Continuous In Vitro Culture of Babesia duncani in a Serum-Free Medium.

Human babesiosis is an emerging tick-borne disease, caused by haemoprotozoa genus of Babesia. Cases of transfusion-transmitted and naturally acquired Babesia infection have been reported worldwide in recent years and causing a serious public health problem. Babesia duncani is one of the important pathogens of human babesiosis, which seriously endangers human health. The in vitro culture systems of B. duncani have been previously established, and it requires fetal bovine serum (FBS) to support long-term proliferation. However, there are no studies on serum-free in vitro culture of B. duncani. In this study, we reported that B. duncani achieved long-term serum-free culture in VP-SFM AGTTM (VP-SFM) supplemented with AlbuMaxTM I. The effect of adding different dilutions of AlbuMaxTM I to VP-SFM showed that 2 mg/mL AlbuMaxTM I had the best B. duncani growth curve with a maximum percentage of parasitized erythrocytes (PPE) of over 40%, and it can be used for long-term in vitro culture of B. duncani. However, the commonly used 20% serum-supplemented medium only achieves 20% PPE. Clearly, VP-SFM with 2 mg/mL AlbuMaxTM I (VP-SFMA) is more suitable for the in vitro proliferation of B. duncani. VP-SFM supplemented with CD lipid mixture was also tested, and the results showed it could support the parasite growth at 1:100 dilution with the highest PPE of 40%, which is similar to that of 2 mg/mL AlbuMaxTM I. However, the CD lipid mixture was only able to support the in vitro culture of B. duncani for 8 generations, while VP-SFMA could be used for long-term culture. To test the pathogenicity, the VP-SFMA cultured B. duncani was also subjected to hamster infection. Results showed that the hamster developed dyspnea and chills on day 7 with 30% PPE before treatment, which is similar to the symptoms with un-cultured B. duncani. This study develops a unique and reliable basis for further understanding of the physiological mechanisms, growth characteristics, and pathogenesis of babesiosis, and provides good laboratory material for the development of drugs or vaccines for human babesiosis and possibly other parasitic diseases.

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.

In vitro screening of novel anti-Babesia gibsoni drugs from natural products.

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoniin vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC50) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.

Pre-clinical evaluation of a whole-parasite vaccine to control human babesiosis.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis.

BACKGROUND: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. METHODS: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. RESULTS: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. CONCLUSIONS: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Vibrational Spectroscopic Based Approach for Diagnosing Babesia bovis Infection.

Babesia bovis parasites present a serious and significant health concern for the beef and dairy industries in many parts of the world. Difficulties associated with the current diagnostic techniques include the following: they are prone to human error (microscopy) or expensive and time-consuming (polymerase chain reaction) to perform. Little is known about the biochemical changes in blood that are associated with Babesia infections. The discovery of new biomarkers will lead to improved diagnostic outcomes for the cattle industry. Vibrational spectroscopic technologies can record a chemical snapshot of the entire organism and the surrounding cell thereby providing a phenotype of the organism and the host infected cell. Here, we demonstrate the applicability of vibrational spectroscopic imaging techniques including Atomic Force Microscopy Infrared (AFM-IR) and confocal Raman microscopy to discover new biomarkers for B. bovis infections. Furthermore, we applied Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) to detect B. bovis in red blood cells (RBCs). Based on changes in the IR spectral bands, with ATR-FTIR in combination with Partial Least Squares-Discriminant Analysis we were able to discriminate infected samples from controls with a sensitivity and specificity of 92.0% and 91.7%, respectively, in less than 2 min, excluding sample extraction and preparation. The proposed method utilized a lysis approach to remove hemoglobin from the suspension of infected and uninfected cells, which significantly increased the sensitivity and specificity compared to measurements performed on intact infected red blood cells (intact infected RBC, 77.3% and 79.2%). This work represents a holistic spectroscopic study from the level of the single infected RBC using AFM-IR and confocal Raman to the detection of the parasite in a cell population using ATR-FTIR for a babesiosis diagnostic.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Phytochemical Screening and Antiprotozoal Effects of the Methanolic Berberis vulgaris and Acetonic Rhus coriaria Extracts.

