- Research Article
- Open Access
Repurposing of antiparasitic drugs: the hydroxy-naphthoquinone buparvaquone inhibits vertical transmission in the pregnant neosporosis mouse model
© Müller et al. 2016
- Received: 11 December 2015
- Accepted: 2 February 2016
- Published: 17 February 2016
The three anti-malarial drugs artemiside, artemisone, and mefloquine, and the naphthoquinone buparvaquone known to be active against theileriosis in cattle and Leishmania infections in rodents, were assessed for activity against Neospora caninum infection. All four compounds inhibited the proliferation of N. caninum tachyzoites in vitro with IC50 in the sub-micromolar range, but artemisone and buparvaquone were most effective (IC50 = 3 and 4.9 nM, respectively). However, in a neosporosis mouse model for cerebral infection comprising Balb/c mice experimentally infected with the virulent isolate Nc-Spain7, the three anti-malarial compounds failed to exhibit any activity, since treatment did not reduce the parasite burden in brains and lungs compared to untreated controls. Thus, these compounds were not further evaluated in pregnant mice. On the other hand, buparvaquone, shown earlier to be effective in reducing the parasite load in the lungs in an acute neosporosis disease model, was further assessed in the pregnant mouse model. Buparvaquone efficiently inhibited vertical transmission in Balb/c mice experimentally infected at day 7 of pregnancy, reduced clinical signs in the pups, but had no effect on cerebral infection in the dams. This demonstrates proof-of-concept that drug repurposing may lead to the discovery of an effective compound against neosporosis that can protect offspring from vertical transmission and disease.
- Vertical Transmission
- Parasite Load
- Pregnant Mouse
- Human Foreskin Fibroblast
Neospora caninum is a cyst-forming apicomplexan parasite closely related to Toxoplasma gondii. N. caninum is one of the most important infectious causes of bovine abortion, stillbirth, and the birth of weak calves, with an economic impact of over 1.3 billion US dollars [1–3]. N. caninum infection can also result in birth of clinically healthy, but persistently infected calves transmitting the parasite to the next generation. In addition, N. caninum causes neuromuscular disease in dogs, and neosporosis has also been detected in a wide range of other species of livestock and wild animals worldwide.
Possible strategies to limit the economic impact of neosporosis include testing and culling of seropositive animals, discontinued breeding with offspring from seropositive cows, vaccination of susceptible and infected animals, and chemotherapeutical treatment of calves from seropositive cows [4, 5]. However, the most effective option is not always the most economic one  and the suitability of any of these options has to be assessed [6, 7].
As is the case for neglected diseases, one option for chemotherapy of neosporosis in cattle is the repurposing of drugs with well-documented antiparasitic activities [8–10]. The most prominent compounds in this context are certainly antimalarial drugs, for which enormous screening campaigns have been initiated, such as the high-throughput screening efforts of St. Jude Children’s Research Hospital (TN, USA), Novartis and GlaxoSmithKline [11–13]. While such large screening campaigns cannot be carried out for N. caninum, simply due to the lack of resources, one possibility is to use a piggy-back approach and perform efficacy testing of selected drugs that are already either marketed or in clinical trials for human and/or veterinary use. By screening a larger panel of such compounds, we here report on the results obtained with the three antimalarials artemisone, artemiside, and mefloquine, and the naphthoquinone buparvaquone.
Artemisone and artemiside are artemisinin derivatives  that have shown promising efficacy against experimentally induced toxoplasmosis in mice . Moreover, artemisone is highly active against N. caninum tachyzoites in vitro  and was shown to protect gerbils against cerebral infection . Artemiside exhibits excellent efficacy against malaria in a murine model , but has not been tested against neosporosis so far. Mefloquine, one of the most common antimalarials , is active in a mouse model of Schistosoma mansoni infection  and shows promising results in Echinococcus multilocularis infected mice , but its effect against N. caninum has never been assessed. Buparvaquone was also originally developed as an anti-malarial compound  and is now commercially available (Butalex ® ), for use in endemic regions against theileriosis in cattle. In other regions of the world, e.g., the EU, it is, however, not registered. In most cases only a single dose injection is required to cure Theileria-infected animals within a few days, with cure rates near to 100% when administered at the early stages of clinical disease . The mode of action of buparvaquone has not been clarified, but there are indications that it blocks the parasite respiratory chain , and resistance against buparvaquone has been associated with mutations in the cytochrome b gene of the parasite [24, 25]. Besides exhibiting an outstanding activity against Theileria spp. [26, 27], buparvaquone is also active against other protozoan parasites including Leishmania spp.  and Babesia spp. . Moreover, buparvaquone is highly active against N. caninum in vitro, and in a non-pregnant mouse model for acute disease we have recently shown that buparvaquone treatment protects mice against infection in the lungs, but not the brain, and prevents clinical signs of acute disease .
