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The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to 'activation' of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. However, in contradistinction to the heme-activation mechanism, the amino-artemisinins are effective in vitro against non-heme-degrading apicomplexan parasites including T. gondii, with IC 50 values of 50-70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 μM) of these two compounds within few days. Thus, T. gondii strains that were resistant against artemisone and artemiside were generated by treating the T. gondii reference strain ME49 with stepwise increasing amounts of these compounds, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Interestingly, eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and - to a lesser extent - in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. These findings suggest that amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC ‘click’ (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii β-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

The apicomplexan parasite Neospora caninum is an important causative agent of congenital neosporosis, resulting in abortion, birth of weak offspring and neuromuscular disorders in cattle, sheep, and many other species. Among several compound classes that are currently being developed, two have been reported to limit the effects of congenital neosporosis: (i) bumped kinase inhibitors (BKIs) target calcium dependent protein kinase 1 (CDPK1), an enzyme that is encoded by an apicoplast-derived gene and found only in apicomplexans and plants. CDPK1 is essential for host cell invasion and egress; (ii) endochin-like quinolones (ELQs) are inhibitors of the cytochrome bc1 complex of the mitochondrial electron transport chain and thus inhibit oxidative phosphorylation. We here report on the in vitro and in vivo activities of BKI-1748, and of ELQ-316 and its respective prodrugs ELQ-334 and ELQ-422, applied either as single-compounds or ELQ-BKI-combinations. In vitro, BKI-1748 and ELQ-316, as well as BKI-1748 and ELQ-334, acted synergistically, while this was not observed for the BKI-1748/ELQ-422 combination treatment. In a N. caninum-infected pregnant BALB/c mouse model, the synergistic effects observed in vitro were not entirely reproduced, but 100% postnatal survival and 100% inhibition of vertical transmission was noted in the group treated with the BKI-1748/ELQ-334 combination. In addition, the combined drug applications resulted in lower neonatal mortality compared to treatments with single drugs., Graphical abstract Image 1, Highlights • BKI-1748 is a bumped kinase inhibitor with anti-Neospora activity in vitro and in vivo • Endochin-like quinolones target the cytochrome bc1 complex and exhibit anti-Neospora properties. • Single and combined applications of these two compound classes do not interfere in pregnancy outcome in mice • BKI-1748/ELQ-334 combination treatment results in 100% postnatal survival and blocks vertical transmission in the neosporosis mouse model

In the past 50 years, enormous progress has been made in the diagnosis, treatment and control of taeniid cestode infections/diseases and in the scientific understanding thereof. Most interest in this group of parasites stems from the serious diseases that they cause in humans. It is through this lens that we summarize here the most important breakthroughs that have made a difference to the treatment of human diseases caused by these parasites, reduction in transmission of the taeniid species associated with human disease, or understanding of the parasites’ biology likely to impact diagnosis or treatment in the foreseeable future. Key topics discussed are the introduction of anti-cestode drugs, including benzimidazoles and praziquantel, and the development of new imaging modalities that have transformed the diagnosis and post-treatment monitoring of human echinococcoses and neurocysticercosis. The availability of new anti-cestode drugs for use in dogs and a detailed understanding of the transmission dynamics of Echinococcus granulosus sensu lato have underpinned successful programs that have eliminated cystic echinococcosis in some areas of the world and greatly reduced the incidence of infection in others. Despite these successes, cystic and alveolar echinococcosis and neurocysticercosis continue to be prevalent in many parts of the world, requiring new or renewed efforts to prevent the associated taeniid infections. Major advances made in the development of practical vaccines against E. granulosus and Taenia solium will hopefully assist in this endeavour, as might the understanding of the parasites’ biology that have come from an elucidation of the nuclear genomes of each of the most important taeniid species causing human diseases.

Aiming to generate novel compounds with improved antitoxoplama activity by exploiting the parasite auxotrophies, a library of nucleic base-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates - was synthesized and evaluated. Various structural features as the nucleobase (adenine, uracil, cytosine, thymine, and xanthine) and the linker between the two units were progressively modified. To substantiate the concept, hybrid molecules comprising the diruthenium moiety and other type of pendant molecules were also synthesized and assessed. The use of the CuAAC (copper catalyzed azide-alkyne cycloaddition) approach for the obtainment of conjugates bearing the trithiolato diruthenium scaffold was validated. 37 Compounds (diruthenium conjugates and corresponding intermediates) were assessed in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) at 0.1 and 1 μM. In parallel the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. 20 Compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii β-gal dose-response studies (IC50, half maximal inhibitory concentration determination) and their toxicity for HFF was assessed at 2.5 μM. Two compounds showing promise for further development were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 μM respectively, significantly lower compared to the standard drug pyrimethamine (IC50 = 0.326 μM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 μM and are candidates as potential treatment options in a suitable in vivo model.

The synthesis, photophysical properties and antiparasitic efficacy against Toxoplasma gondii β-gal (RH strain tachyzoites expressing β-galactosidase) grown in human foreskin fibroblast monolayers (HFF) of a series of 15 new conjugates BODIPY-trithiolato-bridged dinuclear ruthenium(II)-arene complexes are reported (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza- s -indacene, derivatives used as fluorescent markers). The influence of the bond type (amide vs ester), as well as that of the length and nature (alkyl vs aryl) of the spacer between the dye and the diruthenium(II) complex moiety, upon the fluorescence and the biological activity were evaluated. The assessed photophysical properties revealed that despite an important fluorescence quenching effect observed after conjugating the BODIPY to the diruthenium unit, the hybrids could nevertheless be used as fluorescent tracers. Although the antiparasitic activity of these series of conjugates appears limited, the compounds demonstrate potential as fluorescent probes to be used for investigating the intracellular trafficking of trithiolato-bridged dinuclear Ru(II)-arene complexes in vitro .

Herein, we developed a single and a duplex TaqMan quantitative qPCR for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wt-dhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

As there is a continuous need for novel anti-infectives, the present study aimed to fuse two modes of action into a novel 3-nitroimidazo[1,2-b]pyridazine scaffold to improve antiparasitic efficacy. For this purpose, we combined known structural elements of phosphodiesterase inhibitors, a target recently proposed for Trypanosoma brucei and Giardia lamblia, with a nitroimidazole scaffold to generate nitrosative stress. The compounds were evaluated in vitro against a panel of protozoal parasites, namely Giardia lamblia, Trypanosoma brucei, T. cruzi, Leishmania infantum and Plasmodium falciparum and for cytotoxicity on MRC-5 cells. Interestingly, selective sub-nanomolar activity was obtained against G. lamblia, and by testing several analogues with and without the nitro group, it was shown that the presence of a nitro group, but not PDE inhibition, is responsible for the low IC50 values of these novel compounds. Adding the favourable drug-like properties (low molecular weight, cLogP (1.2–4.1) and low polar surface area), the key compounds from the 3-nitroimidazo[1,2-b]pyridazine series can be considered as valuable hits for further anti-giardiasis drug exploration and development.

