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Trop Anim Health Prod (2013) 45:941–946 DOI 10.1007/s11250-012-0311-1

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Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India Prem Sagar Maurya & Radhamma Lakshmipathy Rakesh & Balaraju Pradeep & Saroj Kumar & Krishnendu Kundu & Rajat Garg & Hira Ram & Ashok Kumar & Partha Sarathi Banerjee

Accepted: 29 October 2012 / Published online: 7 November 2012 # Springer Science+Business Media Dordrecht 2012

Abstract A total of 938 faecal samples (461 cattle calves, 264 buffalo calves, 55 lambs, 116 kids and 42 piglets) from different livestock farms and individual small holdings in six targeted states of India were collected and screened by modified Ziehl–Neelsen staining technique to determine the prevalence of Cryptosporidium spp. and its association with age, sex, season and faecal consistency in domesticated animals. Overall, 16.2 % of the animals were positive for Cryptosporidium infection with prevalence of 16.3, 24.2, 1.8, 3.5 and 19.1 % in cattle calves, buffalo calves, lambs, kids and piglets, respectively. The prevalence of infection was significantly higher (p0.05) was recorded in females than in males. Seasons had a significant effect (p0.05) in post-monsoon than in monsoon season. A high degree of association was noticed between Cryptosporidium infection and diarrhoea in ruminants screened during the present study. But, in case of pigs, the prevalence was higher in non-diarrhoeic than in diarrhoeic animals. Genotyping of Cryptosporidium spp. based on nested PCR amplification of partial 18S rRNA and its subsequent digestion with SspI, VspI and MboII restriction enzymes P. S. Maurya : R. L. Rakesh : B. Pradeep : S. Kumar : K. Kundu : R. Garg : H. Ram : A. Kumar : P. S. Banerjee (*) Division of Parasitology, Indian Veterinary Research Institute, Izatnagar Bareilly 243122 Uttar Pradesh, India e-mail: [email protected]

revealed prevalence of Cryptosporidium parvum in representative number of positive samples of cattle, buffalo and goats. Keywords Prevalence . Cryptosporidiosis . Domesticated young animals . India

Introduction Cryptosporidiosis is an emerging protozoan disease of public health significance, causing gastrointestinal illness in a wide variety of fish, amphibians, reptiles, birds and mammals including humans, cattle, buffalo, sheep, goat, pig, dog, cat and horses throughout the world. Currently 23 species of Cryptosporidium have been recognised as valid species (Xiao 2010). Cryptosporidiosis in animals is seen as a zoonotic threat to humans because of the shedding of huge numbers of resistant oocysts, thus contaminating the surface water. Apart from its zoonotic importance, cryptosporidiosis drew the attention of researchers in veterinary field due to the fact that it may assume a harmful, difficult to control disease in many farm animals and results in significant economic losses. The pathogenesis and sequel of the infection depend on the immune status of the host. The fate may range from severe but self-limiting diarrhoea in immunocompetent individuals to a debilitating, lifethreatening, prolonged infection in immunocompromised individuals, such as AIDS patients (Chalmers and Davies 2010). It is important to study the prevalence of Cryptosporidium prevalent in different agro-climatic regions, in order to understand the transmission dynamics of the infection and for designing effective control measures. Varying degrees of prevalence of Cryptosporidium spp. have been reported from various countries around the globe in domesticated animals (Fayer and Xiao 2008). The studies on prevalence of Cryptosporidium spp. in India suggest a

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prevalence of 30–50 % in diarrhoeic neonatal calves with gradual reduction in infection as the age advances (Roy et al. 2006; Paul et al. 2008). This paper describes the prevalence of cryptosporidiosis in young domestic animals in different parts of India and reports its association with various risk factors viz. age, sex, season and faecal consistency.

