The transmissibility of Trypanosoma congolense seems to be associated with its level of resistance to isometamidium chloride

In large parts of Africa the control of livestock trypanosomiasis relies on the use of trypanocidal drugs. Resistance against the available compounds is developing rapidly in the trypanosome population. The effect of the development of drug resistance on the fitness of the trypanosome is not well known. To determine the effect of the development of resistance to isometamidium chloride on the trypanosome's transmissibility, transmission experiments were conducted. Use was made of three isogenic clones of Trypanosoma congolense with different susceptibility to the drug. The infection rate in Glossina morsitans morsitans differed significantly between clones and was significantly higher in tsetse flies infected with the T. congolense clone with the highest level of drug resistance.     

Effect of isometamidium on Trypanosoma congolense infectivity

Isometamidium chloride (Samorin, RMB, England) is a widely used and highly effective trypanocide for the treatment of bovine trypanosomiases. However, the appearance of isometamidium-resistant populations of T. congolense in Africa makes it necessary to develop methods for the rapid and reliable detection of drug resistance in the laboratory. Currently available tests are time-consuming and/or expensive. In the present study, the short-term in vitro incubation of trypanosomes in a range of isometamidium concentrations and the infectivity of the parasites in mice has been assessed. A series of T. congolense isolates were used which were known to differ in their in vivo sensitivity to the drug. The results showed a close correlation between the known level of resistance and the capability of trypanosomes to remain infective after incubation in isometamidium. Thus isolates displaying a high level of resistance in vivo remained infective following incubation in higher concentrations of drug. This assay may provide a simple and reliable method for detecting drug resistance in T. congolense.     

Protective efficacy of isometamidium chloride and diminazene aceturate against natural Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax infections in cattle under a suppressed tsetse population in Uganda

The protective efficacy of isometamidium chloride (ISMM) and diminazene aceturate (DIM) against Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax infections in cattle under a suppressed tsetse population was assessed in southeast Uganda. A total of 66 and 57 trypanosome-infected cattle were treated with ISMM and DIM, respectively together with 177 trypanosome-free animals not treated were followed for 12 months, checked every 4 weeks. There was no statistical difference in the mean time to infection with any trypanosome species in animals treated with ISMM or DIM. However, the mean time to trypanosome infection was significantly longer for treated animals than controls. The mean time to infection with each of the three trypanosome species differed significantly, with the average time to T. vivax infection the lowest, followed by T. congolense and then T. brucei. The protective efficacy of DIM was as good as that of ISMM; implying curative treatments against trypanosomosis are sufficient for combination with tsetse control. Isometamidium chloride or DIM had the highest impact on T. brucei and T. congolense infections in cattle.     

Factors influencing the duration of isometamidium chloride (Samorin) prophylaxis against experimental challenge with metacyclic forms of Trypanosoma congolense

The duration of a single isometamidium chloride (Samorin) prophylactic treatment against Trypanosoma congolense ILNat. 3.1 and T. congolense IL 285 was examined in 24 Boran steers with regard to (1) the dose of drug, (2) the level of metacyclic challenge and (3) the influence of infection with an unrelated serodeme at the time of treatment. The cattle were repeatedly challenged at monthly intervals between 2 and 7 months following treatment, either by five infected Glossina morsitans centralis or by intradermal inoculation of 5 X 10(3) or 5 X 10(5) in vitro-derived metacyclic trypanosomes. A dose of 1 mg kg-1 afforded complete protection for 4 months and 0.5 mg kg-1 for 3 months against the two T. congolense serodemes examined, irrespective of the method or weight of challenge. In another group of cattle, which had an established infection at the time of treatment, the duration of chemoprophylaxis against an unrelated serodeme was the same as the other groups which had no previous experience of trypanosome infection. Antibodies to metacyclics did not appear in any of the cattle as long as the chemoprophylaxis was effective. An exception to this was the group challenged with 5 X 10(5) in vitro-derived metacyclic parasites, in which low antibody titres were detected. In all cases these proved to be non-protective. It was concluded that, under the experimental conditions employed, (1) there was a direct relationship between drug dosage and the duration of chemoprophylaxis, (2) the weight of metacyclic challenge did not affect the duration of chemoprophylaxis and (3) when used to treat an existing infection, isometamidium chloride exerted the same degree of chemoprophylactic activity.     