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant-free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)-restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell-penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor-specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat12.5 nanofiber) increased antigen cross-presentation by bone marrow-derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat12.5 nanofibers exhibited increased presentation of the H2kb-epitope (reminiscent for cross-presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor-infiltrating IFN-γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber-based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.

The synthesis, characterization, photophysical and biological properties of 13 new conjugate coumarin‐diruthenium(II)⋅arene complexes against Toxoplasma gondii are presented. For all conjugate organometallic unit/coumarins, an almost complete loss of fluorescence efficacy was observed. However, the nature of the fluorophore, the type of bonding, the presence and length of a linker between the coumarin dye and the ruthenium(II) moiety, and the number of dye units influenced their biological properties. The in vitro activity against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF) leads to IC50 values for T. gondii β‐gal from 105 to 735 nM. Of note is that nine compounds displayed lower IC50 than the standard drug pyrimethamine. One compound applied at its IC50 did not affect B‐cell proliferation but had an impact on T‐cell proliferation in murine splenocyte cultures. Transmission electron microscopy of T. gondii β‐gal‐infected HFF showed that treatment predominantly affected the parasites’ mitochondrion., The (para)site for treatment: Thirteen coumarin‐diruthenium(II)⋅arene conjugates were evaluated against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF). The IC50 values ranged from 105 to 735 nM, with nine compounds displaying a lower IC50 than the standard drug pyrimethamine. TEM of T. gondii‐infected HFF showed that treatment predominantly affected the parasites’ mitochondrion.

Background Cutaneous leishmaniasis (CL) is a Neglected Tropical Disease (NTD) that causes high morbidity in the tropics and sub-tropics. Despite the remarkable advancements in the treatment of CL, the available therapeutics are far from ideal and also cause serious adverse side effects. Negative air ions (NAIs) generators are widely available for domestic and industrial uses. Several studies have reported on positive effects of NAIs therapy on human health as a non-pharmaceutical treatment for respiratory disease, allergy, or stress-related health conditions, including infectious diseases. To our knowledge, no studies have examined the effectiveness of the NAIs therapy against Leishmania parasites. The aims of this study were to investigate the effect of NAIs therapy on Leishmania major (L. major) the causative agent of CL in in vitro and in a murine model. Methodology/Principal findings In vitro anti-leishmanial effects of NAIs therapy were measured by parasitological methods. NAIs therapy was assessed in vivo in L. major infected BALB/c mice by measuring the footpad (FP) lesion size and parasite load using metric caliper tool and qPCR, respectively. Immune responses in treated and non-treated mice were assessed by measuring the levels of IFN-γ, IL-4, NO and arginase activity. In vitro NAIs therapy significantly decreased the viability of Leishmania promastigotes and of amastigotes cultured in macrophages, but did not affect the host cells. NAIs therapy of L. major infected BALB/c mice resulted in reduced FP lesion size, diminished parasite burden, and importantly decreased induction of IL-4 and arginase activity in the presence of NAIs. In contrast IFN-γ and NO levels were significantly enhanced. NAIs therapy significantly diminished the progression of disease compared to the control group, but was less effective than amphotericin B treatment. Conclusions Our study shows that NAIs treatment was effective in vitro and in Leishmania-infected mice, elicited a T-helper 1 (Th1) response and increased efficient cellular immunity, resulting in a diminished parasite load. Therefore, NAIs therapy can be considered as a useful and safe tool that can contribute to clearing L. major infections without inducing toxicity in host cells. The applications and mechanisms of NAIs therapy warrant further investigation especially in humans suffering from CL.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.

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 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 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., Graphical abstract Image 1, Highlights • BKI-1294 triggers a transiently decrease in anti-N. caninum IgG levels in sheep. • BKI-1294 does not protect against abortion and vertical transmission of N. caninum. • The efficacy of BKI-1294 is different in pregnant mice and sheep models of neosporosis.

OBJECTIVES Group B Streptococcus (GBS) increasingly causes invasive disease in non-pregnant adults, particularly in elderly persons and those with underlying diseases. Combination therapy with penicillin plus gentamicin has been suggested for periprosthetic joint infection. The postulated synergism of this combination is based on experiments with planktonic bacteria. We aimed to assess the efficacy of this combination against sessile bacteria. METHODS Four different GBS strains were used. We compared results of MICs with those of minimal biofilm eradication concentrations (MBECs), applied chequerboard assays to the MBEC device and calculated the fractional inhibitory concentration index. Synergism was evaluated with time-kill assays against bacteria adherent to cement beads, using penicillin (0.048, 0.2 and 3 mg/L), gentamicin (4 and 12.5 mg/L) and a combination thereof. Results were evaluated via colony counting after sonication of beads and scanning electron microscopy. RESULTS MBEC/MIC ratios were 2000-4000 for penicillin and 1-4 for gentamicin. In chequerboard assays, synergism was observed in all four isolates. In time-kill assays, penicillin and 12.5 mg/L gentamicin showed synergism in two isolates. In the other two isolates 12.5 mg/L gentamicin alone was as efficient as the combination therapy. CONCLUSIONS These in vitro investigations show activity of 12.5 mg/L gentamicin, alone or as an adjunct to penicillin, against four strains of biofilm GBS. This concentration cannot be achieved in bone with systemic administration, but can be reached if administered locally. The combination of systemic penicillin plus local gentamicin indicates a potential application in orthopaedic-device-associated GBS infections. Studies with a larger number of strains are required to confirm our results.

The essential oil (EO) of Thymus capitatus, seven fractions (F1–F7) obtained from silica gel chromatography, and several pure EO components were evaluated with respect to in vitro activities against Echinococcus multilocularis metacestodes and germinal layer (GL) cells. Attempts to evaluate physical damage in metacestodes by phosphoglucose isomerase (PGI) assay failed because EO and F1–F7 interfered with the PGI-activity measurements. A metacestode viability assay based on Alamar Blue, as well as transmission electron microscopy, demonstrated that exposure to EO, F2 and F4 impaired metacestode viability. F2 and F4 exhibited higher toxicity against metacestodes than against mammalian cells, whereas EO was as toxic to mammalian cells as to the parasite. However, none of these fractions exhibited notable activity against isolated E. multilocularis GL cells. Analysis by gas chromatography-mass spectrometry showed that carvacrol was the major component of the EO (82.4%), as well as of the fractions F3 (94.4%), F4 (98.1%) and F5 (90.7%). Other major components of EO were β-caryophyllene, limonene, thymol and eugenol. However, exposure of metacestodes to these components was ineffective. Thus, fractions F2 and F4 of T. capitatus EO contain potent anti-echinococcal compounds, but the activities of these two fractions are most likely based on synergistic effects between several major and minor constituents.

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., Graphical abstract Image 1, Highlights • Oral application of BKI 1369 effectively reduced oocyst excretion and diarrhea in Cystoisospora suis infected piglets. • 200 nM of BKI 1369 almost completely suppressed parasite proliferation in vitro. • IC50 and IC95 concentrations of BKI 1369 did not induce morphological alterations in in vitro cultured merozoites. • Cystoisospora suis CDPK1, the putative target of BKI 1369, has glycine as gatekeeper residue.

