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Background   Neospora caninum , a coccidian protozoan, represents an important cause of bovine abortion. Available  N. caninum  strains show considerable variation  in vitro  and  in vivo , including different virulence in cattle. To which extent sexual recombination, which is possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contributes 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 using ten previously established microsatellite markers. In addition to our own data, those from a recent study providing data on five markers from other northern Italian regions were included and analysed.    Results  Of the 55 samples finally subjected to genotyping, 35 were typed at all or 9 out of 10 loci and their individual multilocus-microsatellite genotype (MLMG) determined. 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 this and a previous North Italian study into eBURST analysis revealed that several 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 microsatellite typing. 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.

Spatial distance between sites of sampling associated with genetic variation among Neospora caninum in aborted bovine foetuses from northern Italy