Molecular identification and genetic characterization of Cryptosporidium spp. in patients with diarrhea in Ningbo City

doi:10.12140/j.issn.1000-7423.2023.02.012

Abstract

Abstract: Objective To analyze the Cryptosporidium infection and genetic characterization of outpatients with diarrhea in a sentinel hospital in Ningbo, to provide new ideas for clinical investigation on etiology of diarrhea. Methods A total of 650 fecal samples were collected from clinical diarrhea patients in NingBo City from November 2019 to July 2021. The genomic DNA from these samples was extracted, and the small subunit ribosomal ribonucleic acid gene (SSU rRNA) of Cryptosporidium was amplified by nested PCR. The products were analyzed by 1.5% agarose gel electrophoresis. The DNA from above positive sample was used as the template to amplify the 60-kDa glycoprotein (gp60) gene by nested PCR, of which the product was analyzed by 1.5% agarose gel electrophoresis. PCR amplification products of similar size to the target bands were sequenced in both directions, and blasted to identify the species or genotype according to the GenBank public database resources using Clustalx 2.1 software. Use Mega 11.0 software to draw the phylogenetic tree by the neighbour-joining (N-J) method. Results Among the fecal samples collected, 369 (56.8%) were from male diarrhea cases, and 281 (43.2%) female cases; of them the ages ranged from 1 to 99 years. After amplifying of the SSU rRNA gene, only one DNA sample showed specific bands at around 830 bp in electrophoresis, indicating Cryptosporidium spp. positive. The patient was a 53-year-old male, farmer with watery stool. According to the analysis of DNA sequence, it was identified as C. meleagridis, which has 100% homology with those of a human-derived isolate from Shanghai, a dairy-derived isolate from Heilongjiang Province, a chicken-derived isolate from Yunnan Province. The phylogenetic tree showed that these isolates locate in the same branch. The product of gp60 gene amplification displayed a specific band at around 955 bp, which was identified as the Ⅲb subtype family, with the highest homology (99.8%) to the human derived ⅢbA24G1R1 subtype, sequence alignment indicate it is a new subtype ⅢbA26G1 of C. meleagridis (GenBank accession No. OQ032548). Conclusion Zoonotic C. meleagridis was identified from a clinical diarrhea case in Ningbo area, suggesting that surveillance on Cryptosporidium infection in people with diarrhea and epidemiological tracing for infection source should be strengthened.

Key words: Clinical diarrhea patient, Cryptosporidium spp., C. meleagridis, Subtype

CLC Number:

R382.3

QIN Yuan, LIU Hua, JIANG Yanyan, CAO Jianping, SHEN Yujuan. Molecular identification and genetic characterization of Cryptosporidium spp. in patients with diarrhea in Ningbo City[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(2): 202-208.