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasmagondii tachyzoites constitutively expressing &beta, galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T.gondii proliferation with IC50 values ranging from 90 to 539 nM, and seven derivatives displayed IC50 values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC50 values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC50 values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6&ndash, 10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11&ndash, 14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.

Sphingosine-1-phosphate is a signaling molecule involved in the control of cell migration, differentiation, survival and other physiological processes. This sphingolipid metabolite can be degraded by the action of sphingosine-1-phosphate lyase (SPL) to form hexadecenal and ethanolamine phosphate. The importance of SPL-mediated ethanolamine phosphate formation has been characterized in only few cell types. We show that in the protozoan parasite Trypanosoma brucei, expression of TbSpl is essential for cell survival. Ablation of TbSpl expression increased sphingosine-1- phosphate levels and reduced de novo formation and steady-state levels of the glycerophospholipid phosphatidylethanolamine (PE). Growth of TbSpl-depleted parasites could be in part rescued by ethanolamine supplementation to the growth medium, indicating that the main function of TbSpl is to provide ethanolamine phosphate for PE synthesis. In contrast to most cell types analyzed, where SPL localizes to the endoplasmic reticulum, we found by high-resolution microscopy that TbSpl is a mitochondrial protein. In spite of its mitochondrial localization, TbSpl depletion had no apparent effect on mitochondrial morphology but resulted in aggregation of acidocalcisomes. Our results link mitochondria to sphingolipid metabolism and suggest possible roles for PE in acidocalcisome function.

Alveolar echinococcosis (AE) is a deadly parasitic disease that requires lifelong treatment with albendazole. Development of host immunity is pivotal with regard to the clinical outcome of AE, but its influence on conventional albendazole treatment is unknown. Using T-cell deficient athymic nude mice, we demonstrated that functional immunity is required for albendazole to be efficacious against murine AE. These results call for attention given the increasing number of immunocompromised patients with AE.

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 applied by gavage for 5 days. Five BKIs did not affect pregnancy, 5 BKIs exhibited 15-35% of neonatal mortality, and 5 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 fertilized eggs to 0.2-50μM of BKIs and microscopical monitoring of embryo development in a blinded manner during 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows to calculate an impact score (Si) that indicates at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for 9 compounds no clear correlation between Si and pregnancy outcome was visible. However, those 3 BKIs affecting zebrafish embryos only at high concentrations (40μM or higher) did not impair mouse pregnancy at all, and those 3 compounds that inhibited zebrafish embryo development already at 0.2μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has a 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.

A case of Acanthocephala infection in a 5-year-old female rehabilitated and released black-bellied pangolin (Phataginus tetradactyla), which was part of a post-release monitoring program of a pangolin research operation in the Central African Republic, is described. This represents the first report of Acanthocephala infection in this species, which lead to intestinal perforation, secondary peritonitis and ultimately to the death of the animal concerned. It is of relevance to alert the pangolin conservation and research community to a so far unreported cause of death. A case history and necropsy findings, as well as preliminary parasite identification and genetic characterization which potentially revealed a new gigantorhynchid taxon are presented.

Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface. IMPORTANCETheileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

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.

Endochin-like quinolones (ELQs) potently inhibit the proliferation of Plasmodium, Toxoplasma, Neospora, and Babesia by targeting the cytochrome b Qo and Qi sites and interfering with oxidative phosphorylation and pyrimidine biosynthesis. The activities of 14 different ELQs were assessed against B. besnoiti tachyzoites grown in human foreskin fibroblasts (HFF) by quantitative real time PCR. The values for 50% proliferation inhibition (IC50) of five ELQs were determined in a 3-days growth assay after an initial screen of 12 ELQs at 0.01, 0.1, and 1 μM. The IC50s of ELQ-121, -136, and -316 were 0.49, 2.36, and 7.97 nM, respectively. The IC50s of ELQs tested against B. besnoiti were higher than IC50s previously observed for P. falciparum and T. gondii. However, the B. besnoiti cytochrome b sequence and the predicted Qo and Qi ELQ binding sites in the Toxoplasma, Neospora, and Besnoitia cytochrome b are virtually identical, suggesting that the differences in ELQ susceptibility are not due to variations in the substrate binding sites. TEM of ELQ-treated parasites primarily demonstrated alterations within the parasite mitochondrion, profound thickening of the nuclear membrane, as well as increased vacuolization within the tachyzoite cytoplasm. Long-term treatment assays of intracellular B. besnoiti with ELQs for up to 20 days followed by the release of drug pressure caused a substantial delay in parasite growth and proliferation while ELQs were present, but parasite proliferation resumed days after ELQs were removed. Interestingly, structural alterations persisted after ELQ removal and parasite proliferation was slowed. These findings provide a basis for further in vivo studies of ELQs as therapeutic options against B. besnoiti infection.

The current chemotherapeutical treatment against alveolar echinococcosis relies exclusively on benzimidazoles, which are not parasiticidal and can induce severe toxicity. There are no alternative treatment options. To identify novel drugs with activity against Echinococcus multilocularis metacestodes, researchers have studied potentially interesting drug targets (e.g. the parasite's energy metabolism), and/or adopted drug repurposing approaches by undertaking whole organism screenings. We here focus on drug screening approaches, which utilize an in vitro screening cascade that includes assessment of the drug-induced physical damage of metacestodes, the impact on metacestode viability and the viability of isolated parasite stem cells, structure-activity relationship (SAR) analysis of compound derivatives, and the mode of action. Finally, once in vitro data are indicative for a therapeutic window, the efficacy of selected compounds is assessed in experimentally infected mice. Using this screening cascade, we found that the anti-malarial mefloquine was active against E. multilocularis metacestodes in vitro and in vivo. To shed more light into the mode of action of mefloquine, SAR analysis on mefloquine analogues was performed. E. multilocularis ferritin was identified as a mefloquine-binding protein, but its precise role as a drug target remains to be elucidated. In mice that were infected either intraperitoneally with metacestodes or orally with eggs, oral treatment with mefloquine led to a significant reduction of parasite growth compared to the standard treatment with albendazole. However, mefloquine was not acting parasiticidally. Assessment of mefloquine plasma concentrations in treated mice showed that levels were reached which are close to serum concentrations that are achieved in humans during long-term malaria prophylaxis. Mefloquine might be applied in human AE patients as a salvage treatment. Future studies should focus on other repurposed anti-infective compounds (MMV665807, niclosamide, atovaquone), which showed stronger in vitro activity against E. multilocularis than mefloquine., Graphical abstract Image 1, Highlights • Anti-infective drugs are repurposed for identification of novel drugs against AE. • Mefloquine is active against E. multilocularis in vitro and in mice. • The energy metabolism of E. multilocularis is a target for future drug development. • MMV665807 and niclosamide are highly active against E. multilocularis in vitro.

