Drug-resistant Trypanosoma congolense in naturally infected donkeys in north Omo Zone, southern Ethiopia

A three-part study was conducted to determine the efficacy of isometamidium chloride in donkey populations naturally infected with trypanosomes in north Omo Zone, southern Ethiopia. In the first, 373 randomly selected donkeys from four villages were examined for trypanosome infections by the dark ground/phase contrast buffy coat technique (BCT) in November 1999. The trypanosome prevalence was 18.2% (95% confidence interval (CI): 14.4, 22.5) and Trypanosoma congolense was the most common species accounting for 66.2% of the overall infections. In the second part, 40 infected donkeys were selected and treated with a prophylactic dose of 1.0mg/kg of isometamidium chloride and thereafter monitored every 14 days for 90 days. Trypanosomes were detected in eight donkeys within 1 month and in 20 donkeys within 2 months of treatment. About 16% (5/32) of donkeys infected with T. congolense were detected parasitemic 1 month after treatment. In addition, the result also revealed that all relapse/breakthrough infections were due to T. congolense. In the third part of this study mice were infected with two T. congolense field isolates from donkeys that were found to be parasitemic within 1 or 2 months after isometamidium treatment. The mice were treated with ranges of doses of isometamidium chloride or diminazene aceturate and thereafter followed for relapse infection. Isometamidium chloride at doses 0.5-4 mg/kg body weight and diminazene aceturate at doses of 3.5-28 mg/kg body weight failed completely to cure T. congolense infections in any of the mice.     

Response of Trypanosoma congolense in goats to single and double treatment with diminazene aceturate.

Diminazene aceturate is one of a limited number of compounds currently marketed for treatment of trypanosomiasis in cattle, sheep and goats. The pharmacokinetics of the compound in goats suggest that double treatment with diminazene aceturate might enhance the compound's therapeutic activity. A study was therefore conducted in goats using two clones of Trypanosoma congolense, IL 3274 and IL 1180, which were previously shown to be resistant and sensitive, respectively, to single treatment with diminazene aceturate. The results indicated that, as compared to single treatment, double treatment with diminazene aceturate at a dose of 7.2 mg kg-1 bodyweight, at either eight or 24 hour intervals, did not greatly enhance the therapeutic activity of the drug. Furthermore, treatment with the same drug dose eliminated infections with T congolense IL 3274 when treatment was administered 24 hours after infected Glossina morsitans centralis had fed, but failed to do so if treatment was delayed until after goats were detected to be parasitaemic. This suggests that failure of T congolense IL 3274 to respond to treatment with diminazene may not be due to drug resistance per se.     

Treatment of experimental trypanosomiasis in pigs.

The therapeutic activity of difluoromethylornithine (DFMO), diminazene aceturate (Berenil) and their combination against chronic trypanosomiasis was investigated in experimental Trypanosoma brucei brucei infections of growing pigs. DFMO (300 mg/kg/day orally for 10 days), diminazene aceturate (7 mg/kg in single intramuscular injection) and a combination of the two agents at the above dosages produced varied periods of aparasitaemia in the treated pigs. Relapse parasitaemia occurred in all treatment groups, with diminazene aceturate providing the longest relief period of 17 days, combination treatment 11 days and DFMO 6 days. The packed cell volume, blood haemoglobin concentration and red cell count values decreased after the pigs were infected with the parasites. The values improved following treatment with the agents and their combination.     

Development of an enzyme-linked immunosorbent assay for the detection and measurement of the trypanocidal drug isometamidium chloride in cattle

An enzyme-linked immunosorbent assay (ELISA) was developed to measure accurately levels of the trypanocidal drug isometamidium in the serum of treated cattle. The assay requires only 5 microliters of test serum, is sensitive to a level of 0.5 pg ml-1 and is highly specific. Cross reactivity does not occur with the two other widely used trypanocidal drugs diminazene aceturate and homidium bromide. Serum drug levels are detectable for up to six months in cattle after a single dose of 1 mg kg-1 intramuscularly, the maximum period under field conditions for which effective prophylaxis can be maintained against tsetse challenge. Application of the assay will aid the rationalisation of treatment campaigns and assist in assessing the occurrence of drug-resistant trypanosome populations.     

