Seroprevalence of Neospora caninum in Sheep and Goats from Grenada , West Indies

Neospora caninum (N. caninum), an intracellular protozoan parasite, causes abortion and neonatal mortality in many herbivores including sheep and goats. The aim of this study was to estimate the Seroprevalence of N. caninum in sheep and goats in Grenada, West Indies. Sera were collected from 138 sheep and 138 goats from all six parishes of Grenada and Carriacou, a small island within Grenada. Animals were identified by gender. Sera were tested for antibodies for N. caninum using an indirect Enzyme Linked Immunosorbant Assay (ELISA) multispecies Kit from Pourquire Laboratorie (IDvet, France). Antibodies to N. caninum were detected in 18 of 138 sheep (13%, 95% confidence interval (CI): 7.39% to 18.61%) and 8 of 138 goats (5.8%, 95% CI: 1.9% to 9.7%). Although seropositivity was high in female sheep and in male goats, there was no statistical difference in seropositivity of males and females in both species of animals (p > 0.05, χ2). The results of this study revealed that sheep and goats in Grenada were exposed to N. caninum and dogs might be considered as a source of infection. There was a need to educate the farmers, possessing small ruminants, about the transmission cycle of N. caninum and methods of prevention, to minimize the exposure of their flocks. This is the first report on seroprevalence of N. caninum in Grenada, West Indies.


Introduction
Neospora caninum is an intracellular protozoan parasite.Dogs and coyotes are definitive hosts which shed infective oocyst in feces.Infected dogs suffer from neuromuscular disorders [1].Intermediate hosts, mainly herbivores (cattle, sheep, goat, horse, bison and deer), get infected by ingestion of oocysts.The parasite can be transmitted transplacentally in several hosts including cattle, in which it is a major route of transmission.The parasite causes economic losses in intermediate hosts through abortion and neonatal mortality.It is a major cause of abortion in cattle, in many countries.Pregnant sheep are highly susceptible to experimental infection by N. caninum [2].
Seroprevalence of N. caninum in sheep and goats has been reported from a few countries of the world.In sheep it has been reported from Slovakia, [3], Brazil [4] [5], and New South Wales [6].In goats it has been reported from Poland [7], Brazil [8] and Romania in dairy goats [9].As far as the authors are aware, there is no published research on N. caninum in sheep and goats from the Caribbean region including Grenada.
The aim of this study is to report for the first time the seroprevalence of N. caninum in sheep and goats from Grenada, West Indies.

Ethical Approval
The research was approved by Institutional Animal Care and Use Committee (IACUC) of the St. George's University.

Collection of Samples
Grenada is the southern most of the Windward Islands of Eastern Caribbean.Its area is approximately 344 Km 2 .It is divided in 6 parishes.Blood was collected from sheep and goats from all 6 parishes of Grenada and Carriacou.Carriacou is largest island of Grenadines, approximately 34 Km 2 with dependency on Grenada.To cover the representative samples, blood was collected from sheep and goats from all six parishes of Grenada and Carriacou.Blood was centrifuged at 3000 g for 5 minutes at 25˚C and sera were stored at −20˚C until tested.
A total of 138 blood samples from goats, and same number of samples from sheep, were collected for assay.It included 35 blood samples from goats and 35 from sheep, from Carriacou.Both species of animals were identified by gender.

Methodology
A commercial ELISA N. caninum multispecies kit from Pourquier Laboratories (IDvet, France) was used, for detection of N. caninum antibodies, according to manufacturer's instructions.According to manufacturer N. caninum multispecies kit has 100% sensitivity and 100% specificity.

Statistical Analysis
A Chi-square test (χ 2 ) of significance for comparison of seroprevalence of N. caninum in sheep and goats was used (http://www.graphpad.com/quickcalcs/contingency).

Results
As shown in Table 1, antibodies to N. caninum were detected in 18 of 138 sheep (13%, 95% confidence interval (CI): 7.39% to 18.61%) and 8 of 138 goats (5.8%, 95% CI: 1.9% to 9.7%).Chi-square value for comparison of seroprevalence in sheep and goats is as follows: Chi-squared equals 3.439 with 1 degree of freedom.The two tailed P value corresponding to chi-squared value is equal to 0.0637.
Seroprevalence of N. caninum according to parish is shown in Table 3.
We reported higher prevalence of N. caninum antibodies in sheep (13%) compared to goats (5.8%) in Grenada.However, the difference was not statistically significant (p > 0.05, χ 2 ).Anastasia et al. [12] also reported similar results while comparing the seroprevalence of N. caninum in these two species of animals.
In our study, there was no significant difference between the gender of sheep and goats for N. caninum antibody (Table 2).In sheep 15.3% females and 5.88% males had antibodies for N. caninum (Chi-square = 0.153, p-value 0.70), while in goats 11.1% males and 3.2% females had antibodies for N. caninum (Chi-Square = 0.004, p-value = 0.95).Eduardo et al. [15] also found no association between sex of goats and prevalence of anti-N.caninum antibodies.Similar studies comparing seroprevalence of N. caninum with gender was not available for sheep.
A few studies indicated the correlation between the presence of dogs on the farm and seroprevalence for N. caninum in small ruminants [16].Herbert et al. [5] and Gustavo et al. [17] suggested positive correlation be-tween the presence of dogs on the farm and seroprevalence of antibodies to N. caninum in sheep and goats.However, Figliuolo et al. [4] reported no association between dogs and prevalence of N. caninum antibodies in sheep.
There was paucity of information on neosporosis in dogs in the Caribbean region.A study conducted 6 years ago, on a small number of dogs, in Grenada revealed 1.8% seropositivity using indirect immunofluorescent antibody test (IFAT) [18].Recent study by Sharma et al. [19] using ELISA revealed low seroprevalence (1.4%) of N. caninum in Grenadian dogs.Since dogs were positive for N. caninum antibodies and the results of this study revealed antibodies to N. caninum in of sheep and goats, therefore in Grenada, dogs might be considered as a source of infection to small ruminants.Further studies were warranted to assess this hypothesis.
N. caninum was considered as a major pathogen for cattle and dogs.It occasionally caused clinical infections in horses, sheep, goats and deer [1].There was a poor documentation of reproductive disorders in Grenada, by farmers, in their sheep and goats.There was an urgent need for campaign to educate farmers about keeping a good record of reproductive disorders in their sheep and goat flocks in order to assess the role of N. caninum in clinical cases of reproductive disorders.Farmers might further be made aware to maintain hygienic conditions without presence of dogs on the small ruminant flocks.

Table 1 .
Seroprevalence of Neospora caninum in sheep and goats from Grenada (West Indies).

Table 2 .
Seroprevalence of Neospora caninum in sheep and goats from Grenada (West Indies) according to gender.

Table 3 .
Proportion of sheep and goats seropositive to Neospora caninum according to parish in Grenada, WI.