Compounds from the Medicines for Malaria Venture Box Inhibit In Vitro Growth of Babesia divergens, a Blood-Borne Parasite of Veterinary and Zoonotic Importance.

Babesiosis is an infectious disease with an empty drug pipeline. A search inside chemical libraries for novel potent antibabesial candidates may help fill such an empty drug pipeline. A total of 400 compounds (200 drug-like and 200 probe-like) from the Malaria Box were evaluated in the current study against the in vitro growth of Babesia divergens (B. divergens), a parasite of veterinary and zoonotic importance. Novel and more effective anti-B. divergens drugs than the traditionally used ones were identified. Seven compounds (four drug-like and three probe-like) revealed a highly inhibitory effect against the in vitro growth of B. divergens, with IC50s ≤ 10 nanomolar. Among these hits, MMV006913 exhibited an IC50 value of 1 nM IC50 and the highest selectivity index of 32,000. The atom pair fingerprint (APfp) analysis revealed that MMV006913 and MMV019124 showed maximum structural similarity (MSS) with atovaquone and diminazene aceturate (DA), and with DA and imidocarb dipropionate (ID), respectively. MMV665807 and MMV665850 showed MMS with each other and with ID. Of note, a high concentration (0.75 IC50) of MMV006913 caused additive inhibition of B. divergens growth when combined with DA at 0.75 or 0.50 IC50. The Medicines for Malaria Venture box is a treasure trove of anti-B. divergens candidates according to the obtained results.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale.

Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.

Alterations of blood parameters after intramuscular administration of imidocarb in healthy lambs.

The aim of this study was to determine the safety of imidocarb dipropionate in sheep. Imidocarb dipropionate (IMDP) was administered (2.4 mg/kg, intramuscular; i.m.) to 10 sheep, and blood samples were obtained 0, 1, 6, and 9 days after treatment. Hemacell counts, serum biochemical values, coagulation values, and serum oxidative status were measured. IMDP caused transient decreases in pH, actual bicarbonate, standard bicarbonate, total carbon dioxide, base excess in vivo, base excess in vitro, oxygen saturation, lactate dehydrogenase, and retinol levels and transient increases in serum creatine kinase-MB, blood urea nitrogen, and creatinine levels. IMDP decreased adenosine deaminase activity, antithrombin III, and superoxide dismutase activity and increased white blood cell counts. In conclusion, IMDP may change serum oxidative status and cause coagulation disorders during treatment in sheep.

Induced mitotic homologous recombination by the babesicide imidocarb dipropionate in Aspergillus nidulans diploid cells.

Imidocarb dipropionate (IMD) is a chemotherapeutic agent prescribed for the treatment and control of babesiosis; it is known to be a nucleic acid synthesis inhibitor. Although it is an effective babesicide, there are reports of persistent IMD residues retained at high levels in edible tissues of cattle, swine and sheep, raising concerns about potential effects on humans. Since the carcinogenic potential of a chemical compound can be assessed through its effect on the homologous recombination, we investigated whether IMD is recombinogenic in Aspergillus nidulans diploid cells and whether it is capable of inducing homozygosis in genes that were previously heterozygous. This analysis was done with a homozygotization assay applied to a heterozygous diploid strain of A. nidulans. IMD used at non-toxic concentrations (2.5 to 10.0 µM) was recombinogenic, demonstrated by homozygotization indices higher than 2.0 for diploid markers. A diploid homozygous for genetic markers from chromosomes I and II was also produced. Since DNA replication blockers that induce DNA strand breaks have been classified as potent inducers of homologous recombination, the recombinogenic potential of IMD may be due to induction of recombinational repair.

Comparison of therapeutic efficacy of different drugs against Trypanosoma vivax on experimentally infected cattle.

