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Survey of canine babesiosis in South Africa.

A questionnaire, designed to obtain qualitative information on a number of variables concerning canine babesiosis (biliary fever) in South Africa, was sent to 510 veterinary practices in late 1993. Of the 157 practices that responded, all were presented with cases of babesiosis and most were situated in Gauteng, the Western Cape and KwaZulu-Natal. Apart from the Western Cape, a winter-rainfall region, the prevalence of babesiosis cases in dogs was highest in summer. Most of the respondent practices treated between 1,000 and 5,000 sick dogs that included 100 to 500 babesiosis cases each year. Respondents identified cerebral babesiosis, enterorrhagia, 'red' or haemoconcentrated babesiosis, acute renal failure and pulmonary babesiosis or 'shock lung', amongst others, as the most prevalent forms of complicated ('atypical') babesiosis. Diminazene, imidocarb and trypan blue were the most popular antibabesials. Trypan blue was most often used in shocked patients, whereas diminazene and imidocarb were preferred when there was a high parasitaemia in the absence of shock. At least 19 antibabesial treatment regimens were used in practices. These comprised the use of single doses of antibabesial drugs; split doses with repeat injections, and combined drug variations, some of which are undesirable due to possible sterilisation of Babesia infection or potential toxicity. Side-effects were most commonly associated with imidocarb use. Ninety-six percent of respondents used supportive treatment (e.g. corticosteroids, vitamins and 'liver support') in all cases of babesiosis. The use of blood transfusion as supportive treatment varied according to practice and severity of the case. Most practices never cross-matched blood to be transfused, and transfusion reactions were rare. Diminazene was most frequently incriminated in cases where drug 'resistance' or relapses occurred. Cerebral and 'red' cases resulted in high mortality. Treatment of babesiosis costs the dog-owning public in South Africa more than R20 million each year. Information on the distribution and possible complicating role of Ehrlichia canis was obtained. Development of a vaccine was the first research priority identified.

Animals↗

In vitro drug sensitivity of Trypanosoma congolense isolates.

The sensitivity of Trypanosoma congolense isolates to diminazene and isometamidium was determined using an incorporation assay based on the uptake of [3H]-hypoxanthine in the presence of serial drug dilutions. The bloodstream forms of the different isolates exhibited variation in their sensitivity to the drugs that correlated well with the in vivo drug response. For diminazene, the sensitivity of the most sensitive population was 40 times that of the least sensitive population. For isometamidium, the IC50 values (the drug concentrations that decreased radiolabel incorporation by 50%) lay in a similar range, except for those found for two isolates from lions, which were 10(3)-10(4) times more sensitive than the isolates from cattle. The sensitivity of procyclic forms differed markedly from that of the bloodstream stages. Therefore, it must be concluded that the procyclic stage does not reflect the sensitivity of the bloodstream forms of T. congolense and that the former should not be used for determinations of in vitro drug sensitivity.

Animals↗

Isolation of drug-resistant strains of Trypanosoma congolense from the lower Shabelle region of southern Somalia.

Drug resistance by pathogenic trypanosomes in Somali livestock has been suspected for some time but there have been few attempts to examine this problem in detail. Field isolations from two areas in the Lower Shabelle Region were obtained by injecting blood from trypanosome infected cattle into a recipient calf. Once the calf became parasitaemic it was treated with a standard dose of isometamidium chloride (Samorin, RMB) at 0.5 mg/kg. When a subsequent relapse infection developed, indicative of drug resistance, blood was taken and injected into groups of cattle and mice and these were treated with a range of doses of isometamidium chloride and diminazene aceturate (Berenil, Hoechst AC) to determine the degree of drug resistance. Both isolates showed remarkably high levels of drug resistance to both isometamidium chloride and diminazene aceturate, with minimum curative doses in cattle of > 2.0 mg/kg and 7.5 mg/kg for the two drugs respectively. Minimum curative doses in mice were approximately ten-fold those in cattle. Fortunately there have been a very few reports from Africa of such high levels of resistance of Trypanosoma congolense to this normal "sanative pair" of drugs. The results indicate that drug resistance could be an important constraint on the use of trypanocidal drugs to control trypanosomosis in Somalia.

Animals↗

Trypanosomiasis control in Boran cattle in Kenya: a comparison between chemoprophylaxis and a parasite detection and intravenous treatment method using isometamidium chloride.