Bumped Kinase Inhibitors (BKIs), targeting Calcium-dependent Protein Kinase 1 (CDPK1) in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology, that have shaped our selection of pre-clinical candidates.

A new species of the Campylobacter genus is described, isolated from the preputial mucosa of bulls (Bos taurus). The five isolates obtained exhibit characteristics of Campylobacter, being Gram-negative non-motile straight rods, oxidase positive, catalase negative and microaerophilic. Phenotypic characteristics and nucleotide sequence analysis of 16S rRNA and hsp60 genes demonstrated that these isolates belong to a novel species within the genus Campylobacter. Based on hsp60 gene phylogenetic analysis, the most related species are C. ureolyticus, C. blaseri and C. corcagiensis. The whole genome sequence analysis of isolate FMV-PI01 revealed that the average nucleotide identity with other Campylobacter species was less than 75%, which is far below the cut-off for isolates of the same species. However, whole genome sequence analysis identified coding sequences highly homologous with other Campylobacter spp. These included several virulence factor coding genes related with host cell adhesion and invasion, transporters involved in resistance to antimicrobials, and a type IV secretion system (T4SS), containing virB2-virB11/virD4 genes, highly homologous to the C. fetus subsp. venerealis. The genomic G+C content of isolate FMV-PI01 was 28.3%, which is one of the lowest values reported for species of the genus Campylobacter. For this species the name Campylobacter portucalensis sp. nov. is proposed, with FMV-PI01 (= LMG 31504, = CCUG 73856) as the type strain.

No effective human vaccine against Toxoplasma gondii (T. gondii) has yet been developed; however, a protective vaccine using immunogenic peptides in a safe delivery vehicle system offers promise. Here, we employed bioinformatics to design a multimeric recombinant T. gondii vaccine using predicted T and B cell epitopes of SAG1, AMA1, ROP2, and GRA4 proteins based on their binding capabilities to common major histocompatibility complex (MHC) molecules. Furthermore, we encapsulated the expressed protein in poly lactic-co-glycolic acid (PLGA) nanoparticles as a delivery vehicle and also used alum as an adjuvant to determine the vaccine potency of this multimeric antigen. BALB/c mice were vaccinated and then challenged with T. gondii RH strain, and the survival rate and cytokine profiles were studied. Mice vaccinated with the multi-epitope-based vaccine, both with and without PLGA, had greater Th1 immune responses, survival rates, specific antibody titers, and IFN-γ and IL-2 levels than controls, while the alum-adsorbed vaccine stimulated a Th2-type humoral immune response.

Diarrhea in calves is one of the most important cattle diseases in Switzerland. The diagnosis and treatment of calf diarrhea represent a major challenge. Single-celled Cryptosporidium parasites are the most prevalent causative agents of calf diarrhea besides rotavirus in the first weeks of life, and are responsible for about 50% of diarrheal cases. Cryptosporidium parvum has been described as a cause of diarrhea in one to three weeks old calves since the 1970s. Oral ingestion of persistent environmental oocysts results in severe diarrhea lasting four to six days and shedding of large numbers of infectious oocysts. A tiny amount of 10 oocysts is already sufficient to cause disease. Detailed knowledge about the epidemiology and virulence of the different C. parvum strains is still lacking. In addition, current diagnostic tests cannot reliably distinguish between non-pathogenic (e.g. C. bovis) and pathogenic Cryptosporidium species. Until now, no effective therapeutic drug or vaccine against calf cryptosporidiosis has been found. Water-borne epidemics and the zoonotic potential of Cryptosporidium in immunodeficient patients are of great medical importance. The increasing number of cryptosporidiosis cases associated with high infant mortality in less industrialized and impoverished regions (including South-East Asia and sub-Saharan Africa) has intensified the research in recent years. The recent discoveries of new therapeutics against C. parvum may benefit calf medicine in the near future. This review article reports on these new developments, highlights calf cryptosporidiosis in Switzerland and draws attention to a new research project.Durchfall bei Kälbern ist eine der häufigsten Rinderkrankheiten in der Schweiz. Die ursächliche Diagnose und Behandlung des Kälberdurchfalls stellen eine grosse Herausforderung dar. Cryptosporidien sind in der Schweiz neben Rotaviren die am häufigsten vorkommenden Erreger des Kälberdurchfalls in den ersten Lebenswochen und für etwa 50% der Fälle verantwortlich. Der einzellige Parasit Cryptosporidium parvum wird seit den 1970er Jahren als Durchfallerreger bei ein bis drei Wochen alten Kälbern beschrieben. Nach oraler Aufnahme von in der Umwelt persistierenden Oocysten kommt es nach wenigen Tagen zu einem vier bis sechs Tage dauernden starken Durchfall mit massiver Ausscheidung von bereits infektiösen Oocysten. Bereits wenige in der Umwelt persistierende Oocysten können krankheitsauslösend sein. In der Epidemiologie herrschen noch grössere Erkenntnislücken zu der vermuteten unterschiedlichen Virulenz verschiedener C. parvum-Stämme. Zudem kommen auch als apathogen angesehene Spezies (unter anderem Cryptosporidium bovis) in Kälbern vor, die von den gängigen diagnostischen Tests nicht verlässlich unterschieden werden können. Bisher wurden kein therapeutisch wirksames Medikament und keine Vakzine gegen die Kälbercryptosporidiose gefunden. In der Human­medizin spielen wasservermittelte Epidemien und die zoonotische Bedeutung von Cryptosporidien bei im­mundefi­zienten Menschen eine grössere Rolle. Die hohe Krank­heitslast der Cryptosporidiose assoziiert mit einer hohen Kindersterblichkeit in wenig industrialisierten und verarmten Regionen (u.a. in Süd-Ostasien und in Afrika südlich der Sahara) hat in den letzten Jahren erneut die Forschung an dem Parasiten befeuert. Besonders die Entdeckung neuer Wirkstoffe gegen C. parvum dürfte in näherer Zukunft auch der Kälbermedizin zu Gute kommen. In diesem Übersichtsartikel wird über diese neuen Entwicklungen berichtet, vor allem aber die Kälbercryptosporidiose in der Schweiz beleuchtet und auf ein neues Forschungsprojekt aufmerksam gemacht.La diarrhée chez les veaux est l’une des maladies du bétail les plus courantes en Suisse. Le diagnostic de la cause et le traitement de la diarrhée des veaux représentent un défi majeur. En Suisse, les cryptosporidies sont, avec les rotavirus, l’agent causal le plus fréquent de diarrhée du veau dans les premières semaines et elles sont responsables d’environ 50% des cas. Le parasite unicellulaire Cryptosporidium parvum a été décrit depuis les années 1970 comme un agent de diarrhée chez les veaux d’une à trois semaines. Après ingestion orale d’oocystes persistants dans l’environnement, il se produit après quelques jours une diarrhée sévère de quatre à six jours avec excrétion massive d’oocystes déjà infectieux. Même quelques oocystes persistants dans l’environnement peuvent être pathogènes. Du point de vue épidémiologique, il existe encore de grandes lacunes dans la connaissance de la variabilité suspectée dans la virulence de diverses souches de C. parvum. En outre, des espèces non pathogènes (entre autres Cryptosporidium bovis) peuvent être présentes chez les veaux, qui ne se distinguent pas de C. parvum avec les tests diagnostiques actuels. Jusqu’à présent, aucun médicament efficace sur le plan thérapeutique et aucun vaccin contre la cryptosporidiose du veau n’ont été trouvés. En médecine humaine, les épidémies transmises par l’eau (en particulier aux États-Unis) et l’importance zoonotique des cryptosporidies comme pathogènes opportunistes chez les personnes immunodéficientes jouent un rôle de premier plan. La forte morbidité de la cryptosporidiose associée à une forte mortalité infantile dans les régions les moins industrialisées et les plus pauvres (entre autres en Asie du Sud-Est et en Afrique subsaharienne) ont relancé la recherche sur ces parasites au cours des dernières années. En particulier, la découverte de nouveaux médicaments contre C. parvum est susceptible de bénéficier à la médecine du veau dans un proche avenir. Cet article de synthèse fait le point sur ces nouveaux développements mais surtout sur la cryptosporidiose du veau en Suisse et attire l’attention sur un nouveau projet de recherche.La diarrea nei vitelli è una delle più frequenti malattie dei bovini in Svizzera. La diagnosi causale e il trattamento della diarrea dei vitelli sono una grande sfida. I criptosporidi con i rotavirus sono i più comuni agenti patogeni della diarrea dei vitelli nella prima settimana di vita e sono la causa del 50% dei casi. Il parassita unicellulare Cryptosporidium parvum viene descritto sin dagli anni 70 come l’agente patogeno della diarrea nei vitelli di età tra 1-3 settimane. Dopo alcuni giorni dall’ingestione orale di persistenti oocisti dell’ambiente circostante si verifica una forte diarrea della durata di quattro a sei giorni con una massiccia escrezione di oocisti già infetti. Solo poche oocisti persistenti nell’ambiente possono causare malattie. In epidemiologia ci sono ancora molte lacune nelle conoscenze della sospetta diversa virulenza dei diversi ceppi di C. parvum. Inoltre, nei vitelli si aggiungono delle specie non patogene (tra cui il Cryptosporidium bovis) che non si distinguono con i test diagnostici. Finora non si sono trovati né farmaci efficaci dal punto di vista terapeutico né vaccini contro la criptosporidiosi nel vitello. Nella medicina umana, le epidemie mediate dall’acqua (specialmente negli Stati Uniti) e l’importanza zoonotica dei criptosporidi come agenti patogeni opportunisti svolgono un ruolo importante per gli esseri umani immunodeficienti. L’alto onere della patologia della criptosporidiosi associato all’alto tasso di mortalità infantile nelle regioni meno industrializzate e povere (tra cui l’Asia sud-orientale e l’Africa sub-sahariana) ha stimolato negli ultimi anni la ricerca sul parassita. In particolare la scoperta di nuovi farmaci contro C. parvum dovrebbe portare in un futuro prossimo dei benefici anche nella medicina per i vitelli. In questa recensione si riporta di questi nuovi sviluppi e in particolare elucida sulla criptosporidiosi del vitello in Svizzera e richiama l’attenzione su un nuovo progetto di ricerca.