Materials and methods Sample collection During the years 2009–2012, a total of 938 faecal samples from various domesticated animals viz. cattle (461), buffalo (264), sheep (55), goat (116) and pigs (42) below 3 months of age were collected per rectum from different livestock farms and individual small holding farms of different districts of six targeted states of India viz. Bareilly (28.35°N, 79.42° E), Lucknow (26.85° N, 80.92° E) and Jhansi (25.46°N, 78.57°E) districts of Uttar Pradesh State (cattle— 234, buffalo—220, sheep—43, goat—104 and pigs—42); Udham Sigh Nagar (28.98°N, 79.40°E), Nainital (29.5°N, 79.7° E) and Dehradun (30.32° N, 78.04° E) districts of Uttarakhand State (cattle—107, buffalo—5 and goat—12); Nadia (22.98°N, 88.48°E) district of West Bengal State (cattle—21 and buffalo—24); Bhojpur (25.34°N, 84.40°E) district of Bihar State (cattle—7 and buffalo—13); Bangaluru (12.98°N, 77.58°E), Chickballapur (13.43°N, 77.72°E) and Mysore (12.30°N, 76.64°E) districts of Karnataka State (cattle—74, buffalo—2 and sheep—12); and Kollam (8.88°N, 76.58°E) district of Kerala State (cattle—18). At the time of sample collection, each animal was clinically examined and their age, sex, breed and faecal consistency (diarrhoeic/ non-diarrhoeic) were recorded. After collection, the samples were immediately brought to the laboratory and processed. When immediate processing was not possible, the samples were immediately put in 2.5 % potassium dichromate solution and kept at 4 °C.

Table 1 Overall prevalence of cryptosporidiosis in different species of animals

State

Direct faecal smears were prepared on clean grease-free microscopic glass slides, air dried and stained by the modified Ziehl–Neelsen technique (MZN) (Henricksen and Pohlenz 1981). Briefly, the air-dried smears were first fixed with methanol for 5 min, air dried and then the smears were transiently fixed over a flame and kept on staining rack. The smears were flooded with concentrated Carbol fuchsin and allowed to stain for 40 min. The slides were then washed thoroughly under running tap water for 5 min, decolorized using 10 % H2SO4 for 10–15 s and then washed again in water. The smears were counterstained with 5 % Malachite green for 5 min and then washed in running tap water for 5 min. After drying, the smears were screened under ×40 and ×100 objectives of microscope for the presence of Cryptosporidium oocysts. The faecal samples which were screened as negative by direct smear examination were further processed by modified Sheather’s sucrose floatation technique (Current et al. 1983). The resulting supernatant was used to make smears which were stained by MZN technique as described above. The smears thus prepared were screened under ×40 and ×100 objectives of microscope for the presence of Cryptosporidium oocysts. Molecular identification of Cryptosporidium at species level For identification of species of Cryptosporidium involved, genomic DNA was extracted from positive faecal samples of 20 cattle calves, 16 buffalo calves and 4 goat kids by using Qiagen®faecal stool DNA extraction kit (Qiagen, GmbH, Germany) as per the manufacturer’s protocol, with the addition of eight freeze–thaw (freezing in liquid nitrogen and immediate thawing at 90 °C) cycles prior to resuspension in lysis buffer. Nested PCR protocol as described by Xiao et al. (2001) was adopted for the amplification of partial 18S rRNA of Cryptosporidium species. For genotyping of Cryptosporidium sp., a restriction fragment length

Number positive/number examined (%) Cattle

Uttar Pradesh Uttarakhand West Bengal Bihar Karnataka Kerala Total

Screening of samples for Cryptosporidium spp. oocysts

58/234 9/107 4/21 0/7 4/74 0/18 75/461

(24.8) (8.4) (19.1) (0) (5.4) (0) (16.3)

Buffalo

Sheep

Goat

Pig

56/220 (25.5) 1/5 (20.0) 3/24 (12.5) 4/13 (30.8) 0/2 (0) – 64/264 (24.2)

1/43 (2.3) – – – 0/12 (0) – 1/55 (1.8)

4/104 (3.9) 0/12 (0) – – – – 4/116 (3.5)

8/42 (19.1) – – – – – 8/42 (19.1)


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40 35 Prevalence (%)