Here we applied a standardized Balb/c mouse model infected with the N. caninum NcSpain-7 isolate  to demonstrate that of these four compounds buparvaquone may have the potential to limit vertical transmission in N. caninum infected animals without inducing detrimental effects on pregnancy.
Tissue culture media, biochemicals, and drugs
If not stated otherwise, all tissue culture media were purchased from Gibco-BRL (Zürich, Switzerland), and biochemical reagents were from Sigma (St. Louis, MO, USA). Kits for molecular biology were purchased from Qiagen (Hilden, Germany). Buparvaquone was provided by Cross Vetpharm Group Limited (Dublin, Ireland). Mefloquine was kindly supplied by Mepha Pharma AG (Aesch BL, Switzerland). Artemiside was synthesized according to the literature procedure  and artemisone was purified and supplied by Cipla Mumbai Ltd.
Host cell cultivation and parasite cultures
Tachyzoites of the N. caninum Nc-Spain7 isolate were cultured and prepared for infection as described .
In vitro efficacy
In vitro efficacies of the compounds were determined using a N. caninum beta-galactosidase reporter strain (Nc-beta-gal) and human foreskin fibroblasts (HFF) as host cells. Briefly, HFF were seeded into 96-well-plates, grown to confluence, and infected with 103 Nc-beta-gal-tachyzoites per well in the presence of the compound to be tested or DMSO as a solvent control. After 3 days, medium was removed, and after one wash with PBS, cells were overlaid with 0.1 mL PBS containing Triton-X-100 (0.05%) and chlorophenyl-red-beta-galactoside (Roche, Rotkreuz, Switzerland). Absorption was continuously read at 570 nm using a 96-well-plate spectrophotometer (Versamax, Molecular Devices, Sunnyvale CA) . Host cell toxicity was determined by Alamar blue as described .
All protocols involving animals were approved by the Animal Welfare Committee of the Canton of Bern under the license BE115/14. All animals used in this study were handled in strict accordance with practices made to minimize suffering. Female and male BALB/c mice, 8 weeks of age, were purchased from a commercial breeder (Charles River, Sulzberg, Germany), and were maintained in a common room under controlled temperature and a 14 h/10 h light cycle according to the guidelines set up by the animal welfare legislation of the Swiss Veterinary Office (approval No. BE 105/14).
Assessment of the effects of mefloquine, artemisone, and artemiside on male BALB/c mice infected with N. caninum Spain-7
Male Balb/c mice were randomly distributed in four groups of six mice each and subcutaneously infected with 105 tachyzoites of the Nc-Spain7 isolate. At day 2 post infection (pi), one group received corn oil alone (placebo control), the others received mefloquine, artemiside, or artemisone (50 mg/kg/day suspended in 100 µL corn oil) for a period of 6 days by gavage once a day. At day 21 pi, the mice were euthanized. Blood was recovered by cardiac puncture and sera were obtained to assess humoral immune responses. Brains and lungs were removed for subsequent determination of parasite load and stored at −20 °C until further processing.
Assessment of the effects of buparvaquone on non-pregnant and pregnant BALB/c mice infected with N. caninum Spain-7
Overview of animal experiments presented in this study
4, 6 mice per group
3, 16 mice per group
Nc-Spain 7 (105)
Nc-Spain 7 (105), 7 days post mating
ARI, ARO, MEF, placebo; days 2–7 pi
BPQ, placebo; days 2–7 pi
21 days pi
non-pregnant: 21 days pi dams: 30 days after birth, i.e., 44 days pi
N. caninum load in brains and lungs, serum titer
Number of pups, N. caninum brain load, serum titer
Analysis of biological samples from in vivo experiments
To quantify the parasite load in brains and lungs, DNA purification was performed employing the DNeasy Blood & Tissue Kit (Qiagen, Basel, Switzerland) according to the standard protocol suitable for animal tissues. The DNA concentrations in all samples were determined using the QuantiFluor dsDNA System (Promega, Madison, Wi., USA) according to the manufacturer’s instructions and adjusted with sterile DNAse free water to 5 ng/µL. Quantification of parasite loads in brains and lungs was performed as described [30, 32]. Serum titers for N. caninum were assessed by ELISA as described [33, 34].