Response of Trypanosoma vivax and Trypanosoma congolense in zebu cattle in North Cameroon to prophylactic treatment with two formulations of isometamidium

We tested the efficacy of two formulations of isometamidium in a tsetse-infested farm in North Cameroon from 20 August 2000 to 5 January 2001. A total of 90 adult cattle were used in three groups of 30 each corresponding to two treated and one untreated control. Drug efficacies were evaluated in terms of reduction of parasite incidence in the host's blood, maintenance of packed-cell volume (PCV) and weight gains. Both drugs reduced the incidence of parasites even though re-infections 2 weeks after treatment were common. PCV values were similar in both treated groups but higher than in the untreated control. Body weight changes followed a similar trend with the control losing weight from a mean of 427+/-119kg at the beginning to 398+/-93kg in 4 months. Weights increased from 375+/-76 and 396+/-110 to 396+/-69 and 418+/-112kg in the Veridium and Trypamidium groups, respectively. Efficacy was similar between the two formulations of isometamidium in the prophylaxis of bovine trypanosomosis. However, the presence of parasites in some animals barely 2 weeks after treatment suggested that either infections were not cleared or residual drug effects were not sufficient to prevent re-infections.     

Action of isometamidium chloride on the insect vector form of Trypanosoma vivax

The effect of isometamidium chloride upon developing and mature Trypanosoma vivax occurring in Glossina palpalis palpalis flies was evaluated. Newly emerged G p palpalis flies were infected with T vivax by allowing them to feed on parasitaemic animals. Two experiments were conducted and in each the flies were divided into two groups. One group of infected flies was fed in vitro through a membrane on defibrinated cow blood containing isometamidium chloride at 0.1 mg ml-1, after which they were dissected and examined for trypanosomes, and the other group was fed in the same way on unmedicated blood. The results showed that out of a total number of 129 flies which fed on medicated blood, none was infected, while 55 out of 127 flies which fed on unmedicated blood were infected. The results indicate that isometamidium chloride eliminated the insect vector form of T vivax. These findings are of potential significance in the control of trypanosomiasis in the field, particularly in the operation of the sterile insect technique.     

Cytokine mRNA profiles in bovine macrophages stimulated with Trypanosoma congolense

It is known that different breeds of cattle display differential susceptibilities to Trypanosome congolense infections, and that N'Dama cattle remain more productive after infection than Boran cattle which are more susceptible to T. congolense. Macrophages from both breeds were cultured in vitro and the expressions of a number of cytokines and iNOS mRNA were analyzed using real time RT-PCR after stimulation with antibody-opsonized trypanosomes. No significant difference was seen between the responses of the two breeds. However, RNA levels of TNF-alpha in the IFN-gamma-primed macrophages were about 100-fold higher than those in the non-primed macrophages. A significant ten-fold decrease was seen for the anti-inflammatory cytokine IL-10. These results indicate that priming of the cells with IFN-gamma cause a serious shift toward an inflammatory response.     

Therapeutic activity of isometamidium chloride in Boran cattle against a tsetse-transmitted clone ofTrypanosoma congolense with a low level of drug resistance

Summary Experiments were conducted with a clone ofTrypanosoma congolense, IL 3580, which exhibited a low level of resistance to isometanidium chloride. Five cattle were treated intramuscularly with isometamidium chloride at a dose rate of 0·5mgkg⁻¹ body weight (BW) and challenged 28 days later with 5Glossina morsitans centralis infected withT. congolense IL 3580. All 5 cattle and 15 untreated steers challenged on the same day became parasitaemic by day 15 post-infection. Thus, at a dose of 0·5 mg kg⁻¹ BW, the prophylactic action of isometamidium chloride did not extend to 28 days following treatment. Subsequently, the 20 steers were divided into 4 groups of 5 animals each and treated with isometamidium chloride at one of the following dose rates; 0·5 or 1·0 mg kg⁻¹ BW intramuscularly and 0·5 or 1·0 mg kg⁻¹ BW intravenously (Groups A, B, C and D, respectively). Group A consisted of the 5 animals that had previously been treated with isometamidium chloride. Animals relapsed in all groups except those in Group B, treated intramuscularly with isometamidium chloride at a dose of 1·0 mg kg⁻¹ BW. Four of the 5 animals in Group A, treated intramuscularly with isometamidium chloride at a dose of 0·5 mg kg⁻¹ BW relapsed following a mean interval of 16 days post-treatment. Similarly, infections in all animals in Groups C and D, given intravenous injections of isometamidium chloride at a dose of 0·5 and 1·0mg kg⁻¹ BW, respectively, were not eliminated as a result of treatment. The mean intervals to first detection of parasitaemia in these 2 groups following treatment were 14 and 20 days, respectively. The results therefore indicate that intravenous administration of isometamidium chloride does not enhance the drug’s therapeutic efficacy in the treatment of aT. congolense clone which expresses a low, but significant, level of resistance to isometamidium.