Two methods of trypanosome control in Boran cattle kept under very high trypanosomiasis risk were compared: the traditional intramuscular isometamidium chloride prophylaxis with a parasite detection and intravenous isometamidium chloride treatment method. The results were related to a control group under diminazene aceturate treatment. Isometamidium chloride at 0.25 mg/kg as routinely used by the ranch was of little benefit by either method, with breakthrough infections occurring as early as one week after treatment. When isometamidium chloride at 1 mg/kg was used, the curative intravenous method appeared to be superior to the intramuscular prophylaxis with regard to cost of drugs and to a 31% higher weight gain over a 30 week period. Weekly infection rates in the intravenous group decreased over time, despite an increasing trypanosomiasis challenge, with a mean interval of 6.4 weeks between treatments as compared with 4.3 weeks in a diminazene aceturate control group. It was concluded that isometamidium chloride given intravenously had not only a very good therapeutic but also a considerable prophylactic effect of not less than four weeks.

Animals↗

The effect of verapamil alone and in combination with trypanocides on multidrug-resistant Trypanosoma brucei brucei.

Following previous studies of verapamil reversal of multidrug resistance in cancer cells and chloroquine resistance in malaria, the effect of the calcium channel blocker verapamil was investigated on multidrug-resistant and susceptible Trypanosoma brucei brucei. Resistance of cloned parasites to diminazene aceturate (Berenil) and isometamidium chloride (Samorin) was expressed in a cell-free in vitro culture system. Verapamil showed antitrypanosomal activity against both, multidrug-resistant and susceptible trypanosomes at concentrations above 1 micrograms/ml. Verapamil did not reverse multidrug resistance when used at concentrations of 0.1 or 1.0 micrograms/ml in combination with diminazene aceturate or isometamidium chloride. Results obtained in vitro correlate with observations in mice. It is suggested that multidrug resistance in African trypanosomes is due to mechanisms other than those occurring in cancer cells, malaria or South-American trypanosomiasis.

Animals↗

The rapid development of drug-resistance by Trypanosoma evansi in immunosuppressed mice.

The effect of immunosuppression on the development of drug resistance by trypanosomes was investigated in mice infected with Trypanosoma evansi. As a result of frequent passage in immunosuppressed mice given subcurative drug treatments clones of T. evansi rapidly developed high levels of resistance to mel Cy, diminazene aceturate and isometamidium chloride. Similar protocols in normal immunocompetent mice infected with the same parent clones did not lead to the development of drug-resistance. The resistant populations developed in immunosuppressed mice maintained their high levels of resistance when tested in normal mice. The mel Cy resistant clone was tested for cross-resistance to other trypanocides and was found to be also highly resistant to diminazene and pentamidine. The results indicate that impairment of the host immune system may lead to the rapid development of drug-resistance by T. evansi under experimental conditions in mice and may possibly play a role in the development of drug resistance by trypanosomes in the field.

Animals↗

Epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia. 2. Factors associated with variations in trypanosome prevalence, incidence of new infections and prevalence of recurrent infections.

An average of 840 East African Zebu cattle from nine herds in the Ghibe valley, southwest Ethiopia were monitored from January 1986 to April 1990. Each month blood samples were collected for analysis of packed red cell volume (PCV) and detection of trypanosomes. Animals found to be parasitaemic and with a PCV less than 26% were treated with diminazene aceturate at a dose of 3.5 mg/kg body weight. The majority of infections were associated with Trypanosoma congolense (84% of infections in adult cattle and 71% in cattle less than 24 months of age), and the mean percentage of adult animals detected parasitaemic 1 month after treatment of an infection with T. congolense was 27%. In order to assess possible existence of drug resistance, a model was applied which allowed monthly incidences of new infections to be distinguished from recurrent infections. This model showed that the monthly incidence of new infections of T. congolense in adult cattle increased significantly from 11% in 1986 to 24% in 1989 following a concomitant increase in the tsetse challenge. The corresponding increase in overall prevalence of T. congolense was from 17% to 38% and the mean prevalence of recurrent infections increased significantly from 6% to 14%. These findings ruled out the possibility that the high prevalence of trypanosome infections in cattle was due only to a high tsetse challenge and pointed to the existence of T. congolense populations which expressed resistance to diminazene. There were variations associated with season, herd, age and sex in the incidence of new infections, prevalence of recurrent infections and relapse to treatment.

Animals↗

Drug sensitivity screening in vitro of populations of Trypanosoma congolense originating from cattle and tsetse flies at Nguruman, Kenya.

