Veterinary Parasitology 119 (2004) 125–135
Trypanocidal drug resistance in eastern province of Zambia L. Sinyangwe a,1 , V. Delespaux b,∗,1 , J. Brandt b , S. Geerts b , J. Mubanga a , N. Machila a , P.H. Holmes c,d , M.C. Eisler c,d a
Central Veterinary Research Institute, Balmoral, P.O. Box 33980 Lusaka, Zambia Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK d International Livestock Research Institute, Nairobi, Kenya b
c
Received 9 July 2003; received in revised form 28 October 2003; accepted 28 November 2003
Abstract 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.
∗ Corresponding author. Tel.: +32-3-247-6262; fax: +32-3-247-6268. E-mail address:
[email protected] (V. Delespaux). 1 L. Sinyangwe and V. Delespaux contributed equally to this article as co-first authors.
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.11.007
126
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
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. © 2003 Elsevier B.V. All rights reserved. Keywords: Trypanosoma sp.; Cattle-protozoa; Isometamidium; Diminazene; Drug resistance; Zambia
1. Introduction Trypanosomosis is considered as an important disease constraint to livestock production in eastern province of Zambia, preventing full use of the land to feed the rapidly increasing human population (Doran, 2000). Although a vector control operation is taking place in the area (Delespaux, 1998), trypanocidal drugs remain widely available and also affordable for farmers through a drug delivery system (Van den Bossche et al., 2000; Delespaux, 2000). Trypanocidal drug treatment will probably remain the mainstay of control of African bovine trypanosomiasis for the foreseeable future, and the development of resistance to the small number of available compounds is generally regarded as a cause for considerable concern (Geerts and Holmes, 1998). Information on the extent and significance of the problem of drug resistance is still scant. Although some firm evidence of resistance to trypanocidal drugs exists in several areas of East and Southern Africa (Chitambo and Arakawa, 1992; Geerts and Holmes, 1998; Sinyangwe et al., 1999), the situation in the eastern province of Zambia has not previously been assessed in detail. Almost all regions of Zambia are infested with tsetse flies (Ford and Katondo, 1977; Van den Bossche and Vale, 2000). Eastern province is worst affected with, according to the Veterinary Department of Zambia, approximately 200,000 cattle at risk from the disease. A survey of bovine trypanosomosis conducted in this province in 1995 by the Regional Tsetse and Trypanosomosis Control Program (RTTCP) showed a prevalence of 13.35% (Hopkins et al., 1995). Consequently there is a high level of trypanocidal drug usage in this province. In this province, Petauke district has the largest cattle population of approximately 77,000. The objective of this study was to assess the extent and the significance of trypanocidal drug resistance in eastern province of Zambia.
2. Materials and methods 2.1. Cross-sectional study In 1996, a cross-sectional study of bovine trypanosomosis was carried out in Lundazi, Petauke and Katete districts of the eastern province of Zambia, based on village crushpens included in the 1995 RTTCP survey (Hopkins et al., 1995). The selection of sampling sites was made from 128 crushpens where the 1995 survey showed greater than 6% prevalence
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
127
Fig. 1. Sampling sites in Petauke, Katete and Lundazi districts, eastern province of Zambia.
