Repurposing of the Malaria Box for Babesia microti in mice identifies novel active scaffolds against piroplasmosis.

BACKGROUND: An innovative approach has been introduced for identifying and developing novel potent and safe anti-Babesia and anti-Theileria agents for the control of animal piroplasmosis. In the present study, we evaluated the inhibitory effects of Malaria Box (MBox) compounds (n = 8) against the growth of Babesia microti in mice and conducted bioinformatics analysis between the selected hits and the currently used antibabesial drugs, with far-reaching implications for potent combinations. METHODS: A fluorescence assay was used to evaluate the in vivo inhibitory effects of the selected compounds. Bioinformatics analysis was conducted using hierarchical clustering, distance matrix and molecular weight correlation, and PubChem fingerprint. The compounds with in vivo potential efficacy were selected to search for their target in the piroplasm parasites using quantitative PCR (qPCR). RESULTS: Screening the MBox against the in vivo growth of the B. microti parasite enabled the discovery of potent new antipiroplasm drugs, including MMV396693 and MMV665875. Interestingly, statistically significant (P < 0.05) downregulation of cysteine protease mRNA levels was observed in MMV665875-treated Theileria equi in vitro culture in comparison with untreated cultures. MMV396693/clofazimine and MMV665875/atovaquone (AV) showed maximum structural similarity (MSS) with each other. The distance matrix results indicate promising antibabesial efficacy of combination therapies consisting of either MMV665875 and AV or MMV396693 and imidocarb dipropionate (ID). CONCLUSIONS: Inhibitory and hematology assay results suggest that MMV396693 and MMV665875 are potent antipiroplasm monotherapies. The structural similarity results indicate that MMV665875 and MMV396693 have a similar mode of action as AV and ID, respectively. Our findings demonstrated that MBox compounds provide a promising lead for the development of new antibabesial therapeutic alternatives.

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.

Efficacy of long-acting oxytetracycline and imidocarb dipropionate for the chemosterilization of Anaplasma marginale in experimentally infected carrier cattle in Argentina.

The expansion of anaplasmosis to non-endemic areas in Argentina has created the need for specific treatments to eliminate Anaplasma marginale from carriers. The most recent studies have failed to chemosterilize A. marginale infections. In this work, we compare the efficacy of long-acting oxytetracycline (OTC) and imidocarb dipropionate (IMD) to chemosterilize the A. marginale infection. For this purpose, twenty steers were randomly clustered into two groups of ten animals each 78 days after A. marginale experimental infection (day 0). Cattle from group 1 (G1) were treated with three doses of 20 mg kg-1 of OTC (Terramycin® LA, 200 mg/ml) 7 days apart by intramuscular injection. Cattle from G2 were treated with two doses of 5 mg kg-1 of IMD (Imizol®, 120 mg/ml) 14 days apart by intramuscular injection. The efficacy of sterilizing treatments was evaluated by detection of DNA by nested PCR, anti-MSP5 antibodies by ELISA and by inoculation of splenectomized calves with blood from the steers 104 days post-treatment (dpt). The results showed 50% efficacy of the OTC treatment to chemosterilize persistent A. marginale infections in cattle and the failure of the IMD treatment under the evaluated conditions. The persistence of specific antibody levels in the sterilized animals (56 dpt) was shorter than the period of DNA detection. The ELISA was the test of choice to confirm the sterilizing outcome after 60 dpt. In spite of its limitations, the sterilization of A. marginale carrier status using OTC, could be useful for high-value bovines in non-endemic areas.

Validated Stability Indicating Chromatographic Methods for Quantification of Imidocarb Dipropionate; Application for the Determination of Its Residues in Bovine Meat and Milk Samples.