An in vitro assay that utilises in vitro-derived metacyclic trypanosomes was used to determine the drug sensitivity of 7 populations of Trypanosoma congolense collected from cattle and tsetse flies at Nguruman; a trypanosomiasis-endemic area in southwest Kenya. The metacyclic trypanosomes used in the assay were obtained from cultures initiated directly from either the blood of cattle with low levels of parasitaemia or from guts of infected tsetse flies. Sensitivities to isometamidium chloride, diminazene aceturate and homidium salts were assessed at various drug concentrations (0.5 ng-50 micrograms/ml). The results were compared with those obtained with two characterised laboratory populations. In spite of the fact that isometamidium chloride had not been widely used at Nguruman, two of the stocks (KE 3302 and KE 3303) expressed high levels of resistance to this drug (resistant to 100 ng/ml and 1000 ng/ml, respectively). In contrast, all of the populations examined were as sensitive, or more so, to diminazene aceturate than the sensitive laboratory clone IL 1180; two stocks (KE 3305 and KE 3306) were more sensitive (resistant to 0.5 microgram/ml, sensitive to 1 microgram/ml) and three stocks expressed the same level of sensitivity as IL 1180 (resistant to 1 microgram/ml, sensitive to 5 micrograms/ml). Since the results of the in vitro assay correlated well with field observations it was concluded that the assay would be a useful tool in epidemiological studies to determine the resistance phenotypes of trypanosome populations in the field, thereby enabling development of appropriate control measures for particular areas.

Animals↗

Variations in packed red cell volume and trypanosome prevalence and relationships with reproductive traits in east African Zebu cows exposed to drug-resistant trypanosomes.

Approximately 320 East African Zebu cows over 36 months of age were monitored monthly from 1986 to 1992 in nine village herds in an area of high trypanosomiasis risk in southwest Ethiopia where there was resistance to all available trypanocidal drugs. Cows were individually treated with diminazene aceturate, either when they were detected parasitaemic and their packed red cell volume (PCV) decreased below 26%, or when they showed clinical signs of trypanosomiasis. The average annual monthly trypanosome prevalence was 24% and the number of treatments of diminazene aceturate per cow per year was 3.1, both of which increased with age. Mean PCV decreased and mean trypanosome prevalence increased during lactation. There was a significant linear association between the time detected parasitaemic during the first 150 days of lactation and calving interval. When corrected for frequency of parasitaemia and treatment there was also an average reduction of 8.4 +/- 2.6 days in calving interval per % unit increase in PCV. Age at first calving decreased by 0.44 +/- 0.26 months per % unit increase in mean PCV maintained between 24 and 30 months corrected for parasitaemia and treatment. The percentage of pregnancies terminating in abortions significantly increased from 6.8 +/- 1.0% to 10.4 +/- 1.3% when cows detected parasitaemic at least once during the last three months of pregnancy were compared with cows not detected parasitaemic. The largest increase to 19.4 +/- 4.3% was in cows with low mean PCVs < 22%. It was concluded that cows which were able to maintain higher than average PCVs when parasitaemic showed superior reproductive performance than those with lower than average PCVs.

Abortion, Veterinary↗

Trypanosoma brucei brucei: expression of drug resistance in vitro.

The susceptibility of five Trypanosoma brucei brucei stocks to diminazene aceturate (Berenil) and isometamidium chloride (Samorin) was determined in vivo and in vitro. Two stocks were resistant in mice to both drugs. Differences in drug-induced growth inhibition occurred between drug-resistant and drug-sensitive stocks when trypanosome bloodstream forms were incubated for 24 hr at 37 degrees C in 4% CO2 in air in a feeder layer-free culture system. Diminazene concentrations which inhibited growth of trypanosomes by 50% (EC50) differed more than 50-fold between drug-resistant and drug-sensitive stocks, but there were only minor differences in EC50 values for isometamidium. However, differences in growth inhibition due to isometamidium occurred when trypanosomes were incubated with drug concentrations (0.001-0.05 micrograms/ml) below the EC50 values. Trypanosome stocks which were propagated in vitro in the presence of drugs for a period of 10 days greatly differed in their viability. In contrast to drug-sensitive trypanosomes, drug-resistant stocks could be maintained without loss of viability in the presence of 0.01 micrograms/ml isometamidium chloride.

