DOI: https://doi.org/10.21203/rs.3.rs-2239872/v1
Mithun (Bos frontalis) is a rare bovine species that is distributed in Yunnan Province, southwestern China. However, the prevalence of that species in mithun is still poorly understood. The prevalence and species of Cryptosporidium spp. in the fecal matters in mithun were examined at 5 locations in Yunnan province, southwestern China. On the basis of the analysis of the 18S rRNA gene by nested PCR (nPCR), followed by DNA sequencing, 39 (6.24%) of 625 fecal specimens collected from 5 locations were positive for Cryptosporidium spp. In different locations, the Cryptosporidium spp. has prevalence rates ranging from 1.27–10.04%, with statistical differences between the five locations (P < 0.05). In four seasons, the Cryptosporidium spp. has prevalence rates ranging from 0 to 15.60%, with obvious differences between the four seasons (P < 0.001). Four Cryptosporidium species were found, known as Cryptosporidium struthionis (n = 18), Cryptosporidium andersoni (n = 10), Cryptosporidium ryanae (n = 1), and Cryptosporidium sp. (n = 10). Until now this is the only report to examine the prevalence and molecular identification of Cryptosporidium spp. in mithun in Yunnan province, southwestern China, which affects public health safety and should be prevented by appropriate measures.
Cryptosporidium spp. is an intestinal parasitic protozoan with a worldwide distribution that infects a diversity of vertebrates, including humans and animals(Gong et al. 2017). The infection mainly transmitted by the fecal-oral route, or through immediate contact with animals, and also through droplet transmission, causing cryptosporidiosis(Pane and Putignani 2022; Putignani and Menichella 2010). Cryptosporidiosis is characterized by symptoms of acute gastroenteritis, including abdominal pain and diarrhea, with a high incidence in infants and young children under the age of 2 years worldwide, it is also a major cause of chronic diarrhea in immunocompromised patients, especially those with AIDS, and increases their mortality(Checkley et al. 2015; Wang et al. 2013; Yang et al. 2021). The occurrence of Cryptosporidiosis can lead to growth retardation, severe diarrhea, and weight loss in cattle, resulting in a serious reduction in their economic value.
Currently, the identification of Cryptosporidium spp. is unreliable based on morphological examination due to the high morphological similarity of their oocysts(Fall et al. 2003), so the identification of Cryptosporidium spp. is mainly based on molecular detection and genotyping(Zhao et al. 2013). Currently, at least 44 different species and above 120 genotypes of Cryptosporidium are reported, 19 of which have been reported on humans(Ryan et al. 2021). When cattle are hosts for Cryptosporidium, they are infected primarily with four species of Cryptosporidium(Gong et al. 2017).
Mithun is a subspecies of Bos gaurus, a large and endangered semi-wild bovine species distributed in China, Bangladesh, India, Myanmar, and Bhutan(Wang et al. 2017). It is mainly distributed in the Nujiang River basin and the N'Mai River basin in Nujiang Prefecture, Yunnan Province, southwestern China, domesticated by the Drung Nationality, and has a high value of economic use and genetic resources as a valuable livestock species for meat. To better protect and utilize the resources of this cattle breed, the breeding scale of mithun in Nujiang Prefecture has been expanded after decades of investment by governments and departments at all levels. As a rare economic resource for herders in Nujiang, mithun is at risk of parasitic infection and parasitic diseases can be devastating to the farming industry. Parasitic infections of mithun have been reported abroad and include trematode, nematode, cestode, Toxoplasma gondii, Neospora caninum, and Cryptosporidium parvum(Chamuah et al. 2016; Chamuah et al. 2015; Rajkhowa et al. 2008; Rajkhowa et al. 2006). However, there are few reports on parasitic infections and diseases in mithun due to the relatively late start of research on parasitic diseases in mithun in China.