Berberis vulgaris (B. vulgaris) and Rhus coriaria (R. coriaria) have been documented to have various pharmacologic activities. The current study assessed the in vitro as well as in vivo inhibitory efficacy of a methanolic extract of B. vulgaris (MEBV) and an acetone extract of R. coriaria (AERC) on six species of piroplasm parasites. The drug-exposure viability assay was tested on three different cell lines, namely mouse embryonic fibroblast (NIH/3T3), Madin-Darby bovine kidney (MDBK) and human foreskin fibroblast (HFF) cells. Qualitative phytochemical estimation revealed that both extracts containing alkaloid, tannin, saponins and terpenoids and significant amounts of flavonoids and polyphenols. The GC-MS analysis of MEBV and AERC revealed the existence of 27 and 20 phytochemical compounds, respectively. MEBV and AERC restricted the multiplication of Babesia (B.) bovis, B. bigemina, B. divergens, B. caballi, and Theileria (T.) equi at the half-maximal inhibitory concentration (IC50) of 0.84 ± 0.2, 0.81 ± 0.3, 4.1 ± 0.9, 0.35 ± 0.1 and 0.68 ± 0.1 µg/mL and 85.7 ± 3.1, 60 ± 8.5, 90 ± 3.7, 85.7 ± 2.1 and 78 ± 2.1 µg/mL, respectively. In the cytotoxicity assay, MEBV and AERC inhibited MDBK, NIH/3T3 and HFF cells with half-maximal effective concentrations (EC50) of 695.7 ± 24.9, 931 ± 44.9, ˃1500 µg/mL and 737.7 ± 17.4, ˃1500 and ˃1500 µg/mL, respectively. The experiments in mice showed that MEBV and AERC prohibited B. microti multiplication at 150 mg/kg by 66.7% and 70%, respectively. These results indicate the prospects of these extracts as drug candidates for piroplasmosis treatment following additional studies in some clinical cases.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 µM concentration as baseline revealed nine effective compounds against four tested parasites. Two "hit" compounds, namely MMV021057 and MMV675968, that showed IC50 < 0.3 µM and a selectivity index (SI)> 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs.

The effects of trans-chalcone and chalcone 4 hydrate on the growth of Babesia and Theileria.

BACKGROUND: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 µM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 µM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 µM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 µM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC-DA and TC-CF were more potent against B. microti infection in mice than their monotherapies. CONCLUSIONS/SIGNIFICANCE: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies.

Artificial feeding of Rhipicephalus microplus female ticks with anti calreticulin serum do not influence tick and Babesia bigemina acquisition.

Ticks are obligate haematophagous ectoparasites considered the principal vectors of disease among animals. Rhipicephalus microplus and R. annulatus ticks are the most important vectors for Babesia bigemina and B. bovis, two of the most important intraerythrocytic protozoan parasites species in cattle, responsible for babesiosis which together with anaplasmosis account for substantial economic losses in the livestock industry worldwide. Anti-tick vaccines are a proved alternative to traditional tick and tick borne diseases control methods but are still limited primarily due to the lack of effective antigens. Subsequently to the identification of antigens the validation is a laborious work often expensive. Tick artificial feeding, is a low cost alternative to test antigens allowing achieving critical data. Herein, R. microplus females were successfully artificially fed using capillary tubes. Calreticulin (CRT) protein, which in a previous study has been identified as being involved in B. bigemina infection in R. annulatus ticks, was expressed as recombinant protein (rCRT) in an E. coli expression system and antibodies raised against rCRT. Anti-rCRT serum was supplemented to a blood meal, offered to partially engorged R. microplus females and their effect in feeding process as well as infection by B. bigemina was analyzed. No significant reductions in tick and egg weight were observed when ticks fed with anti-rCRT serum. Furthermore, B. bigemina infection levels did not show a statistically significant decrease when ticks fed with anti-rCRT antibodies. Results suggest that CRT is not a suitable candidate for cattle vaccination trials.

Molecular detection and characterization of potentially new Babesia and Theileria species/variants in wild felids from Kenya.

