Molecular characterizations of Cryptosporidium spp. in brown rats (Rattus norvegicus) from an animal feedlot in Xinjiang, China

doi:10.21203/rs.3.rs-4266958/v1

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Molecular characterizations of Cryptosporidium spp. in brown rats (Rattus norvegicus) from an animal feedlot in Xinjiang, China

https://doi.org/10.21203/rs.3.rs-4266958/v1

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Cryptosporidiumspp. infection is common in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, and a fecal sample was collected directly from the rectal contents of each rat. The extracted DNA from all fecal samples was examined for Cryptosporidium spp. by PCR at SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was 5.5% (24/435). The infection rates varied across different animal enclosures. Specifically, infection rates were 0% (0/51) in chicken coop, 0% (0/3) in cowshed, 6.1% (6/98) in sheepfold, 7.6% (10/132) in pigsty, 7.0% (5/71) in dovecote, and 3.8% (3/80) in outdoor environments. The study identified three species and one genotype of Cryptosporidium, including C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium Rat genotype IV (n = 9). Of the C. parvum isolates, two were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These findings present fundamental data on the prevalence and genetic evolution of Cryptosporidium in rodents.

Cryptosporidium

detection

identify

subtype

rodents

Cryptosporidium spp. are zoonotic protozoan parasites capable of infecting over 260 vertebrate species, including humans (Ryan et al. 2014). Brown rats (Rattus norvegicus) are a widespread group of rodents found in various environments, including urban, rural, and animal habitats, and play a crucial role in the transmission of Cryptosporidium (Meerburg et al. 2009).To date, at least 17 species/genotypes of Cryptosporidium have been found toinfect rats, including C. parvum, C. ubiquitum, C. muris, C. andersoni, C. proliferans, C. scrofarum, C. meleagridis, C. occultus, C. viatorum, C. tyzzeri, C. canis, and Cryptosporidium Rat genotypes I to IV, Cryptosporidium pika genotype and Cryptosporidium Qinghai vole genotype (Zhao et al. 2019).

Cryptosporidium infection in wild rodents have been studied in several regions of China, including Shanxi, Guangxi Zhuang Autonomous Region, Zhejiang Province, Shandong Province, Inner Mongolia Autonomous Region, Hainan Province and Heilongjiang Province, China. The infection rate of Cryptosporidium in wild rodents was 6.8% in China (Lv et al. 2009). Additionally, Cryptosporidium infection was common in cattle, sheep and pigs in Xinjiang Uygur Autonomous Region (hereinafter referred to as Xinjiang), China (Qi et al. 2019; Qi et al. 2016; Qi et al. 2020). However, there are few reports on investigations of Cryptosporidium infections in rodents in Xinjiang. The objective of this study is to examine the characteristics of Cryptosporidium infections and the species distribution of brown rats in an animal feedlot in Xinjiang, in order to improve our understanding of Cryptosporidium species in rodents in China.

Ethical approval

The Research Ethics Committee of Tarim University reviewed and approved our study (approval no. TUEC 2021-35).

Brown rats collections and rectal contents sample collection

From October 2021 to December 2023, samples of brown rats were collected from an animal feedlot in Alaer city, Xinjiang, where cattle, sheep, pigs, chickens and pigeons were raised. Sticky boards were placed in various locations, including the chicken coop, cowshed, sheepfold, pigsty, dovecote, and outdoor environments. The brown rats captured each morning were sent to the laboratory for further analysis. A total of 291 brown rats were captured in China, including 24 from the chicken coop, 2 from the cowshed, 84 from the sheepfold, 61 from the pigsty, and 44 from the dovecote..

The captured brown rats were anaesthetised by injecting 0.2 mL of 3% pentobarbital sodium solution into their abdominal cavity. Subsequently, they were dissected, and a rectal contents sample of approximately 300 mg was collected and placed in a 1.5 mL centrifuge tube for DNA extraction.

DNA extraction and PCR amplification

The E.Z.N.A.® Stool DNA Kits (Omega Biotek Inc, Norcross, USA) were used to extract DNA from 300 mg of each sample. The extracted DNA samples were stored at -20°C.

PCR was used to detect Cryptosporidium in all DNA samples by targeting the partial SSU rRNA gene. The primers used were forward 18SiCF1: 5′-GACATATCATTCAAGTTTCTGACCC-3′ and reverse 18SiCR1:5′-CTG AAGGAGTAAGGAACAACC-3′, as previously described. The secondary PCR was performed using forward primer 18SiCF2:5′-CCTATCAGCTTTAGACGGTAGG-3′ and reverse primer 18SiCR2: 5′-TCTAAGAATTTCACCTCTGACTG-3′ (Ryan et al. 2003). C. parvum was subtyped using PCR at the 60-kDa glycoprotein (gp60) gene, following the method described by Alves et al. (2003). The primary PCR used forward primer AL3531 (5′-ATAGTCTCCGCTGTATTC-3′) and reverse primer AL3535 (5′- GGAAGGAACGATGTATCT-3′), while the secondary PCR used forward primer AL3532 (5′-TCCGCTGTATTCTCAGCC-3′) and reverse primer AL3534 (5′-GCAGAGGAACCAGCATC-3′) (Alves et al. 2003). The PCR products were detected by electrophoresis on 1.5% agarose gels.