Therapeutic effect of Berenil and Samorin in mice infected with four trypanosome populations isolated from Zambian cattle

Four populations of Trypanosoma congolense and Trypanosoma brucei brucei were isolated from cattle under different management practices and environments in Zambia. All four isolates had varied responses to both diminazene aceturate (Berenil) and isometamidium chloride (Samorin) as curative drugs in infected mice. Trypanosomes from a traditionally managed herd in a high-tsetse-challenge area had the strains most resistant to Berenil, with maximum curative dose of 45 mg kg-1 body weight. Another isolate from a high-tsetse-challenge area was evidently resistant both to Berenil at 40 mg kg-1 and to Samorin at 4 mg kg-1. The strains most susceptible to both Berenil and Samorin were from a commercially managed herd of cattle under medium tsetse challenge. They responded to recommended cattle standard doses of 3.5 mg kg-1 or 7 mg kg-1 Berenil and to as little as 0.25 mg kg-1 Samorin. It is evident that trypanosome strains resistant to Berenil and/or partially resistant to Samorin exist, and that both T. congolense and T. b. brucei are implicated.     

The Effect of Diminazene Aceturate on Spienectomized Dogs with Babesia gibsoni Infection

The effect of diminazene aceturate on splenectomized and nonspienectomized dogs with Babesia gibsoni (B. gibsoni) infection was investigated. In splenectomized dogs, the fissional and multiplicational stages of B. gibsoni were observed in peripheral blood films, and hemoglobinuria was frequently observed. These findings were different from previous reports and were not changed by administration of diminazene aceturate. It is clear that the intramuscular administration of diminazene aceturate at the dose of 3 mg/kg body weight for 3 days is not effective against B. gibsoni infection in splenectomized dogs.     

Chemotherapy of T b brucei infection: Use of DFMO, diminazene aceturate, alone and in combination

The therapeutic activity of diminazene aceturate, difluoromethylornithine (DFMO) and a combination of the two agents was investigated in experimental Trypanosoma brucei brucei infections in mongrel dogs. The criteria used in the assessment of the trypanocidal effect of these compounds included the examination of the blood for the parasite, as well as clinical and haematological changes at intervals following treatment. Diminazene aceturate (7 mg/kg intramuscularly), DFMO (300 mg/kg/day orally in three divided doses for six days) and the combination of diminazene aceturate (7 mg/kg intramuscularly) and DFMO (300 mg/kg/day orally for six days) produced an intermittent aparasitemia in the dogs. Relapse infection occurred in all the three groups, but the period of aparasitemia produced by the combination of the agents was longest. The packed cell volume, haemoglobin concentration and red cell count values decreased after the dogs were inoculated with the parasite. The values improved slightly following the treatments with the agents or their combination. The total white blood cell counts in the infected dogs indicated leucocytosis, but this improved with drug treatment.     

中西药治疗牛环形泰勒虫病的效果观察

托克逊县是牛环形泰勒虫病的疫区之一,准确诊断以及有效治疗是防控该病的要点。论文采用流行病学调查、实验室方法检测和临床诊断的方法综合确诊45头舍饲患牛作为研究对象。将45头阳性牛随机分为西药治疗组、中药治疗组以及空白组,按1、2、3、4、5d以及1周,分别对每组患牛进行临床症状观察、体温测定、血涂片观察、染虫率统计,评价治疗效果。结果表明,托克逊县夏乡5个镇散养户的194头牛血液涂片中62份有环形泰勒虫虫体;PCR检测有57份DNA样品得到与预期结果相同的534bp条带;临床诊断有45头牛表现为牛环形泰勒虫病症状;西药治疗组治愈率为93.33%(14/15)明显高于中药治疗组治愈率为73.33%(11/15)。西药组对于治疗环形泰勒虫病具有显著的效果,可为新疆有效治疗环形泰勒虫病提供参考。     

Chemotherapy of surra in horses and mules with diminazene aceturate

During June–July 2000, an outbreak of surra occurred on an equine breeding farm in Khonkaen Province, Thailand. Forty-two percent of pregnant mares aborted or gave stillbirth and 40% (19/47) of horses and 10% (1/10) of mules died from surra. In August 2000 Trypanosoma evansi were detected in the remaining animals (28 horses and nine mules) on the farm by blood smear and/or the haematocrit centrifuge technique. All animals were treated with diminazene aceturate at 3.5 mg/kg body weight by intramuscular injection on days 0 and 41 of the study. Blood samples of eight randomly selected horses and mules were collected on days 0, 1 and once a week until day 56 and examined for T. evansi by various parasitological techniques. The sera were tested for antibodies against T. evansi using an indirect enzyme linked immunosorbent assay (ELISA).

The results revealed that diminazene aceturate at 3.5 mg/kg appeared to be effective in the first treatment of horses and mules infected with T. evansi. Parasites were cleared from the peripheral blood of horses on days 1 and 7 and mules on days 1 and 14. Thereafter the number of positive animals increased. After the second treatment, 50% of horses and 25% of mules were still positive to surra 24 h after treatment demonstrating that diminazene had no protective effect. Mild to severe toxicity of diminazene was seen in the horses and mules after injection.     

Relapses in dogs experimentally infected with Trypanosoma brucei and treated with diminazene aceturate or isometamidium chloride

Twenty dogs of mixed local East African breeds were used. Five of the dogs were uninfected controls and 15 were infected with T. brucei (ILRAD 273). Five of the infected dogs were untreated controls, five were treated with a high curative dose of diminazene aceturate, (7 mg kg-1 body weight (wt.), and five were given a subcurative dose of isometamidium chloride (1 mg kg-1 body wt.). The drugs, given at 8 days post infection (d.p.i..), led to apparent recovery. The antibody titres, however, remained high in both groups and at 42-49 d.p.i. there was at least one relapse in each treatment group. Parasite populations from relapsed animals were more resistant to the drugs than the original infecting populations. The implications of these findings are discussed.     

The effects of diminazene aceturate on systemic blood pressure in clinically healthy adult dogs

Diminazene aceturate is a commonly used antibabesial agent. It has been postulated that diminazine may induce a decrease in blood pressure and exacerbate the hypotension presented in dogs with babesiosis. This study was undertaken to assess the effect of diminazine aceturate on the blood pressure of healthy dogs. Six healthy German shepherd dogs between 18 and 24 months of age with a mean weight of 30.4 +/- 2.75 kg were used. Blood pressure was directly measured at the following time intervals: -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 90 and 120 minutes after treatment with diminazine aceturate (4.2 mg/kg) intramuscularly. No statistical difference (P > 0.05) was found in blood pressure between any of the time intervals. An increase in heart rate was seen 5 minutes after the administration of diminazine aceturate but no change in blood pressure was evident. This study concluded that diminazene aceturate in its current formulation with antipyrine does not alter blood pressure in healthy adult dogs.     

Bovine trypanosomiasis in southern Tanzania: Investigation into the incidence of infection and duration of chemoprophylaxis

Before implementing chemoprophylaxis to control bovine trypanosomiasis it is essential to have epidemiological data upon which to base control regimes. A study was conducted under natural tsetse challenge with two groups each of 12 calves grazing their first season. Group 1 received isometamidium treatments prophylactically at intervals during the rainy season and calves in group 2 were treated individually with diminazene as they become infected with trypanosomes. Infections were first detected in the unprotected calves and indicated that the onset of challenge was approximately four weeks after the rainy season began. Trypanosoma vivax accounted for 21 of the 30 infections detected in blood smears, and although one infection remained unspeciated, the remaining eight were T. congolense. It was concluded that a prophylactic regime beginning one month after the start of the rains with repeat treatments of isometamidium at 1 mg/kg at intervals of 10 weeks could be expected to give good control of trypanosomiasis at this location.     

In vivo assessment of drug sensitivity of African trypanosomes using the akinetoplastic induction test.

Following treatment of mice infected with Trypanosoma congolense or T brucei brucei with various doses of isometamidium chloride or diminazene aceturate, the induction of akinetoplastic (AK) forms was observed in the trypomastigotes of both species within 10 hours of drug administration. The levels of AK-induction were closely correlated with the levels of resistance to each compound found using a standard in vivo drug assay in mice. In general, ineffective doses of either compound conferred AK-induction rates of less than 30 per cent; relapsing cases had between 30 and 50 per cent while curative doses had AK-induction rates of 50 per cent or more. In vivo determination of AK-induction rates using ordinary light microscopy is thus a potentially feasible alternative indicator to the conventional use of mice infection and treatment methods for assessing drug sensitivity in African trypanosomes.     

A drug incubation infectivity test (DIIT) for assessing resistance in trypanosomes

Blood stream forms of drug-resistant and sensitive Trypanosoma brucei brucei, Trypanosoma brucei evansi and Trypanosoma vivax were incubated in a liquid medium for 24 h at 37 degrees C in the presence of various concentrations of diminazene aceturate (Berenil) or isometamidium chloride (Samorin), and assayed for infectivity in mice. Whereas the infectivity to mice of all Samorin-sensitive trypanosomes was decreased after incubation with 1 ng Samorin ml-1, the Samorin-resistant stocks remained infective for mice. Two of the Samorin-resistant stocks remained infective after incubation with Samorin concentrations of up to 50 ng ml-1. The infectivity of Berenil-resistant trypanosome stocks were also retained after incubation with drug concentrations (0.5 or 1.0 micrograms ml-1) which otherwise inhibited the infectivity of Berenil-sensitive trypanosome stocks. In addition, differences in infectivity were observed when Berenil-resistant and sensitive trypanosome stocks were incubated in medium supplemented with serum from goats previously treated with Berenil. Thus, drug-resistant and sensitive trypanosomes can be clearly distinguished using the drug incubation infectivity test.     

The efficacy of berenil (diminazene aceturate) against Trypanosoma evansi infection in mice

The efficacy of berenil (diminazene aceturate) was studied in experimental Trypanosoma evansi infection in albino mice. The criteria used for the assessment of the anti-trypanosomal effect of berenil included the examination of the blood and tissues for T. evansi and the clinical, pathological and enzyme histochemical changes seen in the lungs, heart, liver, kidney and spleen at intervals after treatment. Single doses of 10 and 20 mg/kg of berenil given intraperitoneally or intramuscularly to infected mice produced a complete elimination of the protozoon and caused a slow tissue recovery mirrored in the persistence of lesions in different organs. Single doses of 3.5 mg/kg of berenil were less effective and none of the three dose levels of the drug used induced toxic effects in albino mice.     

Ghibe river basin in Ethiopia: Present situation of trypanocidal drug resistance in Trypanosoma congolense using tests in mice and PCR-RFLP

A cross-sectional study was carried out in the Ghibe valley from August to October 2010. 411 head of cattle were sampled in eight villages for buffy coat examination (BCE) and blood spots were collected from each animal for trypanosomose diagnosis by 18S-PCR-RFLP and diminazene aceturate (DA) resistance by Ade2-PCR-RFLP. Three villages were selected in a zone where trypanosomosis control operations are currently on-going whereas the other 5 villages were located outside these control operations. Twenty-four samples (5.84%) were diagnosed positive for Trypanosoma congolense by BCE and injected in mice for further characterization. Twelve of those isolates successfully multiplied in mice and were tested by an in vivo mouse test for diminazene (DA) (10 and 20mg/kg B.W.) and isometamidium (ISM) (1mg/kg B.W.) resistance. All were shown to be resistant to both drugs at all doses. The use of the Ade2-PCR-RFLP on these isolates confirmed their DA-resistance profile. Seventy-three of the collected blood spots (17.8%) were diagnosed positive for T. congolense by 18S-PCR-RFLP of which 37 (50.7%) gave amplification products with the Ade2-PCR-RFLP. Here, 35 (94.6%) showed a resistant profile, 1 (2.7%) a sensitive profile and 1 (2.7%) a mixed profile. The data were analysed by logistic regression model and the relapsing time in mice tests was assessed using the Cox regression model. There was no significant intervention effect (P=0.83) with odds ratio equal to 1.21 when using the BCE data. 18S-PCR-RFLP test also showed no significant intervention effect (P=0.60) with odds ratio equal to 1.43. The hazard ratio of getting parasitaemic after treatment with DA at 20mg/kg B.W. compared to the control group was 0.38 which differs significantly from one (P<0.001). Relapsing time after treatment with DA 10mg/kg B.W. or ISM 1mg/kg B.W. was also significantly longer than the prepatent period of the control group. The situation of drug resistance in the Ghibe valley is further discussed.     

Trypanocidal drug resistance in eastern province of Zambia

A survey to investigate resistance to drugs used in the treatment of bovine trypanosomosis was conducted in the eastern province of Zambia between 1996 and 1998. A cross-sectional study was conducted in three districts (Petauke, Katete, Lundazi) at 34 village sampling sites selected at random from villages that had shown greater than 6% prevalence of bovine trypanosomosis during an earlier survey. A longitudinal study was conducted in same three districts over a 1-year period. The study sites were chosen from the cross-sectional study and included eight sites showing high trypanosomosis prevalence and where no control activities were recorded. Use was made of parasitological methods, tests of resistance in cattle and mice and isometamidium-ELISA. Overall mean prevalence of trypanosomosis was 14.4, with 96% of infections caused by Trypanosoma congolense. The remainder was caused by Trypanosoma vivax (2%) and Trypanosoma brucei (2%). Tests in mice showed that of the stabilates collected, 24 (34%) were resistant to only isometamidium chloride, 8 (11.3%) were resistant to only diminazene aceturate, 1 (1.4%) was resistant to both drugs, and 38 (53.5%) were sensitive to both drugs. At least 2 out of 27 stabilates tested in cattle appeared to be resistant to trypanocidal drugs, 1 to isometamidium and 1 to diminazene. Isometamidium could be detected in only 63 (4.1%) of 1526 serum samples from cattle in the study. Only 6 (2.8%) of 212 serum samples from trypanosome-infected cattle had serum levels of the drug above 0.4 ng isometamidium per ml serum which is indicative for drug resistance in the infecting parasite population. Although some drug resistance is apparent, diminazene aceturate and isometamidium chloride can still be expected to be effective as a sanative pair in this area in most cases, since not more than 1 stabilate of 71 investigated showed evidence of resistance to both drugs.     

