2019年某实验猴养殖场食蟹猴犬瘟热暴发的诊断

doi:10.12300/j.issn.1674-5817.2024.160

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (3): 360-367.DOI: 10.12300/j.issn.1674-5817.2024.160

2019年某实验猴养殖场食蟹猴犬瘟热暴发的诊断

王晨娟¹, 杨玲焰², 王立鹏¹, 孙雪萍¹, 李静文¹, 郭连香¹, 荣荣²()(), 时长军¹()()

^1.苏州西山生物技术有限公司, 苏州 215123
^2.西交利物浦大学理学院生物科学系, 苏州 215123

收稿日期:2024-11-04 修回日期:2024-12-30 出版日期:2025-07-07 发布日期:2025-06-25
通讯作者: 荣荣（1967—），女，博士，副教授，研究方向：病毒感染性疾病的机制及控制。E-mail：Rong.Rong@xjtlu.edu.cn。ORCID： 0000-0002-7641-7684；
时长军（1962—），男，博士，高级工程师，研究方向：实验动物疫病学。E-mail：changjun.shi@vrl.net。ORCID：0009-0000-9970-9930
作者简介:王晨娟（1987—），女，硕士，助理研究员，研究方向：实验动物质量检测。E-mail：chenjuan.wang@vrlasia.cn
基金资助:
国家重点研发计划项目“灵长类实验动物育种、繁育、SPF猴培育及资源保护研究”子课题“猴病原检测新方法建立和高品质种群培育”(2022YFF0710903);2021年江苏省重点研发计划（社会发展）项目“灵长类实验动物重大疫病监测技术应用研发”(BE2021668)

Diagnosis of an Outbreak of Canine Distemper in Cynomolgus Monkeys in an Experimental Monkey Farm in 2019

WANG Chenjuan¹, YANG Lingyan², WANG Lipeng¹, SUN Xueping¹, LI Jingwen¹, GUO Lianxiang¹, RONG Rong²()(), SHI Changjun¹()()

^1.Suzhou Xishan Biotechnology Inc. , Suzhou 215123, China
^2.Department of Biological Sciences, School of Science, Xi'an Jiaotong-Liverpool University, Suzhou 215123, China

Received:2024-11-04 Revised:2024-12-30 Published:2025-06-25 Online:2025-07-07
Contact: RONG Rong (ORCID: 0000-0002-7641-7684), E-mail: Rong.Rong@xjtlu.edu.cn;
SHI Changjun n （ORCID: 0009-0000-9970-9930）， E-mail: changjun.shi@vrl.net

摘要/Abstract

摘要：

目的报告2019年某实验猴养殖场中食蟹猴群暴发犬瘟热病毒的诊断情况。方法针对2019年底中国华南地区某实验猴养殖场送检的21只患病食蟹猴（出现面部红疹、皮屑、流鼻涕和腹泻等症状）的血清、皮肤红疹拭子和1只病死猴的抗凝全血、肝、肺、皮肤组织，共46份样品，采用实时荧光定量PCR法检测犬瘟热病毒基因片段，采用免疫组织化学染色法检测肺组织中犬瘟热病毒核蛋白表达。将病死猴的皮肤组织研磨过筛，取滤液接种于单层MDCK细胞系中培养分离病毒，将分离的病毒进行全基因组测序鉴定，采用Clustal Omega工具对亚洲不同犬瘟热病毒分离株进行比对和同源性分析，构建遗传发育树，并对其进行遗传进化分析。结果经临床追溯，患病的食蟹猴出现了与麻疹病毒感染食蟹猴类似的症状，剖检病死猴可见肺部有红色病变，大肠黏膜有明显出血。实时荧光定量PCR检测患病猴血清、皮肤红疹拭子及病死猴各组织样本中的犬瘟热病毒核酸结果均呈阳性，由标准曲线公式计算出皮肤组织中的病毒载量最高；免疫组织化学染色病死猴的肺组织显示犬瘟热病毒核蛋白聚集在肺泡上皮细胞、肺支气管和细支气管处；从病死猴皮肤组织中分离出1株犬瘟热病毒株，遗传进化分析表明其与越南犬中发现的亚洲1型CDV/dog/HCM/33/140816株亲缘关系最近，基因相似度达到98.86%。结论综合临床症状、核酸检测、病毒蛋白免疫组织化学法及全基因组测序分析结果，诊断该猴场的食蟹猴感染了犬瘟热病毒，建议将犬瘟热病毒纳为饲养猴群的监测项目之一。本研究还为进一步分析犬瘟热病毒的分子生物学特性提供了基础。

