Aberrant use and poor quality of trypanocides: a risk for drug resistance in south western Ethiopia.

BACKGROUND: Trypanocidal drugs have been used to control African animal trypanosomosis for several decades. In Ethiopia, these drugs are available from both authorized (legal) and unauthorized (illegal) sources but documentation on utilization practices and quality of circulating products is scanty. This study looked at the practices of trypanocidal drug utilization by farmers and the integrity of active ingredient in trypanocides sold in Gurage zone, south western Ethiopia. The surveys were based on a structured questionnaire and drug quality determination of commonly used brands originating from European and Asian companies and sold at both authorized and unauthorized markets. One hundred farmers were interviewed and 50 drug samples were collected in 2013 (Diminazene aceturate = 33 and Isometamidium chloride = 17; 25 from authorized and 25 from unauthorized sources). Samples were tested at the OIE-certified Veterinary Drug Control Laboratory (LACOMEV) in Dakar, Senegal, by using galenic standards and high performance liquid chromatography. RESULTS: Trypanosomosis was found to be a major threat according to all interviewed livestock keepers in the study area. Diminazene aceturate and isometamidium chloride were preferred by 79% and 21% of the respondents respectively, and 85% of them indicated that an animal receives more than six treatments per year. About 60% of these treatments were reported to be administered by untrained farmers. Trypanocidal drug sources included both unauthorized outlets (56%) and authorized government and private sources (44%). A wide availability and usage of substandard quality drugs was revealed. Twenty eight percent of trypanocidal drugs tested failed to comply with quality requirements. There was no significant difference in the frequency of non-compliance between diminazene-based and isometamidium chloride products (P = 0.87) irrespective of the marketing channel (official and unofficial). However, higher rates of non-compliant trypanocides were detected for drugs originating from Asia than from Europe (P = 0.029). CONCLUSION: The findings revealed the presence of risk factors for the development of drug resistance, i.e. wide distribution of poor quality drugs as well as substandard administration practices. Therefore, it is strongly recommended to enforce regulatory measures for quality control of veterinary drugs, to expand and strengthen veterinary services and to undertake trypanocidal drug efficacy studies of wider coverage.

Cattle breeding, trypanosomosis prevalence and drug resistance in Northern Togo.

African Animal Trypanosomosis (AAT) is a major disease of cattle in Togo and its control is essentially based on chemotherapy. However, because of excessive use of trypanocides during the past decades, chemo-resistance in the parasites has developed. In order to assess the current situation of AAT and resistance to trypanocidal drugs in Northern Togo, a study was conducted on cattle from December 2012 to August 2013 in the regions of Kara and Savanes. An initial cross-sectional survey was carried out in 40 villages using the Haematocrit Centrifugation Technique (HCT). Out of these, 5 villages with a trypanosome prevalence of >10% were selected for a block treatment study (BT) with diminazene diaceturate (DA: 3.5mg/kg for a 14-day follow-up) and isometamidium chloride (ISM: 0.5mg/kg for a 28-day follow-up). Positive blood samples collected during the parasitological surveys and an equivalent number of negatives were further analyzed by PCR-RFLP for trypanosome species confirmation and molecular diagnosis of resistance to DA in Trypanosoma congolense. The results from 1883 bovine blood samples confirmed a high overall trypanosome prevalence of 10.8% in Northern Togo. PCR-RFLP revealed that T. congolense is the dominant pathogenic trypanosome species (50.5%) followed by T. vivax (27.3%), and T. brucei (16.2%). The BT showed varying levels of treatment failures ranging from 0 to 30% and from 0 to 50% for DA and for ISM respectively, suggesting the existence of resistant trypanosome populations in the study area. Our results show that AAT still represents a major obstacle to the development of cattle husbandry in Northern Togo. In areas of high AAT risk, a community-based integrated strategy combining vector control, rational use of trypanocidal drugs and improving the general condition of the animals is recommended to decision makers.

Drug quality analysis through high performance liquid chromatography of isometamidium chloride hydrochloride and diminazene diaceturate purchased from official and unofficial sources in Northern Togo.

