The effect of an injectable toltrazuril - gleptoferron (Forceris®) on Cystoisospora suis oocyst excretion and growth of neonatal piglets pre- and post-weaning.

In this study the efficacy of an intramuscular formulation of toltrazuril combined with gleptoferron for the control of porcine cystoisosporosis caused by Cystoisospora suis was investigated. The study was carried out on three Belgian farms with a confirmed history of C. suis infections. As none of the farms implemented a standardized toltrazuril treatment regimen for their piglets, the presence of resistant C. suis strains seems improbable. In total 90 litters, representing 1249 piglets, were included in the study and randomly allocated to either the treatment or control group. Piglets in the treatment group received a single intramuscular injection, containing 45 mg toltrazuril and 200 mg gleptoferron, between 1 and 3 days of age. Piglets in the control group received a single injection with only 200 mg gleptoferron. The effect of treatment on oocyst excretion, expressed in oocysts per gram of feces (OPG), average daily weight gain (ADG) and mortality was determined both pre- and post-weaning. A significant decrease in OPG as well as a decrease in the number of litters (pre-weaning) and pens (post-weaning) that tested positive for cystoisosporosis, was observed in the treated animals compared to the controls. Furthermore, treatment resulted in an increased ADG during the period from day 1 to day 21 (p-value: 0.03881). There was no significant difference in mortality observed between the treatment group to the control group (p-value: 0.2167). To our knowledge, this is the first report on the effect of toltrazuril on oocyst excretion after weaning. This finding highlights the potential long-term benefits of the treatment beyond the initial administration.

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.

Cystoisospora suis merozoite development assay for screening of drug efficacy in vitro.

Cystoisospora suis is a common diarrheal pathogen of piglets and typically controlled by metaphylactic toltrazuril application. Recently, toltrazuril resistance has been reported in the field; however, both evaluation of toltrazuril efficacy against field isolates and the anticoccidial drug development for pigs is hampered by costs and labor of animal experimentation. Therefore an in vitro merozoite development assay was developed to evaluate the efficacy of compounds against C. suis in vitro. Monolayers of IPEC-1 cells were infected with sporozoites derived from oocysts of defined C. suis laboratory strains and the optimal infection dose as well as concentration, time point and duration of treatment were evaluated by quantitative real-time PCR. Cell cultures were treated with bumped kinase inhibitor (BKI) 1369 at different time points to evaluate the possibility to delineate effects on different developmental stages in vitro during invasion and early infection, and to determine different inhibitory concentrations (IC50, IC95). BKI 1369 had an IC50 of 35 nM and an IC95 of 350 nM. Dose- and duration-dependent efficacy was seen when developing stages were treated with BKI 1369 after infection (days 0-1, 2-3 and 2-5) but not when sporozoites were pre-incubated with BKI 1369 before infection. Efficacies of further BKIs were also evaluated at 200 nM. BKI 1318, 1708, 1748 and 1862 had an efficacy comparable to that of BKI 1369 (which is also effective in vivo). BKI 1862 showed a more pronounced loss of efficacy in lower concentrations than BKI 1369, signifying pharmacokinetic differences of similar compounds detectable in vitro. In addition, the effects of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, on a toltrazuril sensitive and a resistant strain of C. suis were evaluated. Inhibition of merozoite growth in vitro by toltrazuril and its metabolites was dose-dependent only for toltrazuril. Clear differences were noted for the effect on a toltrazuril-sensitive vs. a resistant strain, indicating that this in vitro assay has the capacity to delineate susceptible from resistant strains in vitro. It could also be used to evaluate and compare the efficacy of novel compounds against C. suis and support the determination of the optimal time point of treatment in vivo.

Efficacy of injectable toltrazuril-iron combination product and oral toltrazuril against early experimental infection of suckling piglets with Cystoisospora suis.

BACKGROUND: Toltrazuril is frequently administered for the metaphylactic control of piglet cystoisosporosis. In a previous study, the efficacy of parenteral toltrazuril (45 mg/piglet, Group Forceris®) applied on the 2nd day of life (dol), and of oral toltrazuril (20 mg/kg of body weight, Group Baycox®) applied on the 4th dol was evaluated in an experimental model with Cystoisospora suis infection on the 3rd dol (late infection, LI). In a follow-up study, efficacy and safety were evaluated against infections with C. suis on the 1st dol (early infection, EI). Parameters included oocyst excretion and faecal consistency, body weight development, bacteriological examinations and animal health. RESULTS: All control piglets (n = 12) shed oocysts and had diarrhoea, while parasite excretion was completely suppressed in both treatment groups (n = 13 each) and diarrhoea was reduced to a single animal (Forceris® group), resulting in significant differences for these parameters between the treated groups and the controls without significant differences among the treatment groups. No treatment-related adverse events were noted. Body weight gain was reduced in the control group during the acute phase of infection, resulting in significantly lower body weight on the 15th dol. Sows and piglets shed high numbers of Escherichia coli. Clostridium perfringens type A was only detected in low amounts in pooled litter samples. In comparison to the LI study oocyst shedding was more intense in the control animals in EI, while diarrhea was more frequent in LI. In both infection models a high efficacy of toltrazuril in the control of parasitological and clinical outcomes of experimental C. suis infection could be demonstrated. Since in the LI study high numbers of Cl. perfringens type A were detected, it is hypothesized that colonization with these opportunistic pathogens has synergistic effects with C. suis and may explain variable clinical outcomes in untreated animals as well as the sporadic occurrence of diarrhea in toltrazuril-treated piglets. CONCLUSIONS: Parenteral and oral toltrazuril administered on the 2nd or 4th dol is safe and effective against experimental infections with C. suis on the 1st to 3rd dol. The clinical outcome of experimental infections seems influenced by bacterial coinfections.