Statistical analysis of the parasite burdens in brains was done using the Kruskal–Wallis test followed by Wilcoxon rank-sum-test. Survival analysis of the pups was performed on the corresponding Kaplan–Meier estimator using the log-rank-test. Nominal data were analyzed using the Chi square test. All analyses were performed using the software package R .
In vitro efficacies of artemisone, artemiside, mefloquine and buparvaquone against N. caninum tachyzoites
In vivo efficacies in a Balb/c mouse model for cerebral N. caninum infection
Treatment of pregnant mice with buparvaquone protects pups from vertical transmission of N. caninum
Effects of buparvaquone treatment on clinical signs, mortality, fertility and cerebral N. caninum Nc-Spain7 infection in non-pregnant mice, dams and pups
Dams and pups
Number of dams
Nc positive dams
Total number of pups
Litter size average
Nc positive pups
In order to investigate whether drug repurposing may be a suitable strategy for obtaining suitable chemotherapeutic agents against neosporosis, we have tested four compounds, namely artemiside, artemisone, mefloquine, and buparvaquone, with proven efficacies against apicomplexan parasites in vitro and in mouse models. Three of these four compounds, artemiside, artemisone, and mefloquine did not affect the cerebral parasite load when assessed in a chronic infection model for N. caninum infection. This demonstrates that a low IC50 value against N. caninum tachyzoites obtained in vitro (in the case of artemisone; see also ) is not a predictive parameter for good in vivo effects. Moreover, our results are not in line with previous findings concerning effects of artemisone against neosporosis in gerbils . However, in that study artemisone was administered by intraperitoneal injection as a formulation solubilized in DMSO, and the authors used another isolate (NcIS491) from Israel , which most likely differs in virulence from the Nc-Spain7 isolate used in this study. In a previous study on experimentally induced murine toxoplasmosis, DMSO solutions of artemisone and artemiside had been applied s.c. , but we refrained from this application mode due to potential side effects that is induced by this treatment based on own observations and on previously published results . Oral application of mefloquine, shown to be effective against schistosomiasis  and more recently against Echinococcus infections in mice  did not exhibit any effects against N. caninum in this study.
Buparvaquone drastically limits the proliferation of N. caninum tachyzoites in vitro. In addition, we have previously shown that administration of buparvaquone protected mice that were infected with a high dosage (2 × 106) of N. caninum tachyzoites of the Nc-Liverpool isolate against acute neosporosis . In the present study, by using a 20 times reduced infection dose (105 tachyzoites), which does not lead to acute symptoms but to chronic infection followed by vertical transmission in dams , we demonstrate that in the short term, buparvaquone reduces the cerebral parasite load– as seen in the non-pregnant mice that were euthanized at 3 weeks pi. However, in the longer term, as seen in the dams that were euthanized 30 days after birth, that is, 44 days pi, no reduction in the cerebral parasite load was evident. This confirms earlier observations that buparvaquone is parasiticidal against N. caninum only after long term treatment . Thus, 6 days of treatment is too short to eradicate the parasite from the dams, but the action of the drug during this short time span is sufficient to consistently impair vertical transmission to the offspring as evidenced by a significantly lower cerebral parasite load and a lower mortality in the offspring. Under the conditions in which we run our in vivo experiments (SPF animals, controlled access, qualified personnel), in uninfected, untreated animals, postnatal mortality does not occur. In the vertical transmission model , postnatal mortality occurs only upon dose dependent infection of the dams. Pups of infected dams die within 3 weeks; pups of not infected dams survive. The model is reproducible as shown in a more recent study .
In conclusion, this study shows that repurposing of established drugs facilitates the identification of compounds that may be applied for treatment of neosporosis, thus avoiding a long and costly drug development process. Other examples of potentially interesting compounds identified earlier through this avenue, and providing interesting results both in vitro and in vivo are miltefosine  and compounds belonging to the class of bumped kinase inhibitors (BKIs) that act against calcium-dependent protein kinase 1, a target that is found in most apicomplexans [32, 39]. BKIs were originally developed to combat malaria . Buparvaquone is already in use against Theileria infections in cattle in endemic countries. Thus, it would be of interest to determine the incidence of neosporosis in buparvaquone treated cattle in those regions as compared to a control population that has not been treated. Moreover, studies in cattle will show whether buparvaquone treatments are able to eradicate N. caninum from the mother cows or whether they reduce the parasite to a low level chronic infection which may reactivate as soon as drug treatment is withdrawn. After establishment of a suitable treatment scheme, the effects on vertical transmission in cattle could be determined.
