Indian Journal of Animal Research

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Molecular Characterisation of Cryptosporidium parvum among Cattle and Cattle Handlers from Tripura (India) and the Associated Risk Factors

Prasenjit Das1, Devajani Deka1,*, H. Lalrinkima2
1Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
2Department of Veterinary Parasitology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
Background: Cryptosporidium parvum is major protozoan parasite of both animals and humans with zoonotic significance. There is a paucity of information on its occurrence and associated risk factors in North Eastern states, India. The present study aimed at the molecular characterization of Cryptosporidium from cattle and cattle handlers in West district of Tripura and risk associates analysis.

Methods: Faecal samples were randomly collected from cattle and cattle handlers (100 each), of West district, Tripura. The samples were subjected to sheather’s sucrose flotation, modified Zeihl Neelsen (mZN) staining and PCR assay targeting the 18S SSUr-RNA gene for detection of Cryptosporidium. The polemerised chain reaction- Restriction length polymorphism (PCR-RFLP) was done to detect Cryptosporidium spp. and C. parvum genotypes. Association of the epidemiological variables in relation to host and environment were studied for comparative analysis.

Result: The incidence of Cryptosporidium was 16 per cent and 7 per cent in cattle and cattle handlers, respectively. The molecular characterisation based on PCR-RFLP analysis revealed 14 C. parvum and two C. andersoni and three C. parvum in cattle handlers. All the 14 C. parvum from cattle and one C. parvum from cattle handlers belonged to bovine genotype (genotype II) and other two C. parvum from cattle handlers belonged to the human genotype (genotype I). Cryptosporidiosis in both cattle and cattle handlers were strongly associated with younger age group and diarrhoeic faecal consistency with significantly (p<0.01) higher occurrence. However, significantly (p<0.05) higher predominance of monsoon season in occurrence of the disease was observed in cattle and sex predominance was not observed in both cattle and cattle handlers.
Cryptosporidium is a common enteric protozoan parasite which is one of the leading causes of gastrointestinal illness in vertebrate animals and human worldwide. The Cryptosporidium oocysts are comparatively resistant and ubiquitous in the environment and are mostly transmitted by faeco-oral route. The ocysts are ingested along with contaminated food or water and infect the gastrointestinal tract of the susceptible host (Dankwa et al., 2021). Cryptosporidium parvum is a zoonotic pathogen and a leading cause of neonatal diarrhoea in young animals, mostly in episodes of outbreak. It often results in stunted growth in humans below five years of age (Bhat et al., 2013, Feng and Xiao, 2017; Zhang et al., 2020). The prevalence of zoonotic parasites is likely to be underestimated in India owing to the lack of proper surveillance and the shortage of information about the existence of asymptomatic animal carriers. Due to lack of effective vaccine, treatment or intervention strategies against Cryptosporidium, control of cryptosporidiosis mainly focuses on its prevention (Jex et al., 2011). Detection of the source of infection and mode of transmission is important in identifying a new endemic area for the control of cryptosporidiosis. 

Although, cryptosporidiosis in bovines has been found to be highly prevalent in other parts of India (Paul et al., 2008) there is paucity of information from North Eastern region (NER). Studies on cryptosporidiosis in zoonotic perspective may be of great public health significance in NER considering the facts that human and animals live in close proximity, improper farming system that lacks in hygienic practice, sharing common water sources for animals and human and high numbers of HIV patients.

Cattle rearing in Tripura are mostly practiced by small and marginal farmers and landless agricultural laborers as a part of integrated farming system (Debbarma et al., 2021). Hence, there could be frequent contact of the animals and nearby water bodies. Infective ocysts are passed in the faeces of these animals and the water may contribute significantly to the contamination of the environment for further transmission to other animals and humans. Despite the immense public health and veterinary importance of this parasite, there is limited data on the occurrence and associated risk factors of cryptosporidiosis in the state. Detection of the C. parvum among cattle and related human population and their genotyping may help to determine whether calves serve as a major reservoir for C. parvum infection in humans. The present study aimed to identify C. parvum by PCR based detection of 18S (SSU) r-RNA gene, its genotypes and demographic associates from dairy cattle and cattle handlers in Tripura, India.
The present study was undertaken in the Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences and AH, Aizawl, Mizoram during 2019-20. A total of 100 faecal samples each from both the cattle and cattle handlers were screened from West district of Tripura. The calendar year was distributed into three seasons, summer (March-May), monsoon (June-September) and winter (October-February). The demographic associates,  namely age, sex and faecal consistency in cattle and cattle handlers that determines the occurrence of Cryptosporidium were studied. The distribution of Cryptosporidium in different age groups of cattle (0-6 months and >6 months) and human (0-5, 6-10 and >10 years) was recorded. The consistency of faeces was recorded as diarrhoeic and non diarrhoeic. The faecal samples were collected per rectum in clean, leak-proof containers and examined for the presence of Cryptosporidium oocyst by using mZN staining after concentrating the samples by sheather’s sucrose floatation technique (Casemore et al., 1985).

