Abstract
Exposure of Neospora caninum tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex™ 200 system, and humoral immune responses against tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy.
Highlights
The apicomplexan parasite Neospora caninum is an important causative agent of abortion or birth of weak offspring in cattle, and to a lesser extent in sheep and other ruminants [1]
Bumped kinase inhibitors (BKIs)-1294 was stored as a 20 mM stock solution in dimethyl sulfoxide (DMSO) at −20◦C; for application in mice, BKI-1294 powder was suspended in corn oil, and the suspension was applied by gavage
Live vaccination without further treatments as shown in group D led to survival of all pups, and no vertical transmission occurred, demonstrating the safety of this live vaccination approach
Summary
The apicomplexan parasite Neospora caninum is an important causative agent of abortion or birth of weak offspring in cattle, and to a lesser extent in sheep and other ruminants [1]. The sexual cycle of N. caninum takes place in canine intestinal tissue, and dogs do shed oocysts which become infective after sporulation, but can act as intermediate hosts and become affected by neurological symptoms. Proliferating tachyzoites represent the disease-causing stage that can cross the placenta and infect the fetus. Upon infection of an immunocompetent host, differentiation into tissue-cyst-forming bradyzoites takes place, which remain viable, proliferate slowly, and do not cause inflammation. Infection of fetuses during pregnancy occurs via exogenous transplacental transmission in cases where primary maternal infection takes place during the course of the pregnancy, or endogenous transplacental transmission in cases where persistently infected animals undergo pregnancy-driven immunomodulation that leads to recrudescence and bradyzoite-tachyzoite reconversion [2, 3]. There is no vaccine on the market for the prevention of bovine or canine neosporosis and so far, no immuno- or chemotherapeutic treatments have found their way to the market. Live-vaccines comprised of low-virulence strains of N. caninum strains have shown promising efficacy in both murine and bovine models [4,5,6,7,8]
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