Piroplasms frequently infect domestic and wild carnivores. At present, there is limited information on the occurrence and molecular identity of these tick-borne parasites in wild felids in Kenya. In 2009, a pair of captive lions (Panthare leo) was diagnosed with suspected babesiosis and mineral deficiency at an animal orphanage on the outskirts of Nairobi, Kenya. Blood smears indicated presences of haemoparasites in the erythrocytes, however, no further investigations were conducted to identify the infecting agent. The animals recovered completely following diet supplementation and treatment with anti-parasite drug. In this report, we extracted and detected parasite DNA from the two lions and seven other asymptomatic feline samples; two leopards (Panthera pardus) and five cheetahs (Acinonyx jubatus). Reverse line blot with probes specific for Babesia spp. of felines indicated the presence of new Babesia species or genotypes in the lions and leopards, and unknown Theileria sp. in the cheetahs. Phylogenetic analyses using partial sequences of 18S ribosomal RNA (18S rRNA) gene showed that the parasite infecting the lions belong to the Babesia canis complex, and the parasite variant detected in the leopards clusters in a clade bearing other Babesia spp. reported in wild felids from Africa. The cheetah isolates falls in the Theileria sensu stricto group. Our findings indicate the occurrence of potentially new species or genotypes of piroplams in all three feline species.

The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay.

Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.

Curcuma as a parasiticidal agent: a review.

Members of the Curcuma plant species (Zingiberaceae) have been used for centuries in cooking, cosmetics, staining and in traditional medicine as "omnipotent" remedies. Herbal preparations made with, and molecules extracted from, Curcuma have been shown to possess a wide variety of pharmacological properties against malignant proliferation, hormonal disorders, inflammation, and parasitosis among other conditions. This review evaluates Curcuma and its associated bioactive compounds, particularly focusing on studies examining the parasiticidal activity of these components against the tropical parasites Plasmodium, leishmania, Trypanosoma, Schistosoma and more generally against other cosmopolitan parasites (nematodes, Babesia, Candida, Giardia, Coccidia and Sarcoptes).

Use of a doxycycline-enrofloxacin-metronidazole combination with/without diminazene diaceturate to treat naturally occurring canine babesiosis caused by Babesia gibsoni.

Canine babesiosis is an important worldwide, tick-borne disease caused by hemoprotozoan parasites of the genus Babesia. Babesia gibsoni is the predominant species that causes canine babesiosis in Taipei, Taiwan. It is a small pleomorphic intraerythrocytic parasite that can cause erythrocyte destruction and hemolytic anemia. Efficacy of oral administration of a doxycycline-enrofloxacin-metronidazole combination with and without injections of diminazene diaceturate in the management of naturally occurring canine babesiosis caused by B. gibsoni was evaluated retrospectively. The overall efficacy of this combination of doxycycline-enrofloxacin-metronidazole in conjunction with and without administration of diminazene diaceturate was 85.7% and 83.3%, respectively; with a mean recovery time of 24.2 and 23.5 days, respectively. Concomitant use of intramuscular diminazene diaceturate may not improve the efficacy of a doxycycline-enrofloxacin-metronidazole combination in management of canine babesiosis caused by B. gibsoni.

Inhibitory effects of (-)-epigallocatechin-3-gallate from green tea on the growth of Babesia parasites.

(-)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and accounts for 50-80% of the total catechin in green tea. (-)-Epigallocatechin-3-gallate has antioxidant, anti-inflammatory, anti-microbial, anti-cancer, and anti-trypanocidal activities. This report describes the inhibitory effect of (-)-Epigallocatechin-3-gallate on the in vitro growth of bovine Babesia parasites and the in vivo growth of the mouse-adapted rodent babesia B. microti. The in vitro growth of the Babesia species was significantly (P<0.05) inhibited in the presence of micromolar concentrations of EGCG (IC50 values=18 and 25 microM for B. bovis, and B. bigemina, respectively). The parasites showed no re-growth at 25 microM for B. bovis and B. bigemina in the subsequent viability test. The drug significantly (P<0.05) inhibited the growth of B. microti at doses of 5 and 10 mg/kg body weight, and the parasites completely cleared on day 14 and 16 post-inoculation in the 5 and 10 mg/kg treated groups, respectively. These findings highlight the potentiality of (-)-Epigallocatechin-3-gallate as a chemotherapeutic drug for the treatment of babesiosis.