In this study, we evaluated the therapeutic efficacy of diminazene diaceturate at a dose of 7 mg/kg (DA), imidocarb dipropionate at 4.8 mg/kg (IMD), isometamidium chloride at 0.5 and 1.0 mg/kg (ISM 0.5 and ISM 1.0) and combinations applied through different methods to treat Trypanosoma vivax in experimentally infected calves. Thirty male Girolando calves were kept indoors and infected intravenously with T. vivax trypomastigotes (approximately 1 × 106). On D-1, the calves were randomized based on the quantity of infecting parasites per animal, yielding six groups of five animals each: G1: positive control group without treatment; G2 animals treated with DA on Day 0 intramuscularly (IM); G3 animals treated with IMD on Day 0 and D + 14 subcutaneously; G4 animals treated with ISM 0.5 on Day 0 IM; G5 animals treated with ISM 1.0 on Day 0 IM; G6 animals received DA on Day 0 and ISM 1.0 on D + 14, both IM. Throughout 180 days, blood samples were collected for the evaluation of T. vivax using the Woo, Brener and PCR methods. The results indicated that the treatment protocols with DA and/or ISM 0.5 and ISM 1.0 had high efficacy (100 %) against T. vivax. Interestingly, cattle that received ISM remained free of parasites until D + 180. In contrast, animals treated with IMD had relapsed T. vivax detected on the 10th and 14th days post-treatment (DPT). Cattle that received ISM 1.0 did not exhibit relapsed T. vivax in the blood, even after reinfection performed on the 50th DPT. However, treatment with DA on Day 0 failed to prevent a new infection of T. vivax on the 50th DPT. The animals that received ISM 1.0 had a transient decrease in packed cell volume similar to that found in the control group. The reappearance of T. vivax in herds in Brazil treated with DA likely occurred due to the short half-life of the drug and not necessarily due to T. vivax resistance to DA.

Ultrastructural and fluorochromatic changes of Anaplasma marginale exposed to oxytetracycline, imidocarb and enrofloxacin in short-term erythrocyte cultures.

Anaplasma marginale causes mild to severe hemoparasitic disease resulting in significant morbidity and mortality in cattle worldwide. In the absence of universally efficacious vaccines, antimicrobial therapy combined with biocontainment and biosecurity strategies are critical to control anaplasmosis. Herein, we compared the effect of oxytetracycline, imidocarb and enrofloxacin on A. marginale isolates in short-term erythrocyte cultures. Electron micrographs detailing antimicrobial-induced changes in rickettsial morphology were scored (0-4) based on ultrastructural changes. These were compared to fluorochromatic changes detected by flow cytometry (FACS) using conversion of hydroethidine (HE) to ethidium bromide (EB) by living organisms to assess viability. A. marginale infectivity in selected cultures was confirmed by subinoculation into susceptible calves. Morphology scores were analyzed using Chi-squared tests and compared to FACS data by ANOVA with isolate, drug and concentration as co-variates in the model. Only the Virginia and Oklahoma isolates exposed to 1.0 microg /ml imidocarb and the Oklahoma isolate exposed to 4.0 microg /ml enrofloxacin were sterilized following antimicrobial exposure. Rickettsia with morphology scores of 0 had significantly more EB positive cells than inclusions with morphology scores of 4 (p=0.039). There was also a significant association between ultrastructural changes and infectivity (p=0.0047). Furthermore, the percent EB positive cells in the antimicrobial exposed cultures was highly predictive of the probability of infectivity (p=0.0026). This is the first study describing ultrastructural changes in A. marginale following exposure to enrofloxacin and imidocarb. These findings demonstrate that FACS and electron microscopy are useful tools to screen new antimicrobials for the use in anaplasmosis chemotherapy.

In vitro growth inhibition of Theileria equi by bumped kinase inhibitors.

INTRODUCTION: Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-à-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity. RESULTS: Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC50)) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318). CONCLUSIONS: The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses.

Effect of imidocarb and levamisole on the experimental infection of BALB/c mice by Leishmania (Leishmania) amazonensis.

