Ezetimibe blocks Toxoplasma gondii-, Neospora caninum- and Besnoitia besnoiti-tachyzoite infectivity and replication in primary bovine endothelial host cells.

Coccidia are obligate apicomplexan parasites that affect humans and animals. In fast replicating species, in vitro merogony takes only 24­48 h. In this context, successful parasite proliferation requires nutrients and other building blocks. Coccidian parasites are auxotrophic for cholesterol, so they need to obtain this molecule from host cells. In humans, ezetimibe has been applied successfully as hypolipidaemic compound, since it reduces intestinal cholesterol absorption via blockage of Niemann−Pick C-1 like-1 protein (NPC1L1), a transmembrane protein expressed in enterocytes. To date, few data are available on its potential anti-parasitic effects in primary host cells infected with apicomplexan parasites of human and veterinary importance, such as Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. Current inhibition experiments show that ezetimibe effectively blocks T. gondii, B. besnoiti and N. caninum tachyzoite infectivity and replication in primary bovine endothelial host cells. Thus, 20 µm ezetimibe blocked parasite proliferation by 73.1−99.2%, via marked reduction of the number of tachyzoites per meront, confirmed by 3D-holotomographic analyses. The effects were parasitostatic since withdrawal of the compound led to parasite recovery with resumed proliferation. Ezetimibe-glucuronide, the in vivo most effective metabolite, failed to affect parasite proliferation in vitro, thereby suggesting that ezetimibe effects might be NPC1L1-independent.

The soluble fraction of Neospora caninum treated with PI-PLC is dominated by NcSRS29B and NcSRS29C.

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.

Triclosan inhibits the growth of Neospora caninum in vitro and in vivo.

Neospora caninum is an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide; thus, there is an urgent need to develop novel therapeutic agents to control the neosporosis. Enoyl acyl carrier protein reductase (ENR) is a key enzyme of the type II fatty acid synthesis pathway (FAS II), which is essential for apicomplexan parasite survival. The antimicrobial agent triclosan has been shown to be a very potent inhibitor of ENR. In this study, we identified an E. coli ENR-like protein in N. caninum. Multiple sequence alignment showed all the requisite features of ENR existed in this protein, so we named this protein NcENR. Swiss-Model analysis showed NcENR interacts with triclosan. We observed that ENR is localized in the apicoplast, a plastid-like organelle. Similar to the potent inhibition of triclosan on other apicomplexa parasites, this compound markedly inhibits the growth of N. caninum at low concentrations. Further research showed that triclosan attenuated the invasion ability and proliferation ability of N. caninum at low concentrations. The results from in vivo studies in the mouse showed that triclosan attenuated the virulence of N. caninum in mice mildly and reduced the parasite burden in the brain significantly. Taken together, triclosan inhibits the growth of N. caninum both in vitro and in vivo at low concentrations.

Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites.

We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 µM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 µM; however, morphological changes occurred in cultures treated with >5 µM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 µM BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 µM BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.

Evaluation of methylene blue, pyrimethamine and its combination on an in vitro Neospora caninum model.

Neospora caninum is an apicomplexan parasite strongly related to reproductive problems in cattle. The neosporosis control is not well established and several fronts are under development, predominantly based on immune protection, immunomodulation and chemotherapy. The use of anti-malarial drugs as therapeutic sources has, in theory, considerable potential for any apicomplexan. Drugs such as methylene blue (MB) and pyrimethamine (Pyr) represent therapeutic options for malaria; thus, their use for neosporosis should be assessed. In this work, we tested the effects of MB and Pyr on N. caninum proliferation and clearance, using LacZ-tagged tachyzoites. The drugs inhibited at nanomolar dosages and its combination demonstrated an antagonistic interaction in proliferation assays, according to the Chou and Talalay method for drug combination index. However, the drug combination significantly improved the parasite in vitro clearance. The repositioning of well-established drugs opens a short-term strategy to obtain low-cost therapeutics approaches against neosporosis.

The p38 MAPK inhibitor, SB203580, inhibits cell invasion by Neospora caninum.