关键词: 犬瘟热病毒, 食蟹猴, 非人灵长类, 遗传进化

Abstract:

Objective To report the diagnosis of a canine distemper virus outbreak among a colony of cynomolgus monkeys at an experimental monkey farm in 2019. Methods A total of 46 samples were collected from 21 diseased cynomolgus monkeys (exhibiting symptoms such as facial rash, skin scurf, runny nose, and diarrhea) and from one deceased monkey at an experimental monkey breeding farm in South China in late 2019, including serum, skin rash swabs, and anticoagulated whole blood, liver, lung, and skin tissues were submitted for testing. All submitted samples were tested for canine distemper virus gene fragments using real-time quantitative PCR, while immunohistochemical staining was performed to detect canine distemper virus nucleoprotein in lung tissues. The skin tissue of the deceased monkey was ground and sieved. The filtrate was inoculated into a monolayer MDCK cell line for virus isolation. Then, whole-genome sequencing was performed to identify the isolated virus. The Clustal Omega tool was used to align and analyze the homology of different Asian canine distemper virus isolates. A phylogenetic tree was constructed, followed by genetic evolutionary analysis. Results Clinical retrospective analysis revealed that the diseased cynomolgus monkeys exhibited symptoms similar to those observed in cynomolgus monkeys infected with measles virus. Necropsy findings showed red lesions in the lungs and significant hemorrhage in the colonic mucosa. Real-time quantitative PCR detected canine distemper virus nucleic acid in the serum, skin rash swabs of the infected monkeys, and various tissue samples of the deceased monkey, all of which tested positive. Calculation based on the standard curve formula indicated the viral load was highest in the skin tissue. Immunohistochemical staining of the deceased monkey's lung tissue demonstrated aggregation of CDV nucleoprotein in alveolar epithelial cells, bronchi, and bronchioles. A CDV strain was isolated from the skin tissue of the deceased monkey. Phylogenetic analysis indicated that this strain shares the closest relationship (98.86%) with the Asian-1 type canine distemper virus strain CDV/dog/HCM/33/140816, previously identified in dogs in Vietnam. Conclusion Based on comprehensive analysis of clinical symptoms, nucleic acid detection, viral protein immunohistochemistry, and whole-genome sequencing results, the diagnosis confirms that the cynomolgus monkeys in this facility are infected with canine distemper virus. It is recommended to include canine distemper virus as a routine surveillance target in captive monkey populations. Additionally, this study provides a foundation for further research on the molecular biological characteristics of canine distemper virus.

Key words: Canine distemper virus, Cynomolgus monkey, Nonhuman primates, Phylogenetic analysis

中图分类号:

Q95-33

王晨娟,杨玲焰,王立鹏,等. 2019年某实验猴养殖场食蟹猴犬瘟热暴发的诊断[J]. 实验动物与比较医学, 2025, 45(3): 360-367. DOI: 10.12300/j.issn.1674-5817.2024.160.

WANG Chenjuan,YANG Lingyan,WANG Lipeng,et al. Diagnosis of an Outbreak of Canine Distemper in Cynomolgus Monkeys in an Experimental Monkey Farm in 2019[J]. Laboratory Animal and Comparative Medicine, 2025, 45(3): 360-367. DOI: 10.12300/j.issn.1674-5817.2024.160.