BACKGROUND: Imidocarb dipropionate (IMD) is an immunomodulator agent commonly used for treatment of anaplasmosis in cattle. OBJECTIVE: Thus, two sensitive, specific, and precise stability-indicating chromatographic methods have been developed, optimized, and validated for its determination in presence of its acid, alkaline, and oxidative stressed degradation products. METHOD: The first method is based on separation of IMD and its forced induced degradation products on reversed phase cyano column using isocratic elution system consisted of sodium acetate buffer-methanol-acetonitrile (55: 30:15, v/v/v), pH 4.6 at a flow rate of 1.2 mL/min, and UV detection at 254 nm. The second method utilized TLC combined with densitometric determination of the separated bands at 254 nm. The separation was achieved using silica gel 60 F254 TLC plates with a mixture of ethyl acetate-methanol-ammonia-water (8.5:1:0.5:0.2, v/v/v/v) as a developing system. RESULTS: HPLC analysis was applied in range of 0.25-40 µg/mL with LOD of 0.073 µg/mL. While densitometric measurements showed linearity in the range of 0.1-1.8 µg/band with LOD of 0.02 µg/band. CONCLUSIONS: The suggested methods were validated in compliance with the ICH guidelines and were successfully applied for determination of IMD in its commercial veterinary formulations with good recoveries. Furthermore, the proposed HPLC method was extended to the determination of IMD residues in bovine meat and milk samples. HIGHLIGHTS: Bovine meat, HPLC, Imidocarb dipropionate, Milk, TLC.

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.

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.

Pharmacokinetics of imidocarb dipropionate in white-tailed deer (Odocoileus virginianus) after single intramuscular administration.

This study was designed to investigate the pharmacokinetics of imidocarb, a carbanilide derivative, in white-tailed deer (Odocoileus virginianus). The pharmacokinetic properties of a single intramuscular (IM) dose of imidocarb were determined in 10 deer. A single IM injection of 3.0 mg/kg imidocarb dipropionate was administered, and blood samples were collected prior to, and up to 48 hr after imidocarb administration. Plasma imidocarb concentrations were determined by high-performance liquid chromatography with ultraviolet detection. The disposition of plasma imidocarb was best characterized by a two-compartment open model. The mean ± SE maximal imidocarb concentration in deer was 880.78 ± 81.12 ng/ml at 38.63 ± 5.30 min postinjection. The distribution phase had a half-life (t1/2α ) of 25.90 ± 10.21 min, and plasma imidocarb concentration declined with a terminal elimination half-life (t1/2ß ) of 464.06 ± 104.08 min (7.73 ± 1.73 hr). Apparent volume of distribution based on the terminal phase (VZ /F) was 9.20 ± 2.70 L/kg, and apparent total body clearance (Cl/F) was 15.97 ± 1.28 ml min-1  kg-1 .

Fatal acute babesiosis associated with Babesia venatorum infection (Babesia sp. EU1) in a captive reindeer calf in Switzerland.

Babesia venatorum was isolated from a captive reindeer calf in Switzerland. The clinical signs consistent with acute babesiosis included hemolytic anemia and hemoglobinuria. The diagnosis was made based on visualization of intraerythrocytic parasites in the stained blood smears and confirmed by PCR analysis of the 18S rRNA gene, with subsequent species identification within Babesia confirmed by sequencing. The reindeer calf was initially treated with supportive care and an antiprotozoal drug (imidocarb dipropionate) but died a few days after hospitalization. Babesia venatorum is also known as Babesia sp. EU1 and can infect different mammalian species, including humans. The current case report aims to increase awareness among veterinarians and reindeer owners about the presence and the associated risk of this zoonotic pathogen. Considering the high morbidity and possible mortality associated with acute babesiosis, captive reindeer should receive tick prevention measures and be tested for subclinical infections in endemic area.

Clinical Occurrence of Canine Babesiosis in the Coastal Area of the Black Sea (Dobrogea) in Southeastern Romania and Associated Epidemiological Implications.