13 páginas, 2 tablas, 7 figuras., Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 mu M, and of 1 mu M in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFN. levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed., We gratefully acknowledge Jacobo Sampere from the SALUVETgroup (Complutense University of Madrid, Spain), Teresa Navarro andJosé María González (University of Zaragoza, Spain), Isabel Ayala,Lucas Troya, Clara Colmenero, Dolores González, Natalia Peral,Verónica Pérez, Ignacio Gómez and Isabel Santiago from the Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, and also Rama Subba Rao Vidadala and Dustin Maly (Department of Chemistry, University of Washington) fortheir assistance in further purification of the study drug. The AnimalExperimentation Service (SEA) at the University of Zaragoza is ac-knowledged for providing their facilities to carry out the reproduction programme. Roberto Sánchez Sánchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), asa part of the Program of Training of University Teaching Staff (FPU,grant number FPU13/03438). Patricia Vázquez has a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Andrew Hemphill is supported by the Swiss National Science Foundation grant(No. 310030 165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI111341 and R01 HD 080670), the U.S. Department of Agriculture(grant 2014-67015-22106) and the Community of Madrid, Spain(PLATESA, S2013/ABI2906).

Tethering known drugs to a metalorganic moiety is an efficient approach for modulating the anticancer, antibacterial, and antiparasitic activity of organometallic complexes. This study focused on the synthesis and evaluation of new dinuclear ruthenium(II)–arene compounds linked to several antimicrobial compounds such as dapsone, sulfamethoxazole, sulfadiazine, sulfadoxine, triclosan, metronidazole, ciprofloxacin, as well as menadione (a 1,4-naphtoquinone derivative). In a primary screen, 30 compounds (17 hybrid molecules, diruthenium intermediates, and antimicrobials) were assessed for in vitro activity against transgenic T.&nbsp, gondii tachyzoites constitutively expressing β-galactosidase (T.&nbsp, gondii β-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in noninfected host cells (human foreskin fibroblasts, HFF) was determined by an alamarBlue assay. When assessed at 1 µM, five compounds strongly impaired parasite proliferation by &gt, 90%, and HFF viability was retained at 50% or more, and they were further subjected to T.&nbsp, gondii β-gal dose-response studies. Two compounds, notably 11 and 13, amide and ester conjugates with sulfadoxine and metronidazole, exhibited low IC50 (half-maximal inhibitory concentration) values 0.063 and 0.152 µM, and low or intermediate impairment of HFF viability at 2.5 µM (83 and 64%). The nature of the anchored drug as well as that of the linking unit impacted the biological activity.

Neosporosis caused by the apicomplexan parasite Neospora caninum is an economically important disease that induces abortion in dairy and beef cattle. There are no vaccines or drugs available on the market for control or treatment of the disease in bovines. The peroxide artemisinin and its derivatives used clinically for treatment of malaria are active against N. caninum and other apicomplexan parasites. We have now evaluated the activities of the readily accessible and chemically robust 11-azaartemisinin 5 and selected N-sulfonyl derivatives prepared as described in the accompanying paper against N. caninum tachyzoites grown in infected human foreskin fibroblasts. Azaartemisinin elicited an IC50 value of 150 nm, and the 2',5'-dichloro-3'-thienylsulfonyl-11-azaartemisinin 17 was found to be the most active, with an IC50 value of 40 nm. Comparison with normal human fetal lung fibroblasts HFLF WI-38 revealed relatively benign cytotoxicity. The compounds were also screened in vitro against TK-10 (renal), UACC-62 (melanoma) and MCF-7 (breast) cancer cell lines; overall, in line with activities against HFLF cells, most compounds in the series were found to be inactive.

Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.

