Isolation and genetic characterization of Neospora caninum from naturally infected sheep

Highlights

• In vitro isolation and genotyping of two new Neospora caninum isolates is described.

• Nc-Spain11 and Nc-Spain12 were isolated from two congenitally infected lambs.

• Neospora caninum ovine isolates were implicated in reproductive failure in a flock.

• Both isolates showed identical multilocus microsatellite genotype.

• Ovine genotype showed close genetic relationship with bovine genotypes.

Abstract

Neospora caninum is considered one of the main causes of abortion in cattle but can also cause abortion in sheep. There is limited knowledge of the N. caninum population infecting sheep, and only one N. caninum isolate from a pregnant sheep from Japan has been reported. This study describes the in vitro isolation and genetic characterization of two new sheep isolates of N. caninum implicated in ovine reproductive failure. We used IFN-γ-knockout mice inoculated with PCR-positive brain homogenates from two clinically healthy but congenitally infected lambs at 4.5 months of age for parasite isolation. The lambs were born to dams from a sheep farm that had experienced pregnancy failure caused by N. caninum in successive generations. Tachyzoites were microscopically visualized in peritoneal flushes from all inoculated mice and were also observed in MARC-145 cell cultures within one week after inoculation with peritoneal flushes. Two N. caninum isolates, Nc-Spain11 and Nc-Spain12, were obtained from each lamb. The genotyping of the Nc-Spain11 and Nc-Spain12 isolates based on 9 microsatellite markers showed identical multilocus genotype (MLG). Comparison between a previous N. caninum genotype dataset including 80 MLGs from Argentinean, Spanish, Mexican, German and Scottish bovine isolates and the Japanese sheep isolate showed that the Nc-Spain11 and Nc-Spain12 MLG was unique and differed from the other MLGs. eBURST analyses showed that the Nc-Spain11 and Nc-Spain12 MLG was genetically clustered with other bovine MLGs and one ovine MLG, and the nearest genetic relationship was with an MLG from a bovine abortion collected in the same geographical area of Galicia.

Introduction

Neospora caninum and Toxoplasma gondii are phylogenetically closely related protozoan parasites that cause reproductive failure in ruminants worldwide. N. caninum is considered the main cause of abortion in cattle, whereas T. gondii is one of the main abortifacient agents in small ruminants (Dubey, 2009; Dubey and Schares, 2011). The relevance of neosporosis to abortion in small ruminants is considered limited, but recent studies have suggested that its importance in sheep flocks may be underestimated in terms of reproductive losses (Moreno et al., 2012; González-Warleta et al., 2014, 2018). Endogenous transplacental transmission occurs in infected ewes, and N. caninum reactivation can cause abortion in sheep in successive pregnancies (González-Warleta et al., 2014, 2018). There is limited knowledge of the N. caninum population infecting sheep. N. caninum has been isolated in culture only from one pregnant sheep in Japan (Koyama et al., 2001). N. caninum was isolated in Brazil via a bioassay using dogs that shed oocysts after consuming the brains of seropositive sheep, but no N. caninum isolates were obtained in cell culture from mice, gerbils and large vesper mice infected with these oocysts (Pena et al., 2007). Here, we describe the first isolation and genetic characterization of N. caninum from two congenitally infected lambs from dams of a commercial sheep farm that had previously experienced reproductive failure caused by N. caninum (González-Warleta et al., 2014).