Figures/Tables 6

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Table 2

Distribution of human, animal and water source C. meleagridis in China

来源 Source	地区 Area	年份 Year	检出率/%（n/N） Rate of positive/% (n/N)	基因亚型（n） Subtype (n)	参考文献 References
人 Human
HIV/AIDS患者 HIV/AIDS patiens	河南 Henan	2006	-	-	[14]
		2007—2010	0.7 (5/683)	ⅢbA27G1R1 (1), ⅢbA29G1R1 (1), ⅢbA26G1R1 (1), ⅢeA26G2R1 (1), N^*(1)	[15]
	台湾 Taiwan	1994—2004	0.1 (1/1 044)	-	[16]
腹泻儿童 Diarrhea children	湖北 Hubei	2016	2.0 (10/500)	ⅢbA21G1R1 (1), ⅢbA22G1R1 (8), ⅢbA26G1R1 (1)	[17]
腹泻儿童 Diarrhea children	上海 Shanghai	2007—2009	0.1 (4/3 245)	-	[18]
腹泻成人 Diarrhea adults	浙江 Zhejiang	2019—2021	0.2 (1/571)	ⅢbA26G1 (1)	本研究 This study
动物 Animal
鹌鹑 Quail	河南 Henan	2006—2007	0.1 (2/1 818)	-	[19]
宠物鸟 Pet bird	河南 Henan	2018—2019	0.8 (8/1 005)	-	[20]
狐狸 Fox	吉林 Jilin	2014—2016	0.5 (1/213)	-	[21]
	山东 Shandong	2019	21.7 (65/299)	ⅢeA21G2R1 (20), ⅢeA19G2R1 (6), ⅢeA17G2R1 (1)	[22]
鸡 Chicken	安徽 Anhui	-	19.5 (39/200)	-	[23]
		2018—2019	0.2 (2/829)	ⅢbA26G1R1b (2)	[24]
	广东 Guangdong	2020—2021	7.8 (78/1 001)	ⅢgA25G3R1a (2), ⅢbA23G1R1c (24), ⅢbA24G1R1 (20), ⅢbA21G1R1a (7), ⅢeA17G2R1 (8), ⅢeA26G2R1 (3)	[25]
	河南 Henan	2006—2007	0.2 (3/1 542)	-	[26]
		2011	10.5 (25/238)	-	[27]
	湖北 Hubei	2017	3.2 (15/471)	ⅢbA22G1R1c (6), ⅢbA23G1R1d (2), ⅢbA26G1R1b (1), ⅢeA17G2R1 (1), ⅢeA19G2R1 (1)	[28]
	新疆 Xinjiang	2020—2021		ⅢbA23G1R1 (1), ⅢbA25G1R1 (20), ⅢbA31G1R1 (4)	[29]
		2022	0.2 (35/175)	ⅢgA22G3R1 (1), ⅢbA25G1R1 (10), ⅢgA24G3R1 (9), ⅢgA25G2R1 (1)	[30]
	云南 Yunnan	2020	-	-	[31]
	浙江 Zhejiang	2010—2012	1.0 (4/385)	-	[32]
牛 Catttle	黑龙江 Heilongjiang	2009—2011	3.3 (5/151）	ⅢeA22G2R1 (5)	[33]
水貂 Mink	吉林、辽宁、黑龙江 Jilin, Liaoning, Heilongjiang	2014	1.9 (3/162)	-	[34]
水 Water
河/湖水 River/lake water	上海Shanghai	2013—2014	2.3 (4/178)	ⅢbA24G1 (1)	[35]
	天津 Tianjin	-	1.9 (1/52)	-	[36]
	重庆 Chongqing	2011	1.5 (1/66)	-	[37]
污水 Sewage	广东 Guangdong	2018—2019	1.8 (6/326)	-	[38]
	南京 Nanjing	2007—2009	2.3 (2/87)	-	[39]
	青岛 Qingdao	2008	6.4 (7/109)	-	[39]
	上海 Shanghai	2006—2007	1.1 (7/90)	-	[40]
		2006—2007	7.8 (7/90)	-	[39]
		2012—2014	5.0 (4/80)	ⅢbA22G1R1c (1)	[41]
	武汉 Wuhan	2008	6.0 (6/100)	-	[39]
自来水 Tap-water	上海 Shanghai	2009—2010	1.3 (1/80)	-	[42]

