Neospora caninum Natural Infection in Tunisian Rams: Serological Study and Molecular Identification of Infection in Semen

Purpose
The aim of this study was to estimate the seroprevalence and molecular prevalence and perform a molecular identification of Neospora caninum in semen of Tunisian rams.
Methods
A total of 92 blood samples were collected from four farms located in four Tunisian governorates (Jendouba, Kairouan, Zaghouan and Ben Arous) and samples were screened with a commercial ELISA kit for N. caninum antibodies. For the same rams, semen samples were collected and tested for the presence of N. caninum ITS1 gene using PCR. Five amplicons were randomly selected for sequencing. A phylogenetic tree was constructed to compare the partial sequences of the ITS1 gene with sequences deposited in GenBank.
Results
The seroprevalence of N. caninum infection was 25% (23/92) and PCR revealed that the molecular infection prevalence in semen was 11.95% (11/92). Kappa test showed an average agreement between seroprevalence and parasite prevalence in semen (κ = 0.44). The highest molecular prevalence was for rams that accomplished more than two mating seasons (21.0 ± 12.1%) compared to those performed less than two mating seasons and yearling individuals (4.0 ± 5.5%) (P = 0.01). There were no differences in N. caninum molecular prevalence according to either breed or locality. Comparison of the partial sequences of the ITS1 gene revealed 99–100% similarity with those deposited in GenBank.
Conclusion
To the best of our knowledge, this is the first detection and molecular identification of N. caninum in semen from rams in North Africa. Our findings indicate that N. caninum infection rate was high in rams.