of bovine trypanosomosis. A total of 34 sites were randomly selected from these 128 and approximately 50 cattle sampled at each site. All the sampling sites were geo-referenced by GPS. The study sites are shown in Fig. 1. At each crushpen, cattle above 6 months were examined for the presence of trypanosome infections using the haematocrit centrifugation technique, and the dark-ground buffy-coat method (Murray et al., 1977). Thin and thick blood smears were also prepared and packed red blood cell volumes (PCVs) were determined. Blood samples for use in the isometamidium-ELISA were collected by jugular venipuncture into Vacutainers (Becton Dickinson). Sera were later separated and stored at −20 ◦ C. Blood samples from cattle found to be positive for Trypanosoma congolense and Trypanosoma brucei were inoculated immediately at the crushpen into mice and stabilates were prepared in liquid nitrogen using glycerol (10% v/v) as a cryopreservative. At the time of sampling of cattle, background epidemiological data, including sex, age, weight, disease records, and drug usage were obtained. 2.2. Longitudinal study Eight sampling sites were selected purposively, based on cattle population and disease prevalence information obtained in the 1995 and 1996 cross-sectional studies. These sites
128
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
had high trypanosome prevalence and no control activity had been carried out. Six sites were located in Katete (three sites) and Petauke (three sites) districts as described previously (Machila et al., 2001). A further two sites were located in Lundazi district. At each selected site, 50 cattle were sampled every 2 months for 1 year from November 1997 till June 1998. These animals were sampled at random from those brought to the sampling site. The techniques and methodologies used were the same as for the cross-sectional studies. Cattle found positive for trypanosomes were treated with diminazene aceturate at 3.5 mg/kg b.w. Treated cattle were identified so that they were not included in the next sampling. The owners were questioned about their drug usage practices. Isometamidium-ELISA and antibody-ELISA for bovine trypanosomosis were conducted on all the serum samples collected. 2.3. Staining and examinations of blood smears All the thick and thin smears prepared in the field were Giemsa stained and examined under a microscope in the laboratory. The trypanosomes seen were identified to species level based on morphology following the recommendations of the Office International des Epizooties (1996). 2.4. Trypanocidal drug sensitivity tests in mice The sensitivity of trypanosomes stabilates to isometamidium chloride and diminazene aceturate was established using standardised methods (Eisler et al., 2001). Briefly, groups of 18 mice were inoculated with 5×105 trypanosomes of a particular stabilate of T. congolense by the intra-peritoneal route, followed 24 h later by administration of aqueous solutions of either isometamidium chloride (1.0 mg/kg b.w.) or diminazene aceturate (20 mg/kg b.w.). Six mice were treated with each drug dose, and six control mice were treated with water to confirm the viability of the trypanosome inoculum. Wet tail-blood films from the mice were examined microscopically twice a week for 60 days, or until evidence of relapse of trypanosome infection was observed. For some stabilates, group sizes of five rather than six mice were used. If at least five mice in each group of six were cured, the parasite population under test was considered to be sensitive to the drug dose with which that group was treated. If one mouse in a treated group of six died without becoming parasitaemic, or if a group size of five was used, at least four of the five mice must be cured before the parasite population could be classified as sensitive. At least five of the six control mice had to become parasitaemic, otherwise the test was repeated for all three groups. 2.5. Trypanocidal drug sensitivity tests in calves One month before infection, calves were brought to a fly-proof experimental housing and treated subcutaneously with ivermectin at 200 g/kg b.w. They were then injected intramuscularly with diminazene aceturate at 3.5 mg/kg b.w. and with long-acting oxytetracycline at 20 mg/kg b.w. They were also sprayed/washed with acaricide.
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
129
Beginning 2 weeks prior to inoculation of trypanosome isolate, and continuing until the end of the experiment, PCV and parasitaemia at least were monitored twice a week using the buffy-coat phase-contrast technique (Murray et al., 1977). For reasons of economy, up to five T. congolense stabilates from each individual sampling site were pooled and each pool was inoculated into a calf aged approximately 6 months. At the first detectable peak of parasitaemia the calves were treated with whichever trypanocidal drug (isometamidium chloride at 0.5 mg/kg b.w. or diminazene aceturate at 3.5 mg/kg b.w.) was commonly used at the sampling site from where the pooled stabilates originated. Following treatment cattle were monitored for trypanosome infection for up to 100 days. If a relapse infection was detected during this period, the animal was treated with the other trypanocidal drug. Later the protocol was modified to two calves per stabilate; one calf was treated with isometamidium chloride at 0.5 mg/kg b.w., while the other one was treated with 3.5 mg/kg b.w. diminazene aceturate. 2.6. Antibody-ELISA Serum samples were screened for the presence of trypanosomal antibodies using an indirect anti-trypanosomal antibody-detection ELISA (Hopkins et al., 1998). 2.7. Isometamidium-ELISA Isometamidium concentrations were determined by ELISA as described by Eisler et al. (1993), with the modifications described by Mubanga (1996). The limit of detection of this assay was 0.4 ng isometamidium/ml serum.