Table 2

References 52

[1]	Ryan U, Fayer R, Xiao LH. Cryptosporidium species in humans and animals: current understanding and research needs[J]. Parasitology, 2014, 141(13): 1667-1685. doi: 10.1017/S0031182014001085 pmid: 25111501
[2]	Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[J]. Lancet, 2013, 382(9888): 209-222. doi: 10.1016/S0140-6736(13)60844-2 pmid: 23680352
[3]	Mondal D, Minak J, Alam M, et al. Contribution of enteric infection, altered intestinal barrier function, and maternal malnutrition to infant malnutrition in Bangladesh[J]. Clin Infect Dis, 2012, 54(2): 185-192. doi: 10.1093/cid/cir807 pmid: 22109945
[4]	Checkley W, White AC Jr, Jaganath D, et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium[J]. Lancet Infect Dis, 2015, 15(1): 85-94. doi: 10.1016/S1473-3099(14)70772-8 pmid: 25278220
[5]	Sandhu SK, Priest JW, Lammie PJ, et al. The natural history of antibody responses to Cryptosporidium parasites in men at high risk of HIV infection[J]. J Infect Dis, 2006, 194(10): 1428-1437. doi: 10.1086/jid.2006.194.issue-10
[6]	Ryan U, Zahedi A, Paparini A. Cryptosporidium in humans and animals--a one health approach to prophylaxis[J]. Parasite Immunol, 2016, 38(9): 535-547. doi: 10.1111/pim.12350 pmid: 27454991
[7]	Ryan U, Zahedi A, Feng YY, et al. An update on zoonotic Cryptosporidium species and genotypes in humans[J]. Animals (Basel), 2021, 11(11). DOI: 3307.
[8]	Xu N, Liu H, Jiang YY, et al. First report of Cryptosporidium viatorum and Cryptosporidium occultus in humans in China, and of the unique novel C. viatorum subtype XVaA3h[J]. BMC Infect Dis, 2020, 20(1): 16. doi: 10.1186/s12879-019-4693-9
[9]	Xiao L. Molecular epidemiology of cryptosporidiosis: an update[J]. Exp Parasitol, 2010, 124(1): 80-89. doi: 10.1016/j.exppara.2009.03.018 pmid: 19358845
[10]	Liu AQ, Gong BY, Liu XH, et al. A retrospective epidemiological analysis of human Cryptosporidium infection in China during the past three decades (1987—2018)[J]. PLoS Negl Trop Dis, 2020, 14(3): e0008146. doi: 10.1371/journal.pntd.0008146
[11]	Feng SY, Chang H, Wang Y, et al. Molecular characterization of Cryptosporidium spp. in Brandt’s vole in China[J]. Front Vet Sci, 2020, 7: 300. doi: 10.3389/fvets.2020.00300
[12]	Stensvold CR, Beser J, Axén C, et al. High applicability of a novel method for gp60-based subtyping of Cryptosporidium meleagridis[J]. J Clin Microbiol, 2014, 52(7): 2311-2319. doi: 10.1128/JCM.00598-14 pmid: 24740082
[13]	Wang X, Shen YJ, Cao JP. Epidemic status and prevention and control progress of cryptosporidiosis in China[J]. J Trop Dis Parasitol, 2022, 20(3): 136-148. (in Chinese)
	(王旭, 沈玉娟, 曹建平. 我国隐孢子虫病流行现状与防控进展[J]. 热带病与寄生虫学, 2022, 20(3): 136-148.)
[14]	Zhang XQ, Lu H, Luo Y, et al. Identification of molecule species of the three cryptosporidiosis in Henan Province[J]. Henan J Prev Med, 2006, 17(2): 79. (in Chinese)
	(张晓琴, 路浩, 罗予, 等. 河南省三株人源隐孢子虫分离株鉴定[J]. 河南预防医学杂志, 2006, 17(2): 79.)
[15]	Wang L, Zhang HW, Zhao XD, et al. Zoonotic Cryptosporidium species and Enterocytozoon bieneusi genotypes in HIV-positive patients on antiretroviral therapy[J]. J Clin Microbiol, 2013, 51(2): 557-563. doi: 10.1128/JCM.02758-12 pmid: 23224097
[16]	Hung CC, Tsaihong JC, Lee YT, et al. Prevalence of intestinal infection due to Cryptosporidium species among Taiwanese patients with human immunodeficiency virus infection[J]. J Formos Med Assoc, 2007, 106(1): 31-35. doi: 10.1016/S0929-6646(09)60213-8
[17]	Wang T, Fan Y, Koehler AV, et al. First survey of Cryptosporidium, Giardia and Enterocytozoon in diarrhoeic children from Wuhan, China[J]. Infect Genet Evol, 2017, 51: 127-131. doi: S1567-1348(17)30077-1 pmid: 28284996
[18]	Feng YY, Wang L, Duan LP, et al. Extended outbreak of cryptosporidiosis in a pediatric hospital, China[J]. Emerg Infect Dis, 2012, 18(2): 312-314. doi: 10.3201/eid1802.110666 pmid: 22305484
[19]	Wang R, Wang F, Zhao J, et al. Cryptosporidium spp. in quails (Coturnix coturnix japonica) in Henan, China: molecular characterization and public health significance[J]. Vet Parasitol, 2012, 187(3/4): 534-537. doi: 10.1016/j.vetpar.2012.02.002
