi. 57 pozitif örnekten 6'sı (%10,53) CCoV Tip II ve 51'i (%89,47) CCoV Tip I olarak belirlendi. RT-PCR dizi analizi, biyoenformatik değerlendirme ve filogenetik değerlendirme yapıldı. Bu bulgular, CCoV Tip I'in incelenen köpek popülasyonunda ishale neden olan koronavirüs enfeksiyonlarının çoğundan sorumlu olduğunu göstermektedir.

Anahtar Kelimeler

• Köpek koronavirüs
• ishal
• M gen
• filogenetik analiz

Molecular Detection and Partial Membrane Gene Sequence-Based Characterization of Canine Coronavirus in Diarrheal Shelter Dogs in Sivas, Türkiye

Öz

Canine coronavirus (CCoV) infection is widespread among dogs worldwide and is a known cause of gastroenteritis. It is particularly lethal in puppy populations. The virus spreads easily in environments where dogs are densely populated, such as animal shelters, and is shed through feces. This study aimed to detect the presence and molecular characteristics of canine coronavirus (CCoV) in shelter dogs with diarrhea at the Sivas Municipality Animal Shelter. Fecal samples were collected from 150 shelter dogs (127 adults and 23 puppies) showing diarrheal symptoms. RT-PCR analysis was performed using two primer sets targeting the CCoV M gene (CCV1/CCV2 and CCV1a/CCV2). The CCV1/CCV2 primer pair showed that 38% (57/150) of diarrheic dogs tested positive for CCoV. The positivity rate was 31.50% (40/127) in adult dogs and 73.91% (17/23) in puppies. Typing was performed using the second primer set (CCV1a/CCV2) based on the M gene. Of the 57 positive samples, 6 (10.53%) were CCoV Type II and 51 (89.47%) were CCoV Type I. RT-PCR sequence analysis, bioinformatics evaluation, and phylogenetic assessment were conducted. These findings indicate that CCoV Type I was responsible for the majority of coronavirus infections causing diarrhea in the studied dog population.

Anahtar Kelimeler

• Dog coronavirus
• diarrhea
• M gene
• phylogenetic analysis

Kaynakça

1. 1.Kartal T, Rowan AN. Stray dog population management. In: Field Manual for Small Animal Medicine. 2018;15-28.
2. 2.Alves AGC, Ribeiro MN, Arandas JKG, Alves RRN. Animal domestication and ethnozootechny. In: Ethnozoology-Animals in Our Lives. 2018;151-165.
3. 3.Martella V, Pratelli A, Elia G, Decaro N, Tempesta M, Buonavoglia C. Isolation and genetic characterization of two G3P5A [3] canine rotavirus strains in Italy. J Virol Methods. 2001;96(1):43-49.
4. 4.Mosallanejad B, Shapouri MRS, Avizeh R, Pourmahdi M. Antigenic detection of canine rotavirus group A in diarrheic dogs in Ahvaz district, Southwestern Iran. Comp Clin Pathol. 2015;24:899-902.
5. 5.Ortega AF, Martínez-Castañeda JS, Bautista-Gómez LG, Muñoz RF, Hernández IQ. Identification of co-infection by rotavirus and parvovirus in dogs with gastroenteritis in Mexico. Braz J Microbiol. 2017;48:769-773.
6. 6.Woo PC, de Groot RJ, Haagmans B, et al. ICTV virus taxonomy profile: Coronaviridae 2023: This article is part of the ICTV virus taxonomy profiles collection. J Gen Virol. 2023;104(4):001843.
7. 7.Regan AD, Millet JK, Tse LPV, et al. Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain. Virology. 2012;430(2):90-99.
8. 8.Batra K, Kumar P, Sehrawat A, Agnihotri D, Maan S. Detection and characterization of canine corona virus infection: an enteric approach. Vet Arh. 2024;94(6):475-486.