3. Results 3.1. Prevalence of bovine trypanosomosis During the cross-sectional study, 1597 cattle over 6 months of age were sampled at 34 randomly selected sampling sites. Trypanosome infections were found in cattle in 25 (74%) sampling sites (Fig. 2). Prevalence in individual villages varied between 0 and 64%. Overall mean prevalence was 14.4%, with 96% of infections caused by T. congolense. The remainder was caused by Trypanosoma vivax (2%) and T. brucei (2%). There were highly significant differences among the three districts in terms of trypanosome infection prevalence in the study villages (χ2 = 142, d.f. = 2; P < 0.001). Villages in Petauke district (n = 17) had the highest mean prevalence (24.6%), Lundazi villages (n = 10) had the lowest mean prevalence (0.8%), while the Katete villages (n = 7) were intermediate with a mean prevalence of 10.4%. The parasitological and trypanosome antibody-ELISA results for the six villages in the longitudinal study in Katete and Petauke districts have been reported elsewhere (Machila et al., 2001). In both of the two villages in Lundazi district, trypanosome prevalence was
130
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
Fig. 2. Trypanosome prevalence in cattle in Petauke, Katete and Lundazi districts, eastern province of Zambia.
0% at two visits (April and August 1998) and between 2.3 and 6% at two other visits (February and June 1998). Hence, parasitological prevalence of trypanosomosis varied widely between the eight villages and between visits. Overall, five villages (two each in Katete and Lundazi districts, one in Petauke district) had a minimum prevalence of 0%, and a maximum prevalence not greater than 6%. Three villages (one in Katete district and two in Petauke district) had maximum prevalences between 20 and 30% and minimum prevalences between 2 and 5%. There was no particular season of peak prevalence, which was unpredictable. 3.2. Investigation of trypanocidal drug resistance in mice Seventy-one T. congolense stabilates collected during these studies were tested for sensitivity to isometamidium chloride and diminazene aceturate in mice. Using discriminatory doses of 1.0 mg/kg b.w. isometamidium chloride and 20 mg/kg b.w. diminazene aceturate, 38 (53.5%) were sensitive to both drugs, 24 (34%) were resistant to only isometamidium, 8 (11.3%) were resistant to only diminazene aceturate and 1 (1.4%) was resistant to both drugs. The geographic locations of villages from which evidence of drug resistance was obtained in mice are shown in Fig. 3.
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
131
Fig. 3. Trypanocidal drug resistance in eastern province of Zambia.
3.3. Investigation of trypanocidal drug resistance in calves Twenty-seven T. congolense stabilates were tested for drug sensitivity in 12 calves. Six pools of three, four or five stabilates, each pool comprising stabilates from a single village in either Katete district (Kapeya village, five stabilates) or Petauke district (Njeleweka and Chipika villages, each three stabilates), two pools of four stabilates and one pool of five stabilates) were tested in six calves. Five of these calves were treated with diminazene aceturate at the first peak of parasitaemia. The sixth calf was treated with isometamidium chloride. One relapse infection was detected 100 days after diminazene treatment of a calf inoculated with a pool of three stabilates from Njeleweka village, Petauke district. No relapse infection was detected in this calf, after re-treatment 1 week later with isometamidium chloride, nor in any of the other five calves. Three further stabilates from Petauke district were each tested in two calves. No relapse infection was detected in the three calves treated with diminazene aceturate. One relapse infection was detected 57 days after treatment with isometamidium chloride in a calf infected with a stabilate from Njeleweka village. Relapse infection was not detected in the other two isometamidium-treated calves. 3.4. Investigations using isometamidium-ELISA Isometamidium-ELISA results were available for 1526 serum samples from cattle in the cross-sectional study. Sixty-three (4.1%) of these serum samples contained levels of
132
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
Fig. 4. Percentage of cattle in Petauke, Katete and Lundazi districts, eastern province of Zambia treated with isometamidium chloride.