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Resumen Se condujeron experimentos con un clon deTrypanosoma congolense IL 3580, que exhibió un bajo nivel de resistencia al cloruro de isometamido. Clinco animales fueron tratados vía intramuscular con cloruro de isometamidio a una dosis de 0·5mg kg⁻¹ de peso vivo (PV). Posteriormente fueron enfrentados 28 días después con 5Glossina morsitans centralis infectadas conT. congolense IL 3580. Todos los infectados (n=5) y 15 novillos sin tratar infectados el mismo día, presentaron parasitèmia el día 15 pos-infección. Se desprende entonces, que la dosis de 0·5 mg kg⁻¹ de PV de cloruro de isometamidio, no protegió más allá de 28 días. Subsecuentemente, los 20 novillos fueron divididos en 4 grupos de 5 animales cada uno, siendo tratados con cloruro de isometamidio, con una de las siguientes dosis: 0·5 ó 1·0 mg kg⁻¹ de PV intramuscular y 0·5 ó 1·0 mg kg⁻¹ vía intravenosa (Grupos A, B, C y D, respectivamente). El Grupo A consistió de 5 animales que habían sido tratados previamente con cloruro de isometamidio. Todos los animales recayeron, excepto aquellos del Grupo B, que habían sido tratados vía intramuscular, a una dosis de 1·0 mg kg⁻¹ de PV. Cuatro de los 5 animales en Grupo A, tratados intramuscularmente con 0·5 mg kg⁻¹ deisometamidio, recayeron 16 días promedio después del tratamiento. Similarmente, las infecciones en todos los animales de los grupos C y D, tratados vía intravenosa con dosis de 0·5 y 1·0 mg kg⁻¹ de PV, respectivamente, no fueron eliminados como resultado del tratamiento. El promedio de intervalos para la detección de parasitémia en estos dos grupos después del tratamiento, fueron 14 y 20 días, respectivamente. Los resultados indican, que la administración de cloruro de isometamidio intravenoso, no aumenta la eficacia terapéutica de la droga en el tratamiento del clon deT. congolense, el cual expresa un bajo pero significativo nivel de resistencia al isometamidio.

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Résumé Des expériences ont été conduites avec un clone deTrypanosoma congolense IL 3580, qui a montré un faible niveau de résistance au chlorure d’isométamidium. Cinq bovins ont été traités par voie intramusculaire avec ce médicament à la dose de 0,5mg/kg⁻¹ de poids vif (PV) Ils ont été soumis 28 jours plus tard au contact de 5Glossina morsitans centralis infectées par Trypanosoma congolense IL 3580. Les cinq animaux en totalité, ainsi que 15 taurillons non traités soumis le même contact se sont révélés parasités au 15e jour de l’infection. Ainsi, à la dose de 0,5 mg Kg⁻¹ PV, l’action prophylactique de chlorure d’isométamidium n’a pas duré jusqu’au 28 e jour suivant le traitement. En conséquence, les 20 taurillons ont été divisés en 4 groupes de 5 animaux chacun et traités au chlorure d’isométamidium avec l’une des doses suivantes: 0,5 ou 1,0 mg/kg⁻¹/PV, par voie intramusculaire et 0,5 ou 1,0 mg/kg⁻¹/PV par voie intraveineuse, constituant ainsi les groupes A, B, C et D respectivement. Le groupe A comprenait les 5 animaux précocement traités. Tous les animaux ont fait une rechute, excepté ceux du groupe B traités par voie intramusculaire avec le chlorure d’isométamidium à la dose de 1,0 mg/kg⁻¹/PV. 4 des 5 animaux du groupe A, traités par la même voie mais à la dose de 0,5 mg kg⁻¹/PV ont rechuté après un intervalle moyen de 16 jours suivant le traitement. De même chez tous les animaux des groupes C et D ayant respectivement re?u une injection intraveineuse aux doses de 0,5 et 1,0 mg/kg⁻¹/PV, le traitement n’a pas éliminé l’infection. Les intervalles moyens pour la première détection de la parasitémie dans ces deux groupes ont été respectivement de 14 à 20 jours. De ces résultats, on peut déduire que l’injection intraveineuse du chlorure d’isométamidium n’accro?t pas l’efficacité thérapeutique du produit dans le traitement d’une trypanosomose due à un clone deTrypanosoma congolense qui exprime à un degré faible, mais significatif, sa résistance à ce médicament.