Amidines↗

Resistance to diminazine aceturate by Trypanosoma congolense from cattle in the Zambezi Valley of Zimbabwe.

The susceptibility of 14 stocks of Trypanosoma congolense, recently isolated from cattle, to therapeutic doses of diminazene aceturate and to isometamidium chloride was assessed in laboratory mice. Eight isolates were readily susceptible to the normal therapeutic dose of diminazene, two were resistant to the drug at 14 mg kg-1, and four were totally resistant at 28 mg kg-1. All the isolates were susceptible to isometamidium chloride at 0.5 mg kg-1. These observations highlight the need for regular evaluation of drugs used in the control of trypanosomosis.

Animals↗

Use of extracts of Picralima nitida bark in the treatment of experimental trypanosomiasis: a preliminary study.

The effect of a boiling water extract of Picralima nitida bark against trypanosomes (T. brucei) in rats was compared with that of diminazene aceturate at 8 mg/kg i.p. Results showed that the extract had a definite trypanocidal effect which was statistically comparable to that of diminazene aceturate. Further work is needed to identify the active pharmacological agent and to standardise the usage of the agent in practice.

Animals↗

The effect of inducing agents on the metabolism of trypanocidal diamidines by isolated rat hepatocytes.

This study has investigated the effect of phenobarbitone (PB), 3-methylcholanthrene (3-MC), and deltamethrin (DM) on the metabolism of two trypanocidal diamidines; pentamidine isethionate and diminazene aceturate in freshly isolated Sprague-Dawley rat hepatocytes. There were significant increases in the total cytochrome p450 content of hepatocytes obtained from rats pre-treated with PB and 3-MC, whereas pre-treatment with DM did not produce any significant induction of cytochrome p450. However, pre-treatment of rats with each of the three agents led to inhibition of pentamidine metabolism following a 3h incubation of pentamidine (100 microM) with freshly isolated rat hepatocytes (5 x 10(6) cells ml(-1)). Pre-treatment with 3-MC caused the highest inhibitory effect on pentamidine metabolism (8-fold inhibition), compared with PB (4.8-fold) and DM (2.2-fold). Six previously reported phase I metabolites of pentamidine were identified in cells from all the pre-treated animals as well as controls. When compared to the control group, there were significant differences between the profiles of the three major metabolites of pentamidine, 1,5-di(4'-amidinophenoxy)-2-pentanol, 1,5-di(4'-amidinophenoxy)-3-pentanol and 5-(4'-amidinophenoxy) pentanoic acid, in hepatocytes from the DM and 3-MC pre-treated rats, whereas no significant differences were observed in the cells from the PB pre-treated group. In contrast, diminazene was not metabolised with the same experimental conditions. Differences in the metabolic profiles of pentamidine and its metabolites as a result of concomitant exposure to environmental xenobiotics could have important toxicological and pharmacological implications for patients that receive the drug.

Animals↗

A drug incubation Glossina infectivity test (DIGIT) to assess the susceptibility of Trypanosoma congolense bloodstream forms to trypanocidal drugs (Xenodiagnosis).

Blood was collected from two Sahelian goats, experimentally infected with either a drug-sensitive cloned population of Trypanosoma congolense (IL 1180) or a multiple drug-resistant T. congolense stock (Samorogouan/89/CRTA/267) and incubated at 37 degrees C for 30 min and 12 h, respectively, in the presence of different drug concentrations (0.5, 1.0, 10.0 and 100.0 microg/ml blood) of diminazene aceturate or isometamidium chloride. After that, the trypanosome/blood/drug suspensions were offered to tsetse flies (2100 teneral Glossina morsitans submorsitans) through an in vitro feeding system, using a silicone membrane. All tsetse flies were dissected and examined for the presence of trypanosomes in labrum, hypopharynx and midgut 20 days after their infective blood-meals. Infectivity of the drug-sensitive cloned population was already completely abolished after incubation with 0.5 microg/ml of both drugs; however, 13.6-42.2% of tsetse having been fed on untreated blood had developed an infection. In contrast, no significant differences were observed in the infection rates between the experimental groups and their control groups when fed on blood infected with the multiple drug-resistant stock after incubation for 30 min with up to 10 microg/ml of diminazene or isometamidium. In consequence, tsetse appear to be a useful tool in the assessment of drug susceptibility of typanosome populations.

Animals↗

Babesia canis infection in canine-red blood cell-substituted SCID mice.