For molecular confirmation of the protozoan parasite, the faecal samples which were positive on light microscopy were subjected to PCR based analysis of 18S (SSU) r-RNA gene by using published primers (CRP-DIAG F1:5'-AACCTGGTTGATCCTGCCAGTAGTC-3' and CRP-DIAG R1: 5'TGATCCTTCTGCAGGTTCACCTACG-5). Genomic DNA was extracted from the faecal samples by using conventional technique (Sambrook et al., 2001). The 18S (SSU) rRNA gene confirmed amplicons were subjected to nested PCR and subsequently PCR based restriction fragment length polymorphism (RFLP) analysis to detect Cryptosporidium species and genotypes of C. parvum. In nested PCR, two sets of primers were used in two successive reactions which increased the specificity of DNA amplification. In primary PCR, the 18S (SSU) r-RNA gene was amplified by using the first set of oligonucleotide primers (F: 5'-TTCTAGAGCTAATACATGCG-3'and R: 5'- CCCTAAT CCTTCGAAACAGGA-3') and the primary PCR products were amplified in secondary PCR by using the second set of oligonucleotide primers (F: 5'-GGAAGGGTTGTATTTA TTAGATAAAG-3' and R: 5'-AAGGAGTAAGGAACAA CCTCCA-3') (Xiao et al., 1999). The 25 µl PCR mixture was composed of 12.50 µl PCR master mix, 1µl forward and reverse primer (10 pmol/ µl), each, 1µl DNA template and 9.50 µl nuclease free water. The thermal cycling conditions are mentioned in Table 1. Gel electrophoresis of amplified DNA was done in 1.5 per cent agarose gel for 2 hours at 80V using Tris Acetate EDTA(1X TAE) running buffer (Sambrook et al., 2001).

Table 1: PCR conditions applied for detection of Cryptosporidium.



The RFLP analysis was done using 10 µl purified nested PCR products. The nested PCR products were separately subjected to restriction endo-nuclease enzyme digestion with SspI and VspIenzymes for detection of Cryptosporidium species and its genotypes, respectively. The 20 µl reaction mixture consisted of 10 µl DNA amplicon, 2 µl RE buffer (10X), 1 µl enzyme (10 IU/µl) and nuclease free water up to 20µl and digested at 37°C for 4 hours in humid condition. The digested product was fractionated in 3 per cent agarose gel and visualized by ethidium bromide staining.

Data were analyzed by SPSS 17.0. Qualitative data were compared by chi-square test or Fisher exact test, as applicable.
Detection and molecular characterization of Cryptosporidium
 
For detection of Cryptosporidium in faecal samples of cattle and cattle handlers, sheather’s sucrose flotation, mZN staining and PCR-RFLP tests were performed. In Sheather’s sucrose floatation, the oocysts appeared as round or oval, refractile bodies with a thin cytoplasmic membrane. However, in mZN staining, the oocysts appeared as spherical to ellipsoidal shaped pink to red stained bodies containing four sporozoites against a pale green background. All the faecal samples which were positive on microscopy were also positive for 18S (SSU) r-RNA gene with an amplicon size of 1745 bp (Fig 1). These gene amplicons were subjected to nested PCR and subsequently PCR-RFLP analysis to detect Cryptosporidium species and genotypes of C. parvum considering its zoonotic significance. Nested PCR assay revealed the 1325 bp band in primary PCR and 825 bp bands in the secondary PCR (Fig 2 and 3). The nested PCR product of 825 bp size holds the key information for species differentiation of Cryptosporidium.

Fig 1: PCR amplification of18S (SSU) rRNA gene of Cryptosporidium; Lane M: 100 bp ladder; Lane 1: Positive control); Lane 2: Negative control; Lane 3: Cryptosporidium (1745 bp); (Lane 4, 5, 6 and 8: Blank.



Fig 2: Primary PCR amplification of 18S (SSU) rRNA sample in Nested PCR; Lane M: 100 bp ladder, Lane 1: Positive control; Lane 2: Negative control; Lane 3, 5, 7 and 8: Cryptosporidium (1325bp); Lane 6: Blank.



Fig 3: Secondary PCR amplification of 18S (SSU) rRNA sample in Nested PCR Lane M: 100 bp ladder; Lane 1: Negative control, Lane 3: Positive control; Lane 4, 5, 6, 7 and 8: Cryptosporidium (825 bp); Lane 9, 10: Blank.