     

The response of pigs experimentally infected with Trypanosoma brucei to isometamidium chloride therapy and the relation to nutrition.

Growing pigs were placed on feeds with high (Group A), medium (B) and low (C) dietary energy and were infected with a virulent stock of T. brucei. Eight weeks later, the infected pigs were treated with isometamidium chloride at 1 mg/kg live weight and all pigs were subsequently placed on a high energy diet to investigate their response to therapy. Clearance of T. brucei from blood was completed 72h after treatment. There was no evidence of relapsed infection up to eight weeks after treatment. Red blood cell parameters returned to normal four to six weeks after treatment with responses being fastest in Group A, B and C had gained about two-thirds of the live weight gains of their non-infected pair-fed controls. It appears that the retarded weight gain as a result of the infection persisted after therapy since drug-treated pigs did not gain as much weight as their non-infected controls.     

Field detection of resistance to isometamidium chloride and diminazene aceturate in Trypanosoma vivax from the region of the Boucle du Mouhoun in Burkina Faso

A longitudinal study assessed the chemoresistance to isometamidium chloride (ISM) and diminazene aceturate (DA) in the region of the Boucle du Mouhoun in Burkina Faso. A preliminary cross-sectional survey allowed the identification of the 10 villages with the highest parasitological prevalences (from 2.1% to 16.1%). In each of these 10 villages, two herds of approximately 50 bovines were selected, one being treated with ISM (1mg/kg b.w.) and the other remaining untreated as control group. All animals (treated and untreated herds) becoming infected were treated with DA (3.5mg/kg b.w.). In total, 978 head of cattle were followed up. Fortnightly controls of the parasitaemia and PCV were carried out during 8 weeks. The main trypanosome species was Trypanosoma vivax (83.6%) followed by Trypanosoma congolense (16.4%). In two villages, less than 25% of the control untreated cattle became positive indicating no need to use prophylactic treatment. These two villages were not further studied. Resistance to ISM was observed in 5 of the remaining 8 villages (Débé, Bendougou, Kangotenga, Mou and Laro) where the relative risk (control/treated hazard ratios) of becoming infected was lower than 2 i.e. between 0.89 (95% CI: 0.43-2.74) and 1.75 (95% CI: 0.57-5.37). In contrast, this study did not show evidence of resistance to DA in the surveyed villages with only 8.6% (n=93) of the cattle relapsing after treatment. Our results suggest that because of the low prevalence of multiple resistances in the area a meticulous use of the sanative pair system would constitute the best option to delay as much as possible the spread of chemoresistance till complete eradication of the disease by vector control operations.     

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.     

Interference between drug-resistant and drug-sensitive stocks of Trypanosoma congolense in goats

A study was undertaken in goats to investigate the ability of two unrelated stocks of Trypanosoma congolense, one of which is highly sensitive to isometamidium chloride and one which is drug-resistant, to become established in the presence of an existing infection with the other stock. The goats, which were initially infected with the sensitive strain and were then challenged with the resistant strain, were cured by treatment at 0.1 mg kg-1 isometamidium, indicating that the resistant stock did not establish an infection. Goats initially infected with the resistant stock, which were then challenged with the sensitive stock, experienced temporary remission of infection followed by relapse after treatment at 0.1 mg kg-1 isometamidium. In contrast, the goat infected only with the resistant stock remained parasitaemic following treatment at 0.1 mg kg-1. This suggests that superinfection with the sensitive stock resulted in the establishment of infection, which suppressed the resistant stock to below the limit of detection of the method used. These observations suggest that isometamidium-resistant stocks may be less viable than sensitive strains, and could explain the relative scarcity of isometamidium resistant in the field.     