Investigating the prevalence and the molecular identification of the Cryptosporidium spp. in mithun is of great importance for local public health safety. Public health events caused by parasites cannot be ignored. The mithun live mainly at altitudes of nearly 2,000 meters, in the upstream part of the Nujiang River and N'Mai River, where rain or snow can carry their feces into the streams and sink into the two rivers, however, it is not clear whether mithun is infected with Cryptosporidium, and our study is the initial on this issue. Hence, the main target of our study was to investigate the prevalence and species of Cryptosporidium in mithun in Nujiang Prefecture, Yunnan Province, southwestern China.
Sample collection
A total number of 625 fresh fecal samples were collected from mithun in four seasons from Juemendang village, Yazuoluo village, Guquan village, Cikai township in the Nujiang River basin, and Dulongjiang township in the N'Mai River basin, Nujiang Prefecture, Yunnan Province, southwestern China, of which 557 were collected from the Nujiang River basin and 68 from the N'Mai River basin. All samples were stored separately in sterile plastic containers and sent to the Dali University Parasite Laboratory, divided and stored partly at 4 ℃ for DNA extraction and the rest at -20°C.
DNA extraction
Each fecal sample of approximately 500 mg was washed 3 times by centrifugation in distilled water at 3000 × g for 10 minutes. Total genomic DNA was extracted using the E.Z.N.A. ® Stool DNA Kit (Omega, BioTek Inc. USA) following the recommendations of the manufacturer. Extracted DNA was frozen at -20 ℃ and used for nPCR.
Nested PCR amplification
The Cryptosporidium SSU rRNA gene was amplified using nPCR according to a previously reported study(Ryan et al. 2003). The following primary PCR primers were employed: 18SiCF2: 5′-GACATATCATTCAAGTTTCTGACC-3′ and 18SiCR2: 5′-CTGAAGGAGTAAGGAACAACC-3′. The total volume of the PCR mixing system of 25 µL included 12.5 µL 2×Taq PCR Mix, 1 µL of forward and reverse primers each (10 µmol/L), 1.5 µL template DNA, and the addition of ddH2O to make up to 25 µL. After the initial start at 94 ℃ for 5 min, in all 35 cycles, each consisting of 94 ℃ for 30 s, 58 ℃ for 40 s, 72 ℃ for 1 min, and finished with 72 ℃ for 10 min. The following secondary PCR primers were employed: 18SiCF1: 5′-CCTATCAGCTTTAGACGGTAGG-3′ and 18SiCR1: 5′-TCTAAGAATTTCACCTCTCTGACTG-3′. The secondary PCR was conducted under the same conditions as the primary PCR using 1.5µL primary PCR product as the template. Lastly, the secondary PCR products were electrophoresed on a 1.5% agarose gel containing 4S Green nucleic acid stain.
Sequencing and phylogenetic analysis
All positive PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequences acquired were used to compared to reference sequences in the GenBank database by using Clustal X 1.83 to determine the identity of Cryptosporidium species. Then, these sequences had been deposited in GenBank with the accession numbers OP363660-OP363677, OP363687-OP363703, and OP363705-OP363708. The phylogenetic tree of Cryptosporidium was established by the neighbour-joining (NJ) method of MEGA 11, genetic distances were calculated based on the Kimura two-parameter model, and bootstrap analysis (1000 replicates) was employed to assess the credibility of the tree.
Statistical analysis was conducted by using SPSS 19.0 to analyze the differences in the prevalence of Cryptosporidium among locations, river basins, and seasons in mithun by chi-square (χ2) test. The difference is statistically significant with P < 0.05.