Trypanocidal drugs remain the most accessible and thus commonly used means of controlling tsetse transmitted animal African trypanosomosis. In Togo, trypanocides are sold on official as well as unofficial markets, but the quality of these trypanocides is undocumented so a drug quality assessment study was conducted from May 2013 to June 2014. Trypanocides supplied by European, Indian and Chinese pharmaceutical companies and sold on official and unofficial markets in Togo were purchased. In total fifty-two trypanocides were obtained, 24 of these samples from official markets and 28 from unofficial markets made up of a total of 36 diminazene diaceturate and 16 isometamidium chloride hydrochloride samples. The samples were analysed in the reference laboratory of the OIE (World Organisation for Animal Health), Laboratory for the Control of Veterinary Medicines (LACOMEV) in Dakar which uses galenic testing and high performance liquid chromatography (HPLC) testing as standard reference analysis methods. The results revealed a high proportion of trypanocides of sub-standard quality on the Togolese market: 40% were non-compliant to these quality reference standards. All of the HPLC non-compliant samples contained lower amounts of active ingredient compared to the concentration specified on the packaging. Non-compliance was higher in samples from the unofficial (53.57%) than from the official markets (25%; p=0.04).The main drug manufacturers, mostly of French origin in the study area, supply quality drugs through the official legal distribution circuit. Products of other origins mostly found on illegal markets present a significantly lower quality.

PCR and microsatellite analysis of diminazene aceturate resistance of bovine trypanosomes correlated to knowledge, attitude and practice of livestock keepers in South-Western Ethiopia.

African Animal Trypanosomosis is threatening the agricultural production and cattle breeding more severely than any other livestock disease in the continent, even more since the advent of drug resistance. A longitudinal study was conducted from November 2012 to May 2013 in the Ghibe valley to evaluate diminazene aceturate (DA) resistance and assess livestock owner's perception of trypanocidal drug use. Four Peasant Associations (PAs) were purposively selected and the cattle randomly sampled in each PAs. At the beginning of the study (t0), 106 bovines positive for trypanosomes by the haematocrit centrifugation technique (HCT) and 119 negative control animals were recruited for six months follow-up using HCT, 18S-PCR-RFLP, DpnII-PCR-RFLP and microsatellite analysis. Prevalence of trypanosomosis was 18.1% based on the HCT technique and the mean PCV value was 23.6±5.1% for the 587 sampled cattle. Out of the 106 HCT positive, 64 (60.4%) were positive for the presence of trypanosomes using the 18S-PCR-RFLP. Species detection showed 38 (59.4%) Trypanosoma congolense savannah, 18 (28.1%) Trypanosoma vivax, 5 (7.8%) Trypanosoma theileri and 3 (4.7%) T. congolense Kilifi. Among the T. congolense savannah samples, 31 (81.6%) showed a DA resistant RFLP profile, 2 (5.3%) a mixed profile and 5 did not amplify using the DpnII-PCR-RFLP. A positive HCT had a significant effect on PCV (p<0.001) with the mean PCV value equal to 24.4±0.2% in the absence of trypanosomes and to 20.9±0.3% in the presence of trypanosomes. PCV increased significantly (p<0.001) with 4.4±0.5% one month after treatment. All T. congolense savannah type were analyzed using microsatellite markers TCM1, TCM3 and TCM4. The main events were new infections (40.0%) and relapses (37.5%) with cures lagging at 22.5%. In 10 purposively selected PAs a semi-structured questionnaire was used. The average herd size was the highest in Abelti PA (6.7±1.8 TLU) and the mean herd size was statistically different (p=0.01) in the 10 PAs. Trypanosomosis was designated as the main disease affecting cattle by 97% of the respondents. DA was used by 95.5% of the farmers though more than half of them (51.9%) were not familiar with isometamidium (ISM). There was a trend to overdose young small animals and to underdose large ones. Oxen were treated very frequently (nearly 20 times/year) and calves almost never. To improve the situation in the Ghibe valley, extension messages should be delivered to promote a rational drug use, improved livestock management and the application of strategic vector control methods.

Detection of African animal trypanosomes: the haematocrit centrifugation technique compared to PCR with samples stored on filter paper or in DNA protecting buffer.

The present study aimed at comparing the trypanosome specific 18S-PCR-RFLP using samples stored either on Whatman filter papers (PCR-RFLP-fp) or in a commercial cell lysis and DNA protecting buffer (PCR-RFLP-pb) with the haematocrit centrifugation technique (HCT), a method widely used for the diagnosis of African Animal Trypanosomosis. Out of 411 head of cattle, 49 (11.92%) (CI=8.95-15.45) scored positive for the presence of trypanosomes by HCT whereas 75 (18.25%) (CI=14.63-22.33) and 124 (30.17%) (CI=25.77-34.86) scored positive using PCR-RFLP-fp and PCR-RFLP-pb, respectively. Out of the 49 positives by HCT, 14 (28.57%) (CI=16.58-43.26) and 28 (57.14%) (CI=42.21-71.18) were concordant by PCR-RFLP-fp and PCR-RFLP-pb, respectively. None of the PCR techniques detected parasites from the Trypanozoon group. Although HCT detected more cases of Trypanosoma vivax (33), species identification using PCR-RFLP-fp and PCR-RFLP-pb were significantly different (p<0.001) from the HCT technique. The use of DNA protective buffer is thus recommended as the output of the PCR-RFLP-pb is improved and the risk of contamination between samples is reduced.

