【摘要】 为研究犬细小病毒（CPV）BJ1606株对犬的致病性，本研究将9只未免疫的比格犬随机分为3组，分别经皮下各注射和灌服3×10⁵TCID₅₀、3×10⁴TCID₅₀及3×10³TCID₅₀的病毒液，1只犬经皮下注射及灌服5 mL DMEM。逐日观察幼犬感染后的临床症状，根据评分标准对各组犬的临床症状评分；采集感染前和感染后2 d、4 d、6 d、8 d抗凝血及血清，检测血清磷（P）、碱性磷酸酶（ALP）、白蛋白（ALB）的含量及血液白细胞数（WBC）；采集感染前及感染后24 h各组犬的抗凝血，采用犬外周血淋巴细胞分离试剂盒分离抗凝血内淋巴细胞，采用qPCR检测各组犬淋巴细胞中IL-1B、TNF-α、IL-12A、IFN-γ、IL-6、IL-4和IL-22等7种细胞因子转录水平的变化；感染后14 d剖检死亡/存活的犬，取肠、心、肝、脾、肺与肾，制作各组织病理切片，观察其病理变化；采用免疫组化试验（IHC）检测病毒的抗原信号。结果显示，感染犬均出现便血等典型的临床症状，且均在感染后6 d综合评分达到或接近5分；高中低剂量组犬的死亡率依次为66.6%、66.6%、100%，总死亡率达77.7%，但各组间犬的死亡率均无显著差异；感染犬血液中的P含量及WBC均呈前期升高、后期下降的趋势，ALP含量上升至200 U/L,ALB含量均大幅度下降至25 g/L左右；血液淋巴细胞中IL-12A、IFN-γ、TNF-α的转录水平与感染前相比显著上升（P<0.05），提示诱发了以体液免疫为主的犬的全身性免疫反应；各脏器剖检病变及组织病理切片观察结果显示，感染组犬肠道、肝脏、肺脏和脾脏均出现不同程度的出血及炎性病变；IHC结果显示病毒主要存在于肠道隐窝及绒毛细胞，部分犬肝脏中也可检测到，心、脾和肾组织未发现抗原阳性信号。对照组犬血液生理生化指标、WBC、临床症状均正常，无显著变化，病理切片及IHC无病变和阳性信号，上述结果表明，该病毒株对2月龄幼犬具有100%的致病性，且具有典型的CPV致病特性，主要定植于犬的肝及小肠、诱导犬的全身性体液免疫反应。本研究从多方面研究了CPV BJ1606株的致病性，为犬作为CPV感染的动物模型奠定了基础，同时也为CPV疫苗效果的评估提供了候选病毒株。更多还原

【Abstract】 In order to study the pathogenicity of BJ1606 strain of canine parvovirus（CPV） in dogs, nine unvaccinated beagles were randomly divided into three groups. Each group was administered viral suspensions containing 3×10⁵ TCID₅₀, 3×10⁴ TCID₅₀, and3×10³ TCID₅₀via subcutaneous injection and oral gavage, respectively, while one dog received 5 mL DMEM as a control through the same routes. The clinical symptoms of the pups after infection were observed daily, and the clinical symptoms of each group were scored according to the scoring criteria. Anticoagulant blood and serum samples were collected at 2 days, 4 days, 6 days and 8 days afte rinfection, and serum phosphorus（P）, alkaline phosphatase（ALP）, albumin（ALB） and blood routine indexes were detected. The anticoagulation blood samples were collected before infection and 24 hours post-infection. The lymphocytes in the anticoagulation blood were isolated using canine peripheral blood lymphocyte isolation kit. qPCR was employed to analyze transcriptional changes of 7 cytokines in each group, including IL-1B, TNF-α, IL-12A, IFN-γ, IL-6, IL-4 and IL-22. The intestinal, heart, liver, spleen, lung and kidney tissues of the dogs that either died or survived by day 14 post-infection were dissected, and the histopathological sections were made to observe the pathological changes. The virus antigen signal was detected by immunohistochemistry（IHC）. The results showed that all the infected dogs had typical clinical symptoms such as hematochezia, and their comprehensive scores reached or were close to 5 points by day 6 post-infection. The mortality rates of high, middle and low dose groups were 66.6%, 66.6% and100%, respectively, and the total mortality rate was 77.7%, but there was no significant difference among the groups. In the infected dogs, P content increased in the early stage and decreased in the later stage, ALP content increased to 200U/L, and ALB content decreased to about 25g/L. The transcription levels of IL-12A, IFN-γ, TNF-α were significantly increased compared with those before challenge, suggesting that system icimmunity was induced mainly by humoral immunity. The pathological examination of organs and sections showed that the intestinaltract, liver, lungand spleen showed varying degrees of the morrhage and inflammation. The results of IHC showed that the virus was mainly detected in intestinal crypt and villus cells, and ins ome dog liver tissues. No positive signals were detected in cardiac, splenic, or renal tissues. All physiological and biochemical indicators, complete blood count, and clinical symptoms of the control dog were normal with no significant changes; no pathological lesions or positive signals were observed in the pathological sections and IHC. The results showed that the strain was 100% pathogenicto 2-month-old puppies and had typical pathogenic characteristics of CPV. The virus mainly colonized in the liver and small intestine and induced systemic cellular and humoral immunity in dogs. This study comprehensively investigated the pathogenicity of CPV strain BJ1606 from multiple perspectives, establishing a foundation for canine CPV animal models while providing a candidate viral strain for CPV vaccine validation studies.更多还原