Objectives Establishment of a mouse model for congenital toxoplasmosis based on oral infection with oocysts from Toxoplasma gondii ME49 and its application for investigating chemotherapeutic options against congenital toxoplasmosis. Methods CD1 mice were mated, orally infected with 5, 25, 100, 500 or 2000 oocysts and monitored for clinical signs and survival of dams and pups until 4 weeks post partum . The parasite burden in infected mice was quantified by real-time PCR in lungs, brains and, in the case of surviving pups, also in eyes. Seroconversion was assessed by ELISA. T. gondii cysts in brain were identified by immunofluorescence. In a second experiment, pregnant CD1 mice challenged with 20 oocysts/mouse were treated with buparvaquone or the calcium-dependent protein kinase 1 inhibitor bumped kinase inhibitor (BKI)-1294 and the outcome of infection was analysed. Results T. gondii DNA was detected in the brain of all infected animals, irrespective of the infection dose. Seroconversion occurred at 3 weeks post-infection. Most pups born to infected dams died within 1 week post partum , but a small fraction survived until the end of the experiment. T. gondii DNA was detected in the brain of all survivors and half of them exhibited ocular infection. Chemotherapy with both compounds led to dramatically increased numbers of surviving pups and reduced cerebral infection. Most efficient were treatments with BKI-1294, with 100% survivors and only 7% brain-positive pups. Conclusions BKI-1294 and buparvaquone exert excellent activities against transplacental transmission in pregnant mice.

We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE., Author summary Various infectious diseases in humans have been associated with altered expression patterns of microRNAs (miRNAs), a class of small non-coding RNAs involved in negative regulation of gene expression. Herein, we revealed that significant alteration of miRNAs expression occurred in murine liver subsequently to experimental infection with E. multilocularis eggs when compared to non-infected controls. At the early stage of murine AE, hepatic miRNAs were mainly down-regulated. Respective target genes of the most extensively down-regulated miRNAs were involved in angiogenesis and fatty acid synthesis. Furthermore, we found higher mRNA levels of three angiogenic and two lipogenic genes in E. multilocularis infected livers compared to non-infected controls. Angiogenesis and fatty acid biosynthesis may be beneficial for development of the E. multilocularis metacestodes. In fact the formation of new blood vessels in the periparasitic area may ensure that parasites are supplied with oxygen and nutrients and get rid of waste products. Additionally, E. multilocularis is not able to undertake de novo fatty acid synthesis, thus lipids must be scavenged from its host. More research on the regulation of the hepatic miRNA transcriptome at more advanced stages of AE is needed.

Protozoan parasites can infect the human intestinal tract causing serious diseases. In the following article, we focused on the three most prominent intestinal protozoan pathogens, namely, Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum. Both C. parvum and G. lamblia colonize the duodenum, jejunum, and ileum and are the most common causative agents of persistent diarrhea (i.e., cryptosporidiosis and giardiasis). Entamoeba histolytica colonizes the colon and, unlike the two former pathogens, may invade the colon wall and disseminate to other organs, mainly the liver, thereby causing life-threatening amebiasis. Here, we present condensed information concerning the pathobiology of these three diseases.

16 páginas, 3 tablas, 6 figuras., Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 M and trough concentrations of 0.4 M at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy., We gratefully acknowledge the SALUVET group (Complutense University of Madrid, Spain), Luis Miguel Ferrer, Jose Ventura, José Calasanz Jiménez, Francisco Saura, Teresa Navarro, and José María González from the University of Zaragoza (Spain), and Victor Herrero and Javier Blanco from Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, Dale Kempf and Kennan Marsh, from AbbVie, Inc., for their suggestion of the Phosal vehicle used in the experiments reported here, Rama S. R. Vidadala and Dustin J. Maly, Department of Chemistry, University of Washington, for characterization and further purification of BKI-1294, and advice from Robert Choy and Eugenio L. de Hostos from the PATH Drug Development Program. The Animal Experimentation Service (SEA) at the University of Zaragoza is acknowledged for providing their facilities to carry out the reproduction program. R.S.-S. is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU; grant number FPU13/03438). A.H. is supported by a Swiss National Science Foundation grant (no. 310030-165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI 111341 and R01 HD 080670), the U.S. Department of Agriculture (grant 2014-67015-22106), and the Community of Madrid, Spain (PLATESA2, P2018/BAA-4370). I.F., A.H., K.K.O., W.C.V.V., and L.M.O.-M. conceived the study and participated in its design. R.S.-S. wrote the manuscript, with results interpretation and discussion inputs from I.F., J.R.-C., A.H., M.A.H., L.K.B., W.C.V.V., and L.M.O.-M. J.J.R. selected the animals and executed the reproductive program. R.S.-S., I.F., and M.R. carried out oocyst infection and drug administration. R.S.-S., I.F., M.R., M.P.D., M.G.-H., E.T., and J.B. participated in clinical examination and sampling of animals and performed necropsies and hematological, biochemical, and histopathological analyses. M.H., L.K.B., R.C., G.R.W., and K.K.O. determined the pharmacokinetics of the compound. R.S.-S. performed peripheral blood stimulation assay, serological assays, PCR analyses, statistical analysis, and interpretation of the results. All authors read and approved the final manuscript. W.C.V.V. is the president and co-owner of ParaTheraTech, Inc., a company that is developing BKIs for animal health. W.C.V.V. did not perform the experiments or interpret the results of the experiments, but he did edit the paper and helped plan the experiments. The other authors have no competing interests to declare.

The lethal disease alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. Current chemotherapeutical treatment of AE relies on albendazole and mebendazole, with the caveat that these compounds are not parasiticidal. Drugs have to be taken for a prolonged period of time, often life-long, which can cause adverse effects and reduces the patients' quality of life. In some individuals, benzimidazoles are inactive or cause toxicity, leading to treatment discontinuation. Alternatives to benzimidazoles are urgently needed. Over the recent years, in vivo and in vitro models for low-to-medium throughput drug discovery against AE have been set in place. In vitro drug tests include the phosphoglucose-isomerase (PGI) assay to measure physical damage induced to metacestodes, and viability assays to assess parasiticidal activity against metacestodes and stem cells. In vitro models are also employed for studies on mechanisms of action. In vivo models are thus far based on rodents, mainly mice, and benefits could be gained in future by comparative approaches in naturally infected dogs or captive monkeys. For the identification of novel drugs against AE, a rare disease with a low expected market return, drug-repurposing is the most promising strategy. A variety of chemically synthesized compounds as well as natural products have been analyzed with respect to in vitro and/or in vivo activities against AE. We here review and discuss the most active of these compounds including anti-infective compounds (benzimidazoles, nitazoxanide, amphotericin B, itraconazole, clarithromycin, DB1127, and buparvaquone), the anti-infective anti-malarials (artemisinin, ozonids, mefloquine, and MMV665807) and anti-cancer drugs (isoflavones, 2-methoxyestradiol, methotrexate, navelbine, vincristine, kinase inhibitors, metallo-organic ruthenium complexes, bortezomib, and taxanes). Taking into account the efficacy as well as the potential availability for patients, the most promising candidates are new formulations of benzimidazoles and mefloquine. Future drug-repurposing approaches should also target the energy metabolism of E. multilocularis, in particular the understudied malate dismutation pathway, as this offers an essential target in the parasite, which is not present in mammals., Highlights • Benzimidazoles are used to treat AE, but new drugs are needed. • New drugs against AE can be identified by drug repurposing. • Drugs against other infectious diseases and cancer can be repurposed against AE. • Most promising are new formulations of benzimidazoles and mefloquine. • Future approaches should include targeting the energy metabolism of the parasite.

