Active shedding of Neospora caninum detected in Australian wild canids in a nonexperimental context.

Infection with Neospora caninum parasites is a leading cause of reproduction losses in cattle worldwide. In Australia, this loss is estimated to total AU$110 million every year. However, despite this considerable economic impact, the transmission cycle and the host(s) responsible for the sylvatic transmission of the parasite remain to be defined. Dingoes (Canis familiaris) have been suggested to be a wildlife host of N. caninum in Australia, but this is yet to be proven in a nonexperimental setting. This study aimed to determine the prevalence of natural N. caninum shedding in Australian wild dogs (defined as dingoes, dingo-domestic dog hybrids and feral dogs) by performing molecular analysis of faecal samples collected in wild dog populations in south-east Australia. Molecular analysis allowed host species identification and dingo purity testing, while genetic analysis of Coccidia and Neospora conserved genes allowed for parasite identification. Among the 115 samples collected and determined to belong to dingoes, dingo-domestic dog hybrids and foxes, Coccidian parasites were detected in 41 samples and N. caninum was identified in one sample of canine origin from South East Australia (Mansfield). Across all samples collected in Mansfield only 15 individuals were successfully identified by genotype. Thereby our study determined that 6.7% (1/15, 95% confidence intervals 1.2-29.9) of wild dogs were actively shedding N. caninum oocysts at this site. Further, only four individuals were identified at a second site (Swift Creek), and none were positive. This study conclusively confirms the role of wild dogs in the horizontal transmission of N. caninum parasites in Australia.

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA.

BACKGROUND: Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. RESULTS: Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. CONCLUSION: The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended.