Longitudinal prevalence, oocyst shedding and molecular characterisation of Cryptosporidium species in sheep across four states 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.Page 1 of 28A c c e p t e d M a n u s c r i p t The prevalence of Cryptosporidium in sheep in the eastern states of Australia has not been 21 well described, therefore a study of the prevalence, oocyst concentration, species and subtypes of 22Cryptosporidium were assessed from lamb faecal samples at three sampling periods (weaning, post-23 weaning and pre-slaughter) from eight farms across South Australia, New South Wales, Victoria 24 and Western Australia. A total of 3,412 faecal samples were collected from approximately 1,182 25 lambs across the 4 states and screened for the presence of Cryptosporidium using a quantitative 26 PCR (qPCR) at the actin locus. Positives were typed at the 18S locus and at a second locus using C. 27 parvum and C. hominis specific qPCR primers. The overall prevalence was 16.9% (95% CI: 15.6-28 18.1%) and of the 576 positives, 500 were successfully genotyped. In general, the prevalence of 29Cryptosporidium was higher in WA than the eastern states. Cryptosporidium prevalence peaked at 30 43.9% and 37.1% at Pingelly (WA2) and Arthur River (WA1) respectively during weaning and at 31

Cryptosporidiumspecies in humans and animals: current understanding and research needs

“…Recently, a genus-specific Cryptosporidium qPCR based on the actin gene has been developed. Although it currently can only be used in screening of Cryptosporidium spp., genotyping can be done subsequently using species-specific qPCR (Yang et al 2014).…”

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

“…The selection of animals included in this experiment and sample collection methods have been previously described (Yang et al, 2014a;2014b). In brief, lambs from eight farms were included in this study (Table 1).…”

“…All samples were screened at the actin locus for Cryptosporidium and Giardia at the glutamate dehydrogenase (gdh) locus using quantitative PCR (qPCR) previously described (Yang et al, 2014a;2014b). 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). PCR contamination controls were used including negative controls and separation of preparation and amplification areas.…”

“…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).…”