A library of 18 dinuclear-thiolato bridged arene ruthenium complexes, some of which with demonstrated activity against cancer cells, was screened for activity against a transgenic Neospora caninum strain that constitutively expresses beta-galactosidase. Initial assessments were done at concentrations of 2500, 250, 25 and 2.5 nM, and 5 compounds were further evaluated with regard to their half maximal proliferation-inhibiting concentration (IC50). Among those, [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-CH3)3]Cl (1), [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-But)3]Cl (2) and [(η6-p-MeC6H4Pri)2Ru2(μ2-SCH2C6H4-p-But)2(μ2-SC6H4-p-OH)]BF4 (9) inhibited N. caninum proliferation with low C50 values of 15, 5 and 1 nM, respectively, while [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-OH)3]Cl (3) and [(η6-p-MeC6H4Pri)2Ru2(μ2-SC6H4-p-mco)3]Cl (5, mco = 4-methylcoumarinyl) were less active (IC50 = 280 and 108 nM, respectively). These compounds did not affect human foreskin fibroblast (HFF) host cells at dosages of 5 μM and above, but impaired proliferation of the human ovarian carcinoma cell line A2780 (IC50 values of 130 nM (1), 30 nM (2), 530 nM (3), 7730 nM (5), 130 nM (9)). A2780 cancer cells were treated with complexes 1, 2, and 5, and biodistribution analysis using inductively coupled plasma mass spectrometry (ICP-MS) showed that most of the drugs accumulated in the mitochondrial fractions. Transmission electron microscopy showed that the parasite mitochondrion is the primary target also in N. caninum tachyzoites, but these compounds, when applied at 200 nM for 15 days in vitro, did not act parasiticidal. Complexes 1, 2 and 9 applied orally at 2 and 10 mg kg−1 day−1 during 5 days in a neosporosis mouse model did not reduce parasite load and did not limit parasite dissemination to the central nervous system. In accordance with these results, ICP-MS carried out on different organs of mice orally administrated with complexes 1 and 9, demonstrated that the drugs were readily absorbed, and after 3 and 48 h, were mainly detected in liver and kidney, but were largely absent from the brain. Thus, dinuclear thiolato-bridged arene ruthenium complexes exhibit interesting activities against N. caninum in vitro, but further modifications of these promising molecules are required to improve their bioavailability and pharmacokinetic properties in order to exert a pronounced and selective effect against N. caninum in vivo.

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity.

The mitochondrial signature glycerophospholipid, cardiolipin (CL), binds to transporters of the inner mitochondrial membrane and plays a central role in formation and stability of respiratory supercomplexes. Functional and structural requirement of CL for mitochondrial membrane proteins has been studied in vitro using purified reconstituted proteins or in CL synthesis knockout cells that are viable under specific growth conditions. However, no information is available on mitochondrial function, protein stability, or expression levels in cells during CL depletion. In contrast to yeast and mammalian cells, CL synthesis is essential in Trypanosoma brucei. By stable isotope labeling with amino acids in cell culture and mass spectrometry, we analyzed protein levels in T. brucei procyclic forms at different time points during depletion of CL using tightly controllable conditional CL synthase knockout mutants and identified a set of novel CL-dependent proteins (CLDPs) with unknown functions. Depletion of individual CLDPs using knockout or knockdown technologies showed that although CL synthesis is essential, expression of a given CLDP is not. In addition, ablation of CL synthesis leads to respiratory supercomplex instability and altered mitochondrial ultrastructure and function. Our findings suggest that CL may bind to and affect many more proteins in eukaryotes than previously thought.-Schädeli, D., Serricchio, M., Ben Hamidane, H., Loffreda, A., Hemphill, A., Beneke, T., Gluenz, E., Graumann, J., Bütikofer, P. Cardiolipin depletion-induced changes in the Trypanosoma brucei proteome.

18 páginas, 6 figuras, 3 tablas., Toxoplasma gondii is an apicomplexan parasite that infects almost all warm-blooded animals. Little is known about how the parasite virulence in mice extrapolates to other relevant hosts. In the current study, in vitro phenotype and in vivo behavior in mice and sheep of a type II T. gondii isolate (TgShSp1) were compared with the reference type II T. gondii isolate (TgME49). The results of in vitro assays and the intraperitoneal inoculation of tachyzoites in mice indicated an enhanced virulence for the laboratory isolate, TgME49, compared to the recently obtained TgShSp1 isolate. TgShSp1 proliferated at a slower rate and had delayed lysis plaque formation compared to TgME49, but it formed more cyst-like structures in vitro. No mortality was observed in adult mice after infection with 1–105 tachyzoites intraperitoneally or with 25–2,000 oocysts orally of TgShSp1. In sheep orally challenged with oocysts, TgME49 infection resulted in sporadically higher rectal temperatures and higher parasite load in cotyledons from ewes that gave birth and brain tissues of the respective lambs, but no differences between these two isolates were found on fetal/lamb mortality or lesions and number of T. gondii-positive lambs. The congenital infection after challenge at mid-pregnancy with TgShSp1, measured as offspring mortality and vertical transmission, was different depending on the challenged host. In mice, mortality in 50% of the pups was observed when a dam was challenged with a high oocyst dose (500 TgShSp1 oocysts), whereas in sheep infected with the same dose of oocysts, mortality occurred in all fetuses. Likewise, mortality of 9 and 27% of the pups was observed in mice after infection with 100 and 25 TgShSp1 oocysts, respectively, while in sheep, infection with 50 and 10 TgShSp1 oocysts triggered mortality in 68 and 66% of the fetuses/lambs. Differences in vertical transmission in the surviving offspring were only found with the lower oocyst doses (100% after infection with 10 TgShSp1 oocysts in sheep and only 37% in mice after infection with 25 TgShSp1 oocysts). In conclusion, virulence in mice of T. gondii type II isolates may not be a good indicator to predict the outcome of infection in pregnant sheep., RS-S is supported by a fellowship from the Spanish Ministry of Education, Culture, and Sports (MECD), as a part of the Program of Training of University Teaching Staff (FPU, grant number FPU13/03438) and a mobility grant for predoctoral short stays in R+D centers (EST16/0719). DG-E is the recipient of a postdoctoral contract from the Junta de Castilla y León, partially funded by the European Social Fund (European Union). NA-V is the recipient of a predoctoral contract from the Ministerio de Economía, Industria y Competitividad (Ref. BES-2016-076513). This work was supported by the Ministry of Economy and Competitiveness (AGL2016-75935-C2-1-R and C2-2-R), the Community of Madrid, Spain (PLATESA, S2013/ABI2906), Junta de Castilla y León (LE080U16), and a grant of the Swiss National Science Foundation to AH (project No. 310030_165782).