isometamidium higher than 0.4 ng/ml. The geographical distribution of cattle with isometamidium concentrations above 0.4 ng/ml in the various study villages is shown in Fig. 4. Isometamidium concentrations exceeding 0.4 ng/ml in trypanosome-infected cattle were considered to provide evidence of drug resistance in the infecting parasite population (Eisler et al., 1997). Trypanosome infections were detected in 212 (13.9%) of the 1526 animals tested for serum isometamidium. Of these 212 infected cattle, only 6 (2.8%) had serum levels of the drug above this concentration. There was no significant difference (χ2 = 0.609, d.f. = 1; P = 0.435) between the proportions of male (4.5%) and female (3.7%) cattle treated with isometamidium, or (χ2 = 1.99, d.f. = 1; P = 0.159) between cattle aged less than 2 years (2.4%) and those older than 2 years of age (2.4%). The differences in treatment rates among the three districts were significant (χ2 = 12.4, d.f. = 2; P < 0.01), with relatively fewer cattle with evidence of isometamidium treatment in Lundazi district (1.9%) than in Petauke district (4.3%) and relatively more in Katete district (7.0%). 4. Discussion Trypanocidal drug resistance in eastern province of Zambia was investigated between 1996 and 1998. A cross-sectional study was first conducted in three districts including 34
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
133
sampling sites selected at random from villages that had shown greater than 6% prevalence of bovine trypanosomosis during an earlier survey (Fig. 1). As a 6% threshold selection has been chosen, the results for the study villages in any district should not be extrapolated to that district as a whole. A longitudinal study over a 1-year period was then conducted in the same three districts. The sampling sites were selected from the cross-sectional study based on high trypanosomosis prevalence and absence of control activities. A combination of parasitological methods, tests of resistance in mice and cattle and isometamidium-ELISA was used. The overall mean prevalence of trypanosomosis was 14.4% and the area of Petauke showed the highest prevalence (Fig. 2) which confirms the results of the RTTCP surveys conducted between 1995 and 1997 where 6361 head of cattle were sampled at 128 sites (Van den Bossche et al., 2000). Drug resistance was already reported in Zambia (Chitambo and Arakawa, 1992) but this study represents the first large scale monitoring of drug resistance in the country (Fig. 3). Almost half of the trypanosome stabilates collected from cattle in eastern province of Zambia showed evidence of resistance to either isometamidium (35.0% of stabilates) or diminazene (12.7% of stabilates) when tested in mice. The high prevalence of isometamidium resistance could be explained by the large scale use of isometamidium (block treatments) in the 1980s (Chizyuka, pers. commun.). There is no particular reason to expect that the degree of resistance to isometamidium will worsen over the foreseeable future since isometamidium usage appears to have declined considerably since then (Van den Bossche et al., 2000; Delespaux, 2000). This decline is confirmed by the low number of animals showing evidence of treatment in this study. However, the drug resistance situation in eastern province of Zambia is not as serious as that observed in coastal areas of Kenya (Mdachi, 1999) or Tanzania, where as few as 6% of stabilates tested were sensitive to the same discriminatory drug doses in mice (Eisler et al., 2000). Fortunately, the use of these two drugs as a sanative pair can still be expected to be effective in this area, since only 1 out of 71 stabilates showed evidence of resistance to both. This situation is contrast to that observed in coastal areas of Kenya and Tanzania where resistance to both drugs in commonly observed in individual stabilates (Mdachi, 1999; Eisler et al., 2000), or in Ethiopia’s Ghibe Valley, where demonstration of resistance to both drugs in individual trypanosomes suggests that this sanative pair is no longer efficacious (Mulugeta et al., 1997). Using drug sensitivity tests in calves at least 2 out of 27 