Bumped kinase inhibitor 1369 is effective against Cystoisospora suis in vivo and in vitro.

Cystoisosporosis is a leading diarrheal disease in suckling piglets. With the confirmation of resistance against the only available drug toltrazuril, there is a substantial need for novel therapeutics to combat the infection and its negative effects on animal health. In closely related apicomplexan species, bumped kinase inhibitors (BKIs) targeting calcium-dependent protein kinase 1 (CDPK1) were shown to be effective in inhibiting host-cell invasion and parasite growth. Therefore, the gene coding for Cystoisospora suis CDPK1 (CsCDPK1) was identified and cloned to investigate activity and thermal stabilization of the recombinant CsCDPK1 enzyme by BKI 1369. In this comprehensive study, the efficacy, safety and pharmacokinetics of BKI 1369 in piglets experimentally infected with Cystoisospora suis (toltrazuril-sensitive, Wien-I and toltrazuril-resistant, Holland-I strains) were determined in vivo and in vitro using an established animal infection model and cell culture, respectively. BKI 1369 inhibited merozoite proliferation in intestinal porcine epithelial cells-1 (IPEC-1) by at least 50% at a concentration of 40 nM, and proliferation was almost completely inhibited (>95%) at 200 nM. Nonetheless, exposure of infected cultures to 200 nM BKI 1369 for five days did not induce structural alterations in surviving merozoites as confirmed by transmission electron microscopy. Five-day treatment with BKI 1369 (10 mg/kg BW twice a day) effectively suppressed oocyst excretion and diarrhea and improved body weight gains in treated piglets without obvious side effects for both toltrazuril-sensitive, Wien-I and resistant, Holland-I C. suis strains. The plasma concentration of BKI 1369 in piglets increased to 11.7 µM during treatment, suggesting constant drug accumulation and exposure of parasites to the drug. Therefore, oral applications of BKI 1369 could potentially be a therapeutic alternative against porcine cystoisosporosis. For use in pigs, future studies on BKI 1369 should be directed towards ease of drug handling and minimizing treatment frequencies.

In vitro treatment of Besnoitia besnoiti with the naphto-quinone buparvaquone results in marked inhibition of tachyzoite proliferation, mitochondrial alterations and rapid adaptation of tachyzoites to increased drug concentrations.

We here assessed the in vitro efficacy of the naptho-quinone buparvaquone (BPQ) against Besnoitia besnoiti tachyzoites in vitro. BPQ is currently licensed for the treatment of theileriosis in cattle in many countries, but not in the EU. In 4-day treatment assays, BPQ massively impaired tachyzoite proliferation with an IC50 of 10 ± 3 nm, and virtually complete inhibition was obtained in the presence of nm BPQ. Exposure to 1 µm BPQ leads to ultrastructural changes affecting initially the mitochondrial matrix and the cristae. After 96 h, most parasites were largely distorted, filled with cytoplasmic amylopectin granules and vacuoles containing components of unknown composition. Host cell mitochondria did not appear to be notably affected by the drug. However, upon prolonged exposure (14-16 days) to increased BPQ concentrations, B. besnoiti tachyzoites exhibited the capacity to adapt, and they resumed proliferation at dosages of up to 10 µm BPQ, albeit at a lower rate. These BPQ-adapted parasites maintained this lower susceptibility to BPQ treatment after freeze-thawing, and inspection by the transmission electron microscopy revealed that they underwent proliferation in the absence of structurally intact mitochondria.

Repurposing of commercially available anti-coccidials identifies diclazuril and decoquinate as potential therapeutic candidates against Besnoitia besnoiti infection.

Repurposing of currently marketed compounds with proven efficacy against apicomplexan parasites was used as an approach to define novel candidate therapeutics for bovine besnoitiosis. Besnoitia besnoiti tachyzoites grown in MARC-145 cells were exposed to different concentrations of toltrazuril, diclazuril, imidocarb, decoquinate, sulfadiazine and trimethoprim alone or in combination with sulfadiazine. Drugs were added either just prior to infection of MARC-145 cells (0 h post infection, hpi) or at 6 hpi. A primary evaluation of drug effects was done by direct immunofluorescence staining and counting. Potential effects on the host cells were assessed using a XTT kit for cell proliferation. Compounds displaying promising efficacy were selected for IC50 and IC99 determination by qPCR. In addition, the impact of drugs on the tachyzoite ultrastructure was assessed by TEM and long-term treatment assays were performed. Cytotoxicity assays confirmed that none of the compounds affected the host cells. Decoquinate and diclazuril displayed invasion inhibition rates of 90 and 83% at 0 h pi and 73 and 72% at 6 h pi, respectively. The remaining drugs showed lower efficacy and were not further studied. Decoquinate and diclazuril exhibited IC99 values of 100 nM and 29.9 µM, respectively. TEM showed that decoquinate primarily affected the parasite mitochondrium, whilst diclazuril interfered in cytokinesis of daughter zoites. The present study demonstrates the efficacy of diclazuril and decoquinate against B. besnoiti in vitro and further assessments of safety and efficacy of both drugs should be performed in the target species.