We here assessed thein vitroefficacy of the naptho-quinone buparvaquone (BPQ) againstBesnoitia besnoititachyzoitesin 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 IC50of 10 ± 3 nm, and virtually complete inhibition was obtained in the presence of nmBPQ. Exposure to 1µmBPQ 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. besnoititachyzoites exhibited the capacity to adapt, and they resumed proliferation at dosages of up to 10µmBPQ, 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.

SUMMARYNeospora caninumis a leading cause of abortion in cattle, and is thus an important veterinary health problem of high economic significance. Vaccination has been considered a viable strategy to prevent bovine neosporosis. Different approaches have been investigated, and to date the most promising results have been achieved with live-attenuated vaccines. Subunit vaccines have also been studied, and most of them represented components that are functionally involved in (i) the physical interaction between the parasite and its host cell during invasion or (ii) tachyzoite-to-bradyzoite stage conversion. Drugs have been considered as an option to limit the effects of vertical transmission ofN. caninum.Promising results with a small panel of compounds in small laboratory animal models indicate the potential value of a chemotherapeutical approach for the prevention of neosporosis in ruminants. For both, vaccines and drugs, the key for success in preventing vertical transmission lies in the application of bioactive compounds that limit parasite proliferation and dissemination, without endangering the developing fetus not only during an exogenous acute infection but also during recrudescence of a chronic infection. In this review, the current status of vaccine and drug development is presented and novel strategies against neosporosis are discussed.

We report on the efficacy of selected endochin-like quinolones (ELQs) against N. caninum tachyzoites grown in human foreskin fibroblasts (HFF), and in a pregnant BALB/c mouse model. Fourteen ELQs were screened against transgenic N. caninum tachyzoites expressing β-galactosidase (Nc-βgal). Drugs were added concomitantly to infection and the values for 50% proliferation inhibition (IC50) were determined after 3 days. Three compounds exhibited IC50 values below 0.1 nM, 3 ELQs had IC50s between 0.1 and 1 nM, for 7 compounds values between 1 and 10 nM were noted, and one compound had an IC50 of 22.4 nM. Two compounds, namely ELQ-316 and its prodrug ELQ-334 with IC50s of 0.66 and 3.33 nM, respectively, were previously shown to display promising activities against experimental toxoplasmosis and babesiosis caused by Babesia microti in mice, and were thus further studied. They were assessed in long-term treatment assays by exposure of infected HFF to ELQs at 0.5 μM concentration, starting 3 h after infection and lasting for up to 17 days followed by release of drug pressure. Results showed that the compounds substantially delayed parasite proliferation, but did not exert parasiticidal activities. TEM of drug treated parasites detected distinct alterations within the parasite mitochondria, but not in other parasite organelles. Assessment of safety of ELQ-334 in the pregnant mouse model showed that the compound did not interfere in fertility or pregnancy outcome. In N. caninum infected pregnant mice treated with ELQ-334 at 10 mg/kg/day for 5 days, neonatal mortality (within 2 days post partum) was found in 7 of 44 pups (15.9%), but no postnatal mortality was noted, and vertical transmission was reduced by 49% compared to the placebo group, which exhibited 100% vertical transmission, neonatal mortality in 15 of 34 pups (44%), and postnatal mortality for 18 of the residual 19 pups during the 4 weeks follow-up. These findings encourage more research on the use of ELQs for therapeutic options against N. caninum infection.

OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection.

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.

Infection with the larval stage (metacestode) of the fox tapeworm Echinococcus multilocularis leads to a primary hepatic disease referred to as alveolar echinococcosis (AE). The progressive disease can be lethal if untreated. In cases where complete parasite resection by surgery is not feasible, the current treatment regimens of AE consist of chemotherapy with the parasitostatic benzimidazoles albendazole or mebendazole over decades. Kinase-inhibitors currently administered in various cancer treatments are of increasing interest also as anti-parasitic drugs due to previous promising in vitro results. In order to search for novel drug targets and treatment regimens, nilotinib (AMN107; Tasigna ® ), an Abl-tyrosine kinase inhibitor and everolimus (RAD001; Afinitor ® ), a serine/threonine-kinase inhibitor, were tested for their treatment efficacy against metacestode vesicles of E. multilocularis in vitro and in BALB/c mice. In vitro treatment with 200 μM nilotinib caused drug-induced alterations after 12 days, and everolimus exerted parasite damage at concentrations dosing from 40 to 100 μM after 5 and 12 days of in vitro exposure. Nilotinib (100 mg/kg) + erythromycin (to increase nilotinib plasma levels: 10 mg/kg intraperitoneal) or everolimus (5 mg/kg) were formulated in honey and administered daily for three weeks and subsequently twice a week for an additional three weeks in experimentally infected mice. Treatments did not result in any reduction of parasite growth compared to untreated control groups, whereas oral treatment with albendazole (200 mg/kg) was highly effective. Combined application of the kinase-inhibitors with albendazole did not lead to a synergistic or additive treatment efficacy compared to albendazole treatment alone. These results show that neither nilotinib nor everolimus represent valuable alternatives to the current treatment regimens against AE.

For over 50 years, metronidazole and other nitro compounds such as nitazoxanide have been used as a therapy of choice against giardiasis and more and more frequently, resistance formation has been observed. Model systems allowing studies on biochemical aspects of resistance formation to nitro drugs are, however, scarce since resistant strains are often unstable in culture. In order to fill this gap, we have generated a stable metronidazole- and nitazoxanide-resistant Giardia lamblia WBC6 clone, the strain C4. Previous studies on strain C4 and the corresponding wild-type strain WBC6 revealed marked differences in the transcriptomes of both strains. Here, we present a physiological comparison between trophozoites of both strains with respect to their ultrastructure, whole cell activities such as oxygen consumption and resazurin reduction assays, key enzyme activities, and several metabolic key parameters such as NAD(P)+/NAD(P)H and ADP/ATP ratios and FAD contents. We show that nitro compound-resistant C4 trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and resazurin reduction rates, lower ornithine-carbamyl-transferase activity, reduced FAD and NADP(H) pool sizes and higher ADP/ATP ratios than wildtype trophozoites. The present results suggest that resistance formation against nitro compounds is correlated with metabolic adaptations resulting in a reduction of the activities of FAD-dependent oxidoreductases., Graphical abstract Image 1, Highlights • Physiological comparison between susceptible and nitro drug resistant Giardia lamblia trophozoites. • Resistant trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and lower resazurin reduction rates. • Resistant trophozoites exhibit lower FAD and NADP(H) contents and higher ADP/ATP ratios. • Resistance formation against nitro compounds correlated with metabolic adaptations avoiding nitrosative and oxidative stress.