A total of 39 positive samples from 625 fecal samples of mithun, with a prevalence of 6.24%, identified C. struthionis (n = 18), Cryptosporidium sp. (n = 10), C. andersoni (n = 10) and C. ryanae (n = 1), and C. andersoni has a zoonotic potential. the Cryptosporidium spp. has prevalence rates in mithun among the five locations ranged from 1.27–10.04%, with the highest prevalence in Cikai township, lowest in Yazuoluo village, with statistically significant differences between locations (P < 0.05, χ2 = 16.849). Besides Yazuoluo and Guquan villages, C. andersoni was prevalent in the other three locations, with the highest prevalence of C. andersoni in mithun in Cikai Township. There was no statistically significant difference in the prevalence of Cryptosporidium spp. between the N'Mai River basin and the Nujiang River basin (P > 0.05). The Cryptosporidium spp. has prevalence rates in mithun ranged from 0-15.60% in the four seasons, with the highest prevalence in spring, and lowest in autumn, with obvious differences between seasons (P < 0.001, χ2 = 50.634). C. andersoni was prevalent in spring and winter, and the highest prevalence was observed in spring (Table 1).
| Factors | Categories | No. tested | No. Positive | Prevalence (%) | Species | |||
|---|---|---|---|---|---|---|---|---|
| C.struthionis | Cryptosporidium sp. | C. andersoni | C. ryanae | |||||
| Locations | Jiumendang village | 97 | 9 | 9.28 | 1 | 5 | 3 | |
| Yazuoluo village | 79 | 1 | 1.27 | 1 | ||||
| Guquan village | 152 | 2 | 1.32 | 2 | ||||
| Cikai township | 229 | 23 | 10.04 | 11 | 5 | 6 | 1 | |
| Dulongjiang township | 68 | 4 | 5.88 | 3 | 1 | |||
| Basins | N'Mai River basin | 68 | 4 | 5.88 | 3 | 1 | ||
| Nujiang River basin | 557 | 35 | 6.28 | 15 | 10 | 9 | 1 | |
| Seasons | Spring | 218 | 34 | 15.60 | 14 | 10 | 9 | 1 |
| Summer | 93 | 1 | 1.08 | 1 | ||||
| Autumn | 120 | 0 | 0 | |||||
| Winter | 194 | 4 | 2.06 | 3 | 1 | |||
| Total | 625 | 39 | 6.24 | 18 | 10 | 10 | 1 | |
In this study, three different C. andersoni were identified based on the SSU rRNA gene, located in the same evolutionary branch as those isolated from dogs (MN038058.1), cattle (DQ448631.2), and humans (MW014314.1), respectively (Fig. 1).
Cryptosporidium spp. is recognized globally as an important cause of intestinal diarrhea-related diseases in humans and animals(Pumipuntu and Piratae 2018). Mithun is distributed in the deep mountains of the N'Mai River and Nujiang River basins in Nujiang Prefecture, Yunnan Province, southwestern China. A previous study has shown that remote areas are more prone to infection with Cryptosporidium(Ma et al. 2019), therefore, this is one of the reasons to investigate the prevalence of Cryptosporidium spp. in mithun in this region. The prevalence of Cryptosporidium spp. was 6.24% in mithun in Yunnan Province in this study, to our knowledge, this is the initial time that the prevalence of Cryptosporidium spp. has been found in mithun in Yunnan Province, southwestern China. Compared with other cattle breeds previously studied in Yunnan Province, the Cryptosporidium prevalence was higher in mithun than in Yunling cattle (0.77%) and dairy buffalo (1.1%) and lower than that in Holstein cows (14.7%)(Liang et al. 2021; Meng et al. 2022). In other regions of China, the prevalence of Cryptosporidium spp. in dairy cattle, yellow cattle, and buffaloes was 2.1%, 3.1%, and 1.9%, respectively in Anhui Province, 3.4% in cattle in Shaanxi Province, 2.53% in yaks in Qinghai Province and 2.55% in dairy cattle in Beijing(Li et al. 2016; Liu et al. 2022; Ren et al. 2019; Zhao et al. 2013). The Cryptosporidium prevalence in this study was found higher than the prevalence in all of the above areas. The prevalence of Cryptosporidium spp. in white yaks in Gansu Province was 5.26%, and the prevalence in dairy cows in both Gansu and Ningxia was 5.09%(Qin et al. 2014; Zhang et al. 2015), similar to the prevalence in our study. However, the prevalence of Cryptosporidium spp. in this study was lower than that in Heilongjiang, Jilin, Taiwan, Xinjiang Uygur Autonomous Region, Shandong, Henan, and Hunan, with the prevalence ranging from 16–32.6%(Huang et al. 2014; Ma et al. 2015; Qi et al. 2015; Tao et al. 2018). The findings of this study differ from the prevalence of Cryptosporidium