Concentrations of isometamidium chloride (Samorin®) in sera of Zebu cattle which showed evidence of hepatotoxicity following frequent trypanocidal treatments

The concentrations of isometamidium circulating in poorly nourished Zebu cattle which showed morbidity, mortality, and biochemical and histopathological evidence of hepatotoxicity, following frequent treatments with isometamidium chloride and diminazene aceturate were investigated using the isometamidium-ELISA. As few as two isometamidium treatments one month apart were associated with significant weight loss, and cattle treated with diminazene aceturate after three or four isometamidium treatments suffered a 50% mortality.
Although there were no obvious, marked elevations in isometamidium concentration which might have allowed the use of the ELISA as a predictor of a potential toxicity problem, concentrations did increase significantly with the number of monthly treatments administered, suggesting drug accumulation, and the increases were significantly higher in cattle to which diminazene had also been administered. In cattle treated with both trypanocides, weight loss and serum glutamate dehydrogenase levels were correlated with isometamidium concentrations. These observations, together with the histopathological findings, support the hypothesis that the morbidity and mortality observed were related to the repeated treatment with isometamidium in conjunction with diminazene aceturate, and that the pathogenesis involved a component of hepatic damage.
It is therefore recommended that cattle, particularly those under nutritional stress, are not subjected to repeated treatments with isometamidium at intervals as short as one month, and particularly not with concurrent administration of diminazene.     

The susceptibility of Trypanosoma congolense isolated in Zambézia Province, Mozambique, to isometamidium chloride, diminazene aceturate and homidium chloride

Resistance to trypanocidal drugs has been detected in various African countries and is a serious impediment to the control of livestock trypanosomosis. To determine whether drug resistant trypanosome strains are present in the Zambézia Province of Mozambique a study was initiated. To assess the effect of the farming system and the drug-use regimen on the development of drug resistance, trypanosome isolates were collected from cattle from subsistence and commercial livestock production systems. The susceptibility of seven isolates against isometamidium chloride, diminazene aceturate and homidium chloride was tested in mice using a multiple-dose test. In four of the seven isolates high levels of drug resistance to diminazene aceturate and isometamidium chloride were detected. In most cases the observed levels of drug resistance correlated with the drug-use practices in the particular livestock production system.     

Treatment Efficiency of Combination Therapy With Diminazene Aceturate and Quinapyramine Sulfate in a Horse With Dourine

Dourine is a lethal protozoan disease of equids, and it is caused by Trypanosoma equiperdum infection via coitus. To date, treatment strategies against the dourine are not recommended because of the frequent relapses; therefore, the World Organisation for Animal Health recommends the stamping-out policy for the control of dourine. Our previous studies have revealed a number of horses with dourine in Mongolia that is the fifth largest horse-breeding country. It is difficult to apply the stamping-out policy for cases of dourine in Mongolia because of an inadequate livestock guarantee system. Therefore, the development of effective treatment measures is an urgent need. In this study, an 8-year-old stallion was definitely diagnosed with dourine based on clinical signs, molecular analysis, and microscopic examination of trypanosomes. Combination therapy with diminazene aceturate and quinapyramine sulfate was applied. Before the treatment, the characteristic clinical signs of dourine were observed, and trypanosomes were detected in the urogenital tract mucosal swab samples by microscopic examination and polymerase chain reaction (PCR). Moreover, positive serological results were obtained. After the treatment, we observed an improvement in the health of the treated horse and no trypanosome infection in its urogenital tract by microscopic examination and PCR. Moreover, serological tests showed seronegative results. The horse has showed no relapse for at least 2.5 years after the treatment, and its reproductive ability has improved. Our result suggests that trypanosomes did not invade cerebrospinal fluid when we started the therapy. In conclusion, the combination therapy has therapeutic potential against dourine at an early phase.