The Apicomplexan parasite Neospora caninum is an obligate intracellular parasitic protozoan. It can cause severe diseases in a number of animals throughout the world. Infection with N. caninum leads to abortions in pregnant animals and neuromuscular disorders of newborns which cause great economic losses to animal husbandry. However, the mechanism of cell invasion by N. caninum is still unclear. This paper aims to investigate the impact of SB203580, a p38 MAPK inhibitor, on host cell invasion by N. caninum. The results suggested the presence of putative p38 MAPK homologues in N. caninum, and incubation of N. caninum with SB203580 markedly reduced the tachyzoite motility and microneme exocytosis (NcMIC2, 3, and 6). Furthermore, treatment or pretreatment of MDBK cells with SB203580 effectively reduced cell invasion by N. caninum. Therefore, SB203580 affected both, parasites and host cells, resulting in inhibition of cell invasion by N. caninum.

Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model.

The three anti-malarial drugs artemiside, artemisone, and mefloquine, and the naphthoquinone buparvaquone known to be active against theileriosis in cattle and Leishmania infections in rodents, were assessed for activity against Neospora caninum infection. All four compounds inhibited the proliferation of N. caninum tachyzoites in vitro with IC50 in the sub-micromolar range, but artemisone and buparvaquone were most effective (IC50 = 3 and 4.9 nM, respectively). However, in a neosporosis mouse model for cerebral infection comprising Balb/c mice experimentally infected with the virulent isolate Nc-Spain7, the three anti-malarial compounds failed to exhibit any activity, since treatment did not reduce the parasite burden in brains and lungs compared to untreated controls. Thus, these compounds were not further evaluated in pregnant mice. On the other hand, buparvaquone, shown earlier to be effective in reducing the parasite load in the lungs in an acute neosporosis disease model, was further assessed in the pregnant mouse model. Buparvaquone efficiently inhibited vertical transmission in Balb/c mice experimentally infected at day 7 of pregnancy, reduced clinical signs in the pups, but had no effect on cerebral infection in the dams. This demonstrates proof-of-concept that drug repurposing may lead to the discovery of an effective compound against neosporosis that can protect offspring from vertical transmission and disease.

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.

Flavonoids modulate the proliferation of Neospora caninum in glial cell primary cultures.

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.

Efficacy of the bumped kinase inhibitor BKI-1708 against the cyst-forming apicomplexan parasites Toxoplasma gondii and Neospora caninum in vitro and in experimentally infected mice.

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) - and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum ß-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 µM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 µM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 µM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 µM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.

In vitro effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites.

Upon the screening of 16 antiproliferative compounds against Toxoplasma gondii and Neospora caninum, two hydrolytically stable ruthenium complexes (compounds 16 and 18) exhibited 50% inhibitory concentrations of 18.7 and 41.1 nM (T. gondii) and 6.7 and 11.3 nM (N. caninum). To achieve parasiticidal activity with compound 16, long-term treatment (22 to 27 days at 80 to 160 nM) was required. Transmission electron microscopy demonstrated the rapid impact on and ultrastructural alterations in both parasites. These preliminary findings suggest that the potential of ruthenium-based compounds should thus be further exploited.

Effects of miltefosine treatment in fibroblast cell cultures and in mice experimentally infected with Neospora caninum tachyzoites.

Miltefosine was investigated for its activity against Neospora caninum tachyzoites in vitro, and was shown to inhibit the proliferation of N. caninum tachyzoites cultured in human foreskin fibroblasts (HFF) with an IC50 of 5·2 µM. Treatment of infected cells with 25 µM miltefosine for a period of 10 h had only a parasitostatic effect, while after 20 h of treatment parasiticidal effects were observed. This was confirmed by transmission electron microscopy of N. caninum-infected and miltefosine-treated HFF. Administration of miltefosine to N. caninum-infected Balb/c female mice at 40 mg/kg/day for 14 days resulted in 6 out of 10 mice exhibiting weight loss, ruffled coat and apathy between days 7 and 13 post-infection. In the group that received placebo, only 2 out of 8 mice succumbed to infection, but the cerebral burden was significantly higher compared to the miltefosine treatment group. In a second experiment, the time-span of treatment was reduced to 5 days, and mice were maintained without further treatment for 4 weeks. Only 2 out of 9 mice in the miltefosine treatment group exhibited signs of disease, while 8 out of 10 mice succumbed to infection in the placebo group. These results showed that miltefosine hampered the dissemination of parasites into the CNS during experimental N. caninum infection in mice.

Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs.

The thiazolide nitazoxanide (NTZ) and some derivatives exhibit considerable in vitro activities against a broad range of parasites, including the apicomplexans Neospora caninum and Toxoplasma gondii tachyzoites. In order to identify potential molecular targets for this compound in both parasites, RM4847 was coupled to epoxy-agarose and affinity chromatography was performed. A protein of approximately 35 kDa was eluted upon RM4847-affinity-chromatography from extracts of N. caninum-infected human foreskin fibroblasts (HFF) and non-infected HFF, but no protein was eluted when affinity chromatography was performed with T. gondii or N. caninum tachyzoite extracts. Mass spectrometry analysis identified the 35 kDa protein as human quinone reductase NQO1 (P15559; QR). Within 8h after infection of HFF with N. caninum tachyzoites, QR transcript expression levels were notably increased, but no such increase was observed upon infection with T. gondii tachyzoites. Treatment of non-infected HFF with RM4847 did also lead to an increase of QR transcript levels. The enzymatic activity of 6-histidine-tagged recombinant QR (recQR) was assayed using menadione as a substrate. The thiazolides NTZ, tizoxanide and RM4847 inhibited recQR activity on menadione in a concentration-dependent manner. Moreover, a small residual reducing activity was observed when these thiazolides were offered as substrates.

Experimental treatment of Neospora caninum-infected mice with the arylimidamide DB750 and the thiazolide nitazoxanide.

The cationic arylimidamide DB750 and the thiazolide nitazoxanide had been shown earlier to be effective against Neospora caninum tachyzoites in vitro with an IC(50) of 160nM and 4.23µM, respectively. In this study, we have investigated the effects of DB750 and nitazoxanide treatments of experimentally infected Balb/c mice, by applying the drugs either through the oral or the intraperitoneal route. In experiment 1, administration of DB750 (2mg/kg/day) and nitazoxanide (150mg/kg/day) started already 3 days prior to experimental infection of mice with 2×10(6) tachyzoites. Following infection, the drugs were further administrated daily for a period of 2 weeks, either orally or intraperitoneally. Intraperitoneal injection of DB750 was well tolerated by the mice, but treatment with nitazoxanide resulted in death of all mice within 3 days. Upon intraperitoneal application of DB750, the cerebral parasite load was significantly reduced compared to all other groups, while oral application of DB750 and nitazoxanide were not as effective, and resulted in significant weight loss. In experiment 2, mice were infected with 2×10(6) tachyzoites and at 2 weeks post-infection, DB750 (2mg/kg/day) was applied by intraperitoneal injections for 14 days. In the DB750-treated group, only 2 out of 12 mice succumbed to infection, compared to 7 out of 12 mice in the placebo-group. DB750 treatment also resulted in significantly reduced cerebral parasite burden, and reduced numbers of viable tachyzoites. Our data suggest that DB750 exerted its activity also after crossing the blood-brain barrier, and that this class of compounds could be promising for the control of N. caninum-associated disease.

Atovaquone, chloroquine, primaquine, quinine and tetracycline: antiproliferative effects of relevant antimalarials on Neospora caninum.

Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.

Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.

Hydroxycinnamic acid-spermidine amides from Tetragonisca angustula honey as anti-Neospora caninum: In vitro and in silico studies.