T. congolense stabilates were shown to be resistant to either diminazene or isometamidium. Since stabilate pools were used, it is not possible to identify the exact number of resistant T. congolense. However, these data confirm the presence of resistance both to isometamidium and diminazene as was shown in the test in mice. The isometamidium-ELISA showed evidence of recent treatment in only a small proportion (4.1%) of cattle tested. This may reflect either infrequent treatment or the use of relatively low doses of the drug, since the isometamidium-ELISA has been shown capable of detecting the drug for up to 4 months following treatment of cattle at a dose rate of 1.0 mg/kg b.w. (Eisler et al., 1997). Interestingly, there were significant differences in the proportions of cattle with evidence of isometamidium treatment among the three districts (Fig. 4). The Lundazi district villages had a relatively small proportion of cattle with evidence of treatment (1.9%), suggesting drug-use practices appropriate for the small
134
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
proportion infected (0.8%). A much higher proportion of cattle in study villages in Petauke district was infected (24.6%), but was not reflected in the proportion with evidence of treatment (4.3%). Finally, the study villages with the highest proportion of cattle with evidence of treatment (7.0%) were those in Katete district, where the proportion with infections (10.4%) was less than half of that in Petauke. The simultaneous presence of trypanosomes and serum concentrations of isometamidium above 0.4 ng/ml were observed in only 2.8% of the infected cattle. This figure is likely to underestimate the prevalence of drug resistance because the large majority of the cattle had not been treated at the time of the sampling. Standardised tests in mice as described by Eisler et al. (2001), allowed the screening of a large number of strains for drug resistance. This approach is particularly suited for investigation of drug resistance on an area-wide basis. The combined use of single-dose tests in mice and cattle and of the isometamidium-ELISA allowed a precise description of the prevailing situation in the province. Underdosing is certainly one of the major causes of drug resistance in this area as was shown by Delespaux et al. (2002) who used an isometamidium-ELISA to monitor the correct use of this drug by farmers and veterinary assistants. In conclusion, this investigation of drug resistance on an area-wide basis in eastern province of Zambia was consistent with previous observations of drug resistance but revealed little evidence of a marked and widespread drug resistance problem. Drugs can still be considered as an effective method for controlling trypanosomosis but should be used with caution. Given the fact that roughly one-third of the trypanosome isolates showed evidence of resistance to isometamidium and one-tenth to diminazene, greater efforts should be made to avoid exclusive reliance on drugs. If the efficacy of the current trypanocidal drugs is to be maintained, the number of treatments should be reduced (in order to reduce drug pressure) as much as possible by integrating drug usage with other control measures (Geerts and Holmes, 1998). Finally the data presented here may be used as a reference against which the situation in future may be compared.
Acknowledgements This study was carried out with the financial support of the European Union (INCO-DC project IC18-CT95-006). The support of the ASVEZA project (funded by the Belgian Development Cooperation) is also gratefully acknowledged.
References Chitambo, H., Arakawa, A., 1992. Trypanosoma congolense: manifestation of resistance to Berenil and Samorin in cloned trypanosomes isolated from Zambian cattle. Zentralbl. Bakteriol. 277, 371–381. Delespaux, V., 1998. Tsetse control: a community based approach. Sleeping, sickness, rediscovered. In: Proceedings of the International Colloquium, Institute of Tropical Medicine, Antwerp, 14–18 December 1998 (abstract). Delespaux, V., 2000. Drug delivery system of trypanocides in eastern province of Zambia. ICPTV Newslett., 2, 29.