Canine babesiosis (CB) is a significant tick-borne disease caused by intra-erytrocytic protozoa of the genus Babesia (Apicomplexa: Piroplasmida) and is currently emerging or re-emerging in many European countries. Despite the increasing incidence of clinical CB, little is known of the epidemiology of this disease in Romania. Therefore, the present study was conducted to provide epidemiological data on CB in the coastal area of the Black Sea (Dobrogea), southeastern Romania. For this, 306 owned dogs presented during 2016-2018 at a veterinary clinic in the area and clinically suspected for CB were tested for the presence of intra-erytrocytic piroplasms using Giemsa-stained thin blood smears. Overall, 27.8% (95% CI: 22.83-33.16) of dogs were positive for large piroplasms. The Babesia spp.-positive dogs were 51 males and 34 females aged from 3 mo to 13 yr (mean 4.2 yr; SD = 3.6). A higher rate of infection was registered in Bichon Maltese (n = 13) and mixed-breed (n = 28) dogs. Most cases (76/85; 89.4%) were diagnosed from March to June, with only a few cases registered in November and December (1 case each). Infected dogs displayed different clinical presentations, varying from mild (38.8%) to moderate (29.4%) and severe disease (29.4%). Based on clinical- pathological changes, 38 (44.7%), 34 (40.0%), and 13 (15.3%) dogs were diagnosed with uncomplicated babesiosis, complicated babesiosis with a single organ dysfunction, and complicated babesiosis with multiple-organ-dysfunction syndrome (MODS), respectively. The recovery rate (81.6%, 70.1%, and 38.4%, respectively) was significantly different in these groups. The age of dogs appeared to be a risk factor for both severe disease (mean age of 5.8 yr) and MODS (mean age 6.8 yr). These findings document the common occurrence of CB in Dobrogea, southeastern Romania, and suggest that appropriate control measures need to be taken.

Hepatozoon canis infection causing a strong monocytosis with intra-monocytic gamonts and leading to erroneous leukocyte determinations.

In this case report, a Swedish flat-coated retriever was diagnosed with an extensive Hepatozoon canis infection. The dog had a prominent monocytosis (14.0 × 109 /L) with H canis gamonts detected in most monocytes, but none were found in the neutrophils. On the hematology system ADVIA 2120 peroxidase (PEROX) cytogram, most leukocytes were seen as a distinct cell population above the lymphocytes, which indicated that most of the cells were larger than lymphocytes and had weak myeloperoxidase staining. This distinct cell cluster appeared to be of a single cell type but was incorrectly divided by the ADVIA 2120 into lymphocytes, monocytes, and large unstained cells (LUC). The total leukocyte counts on the ADVIA 2120 WBC basophil (BASO) channel were much higher than that on the WBC PEROX count. The WBC BASO cytogram appeared abnormal with two parallel cell populations, so the BASO WBC count was considered erroneous. Polymerase chain reaction and DNA sequencing verified H canis infection. The dog was treated with subcutaneous imidocarb dipropionate (6 mg/kg) injections every other week. Post-treatment hematology analyses indicated that the percentage of parasitized leukocytes decreased from 40% to 5% about 4 weeks after the start of treatment and were not found in any monocytes 6 weeks after the beginning of the treatment. In conclusion, H canis infection in this dog was associated with a strong monocytosis, and gamonts were present in many monocytes, which caused aberrant automated leukocyte counts to occur.

Hepatozoon canis in three imported dogs: a new tickborne disease reaching the United Kingdom.

An increasing number of non-endemic vectorborne pathogens have been described in dogs imported to the UK in the past two decades. Recently, an outbreak of canine babesiosis in south-east England has raised veterinary awareness with regard to the impact of such diseases on the UK canine population. Canine hepatozoonosis, caused by Hepatozoon canis and transmitted by the ingestion of Rhipicephalus sanguineus ticks, is widespread in the Mediterranean basin. Herein we describe the first three molecularly confirmed clinical cases of canine hepatozoonosis in dogs imported into the UK. Veterinarians in the UK should be aware of H canis as a potential infection in imported dogs, especially in the face of the expanding distribution of R sanguineus ticks in Europe.

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania.

