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Two haemoprotozoan parasites, Babesia caballi and Babesia equi, can cause equine piroplasmosis. Due to the presence of potential tick vectors in areas so far unaffected by equine babesias, import and export regulations often require the serum testing of animals for evidence of infection. Although the complement fixation test (CFT) has been recommended for detecting the presence of antibodies to Babesia spp., it has been demonstrated to have several disadvantages, including false-positive results and low sensitivity for detecting latent infections. An enzyme-linked immunosorbent assay (ELISA) may be an alternative for increased and sensitive detection of acute and latent babesial infections, but its development to date has been hindered by a limited antigen supply and poor specificity. In vitro cultivation of both parasite species and the identification of parasite proteins for diagnostic use has facilitated the development of a highly sensitive and specific ELISA. For the direct detection of the parasites, DNA probes are now available. Several drugs are available for the treatment of equine piroplasmosis. For instance, diminazene diaceturate is effective in the chemosterilization of B. caballi and in the elimination of clinical signs in B. equi infections. Antitheilericidal drugs such as buparvaquone have been demonstrated to be effective in combatting disease due to B. equi and may--in combination with imidocarb--also eliminate the parasite. The control of equine piroplasmosis must include effective tick control, seromonitoring of animals and the application of chemotherapy.

Aim: To study the prevalence of Theileria equi among horses in different age groups, both sexes, months and seasons of the year, and regions of Giza governourate. Studying the changes in the blood picture, blood chemistries, liver enzymes associate with T.equi infections in horses. Evaluating IFA and CFT at different dilutions in the serodiagnosis of T.equi infections in horses. Evaluating four anti-Theileria medication regimens (diminazine aceturate, imidiocarb 7%, buparvaquone and a combination of imidiocarb 7% and buparvaquone) in treatment of T.equi infections in horses. Materials and Methods: Total of 149 horses were examined by clinical signs and blood smears. Fortey whole blood samples from T.equi infected horses were examined to measure haemoglobin, total RBCs count and PCV. Fortey serum samples from T.equi infected horses were examined to measure total bilirubin, direct bilirubin, ALT and AST enzymes. Serum samples from T.equi infected (40) and non infected (14) horses were tested by indirect fluorescent antibody test (IFA) and complement fixation test (CFT) at different dilutions. Four groups of T.equiinfected horses (A,B,C,D), each group was represented by 10 horses and was separately treated with diminazine aceturate, imidiocarb 7%, buparvaquone and a combination of imidiocarb 7% and buparvaquone respectively. Results: the prevalence of T.equi was 41.61% in totally examined horses. The prevalence was higher in males than females. The highest prevalence was among age group ranged from 5-10 years as (22.81%). The highest prevalence was in July and was recorded as (25.81%) and the disease was more prevalent in summer than winter. The highest prevalence was recorded in Nazlet-alsamman as (51.61%). Equine theileriosis was clinically characterized by fever, haemoglobinuria, oedema, anaemia and icterus. The best dilution for IFA was 1/160 where sensitivity, specificity and accuracy were the highest for this test as (98%), (92.86%) and (97.44%) respectively. The best dilution for CFT was 1/32 where sensitivity, specificity and accuracy were collectively the best as (90%), (92.86%) and (90.74%) respectively. Conclusion: It was concluded that T.equi is prevalent among horses in Giza governourate, its prevalence is varied according to the age, sex of horses, months, seasons and regions. T.equiinfections in horses are accompanied with changes in blood pictures, blood chemistries and liver enzymes. Both IFA and CFT could be used for the serodiagnosis of T.equi . The used four anti-Theileria medication regimens have the same ability to eradicate T.equi from the infected horses. [Vet World 2013; 6(2.000): 76-82]

We have previously developed a mouse model which allowed the proliferation of Theileria sergenti in severe combined immunodeficiency (SCID) mice with circulating bovine erythrocytes (Bo-RBC). In the present study, this model was utilized to test the efficacy of anti-theilerial drugs. Bo-RBC-SCID mice were created by giving periodic transfusions of T. sergenti-free Bo-RBC, and subsequently infecting with T. sergenti. Three anti-protozoal compounds, Pamaquine (Yamanouchi Pharmaceutical Co. Ltd), Ganaseg (Japan CIBA-GEIGY Ltd) and Buparvaquone (Coopers Animal Health Ltd), were subcutaneously administered into the mice at doses recommended for cattle therapy. Blood examinations demonstrated that all three drugs significantly reduced the level of parasitemia although Ganaseg was effective only at a dose five times higher than that recommended for cattle therapy. Administration of the drugs neither caused any sign of acute toxicity nor changed the rate of Bo-RBC in the SCID mice's circulating blood cells. The results indicate that the Bo-RBC-SCID mouse model may offer a useful in vivo system for evaluating the efficacy of anti-protozoal drugs against T. sergenti.