[20]	Dong HJ, Cheng R, Li XM, et al. Molecular identification of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in captive pet birds in Henan Province, central China[J]. J Eukaryot Microbiol, 2021, 68(2): e12839.
[21]	Yang ZY, Zhao W, Wang JG, et al. Molecular detection and genetic characterizations of Cryptosporidium spp. in farmed foxes, minks, and raccoon dogs in northeastern China[J]. Parasitol Res, 2018, 117(1): 169-175. doi: 10.1007/s00436-017-5686-5
[22]	Wang WJ, Wei YT, Cao SH, et al. Divergent Cryptosporidium species and host-adapted Cryptosporidium canis subtypes in farmed minks, raccoon dogs and foxes in Shandong, China[J]. Front Cell Infect Microbiol, 2022, 12: 980917. doi: 10.3389/fcimb.2022.980917
[23]	Wei JZ, Li PY, He CS, et al. Investigation on infectious condition of chicken Cryptosporidium in a private farm of Anhui Province[J]. Chin J Vet, 2003, 23(5): 472-473. (in Chinese)
	(魏建忠, 李培英, 何长生, 等. 安徽省某鸡场鸡隐孢子虫感染情况调查[J]. 中国兽医学报, 2003, 23(5): 472-473.)
[24]	Gong Z, Kan ZZ, Huang JM, et al. Molecular prevalence and characterization of Cryptosporidium in domestic free-range poultry in Anhui Province, China[J]. Parasitol Res, 2021, 120(10): 3519-3527. doi: 10.1007/s00436-021-07191-y
[25]	Lin XH, Xin LY, Qi M, et al. Dominance of the zoonotic pathogen Cryptosporidium meleagridis in broiler chickens in Guangdong, China, reveals evidence of cross-transmission[J]. Parasit Vectors, 2022, 15(1): 188. doi: 10.1186/s13071-022-05267-x
[26]	Wang RJ, Jian FC, Sun YP, et al. Large-scale survey of Cryptosporidium spp. in chickens and Pekin ducks (Anas platyrhynchos) in Henan, China: prevalence and molecular characterization[J]. Avian Pathol, 2010, 39(6): 447-451. doi: 10.1080/03079457.2010.518314
[27]	Dai J, Yan WC, Wang TQ, et al. Epidemiological analysis of Cryptosporidium species from intensive chicken farms in Luoyang[J]. J Henan Univ Sci Technol Nat Sci, 2012, 33(1): 63-66, 8. (in Chinese)
	(代娟, 闫文朝, 王天奇, 等. 洛阳地区规模化鸡场隐孢子虫流行病学分析[J]. 河南科技大学学报(自然科学版), 2012, 33(1):63-66, 8.)
[28]	Liao C, Wang T, Koehler AV, et al. Molecular investigation of Cryptosporidium in farmed chickens in Hubei Province, China, identifies “zoonotic” subtypes of C. meleagridis[J]. Parasites Vectors, 2018, 11(1): 484. doi: 10.1186/s13071-018-3056-5
[29]	Feng XW, Xin LY, Yu FC, et al. Genetic characterization of Cryptosporidium spp. in Hotan Black Chickens in China reveals two novel subtypes of Cryptosporidium meleagridis[J]. Parasite, 2022, 29: 50. doi: 10.1051/parasite/2022051
[30]	Feng XW, Tuo HX, Li TT, et al. Longitudinal surveillance of Cryptosporidium spp. in broiler chickens in Xinjiang, northwest China: genetic diversity of Cryptosporidium meleagridis subtypes[J]. Parasitol Res, 2022, 121(12): 3589-3595. doi: 10.1007/s00436-022-07683-5
[31]	Zhao GH, Han JQ, Ge LH, et al. Identification of Cryptosporidium from laying hens in Yunnan Province and bioinformatics analysis of 18S rRNA[J]. Heilongjiang Animal Sci Vet Med, 2022(19): 76-79, 142. (in Chinese)
	(赵国洪, 韩建强, 葛丽红, 等. 云南省蛋鸡源隐孢子虫鉴定及18S rRNA生物信息学分析[J]. 黑龙江畜牧兽医, 2022(19): 76-79, 142.)
[32]	Wang LM, Xue X, Li JQ, et al. Cryptosporidiosis in broiler chickens in Zhejiang Province, China: molecular characterization of oocysts detected in fecal samples[J]. Parasite, 2014, 21: 36. doi: 10.1051/parasite/2014035 pmid: 25075975
[33]	Zhang WZ, Wang RJ, Yang FK, et al. Distribution and genetic characterizations of Cryptosporidium spp. in pre-weaned dairy calves in Northeastern China’s Heilongjiang Province[J]. PLoS One, 2013, 8(1): e54857. doi: 10.1371/journal.pone.0054857
[34]	Zhang S, Tao W, Liu C, et al. First report of Cryptosporidium canis in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) and identification of several novel subtype families for Cryptosporidium mink genotype in minks (Mustela vison) in China[J]. Infect Genet Evol, 2016, 41: 21-25. doi: 10.1016/j.meegid.2016.03.016
[35]	Hu Y, Feng YY, Huang CC, et al. Occurrence, source, and human infection potential of Cryptosporidium and Enterocytozoon bieneusi in drinking source water in Shanghai, China, during a pig carcass disposal incident[J]. Environ Sci Technol, 2014, 48(24): 14219-14227. doi: 10.1021/es504464t