L. Sinyangwe et al. / Veterinary Parasitology 119 (2004) 125–135
135
Delespaux, V., Geerts, S., Brandt, J., Elyn, R., Eisler, M.C., 2002. Monitoring the correct use of isometamidium by farmers and veterinary assistants in eastern province of Zambia using the Isometamidium-ELISA. Vet. Parasitol. 110, 117–122. Doran, M. (Ed.), 2000. Socio-economics of trypanosomosis. In: Implications for Control Strategies within the Common Fly-belt of Malawi, Mozambique, Zambia and Zimbabwe, 1st ed. RTTCP, Harare, pp. 1–156. Eisler, M.C., Gault, E.A., Smith, H.V., Peregrine, A.S., Holmes, P.H., 1993. Evaluation and improvement of an enzyme-linked immunosorbent assay for the detection of isometamidium in bovine serum. Ther. Drug Monit. 15, 236–242. Eisler, M.C., Gault, E.A., Moloo, S.K., Holmes, P.H., Peregrine, A.S., 1997. Concentrations of isometamidium in the sera of cattle challenged with drug-resistant Trypanosoma congolense. Acta Trop. 63, 89–100. Eisler, M.C., Ndung’u, J.M., Murilla, G.A., Mdachi, R.E., Mbwambo, H., Sinyangwe, L., Machila, N., Delespaux, V., Geerts, S., Brandt, J., Peregrine, A.S., McDermott, J.J., Holmes, P.H., 2000. Area-wide appraisal of drug resistance in trypanosomes infecting cattle in east and southern Africa. ICPTV Newslett. 2, 16–18. Eisler, M.C., Brandt, J., Bauer, B., Clausen, P.H., Delespaux, V., Holmes, P.H., Ilemobade, A., Machila, N., Mbwambo, H., McDermott, J., Mehlitz, D., Murilla, G., Ndung’u, J.M., Peregrine, A.S., Sidibe, I., Sinyangwe, L., Geerts, S., 2001. Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle. Vet. Parasitol. 97, 171–182. Ford, J., Katondo, K.M., 1977. Maps of tsetse flies (Glossina) distribution in Africa, 1973, according to sub-generic groups on scale of 1:5,000,000 (plus a set of nine maps in colour). Bull. Anim. Health Prod. Afr. 2, 187–193. Geerts, S., Holmes, P.H., 1998. Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT Tech. Sci. Series No. 1. FAO, Rome, 31 pp. Hopkins, J.S., Robinson, T.P., Harris, R., 1995. Final technical report of the insect pest management initiative project Zambia, IPMI (tsetse). Report to the Overseas Development Administration (UK), Department for Animal Production and Health, Lusaka. Hopkins, J.S., Chitambo, H., Machila, N., Luckins, A.G., Rae, P.F., Van den Bossche, P., Eisler, M.C., 1998. Adaptation and validation of antibody-ELISA using dried blood spots on filter paper for epidemiological surveys of tsetse-transmitted trypanosomosis in cattle. Prev. Vet. Med. 37, 91–99. Machila, N., Sinyangwe, L., Mubanga, J., Hopkins, J.S., Robinson, T., Eisler, M.C., 2001. Antibody-ELISA seroprevalence of bovine trypanosomosis in the eastern province of Zambia. Prev. Vet. Med. 49, 249–257. Mdachi, R., 1999. Epidemiological studies into the impact of trypanocidal drug resistance on the control of trypanosomiasis in coastal Kenya. Ph.D. Thesis. University of Glasgow, Glasgow. Mubanga, J., 1996. Studies on chemoprophylaxis of trypanosomiasis using a competitive enzyme immunoassay for the detection of isometamidium and homidium in cattle and sheep. MVM Thesis. University of Glasgow, Glasgow, 174 pp. Mulugeta, W., Wilkes, J., Mulatu, W., Majiwa, P.A., Masake, R., Peregrine, A.S., 1997. Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe. Ethiopia Acta Trop. 64, 205–217. Murray, M., Murray, P.K., Mcintyre, W.I.M., 1977. An improved parasitological technique for the diagnosis of African trypanosomosis. Trans. R. Soc. Trop. Med. Hyg. 71, 325–326. Office International des Epizooties (Ed.), 1996. Manual of Standards for Diagnostic Tests and Vaccines, supplément au Manuel des normes pour les tests de diagnostic et les vaccins pour les maladies des listes A et B des mammifères, oiseaux et abeilles, Paris, X.6, pp. 105–108. Sinyangwe, L., Machila, N., Mubanga, J., Delespaux, V., Brandt, J., Geerts, S., Holmes, P.H., Eisler, M., 1999. Trypanocidal drug resistance in the eastern province of Zambia. In: Proceedings of the 25th Meeting of the International Scientific Council for Trypanosomiasis & Research Control (ISCTRC), Mombasa, Kenya, 27 September–1 October 1999, 39 (abstract). Van den Bossche, P., Doran, M., Connor, R.J., 2000. An analysis of trypanocidal drug use in the eastern province of Zambia. Acta Trop. 75, 247–258. Van den Bossche, P., Vale, G.A. (Eds.), 2000. Tsetse and Trypanosomosis in Southern Africa, 1st ed. RTTCP, Harare, pp. 1–140.