Groups of calves were infected by the injection of ground-up-tick supernatant from ticks infected with ODE-Anand stock of Theileria annulata, the causative agent of tropical theileriosis. Treatment with long-acting oxytetracycline, at 20 mg kg-1, injected intramuscularly, had no effect against severe Theileria annulata infection when administered either as a single injection on the day of infection or as three injections given on days 8, 10 and 12 after infection. Halofuginone lactate, given orally at 1.2 mg kg-1 was effective but caused anorexia, diarrhoea and debility. Parvaquone at 20 mg kg-1 intramuscularly given on day 11 after infection, had a marked suppressive effect, while buparvaquone was highly effective. A single treatment with buparvaquone, either at 5 mg kg-1 or 2.5 mg kg-1 intramuscularly, rapidly eliminated schizonts and piroplasms of T annulata. At 5 mg kg-1 it resulted in rapid recovery of all the treated calves.

A clinical trial was conducted to test buparvaquone (Butalex; Coopers Pitman-Moore) in the treatment of East Coast fever under field conditions in Kenya. Data from 229 cases were analysed following treatment with one (69), two (142) or three (18) doses at 2.5 mg/kg. The majority of cattle (95.2 per cent) were exotic (Bos taurus) or improved (Bos taurus cross Bos indicus) and 39.3 per cent were infected with Anaplasma marginale. There was an overall recovery rate of 85.6 per cent, with 90.1 per cent recovering following one treatment and 75.4 per cent recovering following two treatments. At a follow-up visit three to six months after completion of the trial data was obtained on 224 cases. Thirty had died, 13 of which were reported to have been from East Coast fever, nine had been sold and six slaughtered. Of the remaining 146, 86.3 per cent were in good condition, 13.7 per cent fair and 2.0 per cent in poor condition. A two dose regimen was most effective and should be recommended except in very early cases or those under direct veterinary supervision.

Theileria annulata-infected cells were cultured in the presence or absence of human recombinant interleukin 2 (hrIL-2). This growth factor proved to be capable of enhancing the growth of the infected cells: its effect was marked, particularly when the cells were seeded at low densities, and it varied from cell line to cell line. The infected cells produced a factor that possessed the biological activities of IL-2, since their supernatants could enhance the proliferation of concanvalin A-stimulated (Con A) blasts. The reactivity of the parasitized cells to hrIL-2 was abolished following their treatment with the antitheilerial drug buparvaquone. In addition, the drug inhibited the binding of 125I-IL-2 to T. annulata-infected cells but failed to suppress its binding to Con A blasts. Northern blot analysis revealed that the drug had no effect on the expression of the alpha chain of the IL-2 receptor (IL-2R). Therefore, it is possible that buparvaquone interferes with the expression of the beta chain of the IL-2R. The role of IL-2 and the IL2R in the permanent proliferation of T. annulata-infected cells is discussed.

The prevalence of haemoprotozoan disease in crossbred cattle was studied by screening 132 clinically suspected blood samples by Geimsa’s stained blood smear method. Among 132 crossbred cattle screened, 57 animals were found positive for haemoprotozoan parasites. Out of 57 positive cases, 41 were found positive for Theileria annulata and the remaining 16 were positive for Babesia bibemina. The highest prevalence was found in 4-6 year age group and in monsoon months. The hematological observation revealed reduced Hb, TEC and PCV in the infected animals. The treatment with buparvaquone and oxytetracyclin for theileriosis and diminazine aceturate for babesiosis are found effective.

Thirty-seven high grade cattle were immunised against Corridor disease (Theileria parva lawrencei infection) on a farm with a history of heavy and often lethal theilerial challenge. Nineteen cattle were immunised by treating with two doses of long-acting oxytetracyclines given at 20 mg/kg on days 0 and 4 after sporozoite stabilate inoculation, while the other 18 were treated with naphthoquinone buparvaquone, given as a single dose of 2.5 mg/kg simultaneously with stabilate inoculation. All the cattle underwent subclinical theilerial reactions with all but two developing high antibody titres on the IFAT test against T. parva schizont antigen by day 35 after the immunisation. Both buparvaquone and long-acting oxytetracycline appeared equally effective in the immunisation. To date, 26 months later, only two cases of theileriosis parasitologically characteristic of T. p. parva have been reported in the immunised cattle. Following the two cases, investigations showed that when uninfected Rhipicephalus appendiculatus nymphal ticks were deliberately fed on healthy resident cattle on the farm, the resultant adult ticks transmitted acute and lethal theilerial infections to five out of five susceptible cattle. The resultant infections were parasitologically characteristic of T. p. parva infections. Furthermore, the monoclonal antibody profiles of schizont infected cell lines from these infections appeared to be characteristic of T. p. parva. It was thus concluded that resident cattle on the farm could be a potential source of T.p. parva infection which had broken through the immunity of T.p. lawrencei immunised cattle and could constitute a reservoir of theilerial infection for ticks and hence to susceptible stock on the farm.