[36]	Xiao SM, Zhang Y, Zhao XY, et al. Presence and molecular characterization of Cryptosporidium and Giardia in recreational lake water in Tianjin, China: a preliminary study[J]. Sci Rep, 2018, 8(1): 2353. doi: 10.1038/s41598-018-20902-3
[37]	Xiao G, Qiu Z, Qi J, et al. Occurrence and potential health risk of Cryptosporidium and Giardia in the Three Gorges Reservoir, China[J]. Water Res, 2013, 47(7): 2431-2445. doi: 10.1016/j.watres.2013.02.019
[38]	Fan YY, Wang XR, Yang RH, et al. Molecular characterization of the waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp. in wastewater and sewage in Guangzhou, China[J]. Parasit Vectors, 2021, 14(1): 66. doi: 10.1186/s13071-020-04566-5
[39]	Li N, Xiao LH, Wang L, et al. Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater[J]. PLoS Negl Trop Dis, 2012, 6(9): e1809. doi: 10.1371/journal.pntd.0001809
[40]	Feng YY, Li N, Duan LP, et al. Cryptosporidium genotype and subtype distribution in raw wastewater in Shanghai, China: evidence for possible unique Cryptosporidium hominis transmission[J]. Medicine (Baltimore), 2009, 47(1): 153-157.
[41]	Huang CC, Hu Y, Wang L, et al. Environmental transport of emerging human-pathogenic Cryptosporidium species and subtypes through combined sewer overflow and wastewater[J]. Appl Environ Microbiol, 2017, 83(16): e00682-17.
[42]	Feng YY, Zhao XK, Chen JX, et al. Occurrence, source, and human infection potential of Cryptosporidium and Giardia spp. in source and tap water in Shanghai, China[J]. Appl Environ Microbiol, 2011, 77(11): 3609-3616. doi: 10.1128/AEM.00146-11
[43]	Slavin D. Cryptosporidium meleagridis (sp. nov.)[J]. J Comp Pathol Ther, 1955, 65: 262-IN23. doi: 10.1016/S0368-1742(55)80025-2
[44]	Zha HB, Jiang JS. The life cycle of Cryptosporidium meleagbidis in quails[J]. Acta Vet Zootechnica Sin, 1994, 25(3): 273-278. (in Chinese)
	(查红波, 蒋金书. 火鸡隐孢子虫在鹌鹑体内发育史的研究[J]. 畜牧兽医学报, 1994, 25(3): 273-278.)
[45]	Elwin K, Hadfield SJ, Robinson G, et al. The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, 2000—2008[J]. Epidemiol Infect, 2012, 140(4): 673-683. doi: 10.1017/S0950268811000860 pmid: 21733255
[46]	Akiyoshi DE, Dilo J, Pearson C, et al. Characterization of Cryptosporidium meleagridis of human origin passaged through different host species[J]. Infect Immun, 2003, 71(4): 1828-1832. doi: 10.1128/IAI.71.4.1828-1832.2003 pmid: 12654797
[47]	Darabus G, Olariu R. The homologous and interspecies transmission of Cryptosporidium parvum and Cryptosporidium meleagridis[J]. Pol J Vet Sci, 2003, 6(3): 225-228. pmid: 14510055
[48]	Huang KH, Akiyoshi DE, Feng XC, et al. Development of patent infection in immunosuppressed C57Bl/6 mice with a single Cryptosporidium meleagridis oocyst[J]. J Parasitol, 2003, 89(3): 620-622. doi: 10.1645/0022-3395(2003)089[0620:DOPIII]2.0.CO;2 pmid: 12880270
[49]	Tůmová E, Skrivan M, Marounek M, et al. Performance and oocyst shedding in broiler chickens orally infected with Cryptosporidium baileyi and Cryptosporidium meleagridis[J]. Avian Dis, 2002, 46(1): 203-207. pmid: 11922336
[50]	Sréter T, Kovács G, da Silva AJ, et al. Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate[J]. Appl Environ Microbiol, 2000, 66(2): 735-738. doi: 10.1128/AEM.66.2.735-738.2000
[51]	Ryan U. Cryptosporidium in birds, fish and amphibians[J]. Exp Parasitol, 2010, 124(1): 113-120. doi: 10.1016/j.exppara.2009.02.002 pmid: 19545515
[52]	Qi M, Wang R, Ning C, et al. Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance[J]. Exp Parasitol, 2011, 128(4): 336-340. doi: 10.1016/j.exppara.2011.04.003 pmid: 21557938

位点 Locus	引物名称 Primer name	引物序列（5'→3'） Primer sequences（5'→3'）	产物长度/bp Product length/bp
SSU rRNA	Cry18sF1	TTCTAGAGCTAATACATGCG	1 100
	Cry18sR1	CCCATTTCCTTCGAAACAGGA	1 100
	Cry18sF2	GGAAGGGTTGTATTTATTAGATAAAG	830
	Cry18sR2	AAGGAGTAAGGAACAACCTCCA	830
gp60	F1	GATGAGATTGTCGCTCGTTATC	1 161
	R1	AACCTGCGGAACCTGTG	1 161
	F2	GAGATTGTCGCTCGTTATCG	955
	R2	GATTGCAAAAACGGAAGG	955