Baseline survey of animal trypanosomosis in the region of the Boucle du Mouhoun, Burkina Faso.

In view of gathering baseline information about the prevalence of animal trypanosomosis, the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) funded a cross sectional survey in the region of the Boucle du Mouhoun which constitutes the Northern limit of the tsetse distribution in Burkina. This cross sectional study was carried out in 53 villages located in the six provinces of the region. A total of 2002 cattle, 1466 small ruminants and 481 donkeys were sampled. This survey showed that about 25% of the cattle had been treated with trypanocidal drugs within 3 months before the survey compared to 3% and 0.42% for the small ruminants and donkeys, respectively. Parasitological prevalence in cattle was low: 0.77% (95% C.I. 0.30-1.95%). No goats and three donkeys were found infected with trypanosomes. Infections were mainly due to Trypanosoma vivax (75.0%) with cases of Trypanosoma congolense (25.0%). In cattle, the serological prevalence of trypanosomosis, for the entire region of the Boucle du Mouhoun, was 34.2% (95% C.I. 26.1-43.4%). For sheep, goats and donkeys, the prevalence were of 20.9% (95% C.I. 12.2-33.5%), 8.5% (95% C.I. 5.7-12.5%) and 5.8% (95% C.I. 3.9-8.6%), respectively. The age and distance to the river were the two main risk factors associated with seropositivity.

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.

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.

Options for the control of tsetse-transmitted livestock trypanosomosis. An epidemiological perspective.

Tsetse-transmitted livestock trypanosomosis affects livestock in large parts of sub-Saharan Africa. In southern Africa two epidemiological situations can be distinguished. The disease can have an endemic nature with high morbidity and low mortality in the livestock population. Endemic livestock trypanosomosis is found mainly in areas where cattle constitute the main host of tsetse and reservoirs of trypanosomes. Epidemic trypanosomosis, with high morbidity and high mortality is found in areas where wildlife persist as main reservoir and where livestock come into contact with tsetse flies transmitting trypanosomes from the sylvatic reservoir. Based on the differences in impact of the disease on livestock health in these two epidemiological settings, the appropriateness of the available trypanosomosis control tools differs. In trypanosomosis endemic areas, trypanocidal drug use could be the most suitable approach. Possible problems associated with the development of resistance in trypanosomes to the drugs need to be investigated further. In epidemic situations, vector control seems the most appropriate long-term solution.

Virulence in Trypanosoma congolense Savannah subgroup. A comparison between strains and transmission cycles.

Trypanosoma congolense strains have been shown to differ in their virulence both between subgroups and within the Savannah subgroup between strains. This review revisits these findings and complements them with information on the virulence of T. congolense Savannah subgroup strains isolated from cattle (domestic transmission cycle) in different geographical areas and of strains isolated in protected areas where trypanotolerant wildlife species are the reservoir of the trypanosomes (sylvatic transmission cycle). The virulence of a total of 62 T. congolense Savannah subgroup strains (50 domestic and 12 sylvatic), determined using a standard protocol in mice, was compared. Virulence varied substantially between strains with, depending on the strain, the median survival time of infected mice varying from five to more than sixty days. The proportion of highly virulent strains (median survival time <10 days) was significantly (P = 0·005) higher in strains from the sylvatic transmission cycle. The analysis highlights repercussions of the domestication of the trypanosomiasis transmission cycle that may have to be taken in consideration in the development of trypanosomiasis control strategies.

New Trypanosoma (Duttonella) vivax genotypes from tsetse flies in East Africa.

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FFLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main clade of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Assessment of the occurrence of trypanocidal drug resistance in trypanosomes of naturally infected cattle in the Adamaoua region of Cameroon using the standard mouse test and molecular tools.