9. 9.MacLachlan NJ, Dubovi EJ, Barthold SW, Swayne DE, Winton JR. Coronaviridae. In: Fenner's Veterinary Virology. 5th ed. Academic Press; 2017:435-461.
10. 10.Lian CY, Yao XY, Lv ZH, Zhang XL, Shao JW. Genetic diversity of canine coronavirus identified in dogs in yulin city, southern China. Virology. 2025;608:110528.
11. 11.Shi K, Shi Y, Shi Y, et al. Genetic and evolutionary analysis of canine coronavirus in Guangxi Province, China, for 2021–2024. Vet Sci. 2024;11(10):456.
12. 12.Mira F, Schirò G, Lanave G, et al. Molecular screening and characterization of canine coronavirus types I and II strains from domestic dogs in Southern Italy, 2019–2021. Transbound Emerg Dis. 2024;7272785.
13. 13.Li N, Bai Y, Liu R, et al. Prevalence and genetic diversity of canine coronavirus in northeastern China during 2019–2021. Comp Immunol Microbiol Infect Dis. 2023;94:101956.
14. 14.Bandai C, Ishiguro S, Masuya N, Hohdatsu T, Mochizuki M. Canine coronavirus infections in Japan: Virological and epidemiological aspects. J Vet Med Sci. 1999;61(7):731-736.
15. 15.Naylor MJ, Harrison GA, Monckton RP, Mcorist S, Lehrbach PR, Deane EM. Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate. J Clin Microbiol. 2001;39:1036-1041.
16. 16.Hussein MA, Al-Graibawi MAA. Clinical and molecular study of canine enteric coronavirus in Iraq. Egypt J Vet Sci. 2024;55(5):1357-1363.
17. 17.Odigie AE, Capozza P, Tempesta M, Decaro N, Pratelli A. Epidemiological investigation of enteric canine coronaviruses in domestic dogs: A systematic review and meta-analysis. Res Vet Sci. 2024;174:105289.
18. 18.Pratelli A, Tinelli A, Decaro N, et al. PCR assay for the detection and the identification of atypical canine coronavirus in dogs. J Virol Methods. 2002;106(2):209-213.
19. 19.Kearse M, Moir R, Wilson A, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647-1649.
20. 20.Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10(3):512-526.
21. 21.Yesilbag K, Yilmaz Z, Torun S, Pratelli A. Canine coronavirus in Turkish dog population. J Vet Med. 2004;51:353-355.
22. 22.Decaro N, Mari V, Elia G, et al. Recombinant canine coronaviruses in dogs, Europe. Emerg Infect Dis. 2010;16:41-47.
23. 23.Ntafis V, Mari V, Decaro N, et al. Canine coronavirus, Greece. Molecular analysis and genetic diversity characterization. Infect Genet Evol. 2013;16:129-136.
24. 24.Akkutay-Yoldar Z, Koç BT, Oğuzoğlu ÇT. Phylogenetic analysis of partial transmembrane protein gene of canine coronaviruses detected in Turkey. Ankara Univ Vet Fak Derg. 2020;67(3):265-271.
25. 25.Timurkan MO, Aydin H, Dincer E, Coskun N. Molecular characterization of canine coronaviruses: an enteric and pantropic approach. Arch Virol. 2021;166:35-42.
26. 26.Doğan F, Köse Sİ. Alt solunum yolu enfeksiyonu olan köpeklerde canine coronavirusun tespiti ve moleküler karakterizasyonu. Vet Hekim Der Derg. 2022;93(2):124-132.
27. 27.Karapınar Z, Timurkan MO. Detection and molecular characterization of canine coronavirus based on partial membrane gene sequences. Van Vet J. 2024;35(1):27-31.
28. 28.Decaro N, Martella V, Ricci D, et al. Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs. J Virol Methods. 2005;130(1-2):72-78.
29. 29.Soma T, Ohinata T, Ishii H, Takahashi T, Taharaguchi S, Hara M. Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan. Res Vet Sci. 2011;90(2):205-207.
30. 30.Costa EM, de Castro TX, de Oliveira Bottino F, Garcia RDCNC. Molecular characterization of canine coronavirus strains circulating in Brazil. Vet Microbiol. 2014;168(1):8-15.
31. 31.Pratelli A, Martella V, Pistello M, et al. Identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype. J Virol Methods. 2003;107(2):213-222.
32. 32.Pratelli A. Genetic evolution of canine coronavirus and recent advances in prophylaxis. Vet Res. 2006;37(2):191-200.
33. 33.Jeoung SY, Ann SY, Kim HT, Kim D. M gene analysis of canine coronavirus strains detected in Korea. J Vet Sci. 2014;15(4):495.
34. 34.Pratelli A, Decaro N, Tinelli A, et al. Two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea. J Clin Microbiol. 2004;42(4):1797-1799.
35. 35.Horsburgh BC, Brierley I, Brown TDK. Analysis of a 9.6 kb sequence from the 3′ end of canine coronavirus genomic RNA. J Gen Virol. 1992;73(11):2849-2862.
36. 36.Radford AD, Singleton DA, Jewell C, et al. Outbreak of severe vomiting in dogs associated with a canine enteric coronavirus, United Kingdom. Emerg Infect Dis. 2021;27(2):517.