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

doi:10.12300/j.issn.1674-5817.2024.160

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 5

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.

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）.

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）.

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

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）

References 29

[1]	TROGU T, CASTELLI A, CANZIANI S, et al. Detection and molecular characterization of canine distemper virus in wildlife from northern Italy[J]. Pathogens, 2022, 11(12):1557. DOI:10.3390/pathogens11121557 .
[2]	SUN Z Z, LI A X, YE H H, et al. Natural infection with canine distemper virus in hand-feeding Rhesus monkeys in China[J]. Vet Microbiol, 2010, 141(3-4):374-378. DOI:10.1016/j.vetmic. 2009.09.024 .
[3]	DUQUE-VALENCIA J, SARUTE N, OLARTE-CASTILLO X A, et al. Evolution and interspecies transmission of canine distemper virus-an outlook of the diverse evolutionary landscapes of a multi-host virus[J]. Viruses, 2019, 11(7):582. DOI:10.3390/v11070582 .
[4]	OLEAGA Á, VÁZQUEZ C B, ROYO L J, et al. Canine distemper virus in wildlife in south-western Europe[J]. Transbound Emerg Dis, 2022, 69(4): e473-e485. DOI:10.1111/tbed.14323 .
[5]	LIANG J, WANG T T, WANG Q, et al. Prevalence of canine distemper in minks, foxes and raccoon dogs from 1983 to 2023 in Asia, North America, South America and Europe[J]. Front Vet Sci, 2024, 11:1394631. DOI:10.3389/fvets. 2024.1394631 .
[6]	CHENG Y N, WANG J K, ZHANG M, et al. Isolation and sequence analysis of a canine distemper virus from a raccoon dog in Jilin Province, China[J]. Virus Genes, 2015, 51(2):298-301. DOI:10.1007/s11262-015-1236-3 .
[7]	WILKES R P. Canine distemper virus in endangered species: species jump, clinical variations, and vaccination[J]. Pathogens, 2022, 12(1):57. DOI:10.3390/pathogens12010057 .
[8]	DI FRANCESCO C E, SMOGLICA C, DI PIRRO V, et al. Molecular detection and phylogenetic analysis of canine distemper virus in marsican brown bear (Ursus arctos marsicanus)[J]. Animals, 2022, 12(14):1826. DOI:10.3390/ani12141826 .
[9]	FRANZO G, DE VILLIERS L, COETZEE L M, et al. Unveiling the molecular epidemiology of canine distemper virus in Namibia: an expected pathogen showing an unexpected origin[J]. Heliyon, 2024, 10(15): e34805. DOI:10.1016/j.heliyon. 2024.e34805 .
[10]	SAKAI K J, NAGATA N, AMI Y, et al. Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008[J]. J Virol, 2013, 87(2):1105-1114. DOI:10.1128/JVI.02419-12 .
[11]	QIU W, ZHENG Y, ZHANG S F, et al. Canine distemper outbreak in rhesus monkeys, China[J]. Emerg Infect Dis, 2011, 17(8):1541-1543. DOI:10.3201/eid1708.101153 .
[12]	陆承平. 兽医微生物学[M]. 3版. 北京: 中国农业出版社, 2001: 525-526.
	LU C P. Veterinary microbiology[M]. 3rd ed. Beijing: China Agricultural Press, 2001:525-526.
[13]	YOSHIKAWA Y, OCHIKUBO F, MATSUBARA Y, et al. Natural infection with canine distemper virus in a Japanese monkey (Macaca fuscata)[J]. Vet Microbiol, 1989, 20(3):193-205. DOI:10.1016/0378-1135(89)90043-6 .
[14]	麦博, 王弘义, 盘宝进, 等. 灵长类动物感染犬瘟热[J]. 中国比较医学杂志, 2012, 22(6):72-76. DOI: 10.3969/j.issn.1671-7856.2012.06.016 .