Novel hydroxynaphthoquinones are reported with outstanding efficacy against Plasmodium, Eimeria and Theileria species. Biochemical evidence is presented for the selective toxicity of these compounds being due to inhibition of parasite respiratory systems.

The antitheilerial activity of buparvaquone (BW 720C) was evaluated in experimentally induced Theileria annulata infections in cross-bred male calves. T. annulata infections were induced by injecting a suspension of infected ground tick tissue suspension (GUTTS) equivalent to two ticks subcutaneously into each calf. Buparvaquone at a dose of 2.5 mg kg-1 body weight was given as a single injection (intramuscularly) on Day 0 (Group 1), Day 8 (Group 2) and Day 12 (Group 3) post-infection. The animals in Groups 4 and 5 were untreated and challenged controls, respectively. All of the recovered animals from Groups 1-4 were challenged with a lethal dose of T. annulata at 6 weeks post-infection. The immunized animals were resistant to the homologous challenge, which killed three of four control animals (Group 5); the controls showed typical antemortem and post-mortem lesions of theileriosis.

Twenty cross-bred (Bos taurus X Bos indicus) calves, 7-21 days old, were infected by a ground-up tick supernate of Hyalomma anatolicum anatolicum infected with the Hisar isolate of Theileria annulata. Six calves acted as untreated controls and they all died of theileriosis within 17 days of infection. The remaining 14 calves were divided into Group A and B, each consisting of seven calves. All the calves of Groups A and B were treated intramuscularly with buparvaquone (BW 720C) on Day 11 post-infection, when clinical signs of theileriosis were apparent. Each calf received 2.5 mg BW 720 C kg-1 body weight as a single injection. In addition, each calf of Group B was given proprietary haematinics by intramuscular injection, daily for 12 days. In Group A, two calves died of cerebral theileriosis and five were clinically cured. However, four of these five calves later died of anaemia. In Group B, all the calves were clinically cured and none died during the observation period of 1 month. The parasitaemia declined to less than 1% within a fortnight of treatment. The initial declines in haemoglobin concentration and packed cell volume were halted and preinfection values were soon restored. No toxic signs attributable to treatment with buparvaquone were observed.

Chronic, undulating Theileria annulata infection was diagnosed as the cause of severely depressed milk yields in a herd of 44 Friesian cows in Egypt. The herd was divided at random into two groups of 22; one group was injected intramuscularly with a single dose of the experimental antitheilerial drug, buparvaquone, while the other remained untreated. Over the subsequent seven weeks milk yield in the treated group increased to double that of the untreated group (P less than 0.05). Pyrexia was controlled within two days of injection of buparvaquone and piroplasm parasitaemia was eliminated in one week. It is suggested that treatment of dairy cattle chronically infected with T. annulata using buparvaquone may have the dual beneficial effect of reducing the pathogenic effects of theileriosis, thereby permitting restoration of an impaired immune system, thus increasing resistance to other infections. If a similar effect could be produced in Bos indicus cattle in T. annulata endemic areas, treatment of indigenous cattle with buparvaquone could be a useful alternative to the introduction of Bos taurus blood as a way of boosting milk production.

About 200 million cattle are believed to be at risk from the debilitating and often fatal effects of tropical theileriosis, caused by Theileria annulata. Currently, there is no very cheap effective drug for treatment of T. annulata infections, although the hydroxynophthoquinones parvaquone and buparvaquone are reported to give good results 1−4 . Control of the parasite principally involves vector control against the ixodid tick vectors — mainly by cattle dipping and spraying with acaricides — and vaccination using attenuated macroschizont-infected leucocytes 1–17 (see Box I). In this article, Roger Hall discusses the nature of immunity that can be achieved against T. annulata , and progress in identifying the main antigens involved in this immunity.

