Prevalence and molecular characterisation of Cryptosporidium and Giardia species in pre-weaned sheep in Australia

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 2 AbstractA total of 477 fecal samples from pre-weaned sheep from 5 different farms in the south west of Western Australia were screened for the presence of Cryptosporidium and Giardia using PCR. There were substantial differences in prevalence between the farms and overall prevalence was 24.5% and 11.1% respectively for Cryptosporidium and Giardia. At the 18S locus, 66 Cryptosporidium positives were identified, the majority of which were C. bovis (n = 52), followed by the cervid genotype (n= 10) and C. parvum (n = 2). At a second diagnostic locus, using C. parvum and C. hominis specific qPCR primers, 63 C. parvum positives were identified, some of which were co-infections with C. bovis. The C. parvum/C. hominis qPCR was more sensitive than the nested 18S PCR at detecting C. parvum. This may be due to the low numbers of oocysts present, as quantitation data indicated that all the C. parvum detected was present in low numbers (1-10 oocysts). It may also be that using C. parvum-specific primers is necessary to determine the true prevalence of C. parvum. Amongst Giardia positive isolates, G. duodenalis genotype E (livestock) was the most prevalent (36/53), with G.duodenalis genotype A detected in 5 positive isolates. There were also 11 mixed A and E infections detected. The findings of the present study indicate that pre-weaned lambs are not an important source of zoonotic Giardia genotypes in Australia but may be an important source of zoonotic Cryptosporidium.

Cryptosporidiumspecies in Australian wildlife and domestic animals

“…Xiao, 2010) but has not been detected in any human cryptosporidiosis cases in Australia to date. Quantification analysis using quantitative PCR (qPCR) and microscopy indicated that oocyst output in sheep feces varies widely and ranges from *1 to 10 6 oocysts per gramme (Yang et al 2009;Ryan et al, unpublished observations). Because sheep can harbour C. parvum, they should be considered a potential source of infection of Cryptosporidium either by direct transmission or by contamination of the environment.…”

Greater intensity and frequency of Cryptosporidium and Giardia oocyst shedding beyond the neonatal period is associated with reductions in growth, carcase weight and dressing efficiency in sheep

“…Cryptosporidium (including C. parvum) and Giardia were identified in all eight flocks included in the present study, and have been widely reported in sheep in Australia (Ryan et al, 2005;Yang et al, 2009;Sweeny et al, 2011;Sweeny et al, 2012a;Yang et al, 2014a;Yang et al, 2014b) and worldwide (Xiao 2010). Therefore the observations of reduced HSCW and dressing % associated with these organisms have important implications for sheep meat industries worldwide.…”

“…An internal amplification control (IAC) which consisted of a fragment of a coding region from Jembrana Disease Virus cloned into a pGEM-T vector (Promega, USA), was used as previously described (Yang et al, 2013). All Cryptosporidium positives were screened using a C. parvum and C. hominis specific qPCR at a unique Cryptosporidium specific gene (Clec) that codes for a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) previously described (Morgan et al, 1996;Yang et al, 2009;Bhalchandra et al, 2013). Cryptosporidium and Giardia oocysts per gram of faeces (g -1 ) were measured by qPCR as previously described (Yang et al, 2014a;2014b).…”

“…The prevalence of Cryptosporidium and Giardia in sheep varies between studies conducted worldwide but for Cryptosporidium generally ranges from 15%-27% in lambs (Santín et al, 2007;Robertson et al, 2010;Ye et al, 2013;Yang et al, 2014a) and for Giardia generally ranges from 1.5-55.6% (Feng and Xiao, 2011). The genus Cryptosporidium consists of 26 valid species and more than 50 genotypes with C. xiaoi, C. ubiquitum and C. parvum most frequently identified in sheep (Ryan et al, 2005;Santín et al, 2007;Soltane et al, 2007, Geurden et al, 2008, Mueller-Doblies et al, 2008, Quílez et al, 2008, Fayer and Santín, 2009Giles et al, 2009;Paoletti et al, 2009, Yang et al, 2009Díaz et al, 2010;Robertson et al, 2010;Wang et al, 2010;Fiuza et al, 2011;Shen et al, 2011;Sweeny et al, 2011;Sweeny et al, 2012a;Cacciò et al, 2013;Connelly et al, 2013;Imre et al, 2013;Yang et al, 2014a). Giardia duodenalis is the species infecting mammals and consists of eight major genetic groups (assemblages), two of which (A and B) are found in both humans and animals (including sheep) and are considered zoonotic, whereas the remaining six (C-H) are hostspecific and do not infect humans (Feng and Xiao, 2011;Ryan and Caccio, 2013).…”

“…Giardia duodenalis is the species infecting mammals and consists of eight major genetic groups (assemblages), two of which (A and B) are found in both humans and animals (including sheep) and are considered zoonotic, whereas the remaining six (C-H) are hostspecific and do not infect humans (Feng and Xiao, 2011;Ryan and Caccio, 2013). The most commonly reported genotypes in sheep are assemblage E (livestock genotype) and assemblage A (van Keulen et al, 2002;Lalle et al, 2005;Ryan et al, 2005;Yang et al, 2009). Assemblage B has been less commonly reported in sheep (Aloisio et al, 2006).…”