A short-term treatment with BKI-1294 does not protect foetuses from sheep experimentally infected with Neospora caninum tachyzoites during pregnancy

Abstract

The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 10 5 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC 50 for N. caninum for 12–15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro , and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed.

Toxoplasma gondii and Neospora caninum infections in sheep and goats in Switzerland: Seroprevalence and occurrence in aborted foetuses

“…In addition, N. caninum DNA was detected in 2.4% (2/82; 95%CI: 0.3–8.5%) and 1.4% (1/73; 95%CI: 0.0–7.4%) of the tested sheep and goat foetuses, respectively, showing occurrence of vertical transmission of this parasite and suggesting its involvement as cause of abortion. Although the frequency of N. caninum -DNA detection in small ruminants was around ten times lower than that previously reported for Swiss cattle, in which N. caninum DNA was amplified in 29% (24/83) ( Gottstein et al, 1998 ) and 21% (50/242) ( Sager et al, 2001 ) of aborted bovine foetuses, this parasite was shown to cause important reproductive losses in some sheep flocks in Switzerland ( Hässig et al, 2003 ) as well as in other countries ( Della Rosa et al, 2021 ; Sánchez-Sánchez et al, 2021a , Sánchez-Sánchez et al, 2021b ; González-Warleta et al, 2018 ; Moreno et al, 2012 ); therefore, N. caninum should be also considered in the differential diagnosis of reproductive failure in small ruminants.…”

“…Some studies observed cross reactions of N. caninum antibodies against T. gondii TgSAG1 antigen ( Huertas-López et al, 2021 ; Sánchez-Sánchez et al, 2021a ). However, this issue seems to be irrelevant in this study, in which only a very low proportion of seropositive animals to N. caninum were recorded.…”

“…Neospora caninum is well recognised as a major abortifacient in cattle ( Dubey et al, 2017 ) and although it seems to have a lower clinical and economic importance in small ruminants, there is increasing evidence of its involvement in cases of abortion and reproductive failure both in sheep and goats worldwide ( Moreno et al, 2012 ; Della Rosa et al, 2021 ; Sánchez-Sánchez et al, 2021a , Sánchez-Sánchez et al, 2021b ; Unzaga et al, 2014 ; Dubey et al, 2017 ; Hässig et al, 2003 ).…”

“…N. caninum DNA was isolated from aborted foetuses. ( Sánchez-Sánchez et al, 2021a , Sánchez-Sánchez et al, 2021b ) Extremadura: Badajoz, and Andalusia: Córdoba and Jaén ELISA (ID Screen Neospora caninum Indirect Multispecies, ID.vet, France) 4/209 1.9 3/12 25.0 Culled Merino and crossbred ewes with no particular disease, extensive breeding systems ( Astorga et al, 2014 ) Castilla-La Mancha: Ciudad Real province ELISA (cELISA Neospora caninum Antibody Test Kit, VMRD, USA) 7/180 3.9 ns/17 ns Manchega sheep flocks, male and female sheep ( Ruiz-Fons et al, 2014 ) Galicia ELISA (ID Screen Neospora caninum Indirect Multi-species, ID.vet, France) 132/2400 5.5 32/44 72.7 Healthy adult sheep (> 6 months) from lamb-producing flocks ( Díaz et al, 2014 ) Galicia: Lugo Province ELISA (cELISA Neospora caninum Antibody Test Kit, VMRD, USA) 18/177 10.1 ns ns Meat crossbred ewes, semi-extensive breeding system, grazing with hay or silage supplement ( Panadero et al, 2010 ) Switzerland Country-wide ELISA (ID Screen® Neospora caninum Indirect, ID.vet, France) + immunoblot (in house) of inconclusive sera 5/653 0.8 4/143 2.8 Randomly sampled general sheep population, healthy animals, different sex and ages This study Zurich IFAT (in house, cut-off 1:160) 12/117 10.3 1/1 100 Ewes from a flock with abortion problems. N. caninum DNA detected in aborted foetuses ( Hässig et al, 2003 ) United Kingdom England and Wales ELISA (Mastazyme, Mast Diagnostics) + verification of positive sera by IFAT (in house, cut-off 1:50) 3/660 0.45 ns <...…”

Vaccine-Linked Chemotherapy Approach: Additive Effects of Combining the Listeria monocytogenes-Based Vaccine Lm3Dx_NcSAG1 With the Bumped Kinase Inhibitor BKI-1748 Against Neospora caninum Infection in Mice

“…CDPK1 plays an important role in gliding motility, invasion, exocytosis, and egress of the parasite ( 21 – 23 ). Several BKIs have shown promising efficacy against N. caninum in vitro and in experimentally infected pregnant mice ( 23 – 25 ), and some BKIs have been advanced to the pregnant sheep infection model ( 24 , 26 , 27 ). BKI-1748 is a recently introduced 5-aminopyrazol-4-carboxamide-based compound with an in vitro EC 50 of 165 nM against N. caninum and no impairment of human foreskin fibroblasts (HFF) viability up to 20 μM ( 28 , 29 ).…”