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.

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.

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.

The influence of relative resistance and urea-supplementation on deliberate infection with Teladorsagia circumcincta during winter.

The consequences for lambs of infection over the winter with Teladorsagia circumcincta were quantified by deliberate, trickle infection of selected animals at 7 months of age. Infected and control uninfected animals were each allocated into four groups, relatively resistant animals on a normal diet, relatively resistant animals on an isocaloric diet supplemented with urea, and relatively susceptible animals on the same two diets. Resistance and susceptibility was assessed by faecal egg counts following natural infection during the summer preceding the deliberate infection. During the deliberate infection egg counts remained low and most parasites recovered at necropsy were inhibited larvae. Nonetheless, infection reduced weight gain, decreased albumin and fructosamine concentrations and provoked a noticeable pepsinogen and eosinophil response. As most larvae were inhibited these responses may have been largely a consequence of immuno-inflammatory responses in the host rather than the direct action of parasites themselves. Relatively resistant animals on the supplemented diet allowed fewer larvae to establish and had higher fructosamine concentrations, higher albumin concentrations and decreased pepsinogen responses. Therefore, a combination of relatively resistant sheep and nutritional supplementation appears most efficient at controlling infection.

Distribution and innervation of lateral line organs in the channel catfish.

The lateral line system of the channel catfish is formed by mechanoreceptive neuromasts located within five pairs of cephalic and one pair of trunk canals, as well as superficial lines of neuromasts, termed accessory and/or pit lines. Five pairs of pit lines occur on the head, and three pairs of superficial lines occur on the trunk. In addition to these mechanoreceptors, which are found in most teleost fishes, catfish also possess a total of over 4000 electroreceptive ampullary organs scattered over the entire body. The lateral line receptors are innervated by five pairs of lateral line nerves whose rami are secondarily associated with facial and trigeminal fibers that innervate taste buds and the dermis of the skin, respectively. The neuromasts of the trunk canal and the ramules of the posterior lateral line nerve that innervate them seem to be organized in a segmental pattern. The same is true for the intervertebral ramules of the recurrent facial ramus, which innervate the external taste buds on the trunk. The fibers of the gustatory and lateral line systems may use the neural crest, the developing spinal nerves, or both, to establish this segmental pattern. In this context, it may not be surprising that there is an intimate relationship among each of the sensory systems in the trunk.

Impact of nutrition on the pathophysiology of bovine trypanosomiasis.

Trypanosomiasis is a major veterinary problem over much of sub-Saharan Africa and is frequently associated with under-nutrition. There is growing evidence that nutrition can have a profound effect on the pathophysiological features of animal trypanosomiasis. These features include anaemia, pyrexia, body weight changes, reduced feed intake and diminished productivity including reduced draught work output, milk yield and reproductive capacity. Anaemia is a principal characteristic of trypanosomiasis and the rate at which it develops is influenced by both protein and energy intakes. Pyrexia is associated with increased energy demands for maintenance which is ultimately manifested by reductions in voluntary activity levels and productivity. Weight changes in trypanosomiasis are markedly influenced by the levels of protein intake. High intakes allow infected animals to grow at the same rate as uninfected controls providing energy intake is adequate whilst low energy levels can exacerbate the adverse effects of trypanosomiasis on body weight. Reductions in feed intake are less apparent in animals which are provided with high protein diets and where intake is limited by the disease animals will often exhibit preferential selection of higher quality browse. Further studies are required to evaluate the minimum levels of protein and energy supplementation required to ameliorate the adverse effect of trypanosomiasis, the nature and quality of protein supplement to achieve these benefits and the influence these have on digestive physiology.

Some pharmacokinetic parameters of the trypanocidal drug homidium bromide in Friesian and Boran steers using an enzyme-linked immunosorbent assay (ELISA).

Pharmacokinetic studies on the trypanocidal drug homidium bromide using a competitive enzyme immunoassay (detection limit 0.1 ng/mL) are reported for non-infected Friesian and Boran steers following treatment with homidium bromide at a dose of 1.0 mg/kg b.w. Following intravenous (i.v.) treatment of Friesian steers (n = 5), the mean serum drug concentrations were 31.9 +/- 2.1 and 3.9 +/- 0.4 ng/mL at 1 and 24 h, respectively. The decline in serum drug concentration was tri-exponential with half-lives of 0.064 +/- 0.037 h for t1/2 alpha, 7.17 +/- 1.87 h for t1/2 beta and 106.3 +/- 6.6 h for t1/2 gamma for distribution and elimination phases 1 and 2, respectively. Drug was detectable in serum for 17 days following treatment. The mean residence time (MRT) was 63.4 +/- 7.5 h. Following intramuscular (i.m.) treatment of Friesian steers (n = 5), the drug concentration at 1 h after treatment was 72.5 +/- 2.2 ng/mL. This declined to 9.8 +/- 1.8 ng/mL at 24 h. Low concentrations of between 0.1 and 0.3 ng/mL remained in circulation for up to 90 days post-treatment. Following intramuscular treatment of Boran steers (n = 5), the mean serum drug concentration at 1 h after treatment was 112.1 +/- 40.3 ng/mL. By 24 h after treatment, the concentration had fallen to 13.0 +/- 3.3 ng/mL. Thereafter, the serum drug concentration-versus-time profile and the pharmacokinetic parameters obtained following non-compartmental analysis were similar to those obtained following intramuscular treatment of Friesian steers.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle.

