Prevalence and management factors contributing to Cryptosporidium and Giardia infection in pre-weaned and post-weaned calves in Johor, Malaysia

The present study was undertaken to determine the prevalence of Cryptosporidium and Giardia in pre-weaned and post-weaned dairy calves and to identify management factors contributing to infection with these parasites. The role of dairy calves as a source of environmental contamination and identification of the potential risk factors contributing to the prevalence of the organism in farms was also investigated. A total of 240 animal faecal samples were collected from 16 farms in Johor, which is in the southern part of Peninsular Malaysia. The selected farms were situated in two districts within the state with high dairy cattle populations. Faecal samples were screened using PCR amplification and sequence analysis of the 18S ribosomal RNA (rRNA) locus for both parasites. Cryptosporidium isolates were further analysed at a diagnostic qPCR locus and the hypervariable glycoprotein (GP60). Giardia isolates were also analysed at the glutamate dehydrogenase (gdh) locus. PCR amplification detected 65 Cryptosporidium positives giving a total prevalence of 27.1% (95% CI: 21.5%, 32.7). Twelve of the 16 farms were positive for Cryptosporidium with an overall farm prevalence of 75% (95% CI: 53.8%, 96.2%). The pre-weaned calf prevalence at 30.8% (95% CI: 22.6%, 39.1%), was similar to post-weaned calf prevalence at 23.3% (95% CI: 15.8, 30.9%) (χ2=1.92, df=1,1, P=0.17). Phylogenetic analysis of 53 of the 65 positives, identified nine samples as C. parvum, fourteen samples as C. andersoni, sixteen samples as C. bovis, ten samples as C. ryanae and four isolates as mixed infections. Subtyping analysis of eight of the 12 C. parvum isolates at the GP60 locus identified five isolates as IIdA15G1, one was IIa18A3R1 and two isolates as IIa17G2R1. PCR amplification detected 30 Giardia positives giving a total prevalence of 12.5% (95% CI: 8.3%, 16.7%). Eleven out of the 16 farms were positive for Giardia with an overall farm prevalence of 68.8% (95% CI: 46%, 91.5%). The prevalence in pre-weaned calves was 16.8% (95% CI: 10.1, 23.5%) and the prevalence in post-weaned calves was 8.3% (95% CI: 3.4, 13.2%) (χ2=4, df=1,1, P=0.04). Sequence analysis of 25 isolates identified all of them as G. duodenalis Assemblage E (Livestock genotype). The present study identified mixed infections in 3.8%, (95% CI: 1.3, 6.3%) of dairy calves. There was no association between farming systems (intensive or semi intensive) and Cryptosporidium prevalence. Intensive farms had an overall prevalence of 31.7% (95% CI: 23.3, 40%), similar to semi intensive farms; 22.5%, (95% CI: 15, 30%) (χ2=2.55, df=1,1, P=0.11). The prevalence of Cryptosporidium species in pre-weaned calves was 28% for C. parvum and C. bovis respectively, followed by C. andersoni (22%); C. ryanae (13%) and mixed sp. (9%). The prevalence of Cryptosporidium species in post-weaned calves was 35% for C. bovis followed by C. andersoni and C. ryanae (30% each) and mixed sp. (5%). Cryptosporidium parvum was only detected in pre-weaned calves. It was only isolated from calves less than one and four-months old, with the highest peak at 33.3%, (95% CI: 14.5, 52.2%) detected in calves aged less than one-month old (χ2=66.1, df=1,9, P=0.0001). The prevalence of Giardia sp. in semi intensive farms was 14.2% (95% CI: 7.9, 20.4%) and was similar to intensive farms; 10.8% (95% CI: 5.3,16.4%) (χ2=2.55, df=1,1, P=0.11). The prevalence of Giardia infection was twice as high in pre-weaned calves at 16.8% (95% CI: 10.1, 23.5%) compared to weaned calves at 8.3% (95% CI: 3.4, 13.2%) (χ2=4.0, df=1,1, P=0.04). Management factors that increased the risk of Cryptosporidium infection were keeping pre-weaned calves in pens with slatted floors, having other cattle farms close by, keeping post-weaned calves in pens with a sand floor and feeding calves with saleable milk. Management factors that decreased the risk of Cryptosporidium infection included frequent treatment with antibiotics and anti-diarrheal drugs such as kaolin pectin, frequent deworming and washing the feeding utensils with disinfectant. Management factors which increased the risk of Giardia infection included keeping weaned calves in pens with sand floors, calf age (i.e. calves younger than 5 months old were at a higher risk of infection) and more frequent use of anthelmintics. Management factors which decreased the risk of Giardia infection were keeping pre-weaned calves in pens with concrete floors and calving in single cow calving areas. Manipulating these factors should help control the level of infection and may control the disease. This is the first epidemiological study done in Malaysia to identify management factors which contribute to infection and is the first study done to identify the subtype of zoonotic C. parvum in dairy calves.