We have developed a mouse model for Babesia canis infection using severe combined immunodeficiency (SCID) mice whose circulating red blood cells had been substituted with canine red blood cells. Substitution of red blood cells in SCID mice was achieved by repetitive transfusions of canine red blood cells, together with administration of an antimouse red blood cell monoclonal antibody. Following inoculation of canine-red blood cell-SCID mice with B. canis, parasites proliferated in the canine red blood cells that had been transfused into the SCID mice, resulting in much higher parasitaemia than that observed in dogs. In an attempt to demonstrate the utility of this mouse model, three antiprotozoal drugs, diminazene diaceturate, clindamycin and oxytetracycline, were examined for their efficacy to inhibit the growth of B. canis in canine-red blood cell-SCID mice. The mouse model clearly showed that diminazene diaceturate and oxytetracycline were capable of eliminating B. canis from the canine-red blood cell-SCID mice, whereas clindamycin exhibited only a static effect as parasitaemia relapsed upon cessation of drug administration.

Administration, Cutaneous↗

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.

Animals↗

Defining the drug incorporation properties of PLA-PEG nanoparticles.

The drug incorporation and physicochemical properties of PLA-PEG micellar like nanoparticles were examined in this study using a model water soluble drug, procaine hydrochloride. Procaine hydrochloride was incorporated into nanoparticles made from a series of PLA-PEG copolymers with a fixed PEG block (5 kDa) and a varying PLA segment (3-110 kDa). The diameter of the PLA-nanoparticles increased from 27.7 to 174.6 nm, with an increase in the PLA molecular weight. However, drug incorporation efficiency remained similar throughout the series. Incorporation of drug into the smaller PLA-PEG nanoparticles made from 3:5, 15:5 and 30:5 copolymers did not influence the particle size, while an increase was observed for the larger systems comprising 75:5 and 110:5 copolymers. An increase in drug content for PLA-PEG 30:5 nanoparticles was achieved by increasing the theoretical loading (quantity of initially present drug). The size of these nanoparticles remained unchanged with the increasing drug content, supporting the proposed micellar type structure of the PLA-PEG 30:5 nanoparticles. The morphology of these systems remained unchanged both at low and high theoretical drug loadings. Formulation variables, such as an increase in the aqueous phase pH, replacement with the base form of the drug and inclusion of lauric acid in the formulation did not improve the incorporation efficiency of drug into PLA-PEG 30:5 nanoparticles. While poly(aspartic acid) as a complexation agent did not improve the drug incorporation efficiency of procaine hydrochloride, it did so for another water soluble drug diminazene aceturate. This may be attributed to a stronger interaction of diminazene aceturate with poly(aspartic acid) relative to procaine hydrochloride, as confirmed by thermodynamic analysis of isothermal titration calorimetric data. The drug incorporation and physicochemical characterisation data obtained in this study may be relevant in optimising the drug incorporation and delivery properties of these potential drug targeting carriers.

Anesthetics, Local↗

Lipid-drug-conjugate (LDC) nanoparticles as novel carrier system for the hydrophilic antitrypanosomal drug diminazenediaceturate.

The objective of the present study was to incorporate the hydrophilic drug diminazenediaceturate at a high loading into lipid nanoparticles by creating nanoparticles from lipid-drug conjugates (LDC). IR and DSC data showed that the antitrypanosomal drug diminazene is able to react with fatty acids to form water-insoluble salts like diminazenedistearate and -dioleate. The salts could be transformed into nanoparticles using high-pressure homogenization technique, established for solid lipid nanoparticles (SLN). By using polysorbate 80 as surfactant, physically stable LDC nanoparticle dispersions of both salts could be obtained. The mean PCS diameters and polydispersity indices were 364 nm and 0.233 for diminazenedistearate and 442 nm and 0.268 for diminazenedioleate, respectively. Due to the composition of the LDC bulk materials, nanoparticles with a high drug load of 33% (w/w) were obtained even for this highly water-soluble drug diminazenediaceturate. The new carrier system of LDC nanoparticles overcomes one limitation of SLN, i.e. the limited loading capacity for hydrophilic drugs. Transforming water-soluble hydrophilic drugs into LDC and formation of nanoparticles allows prolonged drug release and targeting to specific sites by i.v. injection. These results provide a first basis of using LDC-polysorbate 80 nanoparticles for brain delivery of diminazene to treat second stage human African trypanosomiasis (HAT).

Blood-Brain Barrier↗