The incidence of Cryptosporidium was recorded as 16 per cent and 7 per cent in cattle and cattle handlers, respectively from West district of Tripura. Brar et al., (2017) reported 25 per cent and 33 per cent incidence of cryptosporidiosis in bovine calves by mZN staining and commercial ELISA, respectively. Thakre et al., (2017) also reported higher incidence (41.59%) of Cryptosporidium in bovine faeces.

The PCR-RFLP analysis using restriction enzyme SspI revealed 14 bovine Cryptosporidium as C. parvum and two as C. andersoni while out of the seven human Cryptosporidium, three were C. parvum. The PCR-RFLP analysis using restriction enzyme SspI  revealed distinct band patterns for C andersoni (448 bp and 370 bp) (Fig 4) and C. parvum (108 bp, 267 bp and 449 bp) (Fig 5). Similar band pattern in PCR-RFLP analysis for C. parvum and C. andersoni in faeces from cattle was earlier reported by Xiao et al., (1999) and Feng et al., (2007).

Fig 4: The PCR-RFLP analysis using restriction enzyme SspI representing C. andersoni; Lane M: 50 bp ladder; Lane 2: Positive control; Lane 3: C. andersoni (448 bp, 370 bp).



Fig 5: The PCR-RFLP analysis using restriction enzyme SspI representing C. parvum; Lane 1: positive control; Lane 2: C. parvum (449 bp, 267 bp, 108 bp); Lane M: 50 bp ladder.



Cryptosporidium parvum was previously reported as the most prevalent Cryptosporidium species in India (Khan et al., 2010). However, simultaneous detection of C. andersoni and C. parvum in cattle indicates the possibility of cross contamination and easy transmission of this parasite (Zhao et al., 2014). Rekha et al., (2016) reported 5.71 per cent Cryptosporidium in bovine with occurrence of C. parvum in calves and C. andersoni in adult animals.

Further,  RFLP analysis using restriction enzyme VspI revealed that all the 14 C. parvum of cattle origin and one C. parvum of human origin belonged to bovine genotype (genotype II) and other two human C. parvum belonged to the human genotype (genotype I). In RFLP analysis by using VspI enzyme, two distinct bands of 628 bp and 104 bp (Fig 6) represented bovine genotype (genotype II) and two distinct bands of size 556 bp and 104 bp (Fig 7) represented human genotype (genotype I) of C. parvum. Similar band patterns of C. parvum genotype I and genotype II had been reported by Xiao et al., (1999). However, Feng et al., (2007) reported the possibility of some missing bands in agarose gel electrophoresis subsequent to RFLP analysis of C. parvum due to small band size.

Fig 6: The PCR-RFLP analysis using restriction enzyme VspI representing bovine genotype (genotype II); Lane M: 50 bp ladder; Lane 2: Positive control; Lane 3: C. parvum genotype II (628 bp, 104 bp).



Fig 7: PCR-RFLP analysis using restriction enzyme VspI representing human genotype (genotype I); Lane M: 50 bp ladder; Lane 2: Positive control; Lane 3: C. parvum genotype I (556 bp, 104 bp).


 
Demographic and environmental associates of Cryptosporidiosis
 
Demographic, geographic, seasonal and socioeconomic status has been contributing to infection sources and transmission routes in the distribution of Cryptosporidium spp. in animals and humans. Risk factor association analysis of cryptosporidiosis has revealed that Cryptosporidium infection is strongly associated with age and faecal consistency of infected cattle and cattle handlers.

A significantly (p<0.01) higher incidence of Cryptosporidium was recorded in young calves of below 6 months (32.35%) than above 6 months (7.57%) (Table 2). Age related variations in the distribution of Cryptosporidium had been observed in earlier studies and pre-weaned calves were the major sources of C. parvum. Decreased rate of infection along with increased age might be ascribed to strengthened immunological competence of the host with increased age and thereby suppressing the infection to a latent stage and thus the adult animals might act as asymptomatic carriers and act as a source of infection for young animals (Xiao and Feng, 2008; Das et al., 2015). It has been indicated that bovine cryptosporidiosis is a disease of neonates and the higher susceptibility of calves to Cryptosporidium has been recorded from different states of India, namely Assam (28.41%) (Das et al., 2015), Kashmir (29.37%) (Sheikh et al., 2007), Pondicherry (25.00%) (Kumar et al., 2004), Uttar Pradesh (35.50%) (Jeyabal and Ray, 2005) and Punjab (33%) (Brar et al., 2017). The susceptibility of bovine calves to C. parvum significantly (p<0.05) decreased with increasing age, below one month (67.26%), 1-3 months (37.11%), 4-8 months (30%) and 9-12 months (17.65%) (Thakre et al., 2017).