The authors declare that they have no competing interests.
RKH and NWH synthesized and provided the drugs used in this experiment. JM performed cell culture and in vitro studies. JM, AAM, AH and VM performed the animal experiments. JM, VM and AAM did the euthanasia, isolation of tissues and PCR assays, JM, AH, AAM and RKH wrote the paper. All authors read and approved the final manuscript.
We acknowledge the financial support by the Swiss National Science Foundation (Grant No. 310030 146162). RKH acknowledges funding from the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package. Cipla Mumbai Pty Ltd. is thanked for the supply of purified artemisone, and we also thank Prof. Luis Ortega-Mora, Universidad Complutense Madrid, for providing the N. caninum Spain-7 isolate.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Monney T, Hemphill A (2014) Vaccines against neosporosis: what can we learn from the past studies? Exp Parasitol 140:52–70View ArticlePubMedGoogle Scholar
- Reichel MP, Alejandra Ayanegui-Alcerreca M, Gondim LF, Ellis JT (2013) What is the global economic impact of Neospora caninum in cattle—the billion dollar question. Int J Parasitol 43:133–142View ArticlePubMedGoogle Scholar
- Dubey JP, Schares G, Ortega-Mora LM (2007) Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev 20:323–367PubMed CentralView ArticlePubMedGoogle Scholar
- Häsler B, Stark KD, Sager H, Gottstein B, Reist M (2006) Simulating the impact of four control strategies on the population dynamics of Neospora caninum infection in Swiss dairy cattle. Prev Vet Med 77:254–283View ArticlePubMedGoogle Scholar
- Häsler B, Regula G, Stark KD, Sager H, Gottstein B, Reist M (2006) Financial analysis of various strategies for the control of Neospora caninum in dairy cattle in Switzerland. Prev Vet Med 77:230–253View ArticlePubMedGoogle Scholar
- Reichel MP, Ellis JT (2006) If control of Neospora caninum infection is technically feasible does it make economic sense? Vet Parasitol 142:23–34View ArticlePubMedGoogle Scholar
- Reichel MP, Ellis JT (2009) Neospora caninum–how close are we to development of an efficacious vaccine that prevents abortion in cattle? Int J Parasitol 39:1173–1187View ArticlePubMedGoogle Scholar
- Sateriale A, Bessoff K, Sarkar IN, Huston CD (2014) Drug repurposing: mining protozoan proteomes for targets of known bioactive compounds. J Am Med Inform Assoc 21:238–244PubMed CentralView ArticlePubMedGoogle Scholar
- Andrews KT, Fisher G, Skinner-Adams TS (2014) Drug repurposing and human parasitic protozoan diseases. Int J Parasitol Drugs Drug Resist 4:95–111PubMed CentralView ArticlePubMedGoogle Scholar
- Matthews H, Usman-Idris M, Khan F, Read M, Nirmalan N (2013) Drug repositioning as a route to anti-malarial drug discovery: preliminary investigation of the in vitro anti-malarial efficacy of emetine dihydrochloride hydrate. Malar J 12:359PubMed CentralView ArticlePubMedGoogle Scholar
- Gamo FJ, Sanz LM, Vidal J, de Cozar C, Alvarez E, Lavandera JL, Vanderwall DE, Green DV, Kumar V, Hasan S, Brown JR, Peishoff CE, Cardon LR, Garcia-Bustos JF (2010) Thousands of chemical starting points for antimalarial lead identification. Nature 465:305–310View ArticlePubMedGoogle Scholar
- Guiguemde WA, Shelat AA, Bouck D, Duffy S, Crowther GJ, Davis PH, Smithson DC, Connelly M, Clark J, Zhu F, Jimenez-Diaz MB, Martinez MS, Wilson EB, Tripathi AK, Gut J, Sharlow ER, Bathurst I, El Mazouni F, Fowble JW, Forquer I, McGinley PL, Castro S, Angulo-Barturen I, Ferrer S, Rosenthal PJ, Derisi JL, Sullivan DJ, Lazo JS, Roos DS, Riscoe MK, Phillips MA, Rathod PK, Van Voorhis WC, Avery VM, Guy RK (2010) Chemical genetics of Plasmodium falciparum. Nature 465:311–315PubMed CentralView ArticlePubMedGoogle Scholar