The adverse effects from using currently available drugs for the treatment of leishmaniasis have motivated the search for new therapeutical agents. The aim of this work was to evaluate the effect of imidocarb and levamisole on the treatment of BALB/c mice experimentally infected by Leishmania (Leishmania) amazonensis. BALB/c mice were infected with 10(6) promastigotes of L. (L.) amazonensis (IFLA/BR/67/PH8) and, starting on day 51, mice were treated subcutaneously with imidocarb (IMD, 34 mg/kg), imidocarb plus levamisole (IMD+LVS, 34 and 12 mg/kg, respectively), only levamisole (LVS, 12 mg/kg) or without treatment (control). Lesion size and swelling were weekly monitored for 10 weeks after the beginning of the treatment. On day 121 post-infection, serum levels of specific IgG from infected mice were evaluated, as well as histopathological and morphometric alterations in the footpad, lymph nodes and spleen of these animals. The data obtained in this study demonstrated that, when compared to controls, mice treated with IMD had lower levels of IgG anti-L. (L.) amazonensis (34.45%), smaller vacuolar area in macrophages (3.75%), lower number of megakaryocytes in spleen (63.19%) and lower parasite burden in the footpad (30.2%). Thus, the evaluated parameters suggest the use of imidocarb as a potential drug in the treatment of tegumentary leishmaniasis.

Chemotherapy against babesiosis.

Babesiosis is caused by a haemotropic protozoal parasite of the genus Babesia, member of the phylum Apicomplexa and transmitted by the bite of an infected tick. There are many Babesia species affecting livestock, dogs, horses and rodents which are of economic significance. Infections can occur without producing symptoms, but babesiosis may also be severe and sometimes fatal caused by the intraerythrocytic parasite development. The disease can cause fever, fatigue and haemolytic anemia lasting from several days to several months. There are a number of effective babesiacides, but imidocarb dipropionate (which consistently clears the parasitaemia; often the only available drug on the market) and diminazene aceturate are the most widely used. Some Babesia spp. can infect humans, particularly Babesia microti and Babesia divergens, and human babesiosis is a significant emerging tick-borne zoonotic disease. Clinical manifestations differ markedly between European and North American diseases. In clinical cases, a combination of clindamycin and quinine is administered as the standard treatment, but also administration of atovaquone-azithromycin is successful. Supportive therapy such as intravenous fluids and blood transfusions are employed when necessary. More specific fast-acting new treatments for babesiosis have now to be developed. This should be facilitated by the knowledge of the Babesia spp. genome and increased interest for this malaria-like parasite.

Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs.

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.

Comparison of the efficacy of enrofloxacin, imidocarb, and oxytetracycline for clearance of persistent Anaplasma marginale infections in cattle.

This study compared enrofloxacin and imidocarb dipropionate treatments with an oxytetracycline regimen proposed by the World Organization for Animal Health for elimination of persistent Anaplasma marginale infections in cattle. The effect of therapy on competitive ELISA and polymerase chain reaction (PCR) reactivity was also assessed. Twelve A. marginale-infected carrier calves were randomly assigned to groups receiving either enrofloxacin (5 mg/kg IV q24h for 5 days), imidocarb (5 mg/kg IM twice, 7 days apart), or oxytetracycline (22 mg/kg IV q24h for 5 days). One calf infected with an Oklahoma isolate in the imidocarb group and one infected with a Virginia isolate in the oxytetracycline group failed to infect a splenectomized calf following blood subinoculation. Both became competitive ELISA negative by 44 days after treatment, but the imidocarb-treated calf remained PCR positive. None of the tested treatments reliably eliminated persistent A. marginale infections in all cattle. Furthermore, PCR was not a reliable means of determining the success of chemosterilization in calves.

Clinical and clinicopathological changes in 6 healthy ponies following intramuscular administration of multiple doses of imidocarb dipropionate.