30 25 20 15 10 5 0 Cattle

Buffalo

Sheep

Goat

Pig

Fig. 1 Prevalence of Cryptosporidium spp. in domestic animals below 1 month (black bar) and between 1 and 3 months (gray bar) of age

polymorphism (RFLP) pattern analysis of nested PCR products was conducted by using the restriction enzymes SspI, VspI and MboII (Xiao et al. 2001; Feng et al. 2007). The specific restriction patterns generated were used for identification of the species of Cryptosporidium. Statistical analysis The data generated on the prevalence of cryptosporidial infection in animals were analysed by chi-square test using SPSS 17.0® software.

below 1 month of age, the prevalence of cryptosporidial infection was 2.9 and 6.1 %, respectively. However, none of the sheep and goats aged 1–3 months was found positive. In piglets, the prevalence was higher in the age group of 1 to 3 months (22.6 %) than in younger animals (9.1 %), though it was statistically insignificant. Statistically insignificant higher prevalence of Cryptosporidium spp. was recorded in females than males (Table 2). Seasonal variations in the prevalence of Cryptosporidium spp. in animals were also recorded in the present study (Table 3). In large ruminants, the prevalence was highest in monsoon followed by pre-monsoon and lowest in post-monsoon season. However, in case of sheep and goats, higher prevalence was recorded in post-monsoon than in monsoon season. Statistical analysis revealed that seasons had a significant effect (p0.05) in non-diarrhoeic (23.1 %) than in diarrhoeic (12.5 %) animals (Fig. 2).

Results

Molecular identification of the species of Cryptosporidium

Prevalence of Cryptosporidium infection in animals

Nested PCR amplification of partial 18S rRNA gene of Cryptosporidium sp. yielded a product of approximately 834 bp. RFLP analysis of all the nested products revealed that restriction enzyme SspI yielded three bands at 450, 267 and 108 bp, VspI yielded two bands at 628 and 115 bp, and MboII yielded two clear bands at 771 and 76 bp. These typical RFLP patterns are indicative of the presence of Cryptosporidium parvum in all the 40 samples (20 cattle calves, 16 buffalo calves and 4 goat kids) screened.

In the present study, 16.2 % of the animals screened were positive for Cryptosporidium sp. oocysts with prevalence of 16.3, 24.2, 1.8, 3.5 and 19.1 % in cattle calves, buffalo calves, lambs, kids and piglets, respectively (Table 1). Results revealed high prevalence of infection in animals of Bihar (20.0 %), Uttar Pradesh (19.8 %) and West Bengal (15.6 %) as compared to Uttarakhand (8.1 %) and Karnataka (4.6 %). Age-related prevalence of Cryptosporidium infection is depicted in Fig. 1. The prevalence of infection was significantly higher (p 0.05) of Cryptosporidium spp. was recorded in females (cattle 17.7 %, buffalo 26.0 %, sheep 2.9 %, goat 4.4 % and pigs 23.8 %) than males (cattle 14.9 %, buffalo 22.0 %, goat 2.1 % and pigs 14.3 %). Higher prevalence of infection in females as compared to male calves has been previously reported by Bhat et al. (2012). However, statistically insignificant higher prevalence of Cryptosporidium spp. in males than in female calves was reported by Paul et al. (2008). The findings of the present study revealed that seasons have a significant effect on the prevalence of infection in large ruminants, with highest prevalence in monsoon (cattle 28.8 % and buffalo 36.6 %) followed by pre-monsoon (cattle 17.1 % and buffalo 24.4 %) and lowest in postmonsoon seasons (cattle 10.9 % and buffalo 18.5 %). Higher prevalence of cryptosporidiosis during monsoon months may be attributed to the overcrowding of these animals in shelters in the free range farming system in India. However, such incidences are high in organised farms, as noticed in the present study. This is mainly because of two reasons: (a) Cryptosporidium spp. spread quickly by the faeco-oral route due to optimum temperature and high humidity during monsoon (Caccio et al. 2005) and (b) the higher number of neonates born out from calving in late spring serve as naive susceptible hosts for the oocysts shed by older calves that were born in the previous winters. Moreover, the heavy rain results in overcrowding of animals and hampers the drying of floor and walls of the wet animals sheds, thereby increasing the survival of Cryptosporidium