图/表 5

图1 患病食蟹猴的临床体征注：A，面部有皮屑；B，流鼻涕；C，脓皮症；D，足部皮肤增厚并破溃。

Figure 1 Clinical symptoms of the infected cynomolgus monkeysNote：A， Facial skin scurf； B， Runny nose； C， Pyoderma； D， Thickening and ulceration of foot skin.

图2 免疫组织化学染色法检测病死猴肺组织切片中病毒蛋白表达（DAB染色，×100）注：大量CDV核蛋白抗原聚集于肺泡上皮细胞（A）、支气管和细支气管（B），红色箭头指示CDV核蛋白抗原的特异性染色（棕褐色）。空白对照组肺组织未见阳性表达（C）。

Figure 2 Detection of viral protein expression in lung tissue sections of the deceased monkey by immunohistochemical staining （DAB staining， ×100）Note： A large amount of CDV nucleoprotein antigen aggregated in the alveolar epithelial cells （A）， bronchi and bronchioles （B）. The red arrow indicates the specific staining （brown color） of the CDV nucleoprotein antigen. No positive expression in lung tissue of blank control group （C）.

图3 病死猴皮肤组织滤液接种MDCK细胞后的病毒感染细胞病变效应表现注：A，接种MDCK细胞后第8天出现细胞病变效应（×40）；B，正常MDCK细胞第8天未出现细胞病变效应（×40）。

Figure 3 Observation of cytopathic effect after inoculation of MDCK cells with skin tissue filtrate from the deceased monkeyNote：A， Cytopathic effect observed in MDCK cells on the 8th day after inoculation（×40）； B， Normal MDCK cells without cytopathic effect on the 8th day （×40）.

病死猴皮肤组织中分离的CDV株全长开放阅读框核苷酸序列与亚洲常见CDV株的一致性(相似度)比对 (%)

Table 1 Sequence identity (similarity) comparison between the full-length open reading frame nucleotide sequence of the CDV strain isolated from the skin tissue of the deceased monkey and prevalent Asian CDV strains

病毒株 Strains	CDV/CYN/SUZ/2020	CDV/dog/HCM/33/140816	ZC1310M	CYN07-dV	BJ-01	MKY-KM08	CDV1_TH/2014	MCL-18-Li-1/1	007Lm	CDV3
CDV/CYN/SUZ/2020	100.00	-	-	-	-	-	-	-	-	-
CDV/dog/HCM/33/140816	98.86	100.00	-	-	-	-	-	-	-	-
ZC1310M	98.44	98.73	100.00	-	-	-	-	-	-	-
CYN07-dV	97.05	97.31	97.70	100.00	-	-	-	-	-	-
BJ-01	97.05	97.30	97.69	99.92	100.00	-	-	-	-	-
MKY-KM08	96.93	97.18	97.56	99.64	99.64	100.00	-	-	-	-
CDV1_TH/2014	94.28	94.54	94.84	94.62	94.61	94.57	100.00	-	-	-
MCL-18-Li-1/1	93.54	93.78	94.11	93.87	93.87	93.82	94.06	100.00	-	-
007Lm	93.93	94.25	94.57	94.53	94.51	94.46	94.57	93.95	100.00	-
CDV3	92.14	92.37	92.68	92.43	92.42	92.43	92.34	91.96	92.76	100.00

图4 表1 基于不同CDV株全开放阅读框序列的系统发育分析（黑框标记的为本研究从病死猴皮肤组织中新分离的CDV株）

Figure 4 Phylogenetic analysis based on full-length open reading frame sequences of different CDV strains （the CDV strain newly isolated from the skin tissue of the deceased monkey in this study is marked with a black box）

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2019年某实验猴养殖场食蟹猴犬瘟热暴发的诊断

Diagnosis of an Outbreak of Canine Distemper in Cynomolgus Monkeys in an Experimental Monkey Farm in 2019

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