Haematological variables and selected serum indices, particularly those affected by changes in renal and hepatic function, were examined in 6 healthy ponies following 4 intramuscular doses of 4 mg/kg imidocarb dipropionate administered every 72 hours. This treatment regime has been reported to sterilise experimental Babesia equi infections in horses and may have value in preventing the spread of this disease during exportation of possible carrier horses to non-endemic countries. Serum bile acids and serum gamma glutamyltransferase activity were measured to evaluate the effect of this treatment regime on hepatic function. Owing to the absence of any increase in these variables it was concluded that this treatment regime had no clinically detectable deleterious effect on hepatic function in healthy ponies. Urinary gamma glutamyltransferase : creatinine ratios (IU/g), serum creatinine and fractional clearance of sodium, potassium and phosphate (%) were calculated as a measure of renal function. Urinary GGT and urinary GGT : creatinine ratios were significantly elevated on Day 5 of the trial, with 2 of the trial animals also exhibiting mild azotaemia indicative of changes in renal function. The changes in urine GGT : urine creatinine ratios observed in this study also provides evidence of the value of this ratio for the early detection of renal toxicity, following exposure to nephrotoxic agents.

Preliminary development of a live drug-controlled vaccine against bovine babesiosis using the Mongolian gerbil, Meriones unguiculatus.

This study investigated the practicality and potential of the gerbil, Meriones unguiculatus, as a source of live Babesia divergens vaccine and also as a model for the use of the vaccine in cattle. A series of experiments with gerbils concerning vaccine infectivity, immunogenicity and safety were carried out. It was concluded that the use of RPMI medium/40% foetal calf serum as a diluent improved vaccine infectivity, but that the parasitaemia of the blood obtained from donor gerbils had little or no effect. The immunostimulants levamisole and killed Corynebacterium parvum improved vaccine immunogenicity and it was also shown that the subcutaneous route of infection resulted in the greatest host response. Control of vaccine virulence with drugs was only possible when drugs with prophylactic properties, such as imidocarb and long-acting oxytetracycline, were used. More studies are required on all these topics, particularly with regard to their applicability to cattle, and also concerning the possible attenuation of the parasite by manipulation in the gerbil host.

Activity of buparvaquone against Theileria cervi in white-tailed deer.

Buparvaquone, a naphthoquinone with known efficacy against Theileria parva parva in cattle, was tested for activity against Theileria cervi piroplasms in both an in vitro culture system and in vivo in experimentally infected white-tailed deer. The in vitro data showed a significant decrease in the incorporation of 3H-hypoxanthine by infected red blood cells treated with buparvaquone when compared to that seen with imidocarb and chloroquine treatment. In both intact and splenectomized deer treated with buparvaquone (2.5 mg kg-1) a gradual decrease in piroplasm parasitaemia was observed following treatment. However, in the splenectomized deer, parasitaemia levels returned to near pretreatment values after approximately 2 weeks.

Prophylactic activity of imidocarb against experimental infection with Babesia canis.

A single subcutaneous injection of imidocarb at 6 mg kg-1 gave 2 weeks protection against experimental inoculation of Babesia canis merozoites in Beagle dogs. The prophylactic effect was evaluated by daily measurements of parasitaemia, rectal temperature, haematological values and specific antibodies.

Humoral immunity and reinfection resistance in dogs experimentally inoculated with Babesia canis and either treated or untreated with imidocarb dipropionate.

It is proposed that the chronic asymptomatic carrier state produced by Babesia canis infection could make dogs more resistant against subsequent infections. This suggests that treatment with imidocarb dipropionate, which removes the organism, can make dogs more susceptible to reinfection in a short period of time. Ten male and female dogs of approximately 4-5 months of age were inoculated with B. canis. Half of them received treatment with imidocarb dipropionate (7 mg/kg) on days 15 and 27 post-infection and the other half were untreated. All the animals were examined using clinical and laboratory methods (CBC, platelet counts and serological study by indirect immunofluorescence test) for a 6-month period. Antibodies were first detected on day 7 post-injection and remained at high levels (1:2560) over the period in the non-treated group. This result was significantly different (P<0.001) from the treated group in which antibodies titers declined after day 34 post-infection. Six months later, after a homologous challenge infection only the dogs of treated group showed parasitaemia, thrombocytopenia and splenomegaly, which was significantly different (P<0.05) from the non-treated group. The sterilizing treatment with imidocarb dipropionate was effective in clearing the infection, but inhibited the maintenance of protective antibodies, making the animals more susceptible to reinfection.

