犬腺病毒CAV-BJ02株的分离与鉴定

doi:10.11843/j.issn.0366-6964.2020.08.027

摘要/Abstract

摘要： 本研究旨在分离犬腺病毒（CAV）的北京地区流行株，采集北京地区某宠物医院2~4月龄发生咳嗽等呼吸道症状犬的鼻咽拭子，经胶体金和PCR检测，初筛为犬腺病毒阳性。经过处理后，将其处理液接种于MDCK细胞，进行连续传代培养，出现变圆、脱落、葡萄串样等特征性细胞病变。通过形态学观察、PCR鉴定及动物回归试验等方法对其进行鉴定。PCR扩增片段测序结果表明，该分离株为犬腺病毒Ⅱ型，遂将其命名为CAV-BJ02（GenBank：MN744708）株。用Reed-Muench法测得CAV-BJ02株的TCID₅₀为10^6.7·（100 μL）^-1。电镜下可清晰地观察到呈正六边形的二十面体、直径在86 nm左右的犬腺病毒颗粒。遗传进化分析结果表明，本株病毒为CAV-Ⅱ型。动物回归试验结果表明，CAV-BJ02株可引起犬发热等轻微临床症状，以口鼻分泌物为主要排毒方式，排毒期为5~6 d，不导致犬死亡。上述研究结果为深入了解北京地区CAV的流行情况，为犬腺病毒病的诊断、防治及后续相关研究奠定了理论基础。

关键词: 犬腺病毒, 分离鉴定, 进化分析, 动物试验

Abstract: This study aimed to isolate an epidemic strain of the canine adenovirus (CAV) from Beijing. Nasopharyngeal swabs were collected from 2 to 4 months dogs with cough and other respiratory symptoms in a pet hospital in Beijing. The sample showing positive with canine adenovirus after initial screening by colloidal gold and PCR, was propagated in MDCK cells after processing. Continuous subculture was carried out, and typical cytopathic lesions such as rounding, shedding, and grape stringing appeared. The sample was identified by morphological observation, PCR identification and animal regression test. The PCR amplification results indicated that the isolate was a canine adenovirus type II, and it was named as CAV-BJ02 (GenBank：MN744708) strain. Virus titers were determined according to the method of Reed and Muench, and TCID₅₀ of CAV-BJ02 isolated was 10^6.7·(100 μL)^-1 in MDCK cells. Canine adenovirus particles with a regular hexagonal icosahedron and a diameter of about 86 nm can be observed clearly under electron microscope. Phylogenetic analysis showed that the sequence of the CAV-BJ02 strain was consistent with that of CAV type Ⅱ. Animal regression test results showed that CAV-BJ02 strain can cause mild clinical symptoms such as fever, with oral and nasal secretions as the main routes for viral shedding, and the viral shedding period is about 5-6 days, which does not cause dogs to die. The results above provided insights into the prevalence of CAV in Beijing and laid a theoretical foundation for the diagnosis, prevention and treatment of canine adenovirus disease.

Key words: canine adenovirus, isolation and identification, evolutionary analysis, animal experiment

中图分类号:

S852.659.1

由欣月, 刘畅, 郝雲峰, 梁琳, 秦彤, 崔尚金. 犬腺病毒CAV-BJ02株的分离与鉴定[J]. 畜牧兽医学报, 2020, 51(8): 2016-2021.

YOU Xinyue, LIU Chang, HAO Yunfeng, LIANG Lin, QIN Tong, CUI Shangjin. Isolation and Identification of Canine Adenovirus CAV-BJ02 Strain[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 2016-2021.