SummaryA Theileria parva lawrencei isolate in the form of a sporozoite stabilate, derived by feeding clean Rhipicephalus appendiculatus nymphal ticks on an African buffalo (Syncerus caffer) captured in the Laikipia District, Kenya, was inoculated into groups of cattle at dilutions between 100 and 10-3. Groups of 3 cattle infected with 1 ml inocula at 100, 10-1 and 10-2 dilutions were treated with 2·5 mg/kg body weight of buparvaquone on day 0 and similar groups were left untreated to act as controls. An additional group, given 100 dilution of the stabilate, was treated with buparvaquone on day 8 post-inoculation. It was found that all control cattle inoculated with the stabilate at dilutions between 100 and 10-2 became infected, but only 2 out of 3 cattle developed patent infections at 10-3 dilution. All 3 control cattle receiving 100 dilution died of theileriosis, 2 at 10-1 and 10-2 dilutions, and 1 at 10-3 dilution died. Buparvaquone treatment on day 0 at 100 dilution resulted in the survival of 2 of 3 cattle and all the cattle at 10-1 and 10-2 dilutions. All the surviving cattle eventually developed a significant serological response against T. parva in the indirect fluorescent antibody test, except 1 in the 10-3 dilution group, and were immune to homologous challenge when tested 3 months later with a lethal inoculum of stabilate, except 2 cattle in the 10-3 dilution group. As a result of a theileriosis problem at about day 60 after inoculation in 2 cattle given 10-2 dilution of stabilate and buparvaquone treatment on day 0, an additional 5 cattle were given 10-2 dilution of stabilate and developed a good immunity after buparvaquone treatment. None was shown to develop the carrier state. Treatment with buparvaquone on day 8 after infection with 100 dilution of stabilate was not successful since 2 died. The stabilate used was shown to produce reproducible infection in cattle at different dilutions.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the lethal disease alveolar echinococcosis. Current chemotherapeutic treatment options are based on benzimidazoles (albendazole and mebendazole), which are insufficient and hence alternative drugs are needed. In this study, we screened the 400 compounds of the Medicines for Malaria Venture (MMV) Pathogen Box against E. multilocularis metacestodes. For the screen, we employed the phosphoglucose isomerase (PGI) assay which assesses drug-induced damage on metacestodes, and identified ten new compounds with activity against the parasite. The anti-theilerial drug MMV689480 (buparvaquone) and MMV671636 (ELQ-400) were the most promising compounds, with an IC50 of 2.87 μM and 0.02 μM respectively against in vitro cultured E. multilocularis metacestodes. Both drugs suggested a therapeutic window based on their cytotoxicity against mammalian cells. Transmission electron microscopy revealed that treatment with buparvaquone impaired parasite mitochondria early on and additional tests showed that buparvaquone had a reduced activity under anaerobic conditions. Furthermore, we established a system to assess mitochondrial respiration in isolated E. multilocularis cells in real time using the Seahorse XFp Analyzer and demonstrated inhibition of the cytochrome bc1 complex by buparvaquone. Mice with secondary alveolar echinococcosis were treated with buparvaquone (100 mg/kg per dose, three doses per week, four weeks of treatment), but the drug failed to reduce the parasite burden in vivo. Future studies will reveal whether improved formulations of buparvaquone could increase its effectivity.

Visceral leishmaniasis is a vector-borne protozoan infection that is fatal if untreated. There is no vaccination against the disease, and the current chemotherapeutic agents are ineffective due to increased resistance and severe side effects. Buparvaquone is a potential drug against the leishmaniases, but it is highly hydrophobic resulting in poor bioavailability and low therapeutic efficacy. Herein, we loaded the drug into silicon nanoparticles produced from barley husk, which is an agricultural residue and widely available. The buparvaquone-loaded nanoparticles were several times more selective to kill the intracellular parasites being non-toxic to macrophages compared to the pure buparvaquone and other conventionally used anti-leishmanial agents. Furthermore, the in vivo results revealed that the intraperitoneally injected buparvaquone-loaded nanoparticles suppressed the parasite burden close to 100%. By contrast, pure buparvaquone suppressed the burden only by 50% with corresponding doses. As the conclusion, the biogenic silicon nanoparticles are promising carriers to significantly improve the therapeutic efficacy and selectivity of buparvaquone against resistant visceral leishmaniasis opening a new avenue for low-cost treatment against this neglected tropical disease threatening especially the poor people in developing nations.

The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.

Chemotherapy of East Coast fever, a lymphoproliferative cancer-like disease of cattle causing significant economic losses in Africa, is largely dependent on the use of buparvaquone, a drug that was developed in the late 1980's. The disease is caused by the tick-borne protozoan pathogen Theileria parva. Buparvaquone can be used prophylactically and it is also active against tropical theileriosis, caused by the related parasite Theileria annulata. Recently, drug resistance was reported in T. annulata, and could occur in T. parva. Using a 3H-thymidine incorporation assay we screened 796 open source compounds from the Medicines for Malaria Venture (MMV) to discover novel chemicals with potential inhibitory activity to T. parva. We identified nine malaria box compounds and eight pathogen box compounds that inhibited the proliferation of F100TpM, a T. parva infected lymphocyte cell line. However, only two compounds, MMV008212 and MMV688372 represent promising leads with IC50 values of 0.78 and 0.61 μM, respectively, and CC50 values > 5 μM. The remaining compounds exhibited a high degree of toxicity (CC50 values 10 μM. Our preliminary data suggest that it may be possible to repurpose compounds from the cancer field as well as MMV as novel anti-T. parva molecules.

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.