Neonatal neosporosis in a 2-week-old Bernese mountain dog infected with multiple Neospora caninum strains based on MS10 microsatellite analysis

•Simultaneous vertical transmission of multiple N. caninum strains typed at MS10 microsatellite.•Neonatal death may occur without characteristic neosporosis-like signs.

Neonatal neosporosis is a challenging disease to diagnose in neonatal and young puppies because the first signs of this condition may not be strongly suggestive of an infectious aetiology. Within two weeks of birth, three of four pups died with a subacute clinical course, some with dyspnea, some with diarrhoea and some with neurologic signs. Neosporosis was diagnosed post-mortem, but only after microscopic examination of tissues collected at necropsy. Histological findings consisted of (i) necrotizing, diffuse interstitial pneumonia associated with intralesional protozoa and (ii) necrotizing multifocal myocarditis with mineralization and intralesional protozoa. No significant alterations were found in the cerebrum or cerebellum (spinal cord was not examined). Immunohistochemistry confirmed protozoal stages and cysts were Neospora caninum. Immunohistochemistry for Toxoplasma gondii was negative. Lung and heart were the most severely affected tissues with large numbers of free zoites, BAG5 positive bradyzoites and tissue cysts of N. caninum further confirmed by N. caninum-specific quantitative real-time PCR. One affected pup which displayed knuckling, ataxia and diarrhoea were treated with trimethoprim sulfadiazine and clindamycin, and made a complete recovery. This surviving pup (at 8 weeks-of-age) and dam were both positive for N. caninum antibody (reciprocal titres 4096 and 256, respectively). Three other intact bitches on the same property were seropositive for N. caninum, suggesting horizontal transmission and a common source of infection, possibly due to consumption of infected meat. Analysis using microsatellite-10 (MS10) demonstrated that multiple strains of N. caninum were present. It was likely that all MS10 N. caninum strains were transplacentally transmitted from dam to pups. This is the first time that multiple N. caninum strains have been demonstrated to be vertically transmitted in dogs. N. caninum should be considered in the differential diagnosis for acute to subacute death in neonatal pups even when neurological signs suggestive of neosporosis are absent.