Therapeutic and prophylactic activity of isometamidium chloride in Boran cattle against Trypanosoma vivax transmitted by Glossina morsitans centralis

Ten Boran steers were infected with Trypanosoma vivax, transmitted by Glossina morsitans centralis; five steers with a T vivax clone from Nigeria and five with a T vivax clone from Kenya. Eleven days after infection all 10 animals were treated with 0.5 mg kg-1 isometamidium chloride. Four steers infected with the Nigerian T vivax and all five infected with the Kenyan T vivax were completely cured. When different steers received a single prophylactic dose of 0.5 mg kg-1 isometamidium chloride and subjected to monthly tsetse-transmitted challenge with the same T vivax clones, complete protection was afforded for at least two months against challenge with the Nigerian T vivax, but for less than one month against the Kenyan T vivax. The findings indicate that the level of sensitivity of a T vivax population to the prophylactic activity of isometamidium chloride cannot be concluded from sensitivity studies based on the therapeutic action of the drug.     

Isometamidium chloride prophylaxis against Trypanosoma congolense challenge and the development of immune responses in Boran cattle

Twenty-four Boran cattle were injected with isometamidium chloride (1 mg/kg bodyweight) to investigate the duration of drug-induced prophylaxis against infection by metacyclic forms of Trypanosoma congolense and to determine if specific antibody responses to the organism were mounted by animals under chemoprophylactic cover. Complete protection against either single challenge by five tsetse flies infected with T congolense, or repeated challenge at monthly intervals by five tsetse flies, lasted for five months. Six months after treatment, two-thirds of the cattle were resistant to challenge, irrespective of whether subjected to single or multiple challenge with trypanosome-infected tsetse flies, or titrated doses of in vitro-cultured metacyclic forms of T congolense (5 X 10(2) to 5 X 10(5) organisms), inoculated intradermally. No animal which resisted infection developed detectable skin reactions at the site of deposition of metacyclic trypanosomes or produced trypanosome-specific antibodies. It was concluded that drug residues effectively limited trypanosome multiplication at the site of deposition in the skin, thus preventing subsequent parasitaemia or priming of the host's immune response.     

Quantitative correlation of parasitological and serological techniques for the diagnosis of Trypanosoma congolense infection in cattle

A comparison was made between serological and parasitological techniques for the diagnosis of bovine trypanosomiasis in Zambia. Overall sero-prevalence rates as determined by IFAT and ELISA were respectively 2.7-fold and 2.9-fold greater then the percentage of samples found positive with the dark ground/phase contrast buffy coat technique (DG). The results obtained by the two serological techniques were found to be closely correlated (94.2%) agreement) and titres obtained by ELISA tended to be slightly higher than those obtained by IFAT. Linear regression analysis of the results obtained by the IFAT and DG techniques revealed a highly significant correlation. This finding would permit the use of only one of the techniques in an epidemiological survey and to extrapolate the results from the regression line.     

Quantitative correlation of parasitological and serological techniques for the diagnosis of Trypanosoma congolense infection in cattle

Summary

A comparison was made between serological and parasitological techniques for the diagnosis of bovine trypanosomiasis in Zambia. Overall sero‐prevalence rates as determined by IFAT and ELISA were respectively 2.7‐fold and 2.9‐fold greater then the percentage of samples found positive with the dark ground/phase contrast buffy coat technique (DG). The results obtained by the two serological techniques were found to be closely correlated (94.2% agreement) and titres obtained by ELISA tended to be slightly higher than those obtained by IFAT. Linear regression analysis of the results obtained by the IFAT and DG techniques revealed a highly significant correlation. This finding would permit the use of only one of the techniques in an epidemiological survey and to extrapolate the results from the regression line.     

Effect of novidium (homidium chloride) chemotherapy on genital lesions induced by Trypanosoma vivax and Trypanosoma congolense infections in Zebu bulls

Zebu bulls chronically infected with Trypanosoma vivax and T. congolense were treated at the 12th week post-infection with novidium and slaughtered at different times after treatment to determine histological evidence of healing of the genital lesions. Though trypanosomes disappeared from the blood soon after chemotherapy, there was incomplete resolution of genital lesions even 10-18 weeks later. Where there is severe degeneration of the testes and epididymes chemotherapy may be ineffective in leading to regeneration and therefore infertility problems may persist in bulls with chronic trypanosomiasis which are later subjected to chemotherapy.     

Two enzymically distinct stocks of Trypanosoma congolense

Two stocks of Trypanosoma congolense, isolated from a sheep and a goat on the Kenyan coast, were found to be distinctly different from 112 other stocks of the same species after comparing, by isoenzyme electrophoresis, trypanosomes originating from various parts of Africa. The results show a marked genetic distinction that may be of epidemiological or taxonomic significance.