Tetragonisca angustula honey was fractioned in a SiO2 column to furnish three fractions (A-C) in which four hydroxycinnamic acid-Spermidine amides (HCAAs), known as N', N″, N‴-tris-p-coumaroyl spermidine, N', N″-dicaffeoyl, N‴-coumaroyl spermidine, N', N″, N‴-tris-caffeoyl spermidine and N', N″-dicaffeoyl and N‴-feruloyl spermidine were identified in the fractions B and C by electrospray ionization tandem mass spectrometry. A primary culture model previously infected with Neospora caninum (72 h) was used to evaluate the honey fractions (A-C) for two-time intervals: 24 and 72 h. Parasitic reduction ranged from 38% on fraction C (12.5 µg/ml), after 24 h, to 54% and 41% with fractions B and C (25 µg/ml) after 72 h of treatment, respectively. Additionally, HCAAs did not show any cell toxicity for 24 and 72 h. For infected cultures (72 h), the active fractions B (12.5 µg/ml) and C (25 µg/ml) decreased their NO content. In silico studies suggest that HCAAs may affect the parasite's redox pathway and improve the oxidative effect of NO released from infected cells. Here, we presented for the first time, that HCAAs from T. angustula honey have the potential to inhibit the growth of N. caninum protozoa.

Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 µM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (Si) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between Si and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 µM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 µM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

Toltrazuril treatment of congenitally acquired Neospora caninum infection in newborn mice.

C57BL/6 mice were infected with Neospora caninum tachyzoites during pregnancy, yielding a transplacental infection of developing fetuses. Subsequently, congenitally infected newborn mice were treated either once or three times with toltrazuril (or placebo) at a concentration of 31.25 mg compound per kg body weight. Both toltrazuril and placebo treatment had no negative effect on newborns, as noninfected treated pups developed normally without differences in mortality and morbidity to matching nontreated control animals. Already one application of toltrazuril was significantly (p < 0.01) able to delay the outbreak of neosporosis in newborn mice, when compared to placebo-treated infected controls. We found significantly higher proportion of surviving newborns in one-time-toltrazuril-treated and three-time-toltrazuril-treated groups (34% and 54%, respectively) when compared to one-time-placebo-treated and three-time-placebo-treated groups (14% and 30%, respectively). There was no significant difference (p = 0.2) in the proportion of surviving pups between one-time-toltrazuril and three-time-toltrazuril treatment. However, the number of diseased and Neospora-positive pups (46% and 47%, respectively) was markedly reduced after three-time-toltrazuril treatment compared to all other groups. Three-time-treatment also resulted in the highest antibody (IgG, IgG2a) response. Pharmacokinetic analyses using individual serum samples revealed that, although toltrazuril was absorbed and metabolized to toltrazuril sulfone by newborn mice, medicated animals exhibited an unexpected rapid turn-over (half-life time) of the compound. Toltrazuril and the metabolite were also found in brain tissues, indicating that passage of the blood-brain barrier occurred. In conclusion, we could show that three times treatment with toltrazuril had a high impact on the course of infection in congenitally N. caninum-infected newborn mice.

Detection, quantifications and pharmacokinetics of toltrazuril sulfone (Ponazuril) in cattle.

Toltrazuril sulfone (Ponazuril) is a triazine-based anti-protozoal agent with highly specific actions against apicomplexan group of organisms, which are undergoing intensive investigation. Toltrazuril sulfone may have clinical application in the treatment of Neospora. caninum and other protozoal infections in cattle. To evaluate absorption, distribution, and elimination characteristics of toltrazuril sulfone in cattle, a sensitive validated quantitative high-pressure liquid chromatography method for toltrazuril sulfone in bovine biological fluids was developed. After a single oral dose of toltrazuril sulfone at 5 mg/kg (as 150 mg/g of Marquis; Bayer HealthCare, Shawnee Mission, KS, USA), samples from six cows showed good plasma concentrations of toltrazuril sulfone, which peaked at 4821 ng/mL +/- 916 (SD) at 48 h postadministration. Thereafter, plasma concentration declined to 1950 ng/mL +/- 184 (SD) at 192 h after administration with an average plasma elimination half-life of approximately 58 h. Following oral dose of toltrazuril sulfone, the observed peak plasma concentrations were in relatively close agreement ranging from the lowest 3925 ng/mL to the highest of 6285 ng/mL with the mean peak plasma concentration being 4821 ng/mL. This study shows that toltrazuril sulfone is relatively well absorbed after oral dose in cattle. These results are therefore entirely consistent with and support the reported clinical efficacy of toltrazuril sulfone in the treatment of experimentally induced clinical cases of N. caninum and other protozoal-mediated bovine diseases.