oocysts. However, in case of sheep and goats, higher prevalence was recorded in post-monsoon (3.3 and 5.3 %) than monsoon (4.8 % in goats) in the present study. This may be due to synchronisation of parturition in these small ruminants in this region so that more susceptible newly born stock with low immunity is present in post-monsoon season. A high degree of association was noticed between Cryptosporidium infection and diarrhoea in ruminants screened during the present study. Roy et al. (2006) also reported higher prevalence of C. parvum in diarrhoeic calves (61.6 %) against the non-diarrhoeic calves (47.2 %) up to 1 month age. Similar findings also have been reported by Paul et al. (2008). But in case of pigs, the prevalence was higher (statistically insignificant, p>0.05) in non-diarrhoeic (23.1 %) than in diarrhoeic (12.5 %) animals. Similar observations have been made by few other workers, who reported that Cryptosporidium spp. infection in pigs are mostly asymptomatic (Wieler et al. 2001). In the present study, PCR-RFLP analysis of the approx. 834 bp nested PCR product of 18S rRNA gene revealed that C. parvum is commonly prevalent in bovine and caprine hosts below 3 months of age in north India. In India, few research workers, viz. Roy et al. (2006) and Paul et al. (2008), have identified C. parvum to be the main causative agent of cryptosporidial diarrhoea among bovine calves by PCR-RFLP analysis of nested PCR product of 18S rRNA gene with SspI and VspI restriction enzymes. But, since there are minor differences in 18S rRNA-based RFLP patterns among C. parvum, Cryptosporidium ryanae and Cryptosporidium bovis, their reliable differentiation generally requires DNA sequencing of nested PCR products when only these two restriction enzymes (SspI and VspI) are used. However, the use of an additional restriction enzyme MboII in conjugation with SspI and VspI can overcome this problem (Feng et al. 2007). For these reasons, the PCR-RFLP studies using three restriction enzymes namely, SspI, VspI and MboII for identifying the species of Cryptosporidium in positive faecal samples were carried out in the present study. Genotyping of Cryptosporidium spp. in pigs could not be attempted in the present study, which could have revealed the facts regarding its epidemiology including transmission cycle and zoonotic potential. The findings of the present study further substantiate the fact that Cryptosporidium spp. infection is widely prevalent in young domesticated animals in different parts of India. Looking into the immense zoonotic potential of the parasite, particularly C. parvum, it is important to monitor the transmission dynamics of cryptosporidiosis in different geoclimatic areas. Further studies on this aspect will provide a valuable basis for the predictive epidemiology of cryptosporidial infection in India. Also, necessary preventive and control measures should be strictly implemented in livestock herds all over the country.

945 Acknowledgments The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar for providing necessary facilities. The financial assistance provided by ICAR, New Delhi in the form of outreach programme on zoonotic diseases is also duly acknowledged. Thanks are also due to the officer in charge of various government livestock farms and to the private livestock owners for their cooperation in sample collection.