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the world. However, clinical outbreaks of EP in Romania during the last decades have not been reported Therefore, the aim of this paper is (i) to describe a clinical B. caballi outbreak in horses on several farms in Southern Romania using a diagnostic and therapeutic approach and (ii) the molecular diagnostic of EP in an endemic area of Romania. In the first case, a 10-month-old stallion male was presented with lethargy, anorexia, fever (40.9 °C), pale mucosal/mucous/membranes and a marked anemia. In the subsequent weeks, three horses from other farms located in the same area, displayed similar clinical signs. B. caballi was diagnosed in all the horses based on Giemsa-stained blood smears and the diagnosis was further confirmed by polymerase chain reaction (PCR), using a single-round and multiplex PCR and sequencing. All four horses were treated with imidocarb dipropionate, at a dose rate of 2.2 mg/kg body weight (two injections at 48 h apart), and all horses clinically recovered within 24-48 h, post-treatment. This report presents the first molecularly characterized B. caballi outbreak in Romania in clinically affected horses, confirmed by DNA sequencing.

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.

Verification of post-chemotherapeutic clearance of Theileria equi through concordance of nested PCR and immunoblot.

Certain countries including the United States remain non-endemic for particular infectious diseases such as equine piroplasmosis through import restrictions and surveillance. Endemic regions often employ premunition as the primary method to control disease, however in non-endemic countries, chemosterilization combined with methods to confirm parasite elimination are required to maintain disease-free status. The ability of imidocarb diproprionate (ID) to clear persistent Theileria equi infection from infected horses has been shown through the inability of treated horses to transmit via blood transfer. However, the common lengthy persistence of anti-T. equi antibody causes regulatory tests such as cELISA or IFA to remain positive for extended periods. Persistence of positive testing creates challenges for regulatory veterinary medicine and international trade. Concordance between nested polymerase chain reaction (nPCR) targeting the ema1 gene and immunoblotting (IB) measuring declination in anti-EMA1 and anti-EMA2 antibody were used to verify clearance of T. equi from 179 ID-treated horses. These data support the use of IB to demonstrate declining anti-EMA1 and EMA2 titers in T. equi-infected horses subsequent to successful ID treatment. Such data provide concordant support to a negative nPCR and allow for a more timely determination of effective ID clearance of T. equi. The post ID treatment results indicate that while nPCR was consistently negative by 14 days and cELISA generally remained positive after 1 year, immunoblot was on average negative after 4 months and 100% in agreement with nPCR.

Microscopic and molecular analysis of Babesia canis in archived and diagnostic specimens reveal the impact of anti-parasitic treatment and postmortem changes on pathogen detection.

BACKGROUND: Classification of Babesia parasites has traditionally relied on morphological differentiation based on piroplasm size and shape. Molecular typing has subsequently revealed a more complex taxonomy for these piroplasms than previously thought. To evaluate the factors that influence the morphology of Babesia species upon microscopic examination and hence, their taxonomic classification, we performed detailed characterizations of piroplasms from archival and prospective collections of cytological samples of dogs with piroplasmosis before and after death. Merozoite morphology and time of parasite disappearance following imidocarb dipropionate was also investigated. METHODS: The study was divided into a (i) review of archived cytological slides from confirmed cases of canine piroplasmosis, and (ii) a prospective study of smears and tissue imprints from 15 recently necropsied dogs. The latter group could be further sub-divided into a non-treated group and an imidocarb dipropionate-treated group. Exact times of treatment before death were reviewed. Additional blood smears prepared from the live dogs and taken before therapy were also evaluated in the latter group. Parasite burden per each slide was determined in both studies. The shape and size of merozoites were described from blood smears taken while the dogs were alive and from different organs during necropsy. The results of all measurements were statistically analyzed. RESULTS: The morphology and size of merozoites from live dogs corresponded to that of previously described 'large' Babesia. The morphology and size of merozoites were significantly different (P < 0.001) in postmortem samples, however, and more consistent in shape and size with piroplasm cells previously referred to as 'small' Babesia. PCR and sequencing confirmed B. canis as the causative agent of disease in all investigated dogs, including in postmortem negative tissue imprints from dogs treated at least 24 h before death. CONCLUSIONS: Changes in the morphology of 'large' B. canis to 'small'-like Babesia observed by light microscopy appear to represent a common postmortem change. Classification of Babesia parasites into 'large' and 'small' Babesia using only microscopy of postmortem slides should be treated with caution. PCR-based methodologies for detection and molecular typing of Babesia spp. may prove valuable for investigating suspected cases of babesiosis following necropsy.