	MAI B, WANG H Y, PAN B J, et al. Natural infection with canine distemper virus in Primates[J]. Chin J Comp Med, 2012, 22(6):72-76. DOI: 10.3969/j.issn.1671-7856.2012.06.016 .
[15]	李慧敏. 犬瘟热病毒的分离鉴定及其抗原蛋白的研究[D]. 成都: 四川农业大学, 2006.
	LI H M. Isolation and identification of canine distemper virus and study on its antigenic protein[D]. Chengdu: Sichuan Agricultural University, 2006.
[16]	黄国君, 岳华, 杨发龙, 等. SYBR GreenⅠ荧光定量RT-PCR检测犬瘟热病毒方法的建立及应用[J]. 中国预防兽医学报, 2008, 30(6):450-454. DOI: 10.3724/SP.J.1011.2008.00534 .
	HUANG G J, YUE H, YANG F L, et al. Development of a SYBR GreenⅠ real-time RT-PCR assay for detection of canine distemper virus[J]. Chin J Prev Vet Med, 2008, 30(6):450-454. DOI: 10.3724/SP.J.1011.2008.00534 .
[17]	GOUY M, GUINDON S, GASCUEL O. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[J]. Mol Biol Evol, 2010, 27(2):221-224. DOI:10.1093/molbev/msp259 .
[18]	GUINDON S, GASCUEL O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[J]. Syst Biol, 2003, 52(5):696-704. DOI:10.1080/10635150390235520 .
[19]	NGUYEN D V, SUZUKI J, MINAMI S, et al. Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam[J]. J Vet Med Sci, 2017, 79(1):123-127. DOI:10.1292/jvms.16-0394 .
[20]	QUINTERO-GIL C, RENDON-MARIN S, MARTINEZ-GUTIERREZ M, et al. Origin of canine distemper virus: consolidating evidence to understand potential zoonoses[J]. Front Microbiol, 2019, 10:1982. DOI:10.3389/fmicb.2019.01982 .
[21]	RIVERA-MARTÍNEZ A, RODRÍGUEZ-ALARCÓN C A, ADAME-GALLEGOS J R, et al. Canine distemper virus: origins, mutations, diagnosis, and epidemiology in Mexico[J]. Life, 2024, 14(8):1002. DOI:10.3390/life14081002 .
[22]	SHESHBERADARAN H, NORRBY E, MCCULLOUGH K C, et al. The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies[J]. J Gen Virol, 1986, 67 ( Pt 7):1381-1392. DOI:10.1099/0022-1317-67-7-1381 .
[23]	ROUXEL R N, SVITEK N, VON MESSLING V. A chimeric measles virus with canine distemper envelope protects ferrets from lethal distemper challenge[J]. Vaccine, 2009, 27(36):4961-4966. DOI:10.1016/j.vaccine.2009.05.096 .
[24]	OTSUKI N, SEKIZUKA T, SEKI F, et al. Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor[J]. Virology, 2013, 435(2):485-492. DOI:10.1016/j.virol.2012.10.033 .
[25]	RENDON-MARIN S, RUÍZ-SAENZ J. Universal peptide-based potential vaccine design against canine distemper virus (CDV) using a vaccinomic approach[J]. Sci Rep, 2024, 14(1):16605. DOI:10.1038/s41598-024-67781-5 .
[26]	SWATI, DEKA D, UPPAL S K, et al. Isolation and phylogenetic characterization of Canine distemper virus from India[J]. Virusdisease, 2015, 26(3):133-140. DOI:10.1007/s13337-015-0256-x .
[27]	GUERCIO A, MIRA F, DI BELLA S, et al. Biomolecular analysis of canine distemper virus strains in two domestic ferrets (Mustela Putorius furo)[J]. Vet Sci, 2023, 10(6):375. DOI:10.3390/vetsci10060375 .
[28]	MOCHIZUKI M, HASHIMOTO M, HAGIWARA S, et al. Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan[J]. J Clin Microbiol, 1999, 37(9):2936-2942. DOI:10.1128/JCM.37.9.2936-2942.1999 .
[29]	SHI N, ZHANG L, YU X H, et al. Insight into an outbreak of canine distemper virus infection in masked palm civets in China[J]. Front Vet Sci, 2021, 8:728238. DOI:10.3389/fvets.2021.728238 .