参考文献 33

[1]	HIEBL A, AUER A, BAGRINOVSCHI G, et al. Detection of selected viral pathogens in dogs with canine infectious respiratory disease in Austria[J]. J Small Anim Pract, 2019, 60(10):594-600.
[2]	MABONI G, SEGUEL M, LORTON A, et al. Canine infectious respiratory disease:new insights into the etiology and epidemiology of associated pathogens[J]. PLoS One, 2019, 14(4):e0215817.
[3]	DECARO N, MARTELLA V, BUONAVOGLIA C. Canine adenoviruses and herpesvirus[J]. Vet Clin North Am:Small Anim Pract, 2008, 38(4):799-814.
[4]	林凡娜. 犬腺病毒的分离鉴定及犬腺病毒基因组文库的构建[D]. 武汉:华中农业大学, 2013.LIN F N. Isolation and identification of the canine virus and construction of canine adenovirus genomic library[D]. Wuhan:Huazhong Agricultural University,2013. (in Chinese)
[5]	BALBONI A, DONDI F, AGNOLI C, et al. Novel sequence variants of viral hexon and fibre genes in two dogs with canine adenovirus type 1-associated disease[J]. Vet J, 2017, 223:73-75.
[6]	王海军, 刘存发, 韩志强, 等. 东北虎感染犬腺病毒Ⅰ型病例分析[J]. 经济动物学报, 2019, 23(4):197-200.WANG H J, LIU C F, HAN Z Q, et al. Death case analysis of Panthera tigris altaica infection with canine adenovirus type Ⅰ[J]. Journal of Economic Animal, 2019, 23(4):197-200. (in Chinese)
[7]	VERIN R, FORZAN M, SCHULZE C, et al. Multicentric molecular and pathologic study on canine adenovirus type 1 in red foxes (Vulpes vulpes) in three european countries[J]. J Wildl Dis, 2019, 55(4):935-939.
[8]	孙杰, 秦飞燕, 朱言柱, 等. 犬腺病毒1型对犬北极狐和银狐的致病性比较[J]. 中国兽医科学, 2019, 49(5):625-631.SUN J, QIN F Y, ZHU Y Z, et al. Pathogenicity comparison of canine adenovirus type 1 in dog, arctic fox and silver fox[J]. Chinese Veterinary Science, 2019, 49(5):625-631. (in Chinese)
[9]	NIEDRINGHAUS K D, BROWN J D, TERNENT M A, et al. A Serosurvey of multiple pathogens in american black bears (Ursus americanus) in Pennsylvania, USA Indicates a lack of association with Sarcoptic mange[J]. Vet Sci, 2019, 6(4):75.
[10]	夏咸柱, 王永贤, 赵吉成, 等. 犬传染性肝炎病毒的分离与鉴定[J]. 中国兽医学报, 1984, 4(3):228-233.XIA X Z, WANG Y X, ZHAO J C, et al. Isolation and identification of canine infectious hepatitis virus[J]. Chinese Journal of Veterinary Science, 1984, 4(3):228-233. (in Chinese)
[11]	范泉水, 夏咸柱, 黄耕, 等. 犬Ⅱ型腺病毒的分离鉴定[J]. 中国兽医科技, 1999, 29(11):28-29.FAN Q S, XIA X Z, HUANG G, et al. Isolation and identification of canine type Ⅱ adenovirus[J]. Chinese Journal of Veterinary Science and Technology, 1999, 29(11):28-29. (in Chinese)
[12]	乔军, 孟庆龄, 夏咸柱, 等. 犬I型腺病毒TN株的分离鉴定[J]. 动物医学进展, 2001, 22(4):97-99.QIAO J, MENG Q L, XIA X Z, et al. Isolation and identification of TN strain of Canine adenovirus-Ⅰ[J]. Progress in Veterinary Medicine, 2001, 22(4):97-99. (in Chinese)
[13]	李东勇. 犬传染性肝炎的诊治[J]. 畜牧兽医科技信息, 2019(8):158.LI D Y. Diagnosis and treatment of canine infectious hepatitis[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2019(8):158. (in Chinese)
[14]	DOWGIER G, MARI V, LOSURDO M, et al. A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2[J]. J Virol Methods, 2016, 234:1-6.
[15]	GARCÍA MARÍN J F, ROYO L J, OLEAGA A, et al. Canine adenovirus type 1(CAdV-1) in free-ranging European brown bear (Ursus arctos arctos):a threat for Cantabrian population?[J]. Transbound Emerg Dis, 2018, 65(6):2049-2056.
[16]	WALKER D, ABBONDATI E, COX A L, et al. Infectious canine hepatitis in red foxes (Vulpes vulpes) in wildlife rescue centres in the UK[J]. Vet Rec, 2016, 178(17):421.