Efficacy, safety and tolerance of imidocarb dipropionate versus atovaquone or buparvaquone plus azithromycin used to treat sick dogs naturally infected with the Babesia microti-like piroplasm.

BACKGROUND: Piroplasmosis caused by the Babesia microti-like piroplasm (Bml) is increasingly being detected in dogs in Europe. Sick dogs show acute disease with severe anaemia associated with thrombocytopenia with a poor response to current available drugs. This study assesses the safety and tolerance of three treatments and compares their efficacy over a full year of follow up in dogs naturally infected with Bml. METHODS: Fifty-nine dogs naturally infected with Bml were randomly assigned to a treatment group: imidocarb dipropionate (5 mg/kg SC, 2 doses 14 d apart) (IMI); atovaquone (13.3 mg/kg PO q 8 h, 10 d)/azithromycin (10 mg/kg PO q 24 h, 10 d) (ATO); or buparvaquone (5 mg/kg IM, 2 d apart)/azithromycin (same dosage) (BUP). Before and after treatment (days 15, 45, 90 and 360), all dogs underwent a physical exam, blood tests and parasite detection (blood cytology and PCR). Clinical efficacy was assessed by grading 24 clinical and 8 clinicopathological signs from low to high severity. RESULTS: Before treatment, most dogs had severe regenerative anaemia (88.13%) and thrombocytopenia (71.4%). On treatment Day 45, clinical signs were mostly reduced in all dogs, and by Day 90, practically all dogs under the ATO or BUP regimen were clinically healthy (76.4 and 88%, respectively). Highest percentage reductions in laboratory abnormalities (82.04%) were detected in animals treated with ATO. Over the year, clinical relapse of Bml was observed in 8 dogs (8/17) treated with IMI. However, on Day 360, these animals had recovered clinically, though clinicopathological abnormalities were still present in some of them. Parasitaemia was PCR-confirmed on Days 90 and 360 in 47.05 and 50% of dogs treated with ATO, 68 and 60.08% with BUP, and 94.1 and 73.3% with IMI, respectively. Even after 360 days, 13.3% of the dogs treated with IMI returned a positive blood cytology result. CONCLUSIONS: IMI showed the worse clinical and parasitological, efficacy such that its use to treat Bml infection in dogs is not recommended. The treatments ATO and BUP showed better efficacy, though they were still incapable to completely eliminate PCR-proven infection at the recommended dose. All three treatments showed good tolerance and safety with scarce adverse events observed.

First clinical case report of Cytauxzoon sp. infection in a domestic cat in France.

BACKGROUND: Feline cytauxzoonosis is an emerging infection caused by tick-transmitted apicomplexan parasites of the genus Cytauxzoon. The association of clinical disease with Cytauxzoon infection appears to be limited to C. felis infections in the Americas. Sporadic infections of wild and domestic felids with Cytauxzoon sp. were recently described in European countries but clinical reports of the infection are rare and incomplete. This case report brings new interesting information on cytauxzoonosis expression in Europe. CASE PRESENTATION: A 9-years-old castrated European shorthair cat living in rural area of north-eastern France (Saint Sauveur, Bourgogne-Franche-Comté region), without any travel history was presented for consultation due to hyperthermia, anorexia, depression and prolonged fever that didn't respond to antibiotic therapy. The cat had outdoor access with a history of vagrancy and was adequately vaccinated (core vaccines and FeLV vaccine). During biological investigations, intraerythrocytic inclusions were observed on blood smear and were further investigated by PCR analysis and sequencing. Molecular analyses confirmed Cytauxzoon sp. infection. The cat was treated with a subcutaneous injection of imidocarb dipropionate (3.5 mg/kg). One week after treatment, the cat improved clinically, although parasitic inclusions within erythrocytes persisted, and only a mild lymphocytosis was found. Two weeks after treatment, the cat appeared in excellent health, appetite was normal and parasitemia was negative. However, one month after treatment the cat relapsed with hyperthermia, anorexia, and depression. Blood smears and PCR were once again positive. Subsequently, the cat received an additional dose of imidocarb dipropionate (3.5 mg/kg SC) and recovered rapidly without other clinical signs. Two weeks after the second imidocarb injection, the cat was hit by a car and died. CONCLUSION: This case provides the first clinical description of infection by Cytauxzoon sp. in a domestic cat in France. These findings support the fact that cytauxzoonosis should be considered in the differential diagnosis of acute febrile illness which does not respond to antibiotic in cats with outdoor access especially in areas where populations of wild felids are present.

Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: Effects on enzymes of the antioxidant, cholinergic, and adenosinergic systems.

Anaplasmosis is a worldwide hemolytic disease in cattle caused by a gram-negative obligatory intracellular bacterium, characterized by anemia and jaundice. Among the treatments used for anaplasmosis is a drug called imidocarb dipropionate, also indicated as an immunomodulator agent. However, it causes side effects associated with increased levels of acetylcholine. In view of this, the effects of imidocarb dipropionate on the purinergic system, and antioxidant enzymes in animals naturally infected by Anaplasma marginale were evaluated. Young cattle (n = 22) infected by A. marginale were divided into two groups: the Group A consisted of 11 animals used as controls; and the Group B composed of 11 animals. Imidocarb dipropionate (5 mg/kg) was used subcutaneously to treat both groups (the Group A on day 6 and the Group B on day 0). The treatment reduced acetylcholinesterase (AChE), and adenosine deaminase (ADA) activities, and increased the dismutase superoxide and catalase activities. No changes on lipid peroxidation (TBARS levels) and BChE activities were noticed. These results suggest that imidocarb dipropionate used to treat A. marginale infection in cattle has effect on antioxidant enzymes, and significantly inhibits the enzymatic activities of ADA and AChE.

Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor.

BACKGROUND: The apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed interest in treatment options for infected horses. Imidocarb dipropionate is the current drug of choice, however variation in clinical response to therapy has been observed. METHODS: We quantified the in vitro susceptibility of two T. equi isolates and a lab generated variant to both imidocarb dipropionate and a bumped kinase inhibitor compound 1294. We also evaluated the capacity of in vitro imidocarb dipropionate exposure to decrease susceptibility to that drug. The efficacy of imidocarb dipropionate for clearing infection in four T. equi infected ponies was also assessed. RESULTS: We observed an almost four-fold difference in imidocarb dipropionate susceptibility between two distinct isolates of T. equi. Four ponies infected with the less susceptible USDA Florida strain failed to clear the parasite despite two rounds of treatment. Importantly, a further 15-fold decrease in susceptibility was produced in this strain by continuous in vitro imidocarb dipropionate exposure. Despite a demonstrated difference in imidocarb dipropionate susceptibility, there was no difference in the susceptibility of two T. equi isolates to bumped kinase inhibitor 1294. CONCLUSIONS: The observed variation in imidocarb dipropionate susceptibility, further reduction in susceptibility caused by drug exposure in vitro, and failure to clear T. equi infection in vivo, raises concern for the emergence of drug resistance in clinical cases undergoing treatment. Bumped kinase inhibitors may be effective as alternative drugs for the treatment of resistant T. equi parasites.

Cytauxzoon sp. infection in two free ranging young cats: clinicopathological findings, therapy and follow up.

Two young brother male free-ranging domestic shorthair cats were evaluated for diarrhea. They presented with intraerythrocytic piroplasms on blood smear evaluation. Only the first cat was anemic (mild non-regenerative anemia). A partial segment of the 18S rRNA was amplified and sequenced, revealing a homology of 99% with Cytauxzoon sp. and of 93% with Cytauxzoon felis. The first cat was treated with doxycycline and imidocarb dipropionate and monitored by serial laboratory exams, resulting negative for Cytauxzoon sp. infection after the end of the therapy (follow-up period of 175 days). The second cat received the same therapy, but doxycycline was discontinued by the owner after 1 week. He was monitored for 130 days, remaining erythroparasitemic and asymptomatic. We described cases of Cytauxzoon sp. infection in domestic cats with detailed clinical data, description of two therapeutic protocols, and follow-up after treatment with opposite parasitological responses (parasitological cure versus persistence of infection).