- Meister S, Plouffe DM, Kuhen KL, Bonamy GM, Wu T, Barnes SW, Bopp SE, Borboa R, Bright AT, Che J, Cohen S, Dharia NV, Gagaring K, Gettayacamin M, Gordon P, Groessl T, Kato N, Lee MC, McNamara CW, Fidock DA, Nagle A, Nam TG, Richmond W, Roland J, Rottmann M, Zhou B, Froissard P, Glynne RJ, Mazier D, Sattabongkot J, Schultz PG, Tuntland T, Walker JR, Zhou Y, Chatterjee A, Diagana TT, Winzeler EA (2011) Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery. Science 334:1372–1377PubMed CentralView ArticlePubMedGoogle Scholar
- Ansari MT, Saify ZS, Sultana N, Ahmad I, Saeed-Ul-Hassan S, Tariq I, Khanum M (2013) Malaria and artemisinin derivatives: an updated review. Mini Rev Med Chem 13:1879–1902View ArticlePubMedGoogle Scholar
- Dunay IR, Chan WC, Haynes RK, Sibley LD (2009) Artemisone and artemiside control acute and reactivated toxoplasmosis in a murine model. Antimicrob Agents Chemother 53:4450–4456PubMed CentralView ArticlePubMedGoogle Scholar
- Müller J, Balmer V, Winzer P, Rahman M, Manser V, Haynes RK, Hemphill A (2015) In vitro effects of new artemisinin derivatives in Neospora caninum-infected human fibroblasts. Int J Antimicrob Agents 46:88–93View ArticlePubMedGoogle Scholar
- Mazuz ML, Haynes R, Shkap V, Fish L, Wollkomirsky R, Leibovich B, Molad T, Savitsky I, Golenser J (2012) Neospora caninum: in vivo and in vitro treatment with artemisone. Vet Parasitol 187:99–104View ArticlePubMedGoogle Scholar
- Guo J, Guiguemde AW, Bentura-Marciano A, Clark J, Haynes RK, Chan WC, Wong HN, Hunt NH, Guy RK, Golenser J (2012) Synthesis of artemiside and its effects in combination with conventional drugs against severe murine malaria. Antimicrob Agents Chemother 56:163–173PubMed CentralView ArticlePubMedGoogle Scholar
- Hall AP (1976) The treatment of malaria. Br Med J 1:323–328PubMed CentralView ArticlePubMedGoogle Scholar
- Keiser J, Chollet J, Xiao SH, Mei JY, Jiao PY, Utzinger J, Tanner M (2009) Mefloquine–an aminoalcohol with promising antischistosomal properties in mice. PLoS Negl Trop Dis 3:e350PubMed CentralView ArticlePubMedGoogle Scholar
- Küster T, Stadelmann B, Hermann C, Scholl S, Keiser J, Hemphill A (2011) In vitro and in vivo efficacies of mefloquine-based treatment against alveolar echinococcosis. Antimicrob Agents Chemother 55:713–721PubMed CentralView ArticlePubMedGoogle Scholar
- Hudson AT, Randall AW, Fry M, Ginger CD, Hill B, Latter VS, McHardy N, Williams RB (1985) Novel anti-malarial hydroxynaphthoquinones with potent broad spectrum anti-protozoal activity. Parasitology 90:45–55View ArticlePubMedGoogle Scholar
- McHardy N, Wekesa LS, Hudson AT, Randall AW (1985) Antitheilerial activity of BW720C (buparvaquone): a comparison with parvaquone. Res Vet Sci 39:29–33PubMedGoogle Scholar
- Sharifiyazdi H, Namazi F, Oryan A, Shahriari R, Razavi M (2012) Point mutations in the Theileria annulata cytochrome b gene is associated with buparvaquone treatment failure. Vet Parasitol 187:431–435View ArticlePubMedGoogle Scholar
- Mhadhbi M, Chaouch M, Ajroud K, Darghouth MA, BenAbderrazak S (2015) Sequence polymorphism of cytochrome b gene in Theileria annulata Tunisian isolates and its association with buparvaquone treatment failure. PLoS One 10:e0129678PubMed CentralView ArticlePubMedGoogle Scholar
- Hostettler I, Müller J, Stephens CE, Haynes R, Hemphill A (2014) A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts. Int J Parasitol Drugs Drug Resist 4:201–209PubMed CentralView ArticlePubMedGoogle Scholar
