Prevalence and correlates of antibodies to Neospora caninum in dogs in Portugal

Neosporosis, caused by Neospora caninum, is an important cause of abortion in cattle and of neurological disease in dogs. This study investigated the prevalence and correlates of antibodies to N. caninum in 441 dogs from the five regions of mainland Portugal. A commercial competitive enzyme-linked immunosorbent assay (cELISA) was used and specific antibodies were detected in 35 (7.9%) dogs. Seroprevalence levels were significantly different among some of the studied regions, as well as between stray dogs (13.6%) and hunting dogs (1.7%). The difference between seropositivity in dogs presenting musculoskeletal or neurological signs (21.4%) and that in animals without clinical signs compatible with neosporosis (5.6%) was close to statistical significance. This is the first report on the seroprevalence of N. caninum in dogs in Portugal. Neosporosis should be considered in the differential diagnosis of neurological disorders of dogs.


Introduction
Neospora caninum Dubey et al., 1988 [6] is a protozoan parasite of animals, with dogs (Canis familiaris) having an important role in the epidemiology of infection, as they and other related canids are the definitive hosts of this pathogen, and so shed oocysts into the environment [5,7]. Neosporosis is an important cause of abortion in cattle, with high economic impact, and a neurological disorder in dogs [8].
Serological surveys indicate worldwide canine exposure to the parasite [5,7]. N. caninum has been confirmed as a cause of bovine abortion in dairy herds in the North of Portugal [4,12]. In serosurveys carried out in Holstein-Friesian cows in the Northern and Central regions of the country, seroprevalence was found to range from 28% in a random sample of dairy cattle to 46% in dairy herds with a history of abortion [3]. In Portugal, N. caninum has also been isolated from the faeces of a stray dog in the southern Algarve region [2].
The present study aimed at investigating the prevalence of antibodies to N. caninum in dogs countrywide, and to assess risk factors associated with infection or exposure in this host species, contributing to a better understanding of its epidemiology in Portugal.

Material and methods
From January 2012 to December 2013, a total of 441 dogs from the five statistical regions of mainland Portugal were sampled in veterinary medical centres (388 domestic animals) and shelters (53 stray animals) and available data on correlates of infection were collected (Table 1). Dogs were randomly included after owners' or legal holders' informed consent. Stray animals had been housed to be sterilised for population control or to be given for adoption. This study was ethically approved by the ethical committee of the Universidade de Trás-os-Montes e Alto Douro as complying with the Portuguese legislation for the protection of animals (Law 92/1995).
Blood samples were obtained by cephalic or jugular venipuncture and serum was separated by centrifugation and preserved at À20°C until used. A commercial competitive enzyme-linked immunosorbent assay (cELISA) was used for the detection of antibodies to N. caninum in serum samples according to the manufacturer's instructions (VMRD, Pullman, WA, USA). Positive and negative control samples were provided in the kit. The percentage of inhibition (% I) was obtained by the formula: % I = 100 À [(sample OD · 100)/mean negative control OD]. When % was equal to or more than 30%, the sample was considered positive. Based on published data, the cELISA results correlated well with the indirect fluorescent antibody test [7,8].
The exact binomial test was used to calculate confidence intervals (CI) for the proportions, with a 95% confidence level. The chi-square and Fisher's exact tests compared proportions of seropositivity to N. caninum (no. of positive dogs/no. of dogs tested). A p-value < 0.05 was considered as statistically significant. Analyses were performed with SPSS Ò 22 software for Windows.

Results and discussion
Antibodies to N. caninum were detected in 35 (7.9%) out of the 441 dogs. Seroprevalence levels were significantly different among some of the studied regions (Table 1): the seroprevalence in dogs living in the Lisbon region was significantly higher than that in the Central and Alentejo regions, while seroprevalence in animals in the North and the Algarve was significantly higher than in those in the Centre. Seroprevalence in stray dogs was significantly higher than in hunting dogs (Table 1).
No statistical differences were found among the other independent variables/categories evaluated. However, the difference between seropositivity in dogs without clinical signs compatible with neosporosis (5.6%) and that in animals presenting musculoskeletal or neurological signs (21.4%) was very close to statistical significance (p = 0.05).
This is the first report on the seroprevalence of infection with or exposure to N. caninum in dogs in Portugal. A review of N. caninum infections in dogs worldwide revealed that among the risk factors, the lifestyle and age of the dogs were the most important. In fact, infection levels were higher in strays versus pets and in older versus young dogs [7,8]. The higher prevalences documented in older dogs suggest most of them become infected after birth. In the present study, as well as in some others [10,11,13], no significant differences were observed between young and old animals. Nevertheless, it is important to point out that the number of dogs in the young group (i.e., 2-11 months old) was smaller than the adult group. Although no significant differences were observed in feeding habitats, the number of seropositive dogs was higher in those animals given food, including strictly home-prepared. Dogs are sometimes fed with raw or undercooked meat which might contain parasite cysts [9].
The geographical distribution of positive cases in the North of Portugal shown here is generally in accord with previous findings on bovine neosporosis [3], underlining the role of dogs in the life cycle of the parasite. On the other hand, the lowest prevalence obtained in dogs in the Centre of Portugal, a region where specific antibodies were previously reported in dairy cows [3], could be related to transplacentary transmission in cattle, with no direct participation of dogs in the epidemiological cycle [8]. Congenital transmission in dogs is clinically significant, but from an epidemiological perspective only a small proportion of dogs are congenitally infected [5].
Although the presence of specific antibodies in dogs only indicates that there was contact with the parasite, the presence of seropositive dogs in a particular area should be considered as a potential risk factor of N. caninum infection in cattle [8]. Thus, due to the lack of data, it is important to screen cattle from the regions of Alentejo, Algarve and Lisbon, where specific antibodies have been detected in dogs.
Neospora caninum can cause clinical disease in dogs of all ages [7]. In the present study an almost significant statistical association was found between seropositivity and musculoskeletal or neurological signs, suggesting that the disease should be considered in the differential diagnosis of neurological conditions in dogs [1].

Conclusion
In conclusion, sanitary conditions and animal health must be improved to prevent the transmission risk of N. caninum by dogs. The results suggest including neosporosis in the differential diagnosis of neurological disorders of dogs, along with the need for further surveillance in relation to the influence of dogs on the epidemiology of infections with N. caninum.