The effect of Imidocarb treatment on Babesia in the bovine and the tick (Boophilus microplus).

Treatment of calves with 5 mg/kg Imidocarb (3,3-1-bis-(2 - imidazolin - 2 - yl)carbanilide dipropionate) given intramuscularly 14 days before and 14 days after exposure to Babesia infected Boophilus microplus larvae rendered the next generation of larvae incapable of transmitting Babesia infection. When administered to calves 14 or 28 days before tick exposure, the drug prevented the development of clinical babesiosis; the larval progeny of ticks reared on the calf which was treated 28 days before infestation were infective. Treatment of a calf 42 days before exposure to infective larvae did not prevent the development of a Babesia parasitaemia but appeared to reduce the severity of infection.

Determination of whole blood cholinesterase in different animal species using specific substrates.

Whole blood cholinesterase was measured using acetyl-, butyryl- and propionylthiocholine as substrates in 10 healthy adult dogs, cats, horses, pigs, goats, sheep and cows, in order to determine and characterise the cholinesterase activity in whole blood of the main domestic animals. An in vitro exposure test with two anticholinesterase compounds, the organophosphate insecticide coumaphos and the carbamate insecticide imidocarb, was also performed. In whole blood of ruminants and pigs, acetylthiocholine yielded the highest cholinesterase activity and other substrates were poorly hydrolysed; in dogs and cats, although acetylthiocholine showed the highest cholinesterase activity, butyryl- and propionylthiocholine also produced high cholinesterase values; in horses, propionylthiocholine was the substrate that yielded the highest cholinesterase activity, closely followed by butyrylthiocholine. All within- and between-run coefficients of variation observed in whole blood samples were less than 5 and 7 per cent, respectively, except when butyrylthiocholine was used as substrate in ruminant blood samples. Butyryl- and propionylthiocholine were the substrates that yielded higher inhibitions after coumaphos exposure, whereas the use of acetylthiocholine showed the highest cholinesterase inhibition after imidocarb exposure. The use of at least two substrates (acetyl and butyrylthiocholine) is recommended for whole blood cholinesterase analyses in domestic animals since it will allow monitoring of both acetyl- and butyrylcholinesterase activities, respectively, and a more accurate detection of exposure to anticholinesterase compounds. However, acetylthiocholine could be used as a unique substrate for whole blood cholinesterase determination in porcine and ruminant samples since butyrylcholinesterase activity is very low in these species. Additionally, propionylthiocholine could be used as an alternative substrate to butyrylthiocholine in horse whole blood samples.

In vivo assays for drug resistance in Babesia divergens using the Mongolian gerbil, Meriones unguiculatus.

Two in vivo drug resistance assays were developed using gerbils. Cross resistance, involving related babesicides as well as the chemically unrelated antibiotic, oxytetracycline, was demonstrated, but the suggestion that imidocarb may select for pathogenic strains of parasites was not supported. Limited tests of field strains did not detect resistance. It is suggested that an in vitro assay would be more appropriate for surveys through in vivo assays would be essential for confirmatory studies.

Prospective comparative study of 3 treatment protocols using doxycycline or imidocarb dipropionate in dogs with naturally occurring ehrlichiosis.

This paper reports the clinicopathologic responses of 93 dogs with spontaneously occurring ehrlichiosis to 3 different treatment protocols. Thirty-two dogs were treated with doxycycline (10 mg/kg/day for 28 days), 31 were treated with imidocarb dipropionate (5 mg/kg given 15 days apart in 2 separate injections), and 30 were treated with both drugs simultaneously, at the doses as specified. The dogs underwent clinicopathologic evaluation before and after treatment, and were examined periodically during the 24-month period after the treatment. No differences were found in the clinical responses among the dogs in the 3 treatment groups. As for the clinicopathologic response, in spite of the fact that at the end of the study the results obtained with the 3 protocols were similar, the platelet count and serum protein electrophoresis results returned to normal more slowly in dogs that received imidocarb dipropionate as compared to those given the other 2 treatments.