Sloan et al. Parasites & Vectors (2017) 10:503 DOI 10.1186/s13071-017-2464-2 RESEARCH Open Access High seroprevalance of Neospora caninum in dogs in Victoria, Australia, compared to 20 years ago Sarah Sloan1,2, Jan Šlapeta3, Abdul Jabbar4, Jaimie Hunnam5, Bert De Groef2, Grant Rawlin1 and Christina McCowan1* Abstract Background: Canids are definitive hosts of the apicomplexan parasite Neospora caninum, the leading cause of abortion in cattle worldwide. For horizontal transmission from canids to occur, oocysts of N. caninum must be shed by the definitive host into the environment of susceptible intermediate hosts such as cattle. The purpose of this study was to determine the prevalence of N. caninum in canids in Victoria, Australia’s leading dairy producing state. Results: Neospora-like oocysts were observed in 8% (18/234) of faecal samples from wild dogs, domestic dogs and red foxes from Victoria, Australia. However, none tested positive for N. caninum DNA using a quantitative PCR. In a separate sample population, blood sera from 483 domestic dogs were tested for anti-N. caninum antibodies using competitive ELISA. A subset of cELISA samples were re-tested using indirect fluorescence antibody test (IFAT). A seroprevalence of 29.8% (144/483; 95% CI: 11.7–47.8%) was calculated when using cELISA; whereas it was 32.9% (27/80; 95% CI: 15.8–51.8%) using IFAT. Potential risk factors were evaluated using univariable analyses and then assessed in separate multivariable models. Using ‘aged’ dogs as a reference, the seroprevalence of ‘adolescent’ and ‘adult’ dogs was 88% (P = 0.05) and 91% (P = 0.08), respectively, indicating seroprevalence increases with age. There was a 19% higher likelihood of infection in rural locations (P = 0.10) relative to urban areas. Jack Russell Terriers had a 22% higher risk of a cELISA-positive result (P = 0.05) regardless of geographical location, age or sex. Conclusion: These results demonstrate that exposure to N. caninum in domestic dogs is widespread in Victoria, although faecal oocyst shedding is infrequent. Our results indicate increased N. caninum seroprevalance status in dogs over the past two decades. The results imply that dogs get either exposed to the infected meat more frequently or that vertical dam to foetus transmission is more frequent than previously thought. Our study calls for re-evaluation of historical N. caninum seroprevalance studies, because the attitude to dog diet changes. Keywords: Neospora caninum, Epidemiology, Neosporosis, ELISA, IFAT, Dog, Canid, Fox, Seroprevalence Background occurs [3–5]. The life-cycle of N. caninum is maintained Neospora caninum is an obligate intracellular, tissue between canids and cattle, but there is often a concur- cyst-forming, coccidian parasite regarded as the leading rent sylvatic life-cycle between wild canids and herbivore cause of abortion in cattle worldwide [1, 2]. Canids - do- species, particularly wild ruminants (e.g. deer) [5, 6]. mestic and wild dogs (Canis lupus familiaris), grey Horizontal transmission is facilitated by the shedding of wolves (Canis lupus), coyotes (Canis latrans) and oocysts in the faeces of the definitive host which sporulate dingoes (Canis lupus dingo) - are confirmed definitive and are then infectious when ingested, the primary route hosts in which sexual reproduction of N. caninum of infection for canids [6–9]. Alternatively, carnivores may be infected by ingestion of tissue cysts in the flesh of non- carnivore species [9]. Infection in ruminants can occur * Correspondence: [email protected] vertically from the infected dam to her offspring, or 1Veterinary Pathobiology, Department of Economic Development, Jobs, Transport and Resources, Bundoora, VIC, Australia Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sloan et al. Parasites & Vectors (2017) 10:503 Page 2 of 9 horizontally via ingestion of N. caninum oocysts via the therefore aiding in prevention of the spread of N. faecal contamination of food or water [10]. caninum and reducing its economic impact on the beef Very little is known about the impact of N. caninum in and dairy industries. Victoria, Australia’s leading dairy region. Australia is a major producer of agricultural products worldwide and Methods the value of beef and milk production in 2015 was valued Sample collection at $11.5 billion and $4.7 billion, respectively [11, 12]. The A single faecal sample per animal from wild dogs economic impact of N. caninum-associated abortion in (n = 11) and red foxes (n = 188) was collected across Australian cattle has been estimated to be more than $100 Victoria, Australia (Fig. 1), by registered trappers and million