Comparison of an injectable toltrazuril-gleptoferron (Forceris®) and an oral toltrazuril (Baycox®) + injectable iron dextran for the control of experimentally induced piglet cystoisosporosis.

BACKGROUND: Cystoisospora suis causes diarrhoeal disease and reduced weight gain in suckling piglets, and a toltrazuril-based oral suspension is available for treatment. Recently a combinatorial product with toltrazuril plus iron has been developed for parenteral application. In this study we compared the efficacy of the injectable product with the oral suspension against experimentally induced piglet cystoisosporosis. METHODS: In a randomised controlled study, three groups of piglets (n = 10-13) were treated either with a fixed dose of 45 mg toltrazuril + 200 mg gleptoferron i.m. per piglet (Forceris®) on the second day of life (study day 2; SD 2) or with 20 mg toltrazuril/kg body weight as an oral suspension (Baycox® 5%) on SD 4 or left untreated (Control group). The Baycox® and the Control group received 200 mg of iron dextran/piglet on SD 2. All piglets were infected with 1000 sporulated C. suis oocysts on SD 3. Faecal samples were taken daily from SD 7 to SD 20 to determine faecal consistency, oocyst shedding and other diarrhoeal pathogens. Body weight was recorded on SD 1 and then weekly until SD 29. Animals were observed daily for general health and after treatment for possible adverse events. RESULTS: In the Control group all animals shed oocysts for 3.1 days on average and all animals showed diarrhoea for an average of five days. Excretion peaked on SD 9 (max. 48,618 oocysts per gram of faeces). Treatment with Forceris® completely suppressed oocyst excretion. In the Baycox® group, low levels of excretion could be detected. Diarrhoea was reduced to single piglets in the treated groups. Body weight development was reduced in the Control group compared to the treated groups. Enteropathogenic bacteria (Escherichia coli, Clostridium perfringens) could be detected. All parameters related to oocyst excretion, faecal consistency and weight gain were significantly improved in the treated groups compared to the Control group without significant differences between the treated groups. Both products were safe to use. CONCLUSIONS: Treatment with both the injectable (Forceris®) and the oral (Baycox®) formulation of toltrazuril in the prepatent period were safe and highly effective against experimental infection with C. suis in newborn piglets.

In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.

Experimentally confirmed toltrazuril resistance in a field isolate of Cystoisospora suis.

BACKGROUND: Constant treatment regimens with toltrazuril against Cystoisospora suis infection in piglets are being applied in the intensive production systems for the last two decades, but the possibility of resistance development has not been addressed so far despite limited availability of treatment alternatives. Recently, a pig producer in The Netherlands who routinely used toltrazuril complained about diarrhea in suckling piglets in the absence of bacterial and viral pathogens, and oocysts of C. suis could be isolated from feces of affected litters. METHODS: Piglets from two litters were infected with a field isolate of C. suis, Holland-I, and treated with 0 (Holl-Ctrl), 20 (Holl-20) or 30 (Holl-30) mg/kg of body weight (BW) of toltrazuril (Baycox®). The efficacy of toltrazuril was measured by assessment of oocyst excretion, fecal consistency and BW gain. A separate litter was infected with a toltrazuril-susceptible strain of C. suis, Wien-I, and treated with 0 (Wien-Ctrl) or 20 (Wien-20) mg/kg BW of toltrazuril for comparison. RESULTS: Treatment with the recommended (20 mg/kg) dose of toltrazuril completely suppressed oocyst shedding and diarrhea in group Wien-20. The prevalence of oocyst excretion was 100% in the groups infected with Holland-I and 80% in the group Wien-Ctrl. Most days with diarrhea were observed in group Holl-20 with an average of 6.40%, followed by 5.71% in Wien-Ctrl, while in Holl-Ctrl and Holl-30 diarrhea was only seen in 1.79% of the samples (n = 14/piglet). Oocyst excretion, fecal consistency and BW gain did not differ significantly among groups infected with Holland-I, indicating loss of efficacy to toltrazuril. CONCLUSION: Experimental infections and treatment confirmed toltrazuril resistance against the field isolate even at increased dosage. Such isolates are a potential threat to pig production as no other effective and economically sustainable alternative treatment is currently available. In the absence of a standardized protocol for resistance testing in C. suis, regular parasitological examination and, if possible, experimental confirmation should be considered to evaluate the extent and consequences of toltrazuril resistance.