Stanny Geerts

African bovine trypanosomiasis: the problem of drug resistance

The three trypanocides used to control tsetse-transmitted trypanosomiasis in domestic animals in Africa have been in use for over 40 years and, not surprisingly, resistance of trypanosomes to these drugs has emerged. Because of the relatively limited market in Africa and the high costs of developing and licensing new drugs, international pharmaceutical companies have shown little interest in the development of new trypanocides for use in either animals or humans. Therefore, the current challenge is to achieve optimal use of the relatively old existing drugs, and it is in this context that the problem of drug resistance has to be quantified--as discussed here by Stanny Geerts, Peter Holmes, Oumar Diall and Mark Eisler.

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.

Molecular tools for the rapid detection of drug resistance in animal trypanosomes

There are currently 17 African countries in which animal trypanocidal drug resistance has been reported. Large-scale surveys were carried out in only ten of them. The lack of baseline information is mainly due to the fact that the methods currently available for the detection of drug resistance are laborious, expensive and time consuming. In this review the mechanisms involved in resistance to isometamidium and diminazene will be discussed, together with some new molecular detection tools that have been developed recently enabling faster diagnosis of drug resistance than conventional laboratory or field tests.

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.

Five-fold increase in Trypanosoma congolense isolates resistant to diminazene aceturate over a seven-year period in Eastern Zambia.

Two groups of Trypanosoma congolense isolates collected from cattle in 1996 (n=39) and 2003 (n=38) in the Eastern Province of Zambia were analyzed by BclI-PCR-RFLP to assess the evolution of diminazene aceturate (DA) resistance over a period of seven years. The results show a significant increase of DA resistance in this relatively short period of time. In 1996, among the 39 isolates, 61.5% were found sensitive, 12.8% resistant and 25.7% had a mixed BclI-PCR-RFLP profile. In 2004, among the 38 isolates, 10.5% were found sensitive, 63.2% were resistant and 26.3% showed a mixed BclI-PCR-RFLP profile. In vivo tests in mice showed that isolates with a sensitive or mixed RFLP profile were sensitive to DA whereas isolates with a resistant RFLP profile were resistant. Since there are no indications that the drug pressure has increased between 1996 and 2003, it is suggested that genetic exchange of resistance genes might explain the increased frequency of resistance to DA.

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.

Chemosensitization of Trypanosoma congolense strains resistant to isometamidium chloride by tetracyclines and enrofloxacin

Background Because of the development of resistance in trypanosomes to trypanocidal drugs, the livelihood of millions of livestock keepers in sub-Saharan Africa is threatened now more than ever. The existing compounds have become virtually useless and pharmaceutical companies are not keen on investing in the development of new trypanocides. We may have found a breakthrough in the treatment of resistant trypanosomal infections, through the combination of the trypanocide isometamidium chloride (ISM) with two affordable veterinary antibiotics. Methodology/Principal Findings In a first experiment, groups of mice were inoculated with Trypanosoma congolense strains resistant to ISM and either left untreated or treated with (i) tetracycline, (ii) ISM or (iii) the combination of the antibiotic and the trypanocide. Survival analysis showed that there was a significant effect of treatment and resistance to treatment on the survival time. The groups treated with ISM (with or without antibiotic) survived significantly longer than the groups that were not treated with ISM (P<0.01). The group treated with the combination trypanocide/antibiotic survived significantly longer than the group treated with ISM (P<0.01). In a second experiment, groups of cattle were inoculated with the same resistant trypanosome strain and treated with (i) ISM, (ii) ISM associated with oxytetracycline or (iii) ISM associated with enrofloxacine. All animals treated with ISM became parasitaemic. In the groups treated with ISM-oxytetracycline and ISM-enrofloxacine, 50% of the animals were cured. Animals from the groups treated with a combination trypanocide/antibiotic presented a significantly longer prepatent period than animals treated with ISM (p<0.001). The impact of the disease on the haematocrit was low in all ISM treated groups. Yet, it was lower in the groups treated with the combination trypanocide/antibiotic (p<0.01). Conclusions/Significance After optimization of the administration protocol, this new therapeutic combination could constitute a promising treatment for livestock infected with drug resistant T. congolense.

Seasonal distribution and abundance of tsetse flies (Glossina spp.) in the Faro and Deo Division of the Adamaoua Plateau in Cameroon

Abstract.  Ten years after the large‐scale tsetse control campaigns in the important cattle rearing areas of the Faro and Deo Division of the Adamaoua Plateau in Cameroon, the seasonal distribution and abundance of tsetse flies (Glossina spp.) were determined. During a period of 12 consecutive months (January–December 2005), the tsetse population was monitored along four trap transects consisting of a total of 32 traps and two flyround transects traversing the study area, which comprised the tsetse‐infested valley, a buffer zone and the supposedly tsetse‐free plateau. Throughout the study period, a total of 2195 Glossina morsitans submorsitans and 23 Glossina tachinoides were captured in the traps and 1007 G. m. submorsitans (78.8% male flies) were captured along the flyround transects. All G. tachinoides and almost all G. m. submorsitans were captured in the valley. Five G. m. submorsitans were captured in traps located in the buffer zone, whereas no flies were captured in traps located on the plateau. The index of apparent abundance (IAA) of G. m. submorsitans was substantially higher in the areas close to game reserves. In the remaining part of the valley, where wildlife is scarce and cattle are present during transhumance (dry season), the IAA of tsetse was substantially lower. In this part of the valley, the abundance of tsetse seemed to be associated with the presence of cattle, with the highest IAA during transhumance when cattle are present and the lowest apparent abundance during the rainy season when cattle have moved to the plateau. It is concluded that the distribution of tsetse in a large part of the valley undergoes substantial seasonal changes depending on the presence or absence of cattle. The repercussions of those findings for the control of tsetse in the valley and the probability of reinvasion of the plateau are discussed.

Energy Metabolism of Bloodstream Form Trypanosoma theileri

ABSTRACT Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.

Epidemiology and control of bovine theileriasis.

The International Colloquium on Epidemiology and Control of Bovine Theileriosis, held at the Institute of Tropical Medicine (ITM), Antwerp, Belgium, 10–12 December 1997, was organized by the Veterinary Department of ITM.