per year [13]. A study of N. caninum infection in hunters during their normal hunting and trapping activ- dogs from Melbourne, Victoria, found a seroprevalence of ities under Victorian legislation. A single faecal sample 5% [14], but nothing has been done since then to deter- per animal from domestic dogs (n = 35) was collected by mine the seroprevalence of Victorian dogs. their respective owners or district veterinary officers, The primary objectives of this study were to determine chilled (4 °C), and sent in via direct post for faecal the prevalence of oocyst shedding and seroprevalence of flotation analysis within 7 days from collection, where N. caninum in canids in Victoria, Australia. Oocyst possible. The geographical location of sample collection shedding was evaluated using faecal flotations coupled and the age, sex and breed of each animal was recorded, with molecular diagnostics. Seroprevalence was evalu- where possible. In addition, dog serum samples (n =488) ated using the competitive enzyme-linked immunosorb- were collected following the conclusion of laboratory test- ent assay (cELISA) and indirect fluorescence antibody ing from two veterinary diagnostic laboratories (Australian test (IFAT). The data obtained enabled us to compare Specialised Animal Pathology; the Veterinary Teaching and contrast our data with those reported from 1997 Hospital, The University of Melbourne) located in [14]. Faecal and serological analyses of canids can help Victoria, including five samples from dogs located outside to identify at-risk locations and animal characteristics, of Victoria: three from New South Wales, one from South Fig. 1 Origin of faecal samples of wild dogs, domestic dogs and foxes tested for the presence of N. caninum oocysts. Mark size is indicative of the number of samples collected per location Sloan et al. Parasites & Vectors (2017) 10:503 Page 3 of 9 Australia and one from Tasmania. The home location, TAMRA-3′. A primer/probe mix was made with 10 μl age, breed and sex of each animal was recorded for all of forward primer (10 μM), 10 μl of reverse primer samples. Of the non-Victorian samples, those from the (10 μM), 3 μl of probe (3 μM), and 77 μl of nuclease- mainland were all collected from sites adjacent to or free water. An 18S rRNA extraction control (TaqMan® straddling the state border. Ribosomal RNA Control Reagents; Applied Biosystems, Foster City, CA, USA) was used to ensure correct Faecal flotation extraction of sample DNA and RNA. Using AgPath-ID A flotation solution was made using 375 g/l of NaCl in One-Step RT-PCR Reagents (Applied Biosystems), 5 μl water to create a solution with a specific gravity of 1.18– of sample-extracted DNA was mixed with 12.5 μlof2X 1.20 [15]. Faecal samples were examined microscopically RT-PCR buffer, 1 μl of primer/probe mix, 1 μlof for the presence of N. caninum-like oocysts (10–15 μmin enzyme, 0.375 μl of 18S rRNA primer/probe mix (1:1:1) diameter); oocysts of N. caninum/Hammondia spp. are and 5.125 μl of nuclease-free water, as per the manufac- shed unsporulated, unlike those of Sarcocystis spp., but, turer’s instructions. Samples then underwent the follow- due to the time between excretion and examination, dur- ing thermocycling programme in a 7500 Fast Real-Time ing which sporulation could have occurred, we included PCR System (Applied Biosystems): 1× (50 °C for 2 min), samples with unsporulated oocysts or with oocysts con- 1× (95 °C for 10 min), and 50× (95 °C for 15 s, 58 °C for taining two sporocysts each with four sporozoites. The 1 min) as per Ghalmi et al. [16]. All samples were run in top layer of the flotation solution supernatant was trans- duplicate. A no-template sample served as negative con- ferred to a 15 ml tube, topped up with water and centri- trol, and N. caninum WA-K9 DNA in 1:10,000 and fuged at 1670× g for 5 min to create a pellet. The tube was 1:100,000 dilutions was used as positive control (original stored at -20 °C until required for DNA extraction. concentration 100 ng/μl). In addition, for flotation-positive samples, the apicom- DNA extraction and PCR plexan D2 domain of the large subunit (LSU) rRNA gene Stored faecal flotation samples from above (n = 179) was amplified using the CR1-CR2 primer pairs as published were defrosted at room temperature (23 ± 2 °C) and previously [17–19], MyTaq™Red Mix (Bioline Australia, centrifuged at 3220× g for 15 min and the pellet (< Alexandria, NSW, Australia) and 2 μlofsampleDNAin 200 mg) used for DNA isolation. All samples that were 25-μl reactions, with included controls, as described positive on flotation and a selection of negative samples previously [20].