[17]	WALKER D, FEE S A, HARTLEY G, et al. Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom[J]. Sci Rep, 2016, 6(1):36051.
[18]	BALBONI A, VERIN R, MORANDI F, et al. Molecular epidemiology of canine adenovirus type 1 and type 2 in free-ranging red foxes (Vulpes vulpes) in Italy[J]. Vet Microbiol, 2013, 162(2-4):551-557.
[19]	ALMES K M, JANARDHAN K S, ANDERSON J, et al. Fatal canine adenoviral pneumonia in two litters of Bulldogs[J]. J Vet Diagn Invest, 2010, 22(5):780-784.
[20]	CHATURVEDI U, TIWARI A K, RATTA B, et al. Detection of canine adenoviral infections in urine and faeces by the polymerase chain reaction[J]. J Virol Methods, 2008, 149(2):260-263.
[21]	高春风. 犬传染性肝炎的诊疗[J]. 畜牧兽医科技信息, 2017(7):117.GAO C F. Diagnosis and treatment of canine infectious hepatitis[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2017(7):117. (in Chinese)
[22]	BALBONI A, MOLLACE C, GIUNTI M, et al. Investigation of the presence of canine adenovirus (CAdV) in owned dogs in Northern Italy[J]. Res Vet Sci, 2014, 97(3):631-636.
[23]	HAMELIN C, JOUVENNE P, ASSAF R. Association of a type-2 canine adenovirus with an outbreak of diarrhoeal disease among a large dog congregation[J]. J Diarrhoeal Dis Res, 1985, 3(2):84-87.
[24]	MACARTNEY L, CAVANAGH H M A, SPIBEY N. Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping[J]. Res Vet Sci, 1988, 44(1):9-14.
[25]	BUONAVOGLIA C, MARTELLA V. Canine respiratory viruses[J]. Vet Res, 2007, 38(2):355-373.
[26]	殷震, 刘景华. 动物病毒学[M]. 2版. 北京:科学出版社, 1997.YIN Z, LIU J H. Animal virology[M]. 2nd ed. Beijing:Science Press, 1997. (in Chinese)
[27]	由欣月, 郝雲峰, 崔尚金, 等. 犬腺病毒SYBR Green Ⅰ荧光定量PCR检测方法的建立[J]. 中国动物传染病学报, 2019, 27(6):29-34.YOU X Y, HAO Y F, CUI S J, et al. Development of SYBR Green Ⅰ real-time PCR for the detection of canine adenovirus[J]. Chinese Journal of Animal Infectious Diseases, 2019, 27(6):29-34. (in Chinese)
[28]	邱薇, 郑颖, 张富强, 等. 犬Ⅱ型腺病毒KM株的分离鉴定[C]//第17次全国犬业科技学术研讨会论文集. 太原:中国畜牧兽医学会犬学分会, 公安部南昌警犬基地, 2017:6.QIU W, ZHENG Y, ZHANG F Q, et al. Isolation and identification of canine type Ⅱ adenovirus KM strain[C]//Proceedings of the 17th National Conference on Science and Technology of Dog Industry. Taiyuan:Chinese Academy of Animal Husbandry and Veterinary Society, Nanchang Police Dog Base, 2017:6. (in Chinese)
[29]	KOPTOPOULOS G. Canine adenoviruses:a review[J]. Vet Bulletin, 1981, 51(3):135-142.
[30]	SWANGO L J, WOODING W J JR, BINN L N. A comparison of the pathogenesis and antigenicity of infectious canine hepatitis virus and the A26-61 virus strain (Toronto)[J]. J Am Vet Med Assoc, 1970, 156(12):1687-1696.
[31]	易程, 邓莉, 唐青海, 等. 一株I型犬腺病毒的分离与鉴定[J]. 中国动物检疫, 2019, 36(2):82-87.YI C, DENG L, TANG Q H, et al. Isolation and identification of a strain of type I canine adenovirus[J]. China Animal Health Inspection, 2019, 36(2):82-87. (in Chinese)
[32]	刘长明, 王瑞, 张洪英, 等. 犬瘟热、猫细小病毒、犬腺病毒弱毒株的理化特性及生物学试验[J]. 中国预防兽医学报, 1999, 21(2):89-92.LIU C M, WANG R, ZHANG H Y, et al. Physical chemical characteristics and biological test of attenuated virus strains of canine distemper virus, feline parvovirus and canine adenovirus[J]. Chinese Journal of Preventive Veterinary Medicine, 1999, 21(2):89-92. (in Chinese)
[33]	张洋, 袁子国, 姜秋杰, 等. 犬2型腺病毒的分离鉴定及初步特性分析[J]. 军事医学科学院院刊, 2008, 32(6):541-544.ZHANG Y, YUAN Z G, JIANG Q J, et al. Isolation, identification and preliminary characteristic analysis of a CAV-2[J]. Bulletin of the Academy of Military Medical Sciences, 2008, 32(6):541-544. (in Chinese)