spp. in the different regions mentioned above, and in combination with those studies, the reasons for these prevalence differences may be associated with regional geographic location, environment, climate, sanitary conditions, sampling conditions, sampling season and number of samples examined. In our study, we found that the prevalence of Cryptosporidium spp. in mithun was significantly different among seasons, with the highest prevalence in spring. However, in a previous study, the prevalence of Cryptosporidium spp. in Egyptian cattle was highest in winter owing to the cold season in Egypt is more suitable for Cryptosporidium to reproduce(Abdelaziz et al. 2022). And Cryptosporidium spp. is more likely to spread among yaks during autumn in Qinghai Province, China(Ren et al. 2019). Owing to the unique lifestyle and habits of mithun, which are wild and mostly raised in the deep mountains on a small scale, the prevalence of Cryptosporidium spp. may be relatively low. Therefore, group feeding may also be the main cause of high prevalence of Cryptosporidium spp. As mithun in Yunnan Province are herded in the mountains, basic data such as sex and age are not available, so the prevalence of Cryptosporidium in mithun of different ages and sexes was not reported in our study. However, previous studies have indicated that the prevalence of Cryptosporidium spp. in younger cattle is higher than that in adult cattle(Geng et al. 2021; Li et al. 2020).
As the host of Cryptosporidium spp., cattle are usually primarily infected by C. bovis, C. parvum, C. andersoni and C. ryanae(Gong et al. 2017). However, four Cryptosporidium spp. in mithun were identified in this study, known as C. struthionis, C. andersoni, C. ryanae, and Cryptosporidium sp., The prevalence of C. struthionis is highest in mithun, followed by C. andersoni, Cryptosporidium sp., and C. ryanae. It is rare that C. struthionis is reported to be prevalent in cattle, but there has been a former study found that C. struthionis is prevalent in yaks in Qinghai Province, China(Wang et al. 2018). In addition, no C. bovis was found and three different species of C. andersoni were identified in this study. Besides being the main cause of cryptosporidiosis in cattle, the C. andersoni has also been isolated from diarrhea patients in Jiangsu Province, China, and from pediatric patients in England and Malawi(Jiang et al. 2014; Leoni et al. 2006; Morse et al. 2007).
The two basins in Nujiang Prefecture, Yunnan Province, southwestern China, are remote and surrounded by mixed agricultural and pastoral areas, where a great number of wild animals exist, and the infected mithun share water with those, making it very convenient to spread Cryptosporidium between each other. The zoonotic parasite C. andersoni in mithun may have a risk to the health of the local population as well as to public health. Therefore, the health-related departments in Nujiang Prefecture, Yunnan Province, should prepare for the prevention of cryptosporidiosis and assess the public health risk. This study was the initial to report the prevalence of Cryptosporidium spp. in mithun and to identify C. andersoni in mithun, the findings could provide basic data for locally prevented Cryptosporidiosis.
Acknowledgements
We are thankful to Li Yuan and Luo Xiao for their cooperation in sample collection.
Author contribution
HLL conceived and designed the study, and critically revised the manuscript. EJZ wrote the main manuscript text and performed the experiment. FJY contributed to the sample collection. EJZ and YTC performed the statistical analysis. All authors have read and agreed to the published version of the manuscript.
Funding
Project support was provided by the National Natural Science Foundation of China [31760726].
Data accessibility
The newly described nucleotide sequences have been deposited in the NCBI database under the accession numbers OP363660-OP363677, OP363687-OP363703, and OP363705-OP363708.
Ethical approval and consent to participate
Fecal samples of mithun were collected with the permission of the farm owners. All procedures were performed under the approval and directions of the Administration Committee of Experimental Animals, Dali University.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.