Detection of non-spermatozoal cells of Neospora caninum in fresh semen of naturally infected bulls

ABSTRACT

Background: Neospora caninum (N. caninum) is a cyst-forming coccidian parasite closely related to Toxoplasma gondii which has emerged as an important cause of reproductive failure in cattle worldwide. Routes of Neospora transmission include transplacental infection through tachyzoites, ingestion of tissues harbouring cysts and oral uptake of sporozoitecontaining oocysts. Transplacental transmission seems to be very efficient for N. caninum in naturally infected cattle and plays a major role in the maintenance and spread of the disease. Other sources of vertical transmission, such as cow to calf transmission via pooled colostrum or milk could also be possible but until now this has not been proven in naturally infected cattle. The possibility of N. caninum transmission via semen requires profound repercussions on the trade of cattle's semen. In the present study, fresh non-extended semen of bulls was evaluated with naturally-acquired neosporosis for the presence of N. caninum by means of PCR. Materials, Methods & Results: Serum samples were analyzed for antibody activity to N. caninum by using the commercially available ELISA kit. Sera samples were obtained before the first semen sampling and after the last one (days 0 and 60). Thirteen seropositive and five seronegative bulls were selected for the next steps of the study. Genomic DNA was extracted from non-spermatozoal cells, purified tachyzoites (6 x 108 cells) of N. caninum (NC-1 strain), T. gondii (RH strain) (6 × 108 cells) and fish (rainbow trout) semen (2 x 106 cells) based on Chomezynski extraction method. PCR was performed using DNA with varying concentrations obtained from purified tachyzoites of N. caninum or T. gondii and from the 54 samples collected at the breeding centre. Genomic DNA of the N. caninum tachyzoites was digested with HindIII and 1 µg of digested DNA was labeled using a biotin labeling kit. Using primers Rep-F and Rep-R, a product with expected molecular weight of 213 bp was recorded. No cross-reaction was observed with other DNA tested from fish semen and/or a closely related species, T. gondeii. The result of Southern blot analysis showed no hybridization of N. caninum oligoneucleotide probes to PCR product of prolactin gene. Based on the results of PCR assay developed in this study, the parasite repetitive sequences were detected in the collected semen samples (a total of 39 samples) of all seropositive bulls (13 animals). Discussion: N. caninum is mainly spread by transplacental transmission from cow to calf. Recently, the seroprevalence of N. caninum infection in breeding bulls has been shown to be moderate, and the possibility of bulls playing a role in the horizontal transmission of N. caninum has been raised. In this regard, semen will require extensive removal of such materials from genomic DNA prior to amplification and it might be beyond the potential of the current kits to isolate DNA. In the present work, non-spermatozoal cells were isolated from the fresh semen via a density gradient centrifugation and subsequently washed and then subjected to DNA extraction by a silica based method. In this approach, it was first attempted to isolate the suspected cells carrying the parasite genome, and then minimize the inhibitory effects of the fresh semen and finally increase the concentration of the extracted DNA. Based on the results of this approach, observing the parasite signal in all semen samples from the seropositive bulls became feasible by the aforementioned PCR assay or what was developed in this study. In the present study, the detection of N. caninum DNA in non-spermatozoal cells semen of naturally infected bulls is reported for the first time which can probably be used as a diagnostic tool. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined, since N. caninum is one of the major causes of abortion in cattle.