Table 2: Occurrence of Cryptosporidium in cattle and cattle handlers and its associated risk factors.



In cattle handlers, 17.65 per cent and 5.19 per cent incidence of Cryptosporidium was recorded in 0-5 years and >10 years age group, respectively (Table 2). Age specific distribution of human cryptosporidiosis showed the highest prevalence in 0 to 24 months age group with the consistent peak occurrence in 0 to 12 months age groups (Pal et al., 2010; Das et al., 2011). The majority of the children in this study were the members of the cattle farmer’s family who live in close contact with animals and belonged to low socio economic status with poor personal hygiene. Thus, direct animal to human and person to person transmission probably played an important role in the epidemiology of Cryptosporidium in children. In addition, most of the infected children and adults probably shared the common water sources with the animals like pond, stream and other natural water bodies.

Cryptosporidium infects the intestine of young calves, humans and other animals resulting in acute enteritis and diarrhoea and the intensity of shedding oocyst has been higher in calves with diarrhoea (Khan et al., 2010; Das et al., 2015). There was a positive association between Cryptosporidium infection and diarrhoeic stools in both cattle and human. The occurrence of Cryptosporidium infection was significantly (p<0.01) higher in diarrhoeic cattle (31.25%) than non-diarrhoeic (8.82%) cattle. However, only diarrhoeic cattle handlers (29.16%) were positive for Cryptosporidium (Table 2). Similarly, higher incidence of Cryptosporidium has been reported in diarrhoeic cattle (24.20%, 50.00%, 32.90%, 81.00%, 24.20% and 59.54%) than non-diarrhoeic cattle (16. 60%, 25. 68%, 7.40%, 18.99%, 16.60% and 29.41%) from Karnataka, Punjab, West Bengal, Assam, Bangaluru and Gujarat irrespective of organized and un-organized farming system (Mallinath et al., 2009; Singh et al., 2006;  Das et al., 2011; Das et al., 2015; Rekha et al., 2016; Thakre et al., 2017), respectively. Similarly, 13.80 per cent diarrhoeic and 3.70 per cent non-diarrhoeic human stool samples were found to be positive for Cryptosporidium (Das et al., 2011).

There was no significant (p>0.05) sex predominance in the occurrence of Cryptosporidium in cattle and figured as 12.00 per cent and 17.33 per cent in male and female, respectively (Table 2). However, the lower incidence of cryptosporidiosis in male animals might be attributed to the smaller number of male calves screened since most of them were culled after birth and the female calves were in more contact with the cows. Thakre et al., (2017) and Bhat et al., (2019) also reported that both sexes of ruminants are equally susceptible to cryptosporidiosis. Similarly, there was no sex wise variation in the incidence of Cryptosporidium  in cattle handlers, 5.45 per cent in male and 8.89 per cent in female. The involvement of both man and woman in the small traditional farming activities in NER probably attributes to the less gender wise difference in the occurrence of Cryptosporidium in human.

The seasonal variation of Cryptosporidium infection in cattle and cattle handlers revealed highest incidence in monsoon (26.83% and 15.63%) followed by summer (14.29% and 5.56%), respectively. However, the incidence was significantly (p<0.05) lower in cattle (3.23%) and not detectable in cattle handlers during winter season (Table 2). Similar findings were also recorded by Das et al., (2015) with highest occurrence in monsoon (27.88%) followed by pre-monsoon (20.14%) and post-monsoon (8.38%). The highest prevalence of cryptosporidiosis in cattle (45.15%) and human (6.30%) during rainy season was also recorded by Das et al., (2011) and Thakre et al., (2017), respectively. In India, environmental ecology has major effect on transmission of cryptosporidiosis and high incidence of cryptosporidiosis during monsoon has been reported in earlier studies in both cattle and human (Bhat et al., 2014; Kali, 2014). High temperature and humidity along with frequent rains in monsoon season enabled the faster transmission of the oocysts. Further, due to the longer viability of oocysts in water and its resistance towards chlorine, sporadic cases as well as waterborne outbreaks of Cryptosporidium are common in monsoon season.
The prevalence of Cryptosporidium infection among cattle and cattle handlers of Tripura indicated a potential public health risk. Detection of C. parvum bovine genotype (genotype II) in human living in close contact with animals indicated bovine as a probable source of human infection. The risk factors associated with C. parvum infection has shown significant incidence in young animals and human along with diarrhoeic episodes.
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