Sequence analyses

The PCR amplified products were bi-sequenced by GENEWIZ (Suzhou, China) and searched using the Basic Local Alignment Search Tool (BLAST) available at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/blast/). Based on the results of the sequence analysis, the reference sequences were downloaded and aligned using Clustal X 2.1 software to identify Cryptosporidium species and subtypes.

The sequences of the Cryptosporidium species/genotype/subtype generated in this study have been deposited in the GenBank under the accession numbers PP593614 - PP593617 (species/genotype) and PP601315 (subtype).

Cryptosporidium infection is widespread among brown rats worldwide (see Table 2). In this study of 435 brown rat samples from an animal raising plot in Xinjiang, 24 samples tested positive for Cryptosporidium by PCR, resulting in an infection rate of 5.5% (24/435) (refer to Table 1). The infection rate was higher than that reported for Cryptosporidium in brown rats in Shanxi Province (3.8%; 2/53), Shandong Province (0%; 0/227), Zhejiang Province (3.4%; 4/119), Iran (4.1%; 3/73), and Nigeria (1.5%; 2/134), but lower than that reported for Cryptosporidium in brown rats in Heilongjiang Province (9.1%; 22/242), Henan Province (28.6%; 16/56), (7.1%; 12/196), Spain (29.0%; 29/100), and Japan (38.0%; 19/50). The prevalence of Cryptosporidium infection in brown rats varies across different regions and is closely related to the rodents' living environment in different sampling sites.

At the SSU rRNA gene, 24 positive samples for Cryptosporidium were successfully sequenced, revealing the presence of three species and one genotype of Cryptosporidium, including C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1) and Cryptosporidium Rat genotype IV (n = 9). Among these, C. occultus has a wide host range (cattle, yak, water buffalo and rat), with rats being the main host of C. occultus (Kváč et al. 2018). C. occultus has also been reported to infect humans in Canada (Ong et al. 2002), England (Robinson et al. 2011) and China (Xu et al. 2020). In the present study, C. occultus was the predominant species infected in brown rats, suggesting that brown rats are one of the natural hosts of C. occultus.

Cryptosporidium ditrichi was previously thought to be restricted to European rodents. Sárka et al (Sárka et al. 2019) found the species in Apodemus spp. (mainly yellow-necked rats) in 12 of 16 European countries, with a prevalence rate of 5.6% (22/392). Thus, the present study marked the first time that C. ditrichi has been detected in rodents outside of Europe. Furthermore, it is noteworthy that the SSU rRNA gene sequence of C. ditrichi in brown rats showed the highest similarity to that of the Swedish human isolate. Moreover, a recent study has also shown that infection with this species leads to typical cryptosporidiosis symptoms in humans in Sweden (Beser et al. 2020), implying that it possesses the risk of transmission from rodents to humans and causing disease (Kivistö et al. 2021).

Cryptosporidium parvum is a zoonotic species that predominantly infects rodents worldwide. It has a host range that includes at least 20 rodent species, with the highest prevalence observed in brown rats, bank voles, and yellow-necked field mice. Multiple C. parvum subtypes have been identified in rodents based on the gp60 gene locus, including IIaA15G2R1, IIaA16G2R1, IIaA17G2R1, IIaA18G1R1b, IIaA18G3R1, IIdA15G1, IIiA10, IIpA9, IIpA6, IIoA15G1 and IIoA13G1 (Zhang et al. 2022). It should be noted that previous reports of C. parvum in brown rats worldwide only included a single subtype, namely IIdA15G1. In contrast, the subtype of C. parvum discovered in Xinjiang, China is IIdA19G1, which is dominant in China and can infect neonatal dairy calves, sheep, and children (Chen et al. 2023; Wang et al. 2023). This suggests that brown rats play an important role as vectors in the risk of zoonotic transmission of C. parvum.

Five generic categories of Cryptosporidium Rat genotype are known (Rat genotypes I-V). Cryptosporidium Rat genotype I and Cryptosporidium Rat genotype IV are the main genotypes infecting rats (Zhao et al. 2018). In 2018, the first detection of Cryptosporidium Rat genotype IV in brown rats was reported in Heilongjiang, China (Zhao et al. 2018). Cryptosporidium Rat genotype IV was also detected in brown rats in Henan Province, China (Zhao et al. 2019). In our study, only Cryptosporidium Rat genotype IV was found, which accounted for more than the average number of total Cryptosporidium positives. It is suggested that Cryptosporidium Rat genotype IV is prevalent in the brown rat population in China.

