Spatial Distance Between Sites of Sampling Associated With Genetic Variation Among Neospora Caninum in Aborted Bovine Foetuses From Northern Italy

Background: Neospora caninum , a coccidian protozoan, represents an important cause of bovine abortion. While only a single genotype of N. caninum exists word-wide, available N. caninum strains show considerable variation in vitro and in vivo , including different virulence in cattle. To which extent sexual recombination, possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contribute to this variation is not clear, yet. Methods: Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum samples were subjected to multilocus-microsatellite genotyping (MLMG) using ten previously established microsatellite markers. In addition to own data, those from a recent study providing data on five markers from other northern Italian regions were included and analyzed. Results: Of the 55 samples finally subjected to MLMG, 35 were typed at all or 9 out of 10 loci. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P< 0.001). Including data from a previous North Italian study (eBURST analysis) revealed that part of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. Variations observed between MLMGs were not equally distributed over all loci, but predominantly observed in MS7, MS6A, or MS10. Conclusions: Our findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by MLMGs. Results suggest that multi-parental recombination in N. caninum is a rare event, but does not exclude uniparental mating. More comprehensive studies on microsatellites in N. caninum and related species like Toxoplasma gondii should be undertaken, not only to improve genotyping capabilities, but also to understand possible functions of these regions in the genomes of these parasites.