Longitudinal prevalence, oocyst shedding and molecular characterisation of Eimeria species in sheep across four states in Australia
Graphical abstract
Introduction
Coccidiosis (Eimeriosis) of sheep is a widespread infection caused by the protozoan parasite, Eimeria, which develops in the small and the large intestine and affects young animals in particular (Chartier and Paraud, 2012). Although often asymptomatic in sheep, coccidiosis can be a serious economic enteric disease, resulting in diarrhea, inefficient weight gains, and occasionally death (Chartier and Paraud, 2012). Sheep of all ages are susceptible, although disease outbreaks are typically observed in young lambs 1–3 months old (2–3 weeks after weaning) following an incubation period of ∼14–20 days (Platzer et al., 2005, Taylor et al., 2011), or when sheep are housed in barns or feedlots (O’Callaghan et al., 1987, Foreyt, 1990, Taylor and Catchpole, 1994, Wright and Coop, 2000). Chronic subclinical infections can also occur with animals shedding low numbers of Eimeria oocysts in their faeces and providing a continuous source of infection for other animals (Kaya et al., 2007). In addition, the presence of Eimeria parasites in the animal intestine has been correlated with increased susceptibility to secondary infection, especially bacterial diseases (Taylor et al., 1973).
The principal species of Eimeria in sheep worldwide are Eimeria ahsata, Eimeria bakuensis, Eimeria parva, Eimeria pallida, Eimeria crandallis, Eimeria weybridgensis, Eimeria ovina, Eimeria ovinoidalis, Eimeria granulosa, Eimeria intricata, Eimeria faurei and Eimeria marsica (Chartier and Paraud, 2012). Of these Eimeria species, E. ovinoidalis and E. crandallis are considered pathogenic and cause the most severe disease (Gregory and Catchpole, 1987, Gregory and Catchpole, 1990, Taylor et al., 2003). Species of Eimeria can be distinguished by oocyst morphology, pre-patent period, site of infection or minimum sporulation time, but all of these methods are labour intensive, time consuming and can be very difficult and unreliable with a mixed sample as different species overlap in size and shape (Tenter et al., 2002, Haug et al., 2007), prompting the development of DNA-based molecular methods mainly for the detection and quantitation of Eimeria in poultry (Morgan et al., 2009, Vrba et al., 2010, Raj et al., 2013).
Effective diagnostic tools would be useful for the detection of pathogenic subclinical Eimeria infections in domestic livestock through faecal monitoring. Such technology could be implemented in a basic control strategy where animals harbouring pathogenic subclinical infections could be isolated from uninfected animals preventing disease transmission (Kaya et al., 2007).
The prevalence and species of Eimeria in sheep in Australia has not been well described and therefore, the aim of the present study was to develop and validate a quantitative PCR (qPCR) assay for detecting Eimeria in sheep and to use this assay to determine the prevalence and oocyst shedding concentrations per gram of faeces (g−1) in lambs in Western Australia, (WA), New South Wales (NSW), Victoria (Vic) and South Australia (SA) at three sampling periods (weaning, post-weaning and pre-slaughter) and compare this data between states.
Section snippets
Animals and faecal sample collection
Faecal samples were collected from cross-bred lambs from 8 different farms across 4 states (Table 1). Lambs were sampled on 3 occasions (i.e. the same animals were sampled on each occasion) at weaning (approx. 12 weeks of age), post-weaning (approx. 19 weeks) and pre-slaughter (approx. 29 weeks). A total of 3412 faecal samples were collected directly from the rectum of approximately 1189 lambs over the 3 sampling periods. Lambs were born and reared in paddocks and were not housed indoors at any
Specificity, sensitivity and efficiency testing of the 18S qPCR
Evaluation of specificity of the 18S qPCR assay revealed no cross-reactions with other genera and detected all the Eimeria isolates tested (data not shown). Sensitivity analysis revealed that the assay could reliably detect ∼80 copies of the cloned Eimeria amplicon per μl of faecal DNA extract which is equivalent to a sensitivity of <1 Eimeria oocyst per μl of faecal DNA extract. (i.e. for 100 spiked Eimeria plasmids, the actual mean numbers of target gene detected was 81.2 which equates to
Discussion
In the present study, the prevalence, oocyst concentration and species of Eimeria were assessed from lamb faecal samples at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across four Australian states using a novel qPCR at the 18S locus.
The qPCR assay was very specific for Eimeria, as it detected all the Eimeria species tested and did not cross-react with the non-Eimeria isolates analysed. The sensitivity of the assay was determined by cloning an Eimeria 18S
Acknowledgments
This study was funded by Meat and Livestock Australia (MLA), Australian Wool Innovation Limited (AWI) and the Australian Government. We thank the participating farmers for their support and providing access to sheep for sample collection. We thank Justin Hoad for providing faecal samples from NSW. Samples from the WA farms were collected by Joshua Sweeny.
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