In conclusion, as a common rodent, the brown rat can transmit and carry a wide range of Cryptosporidium species, including some zoonotic Cryptosporidium species such as C. parvum and C. occultus. Research on brown rats to assess their role in the dynamics of Cryptosporidium infection and transmission in humans and animals should be further intensified.

Ethical Approval

The Research Ethics Committee of Tarim University reviewed and approved our study (approval no. TUEC 2021-35). The review was performed in accordance with the Regulations on the Management of Experimental Animals formulated by the National Science and Technology Commission of the People's Republic of China and the Guiding Opinions on the Treatment of Experimental Animals issued by the Ministry of Science and Technology of the People's Republic of China.

Consent to publication

All the authors consent to publication of this article.

Consent to Participate

Not applicable.

Competing interests

The authors have no competing interests to declare.

Author contributions

Zhenjie Zhang and Meng Qi conceived and designed the study. Yongqiang He collected the samples. Min Li, Ping Li and Chenhao Zhao carried out the PCR assays and sequence analyses. Zhenjie Zhang, Min Li, Fuchang Yu, Hui Dong interpreted the results and drafted the manuscript. All authors have read and approved the final version of the manuscript.

Funding

This work was supported by the National College Students Innovation and Entrepreneurship Training Program (202310757002).

Availability of data and materials

The nucleotide sequences reported in this study have been deposited in the GenBank database of the National Center for Biotechnology Information under accession numbers PP593614 - PP593617 (species/genotype) and PP601315 (subtype).

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Table1 Infection tate of Crytosporidium species/genotypes in brown rats from a animal raising plot in Alaer in Xinjiang, China.

Location	No. of samples	No. of Positive (%)	Species/genotypes/subtypes (n)
Chicken coop	51	0 (0)	/
Cowshed	3	0 (0)	/
Sheepfold	98	6 (6.1%)	C. occultus (4), C. parvum (1)/IIdA19G1 (1), Cryptosporidium Rat genotype IV (1)
Pigsty	132	10 (7.6%)	C. occultus (2), Cryptosporidium Rat genotype IV (8)
Dovecot	71	5 (7.0%)	C. occultus (3), C. parvum (2)/IIdA19G1 (1),
Outdoor environment	80	3 (3.8%)	C. occultus (1), C. parvum (1), C. ditrichi (1)
Total	435	24 (5.5%)	C. occultus (10), C. parvum (4)/IIdA19G1 (2), C. ditrichi (1), Cryptosporidium Rat genotype IV (9)

Table 2 Infection tate of Crytosporidium species/genotypes in brown rats (Rattus norvegicus) .

Country/Province	Infection rate % (n/N)	Species/genotypes (n)	Reference
China/Shanxi	3.8% (2/53)	Cryptosporidium sp. novel 2 (n=4)	(Ni et al. 2021)
China/Shandong	0 (0/227)	-	(Ni et al. 2021)
China/Zhejiang	3.4% (4/119)	Cryptosporidium sp. novel 2 (n=4)	(Ni et al. 2021)
China/Heilongjiang	6.7% (2/30)	Rat genotype I (2)	(Zhao et al. 2018)
China/Heilongjiang	15.0% (15/100)	Rat genotype I (9), Rat genotype IV (3)	(Zhao et al. 2018)
China/Heilongjiang	2.7% (1/37)	C. ubiquitum (1)	(Zhao et al. 2018)
China/Heilongjiang	7.3% (4/55)	Rat genotype IV (3), suis-like genotype (1)	(Zhao et al. 2018)
China/Heilongjiang	0 (0/20)	-	(Zhao et al. 2018)
China/Heinan	28.6% (16/56)	Rat genotype IV (13), Rat genotype III (1), C. muris (1), C. occultus (1)	(Zhao et al. 2019)
Spain/Barcelona	0 (0/15)	-	(Galán-Puchades et al, 2021)
Spain/Barcelona	34.5% (29/85)	Cryptosporidium spp. (29).	(Galán-Puchades et al, 2021)
China/Henan	7.1% (12/196)	C. parvum (9), C. muris (3)	(Zhao et al. 2015)
Iran/Ahvaz	4.1% (3/73)	C. parvum (3)	(Saki et al. 2016)
Nigeria	1.5% (2/134)	Cryptosporidium andersoni (1), Rat genotype II (1)	(Ayinmode et al. 2017)

No competing interests reported.

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Molecular characterizations of Cryptosporidium spp. in brown rats (Rattus norvegicus) from an animal feedlot in Xinjiang, China

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