From May to November 2005, a study was carried out to assess the occurrence of trypanocidal drug resistance (DR) in trypanosomes of naturally infected cattle of the Adamaoua region of Cameroon. Two distinct Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) procedures were used together with an Allele specific-PCR (AS-PCR) and the standardized single-dose mouse test. Using the mouse test, 3 of the 13 Trypanosoma brucei isolates and all 14 tested Trypanosoma congolense isolates were resistant to ISM. However, only 11 of the 25 T. congolense isolates were diagnosed as resistant to ISM using the MboII-PCR-RFLP. Resistance to DA was identified in 1 of the 13 T. brucei isolates and all 11 T. congolense isolates which were tested with the mouse test. Using the AS-PCR or BclI-PCR-RFLP, 3 of the 13 T. brucei isolates and all 25 T. congolense isolates respectively were found resistant. The data presented in this study prove that DR is widespread in the Adamaoua Department of Cameroon. The problem appears to be more serious in T. congolense than in T. brucei. Appropriate measures need to be taken in order to control bovine trypanosomosis in this area.

SSCP analysis of the P2 purine transporter TcoAT1 gene of Trypanosoma congolense leads to a simple PCR-RFLP test allowing the rapid identification of diminazene resistant stocks.

Analyses were made on a Trypanosoma congolense contig coding a putative P2-like nucleoside transporter (the contig was named in this study TcoAT1). The sequence includes a start and stop codon and presents a high similarity with the gene TbAT1 of T. brucei (Smallest Sum Probability 2.8e-136). To investigate a possible link between point mutations and diminazene aceturate (DA) resistance in mice, the TcoAT1 putative genes of 26 T. congolense strains, characterised for DA sensitivity in the single dose mouse test, were screened by means of the Single Strand Conformation Polymorphism technique (SSCP). Results showed that the SSCP profiles of 23 out of 26 (88.5%) T. congolense strains were confirmed by the sensitivity test in mice with the commonly accepted criterion for sensitivity to diminazene being a CD80 of 20mg/kg in the mouse test. The remaining T. congolense strains showed a resistant SSCP profile and relapsed in mice after treatment at doses lower than 20mg/kg indicating that the SSCP is more sensitive than the single dose mouse test for the detection of resistance to diminazene. However, none of the strains used in this study showed a sensitive SSCP profile while they were resistant in the single dose mouse test. The sequencing of the TcoAT1 gene of two sensitive, two intermediate and two resistant strains allowed the set up of a PCR-RFLP test for the discrimination between sensitive and resistant strains confirming the SSCP results for the 26 strains of this study.

Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense.

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.

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.

PCR-RFLP using Ssu-rDNA amplification: applicability for the diagnosis of mixed infections with different trypanosome species in cattle.

The use of a single restriction fragment length polymorphism (RFLP)-PCR assay which is able to characterise all important bovine trypanosome species was evaluated for the detection of mixed infections with Trypanosoma brucei brucei, Trypanosoma theileri, Trypanosoma congolense and Trypanosoma vivax. Results showed that mixed infections are detectable at a minimum ratio of 2%/98% of standardised DNA solutions with a concentration of 10 ng ml(-1). All mixed infections gave clear profiles that could be easily differentiated except with T. theileri and T. congolense where the T. theileri band was concealed by the T. congolense profile.

PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle.

A single polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used to characterise all important bovine trypanosome species. This is the first report of a sensitive pan-trypanosome PCR assay amplifying all species including T. vivax to a comparable extent using a single primer pair. A semi-nested PCR approach resulted in the detection of one T. congolense trypanosome genome/40 microl of blood, applied as buffy coat on filter paper. Restriction enzyme analysis using Msp1 and Eco571 gave a clear distinction between T. congolense, T. brucei, T. vivax and T. theileri. Several subgroups within the T. congolense group could be distinguished but no differences between the species belonging to the subgenus Trypanozoon or between T. simiae and T. theileri could be found. The use of MboII restriction enzyme allowed differentiation between T. simiae and T. theileri. The potential of the essay to be used as a suitable diagnostic tool is discussed.

Monitoring the correct use of isometamidium by farmers and veterinary assistants in Eastern Province of Zambia using the isometamidium-ELISA.

A survey to monitor the use of trypanocidal drugs by cattle breeders was conducted in Zambia. Use was made of a questionnaire and of the isometamidium-ELISA technique. One hundred and twenty-two farmers and 50 veterinary assistants were interviewed. The isometamidium-ELISA was used to monitor the isometamidium serum concentration in 72 cattle, 1 week after unsupervised treatment by 56 farmers and 16 veterinary assistants. Although there was no clear indication of underestimation of the weight of the animals and although farmers had adequate knowledge of the correct usage of isometamidium, the results suggest frequent underdosing when considering isometamidium serum concentrations 1 week after treatment. In 76% of the cases, the expected protection period was equal or shorter than 28 days and equal or shorter than 33 days in 90% of the treated cattle.

Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle.

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.