References Banerjee, P. S., Mandal, M., Paul, S., Kumar, P. R., Mitra, A., Gupta, S. C., Raina, O. K., Tewari, A. K. and Rao, J. R., 2010. Clinical cryptosporidiosis in two Black Bengal kids. In: S.K. Gupta, S. Singh, S.S. Chaudhri and S. Vohra (eds), Souvenir-cum-Abstract Compendium of XX National Congress of Veterinary Parasitology, Hisar, 2010 (Indian Association for the Advancement of Veterinary Parasitology, India), 7–8 Bhat, S. A., Juyal, P. D. and Singla, L. D., 2012. Prevalence of cryptosporidiosis in neonatal buffalo calves in Ludhiana district of Punjab, India. Asian Journal of Animal and Veterinary Advances, 7, 512–520 Caccio, S. M., Thompson, R. C. A., McLauchlin, J. and Smith, H. V., 2005. Unraveling Cryptosporidium and Giardia epidemiology. Trends in Parasitology, 2, 430–437 Chalmers, R. M. and Davies, A. P., 2010. Minireview: clinical cryptosporidiosis. Experimental Parasitology, 124, 138–146 Current, W. L., Reese, N. C., Ernst, J. V., Bailey, W. S., Heyman, M. B. and Weinstein, W. M., 1983. Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies on outbreak and experimental transmission. The New England Journal of Medicine, 308, 1252–1258 Fayer, R., Santin, M., Trout, J. M. and Greiner, E., 2006. Prevalence of species and genotypes of Cryptosporidium found in 1 to 2-yearold dairy cattle in the eastern United States. Veterinary Parasitology, 135, 105– 112 Fayer, R. and Xiao, L., 2008. Cryptosporidium and cryptosporidiosis. CRC, Boca Raton Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W., Cama, V. and Xiao, L., 2007. Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Veterinary Parasitology, 144, 1–9 Guselle, N. J., Appelbee, A. J. and Olson, M. E., 2003. Biology of Cryptosporidium parvum in pigs: from weaning to market. Veterinary Parasitology, 113, 7–18 Henricksen, S. A. and Pohlenz, J. F. L., 1981. Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Veterinaria Scandinavica, 22, 594 Morgan, U. M., Monis, P. T., Fayer, R., Deplazes, P. and Thompson, R. C., 1999. Phylogenetic relationships among isolates of Cryptosporidium: evidence for several new species. The Journal of Parasitology, 85, 1126–1133 Paul, S., Chandra, D., Ray, D. D., Tewari, A. K., Rao, J. R., Banerjee, P. S., Baidya, S. and Raina, O. K., 2008. Prevalence and molecular characterization of bovine Cryptosporidium isolates in India. Veterinary Parasitology, 153, 143– 146 Rajendran, P., Ajjampur, S. S. R., Chidambaram, D., Kattula, D., Rajan, D. P., Ward, H. and Kang, G., 2011. Investigation of potential zoonotic transmission of cryptosporidiosis in southern India. American Journal of Tropical Medicine and Hygiene, 85, 657–659 Roy, S. S., Sarkar, S., Batabyal, S., Pramanik, A. K. and Das, P., 2006. Observation on the epidemiology of bovine Cryptosporidiosis in India. Veterinary Parasitology, 141, 330–333

946 Sabry, A., Khodery, E. and Osman, S. A., 2008. Cryptosporidiosis in buffalo calves (Bubalus bubalis): Prevalence and potential risk factors. Tropical Animal Health and Production, 40, 419–426 Venu, R., Latha, B. R., Basith, S. A., Raj, G. D., Sreekumar, C. and Raman, M., 2012. Molecular prevalence of Cryptosporidium spp. in dairy calves in southern states of India. Veterinary Parasitology, 188, 19–24 Wang, R., Qiu, S., Jian, F., Zhang, S., Shen, Y., Zhang, L., Ning, C., Cao, J., Qi, M. and Xiao, L., 2010. Prevalence and molecular identification of Cryptosporidium spp. in pigs in Henan, China. Parasitology Research, 107, 1489–1494

Trop Anim Health Prod (2013) 45:941–946 Wieler, L. H., Ilieff, A., Herbst, W., Bauer, C., Vieler, E., Bauerfeind, R., Failing, K., Klos, H., Wengert, D., Baljer, G. and Zahner, H., 2001. Prevalence of enteropathogens in suckling and weaned piglets with diarrhea in southern Germany. Journal of Veterinary Medicine, Series B, 48, 151–159 Xiao, L., 2010. Molecular epidemiology of cryptosporidiosis: an update. Experimental Parasitology, 124, 80–89 Xiao, L., Bern, C., Limor, J., Sulaiman, I., Roberts, J., Checkley, W., Cabrera, L., Gilman, R. H. and Lal, A. A., 2001. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. Journal of Infectious Diseases, 183, 492–497