- Hashemi-Fesharki R (1991) Chemotherapeutic value of parvaquone and buparvaquone against Theileria annulata infection of cattle. Res Vet Sci 50:204–207View ArticlePubMedGoogle Scholar
- Croft SL, Hogg J, Gutteridge WE, Hudson AT, Randall AW (1992) The activity of hydroxynaphthoquinones against Leishmania donovani. J Antimicrob Chemother 30:827–832View ArticlePubMedGoogle Scholar
- Zaugg JL, Lane VM (1992) Efficacy of buparvaquone as a therapeutic and clearing agent of Babesia equi of European origin in horses. Am J Vet Res 53:1396–1399PubMedGoogle Scholar
- Müller J, Aguado-Martinez A, Manser V, Balmer V, Winzer P, Ritler D, Hostettler I, Solís D, Ortega-Mora LM, Hemphill A (2015) Buparvaquone is active against Neospora caninum in vitro and in experimentally infected mice. Int J Parasitol Drugs Drug Resist 5:16–25PubMed CentralView ArticlePubMedGoogle Scholar
- Arranz-Solis D, Aguado-Martinez A, Müller J, Regidor-Cerrillo J, Ortega-Mora LM, Hemphill A (2015) Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model. Vet Parasitol 211:133–140View ArticlePubMedGoogle Scholar
- Ojo KK, Reid MC, Kallur Siddaramaiah L, Müller J, Winzer P, Zhang Z, Keyloun KR, Vidadala RS, Merritt EA, Hol WG, Maly DJ, Fan E, Van Voorhis WC, Hemphill A (2014) Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy. PLoS One 9:e92929PubMed CentralView ArticlePubMedGoogle Scholar
- Debache K, Alaeddine F, Guionaud C, Monney T, Müller J, Strohbusch M, Leib SL, Grandgirard D, Hemphill A (2009) Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites. Int J Parasitol 39:1373–1384View ArticlePubMedGoogle Scholar
- Debache K, Guionaud C, Alaeddine F, Mevissen M, Hemphill A (2008) Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites. Int J Parasitol 38:1455–1463View ArticlePubMedGoogle Scholar
- R Core Team (2012) R: A language and environment for statistical computingGoogle Scholar
- Fish L, Mazuz M, Molad T, Savitsky I, Shkap V (2007) Isolation of Neospora caninum from dairy zero grazing cattle in Israel. Vet Parasitol 149:167–171View ArticlePubMedGoogle Scholar
- Colucci M, Maione F, Bonito MC, Piscopo A, Di Giannuario A, Pieretti S (2008) New insights of dimethyl sulphoxide effects (DMSO) on experimental in vivo models of nociception and inflammation. Pharmacol Res 57:419–425View ArticlePubMedGoogle Scholar
- Küster T, Stadelmann B, Rufener R, Risch C, Müller J, Hemphill A (2015) Oral treatments of Echinococcus multilocularis-infected mice with the anti-malarial drug mefloquine that potentially interacts with parasite ferritin and cystatin. Int J Antimicrob Agents 46:546–551View ArticlePubMedGoogle Scholar
- Winzer P, Müller J, Aguado-Martínez A, Rahman M, Balmer V, Ortega-Mora L, Ojo KK, Fan E, Maly D, Van Voorhis WC, Hemphill A (2015) In vitro and in vivo effects of the bumped kinase inhibitor 1294 in the related cyst-forming apicomplexans Toxoplasma gondii and Neospora caninum. Antimicrob Agents Chemother 59:6361–6374View ArticlePubMedGoogle Scholar
- Debache K, Hemphill A (2012) Effects of miltefosine treatment in fibroblast cell cultures and in mice experimentally infected with Neospora caninum tachyzoites. Parasitology 139:934–944View ArticlePubMedGoogle Scholar
- Ojo KK, Pfander C, Mueller NR, Burstroem C, Larson ET, Bryan CM, Fox AM, Reid MC, Johnson SM, Murphy RC, Kennedy M, Mann H, Leibly DJ, Hewitt SN, Verlinde CL, Kappe S, Merritt EA, Maly DJ, Billker O, Van Voorhis WC (2012) Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors. J Clin Invest 122:2301–2305PubMed CentralView ArticlePubMedGoogle Scholar