Clinical management of babesiosis in dogs with homeopathic Crotalus horridus 200C.

Homeopathic Crotalus horridus 200C was evaluated in 13 clinical cases of babesiosis in dogs, compared with another 20 clinical cases treated with diminazine. Babesiosis is an important tropical tick-borne haemoprotozoan disease in dogs clinically manifested by anorexia, dehydration, temperature, dullness/depression, diarrhoea/constipation, pale mucosa, hepatomegaly, vomiting/nausea, splenomegaly, distended abdomen/ascites, yellow coloured urine, emaciation/weight loss, and occular discharge. The diagnosis of babesiosis was based on cytological evidence of Babesia gibsoni in freshly prepared blood smears. The dogs were treated with oral C. horridus 200C, 4 pills four times daily for 14 days (n=13) or diminazine aceturate 5 mg/kg single intramuscularly dose (n=20). All the dogs were administered 5% Dextrose normal saline at 60 ml/kg intravenously for 4 days. Initial clinical scores were similar in both groups and showed similar progressive improvement with the two treatments over 14 days. Parasitaemia also improved in both groups, but haematological values showed no change. No untoward reactions were observed. It appears that C. horridus is as effective in causing clinical recovery in moderate cases of canine babesiosis caused by Babesia gibsoni as the standard drug diminazine. Large scale randomized trials are indicated for more conclusive results.

Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp.

A review is given on the Babesia, Theileria, and Anaplasma species infecting sheep and goats. B. ovis is the most important disease agent. It is transmitted by Rhipicephalus bursa, R. turanicus, Hyalomma anatolicum excavatum, and probably by R. evertsi evertsi B. ovis is widely spread in southern Europe, the Middle East, and central Asia. Its geographical distribution in South and East Asia and in Africa is widely unknown. B. motasi obviously represents several nosodemes in separate regions. It is not pathogenic for intact sheep in northern Europe, whereas it is probably more pathogenic than B. ovis in India and northern Africa. The known vectors of B. motasi are Haemaphysalis punctata and R. bursa. Theileria hirci is transmitted by H. a. anatolicum but occurs outside the distribution area of this tick. Malignant theileriosis of sheep and goats is an important disease in Iraq, Iran, and India. An attenuated macroschizont vaccine is successfully being used in Iran. Anaplasma ovis is transmitted by R. bursa and probably other ticks in the Old World and by Dermacentor andersoni in the New World. A. ovis is widely spread in the Old World. Outbreaks occur only under extreme conditions. The identity of the tick-borne disease agents of sheep and goats and of their vector ticks is uncertain in many regions of the Old and the New World.

Evaluation of selected antiprotozoal drugs in the Babesia microti-hamster model.

The presently used therapy for Babesia microti infections, a combination of quinine and clindamycin, does not always result in parasitologic cures. To identify possible alternative chemotherapeutic agents for such infections, we screened, in the hamster-B. microti system, 12 antiprotozoal drugs that have either recently been released for human use or were in experimental stages of development at the Walter Reed Army Institute of Research for the treatment of malaria and leishmaniasis. Several well-recognized antimalarial drugs, such as mefloquine, halofantrine, artesunate, and artelenic acid, exhibited little or no effect on parasitemia. Two 8-aminoquinolines, WR006026 [8-(6-diethylaminohexylamino)-6-methoxy-4-methylquinoline dihydrochloride] and WR238605 [8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5 -(3-trifluoromethylphenoxy-7) quinoline succinate], produced clearance of patent parasitemia. Furthermore, blood from infected hamsters treated with WR238605 via an intramuscular injection failed to infect naive hamsters on subpassage, thus producing a parasitologic cure. These two compounds merit further screening in other systems and may prove useful in treating human babesiosis.