Two enzyme-linked immunosorbent assays (ELISA) for the determination of homidium in serum of treated cattle have been developed and evaluated. One is a direct competition (Assay 1) and the other an indirect competition assay (Assay 2). Both assays are highly sensitive with a limit of detection of 0.1 ng homidium per mL serum. Homidium levels were measurable in serum of cattle for over 2 months following administration of a single intramuscular (i.m.) dose at 1 mg/kg bodyweight. The level of sensitivity afforded by these assays makes them potentially useful tools in the pharmacokinetic evaluation of homidium and for investigating drug resistance or causes of drug failure. Assay 2 was chosen as being most suitable for further studies.

The influence of energy intake on some blood biochemical parameters in Scottish Blackface sheep infected with Trypanosoma congolense.

The intensity of parasitaemia, degree of anaemia, live body weight gains and blood biochemical changes were measured in two groups of Scottish Blackface sheep infected experimentally with Trypanosoma congolense and allowed either a high (9.9 MJ metabolisable energy (ME) per day) or a low (6.1 MJ ME per day) energy intake. It was observed that infected animals on the low energy intake had a longer mean prepatent period, but following patency they developed more severe anaemia and greater growth retardation than those on the high energy intake. Both infected groups exhibited significant reductions in serum total lipids, phospholipids, plasma cholesterol and albumin. However, these changes were more severe in the animals on the low energy intake than in those on the high energy intake. It was concluded that adequate energy nutrition enhances the ability of infected animals to withstand the adverse effects of infection by promoting body weight gains and moderating the severity of the pathophysiological changes associated with ovine trypanosomosis.

Extension of the prophylactic effect of isometamidium against trypanosome infections in cattle using a biodegradable copolymer.

Two trials were carried out in order to compare the prophylactic effect of a subcutaneously implanted sustained release device (SRD) containing a mixture of a biodegradable copolymer, poly(caprolactone-co-L-lactide), and isometamidium (ISMM) with that obtained after intramuscular injection of the drug. In a first experiment under controlled conditions, two groups of cattle were treated with 0.5 mg/kg isometamidium either as a SRD or intramuscularly (i.m.), and exposed at monthly intervals to Glossina morsitans morsitans infected with Trypanosoma congolense. The average protection period was at least 24 months in the SRD treated against 5.7 months in the i.m. treated group. Using an ISMM enzyme-linked immunosorbent assay, the drug could be detected until 140 days post-treatment in the latter group, whereas in the former group, traces of the drug were detectable until 330 days after treatment. Furthermore, a field trial was carried out at the Madina Diassa ranch in Mali involving three groups of N'Dama cattle, each containing 23 or 24 animals. Two groups were treated with 1 mg/kg ISMM either as a SRD or i.m. and a third group served as untreated control. Twelve months after treatment, the cumulative infection rates were 56.5, 87.8 and 91.6% in the SRD implanted, the i.m. treated and the control groups, respectively. The ISMM concentrations were slightly lower than in the laboratory trial, but the overall pattern of drug disappearance from the sera of the SRD treated cattle was very similar in both trials. Statistical analysis showed that the incidence of trypanosomiasis was significantly lower in the SRD treated than in the i.m. treated group.

Trypanosoma brucei: comparison of circulating strains in an endemic and an epidemic area of a sleeping sickness focus.

Human sleeping sickness in East Africa is characterized by periods of long-term endemicity interspersed with short-term epidemics. The factors generating these huge changes are largely uncharacterized but probably reflect complex interactions among socioeconomic factors, ecological factors, and the movement and diversity of trypanosome strains. To investigate the role of trypanosome strains in the generation of these epidemics, we addressed two important questions. (1) Are the trypanosome strains circulating within a focus the same during times of endemicity and during an epidemic? (2) How stable are trypanosome strains within a single animal reservoir host? Using restriction fragment length polymorphism analysis of repetitive DNA, we have examined the relationship between Trypanosoma brucei isolates, taken from the Busoga focus of human sleeping sickness, during an endemic period (Busia, Kenya, 1993-1994) and stocks isolated during an epidemic period (Tororo, Uganda, 1988-1990). We show that similar strains, including human infective strains, are circulating in domestic cattle (the most significant animal reservoir) in both epidemic and endemic areas of the Busoga focus. Furthermore, we show the important finding that individual animals harbor the same genotype of T. brucei for a period of time and may be clonal for a given parasite strain.

The influence of dietary supplementation with urea on resilience and resistance to infection with Haemonchus contortus.

Previous research has indicated that supplementing an apparently adequate diet with additional protein improves both host resistance and resilience in lambs infected with Haemonchus contortus. The present study tested the influence of supplementation with non-protein nitrogen (urea). Helminth-naive Hampshire Down lambs were given an apparently adequate basal diet or a diet supplemented with urea. The lambs were then infected with Haemonchus contortus for 10 weeks. Supplementation with urea had no discernible effect on resistance to infection; faecal egg counts, worm burdens, worm lengths and mean number of eggs per adult female worm did not differ between the 2 groups. However, lambs on the supplemented diet showed better resilience; they had greater packed red cell volumes, higher plasma albumin concentrations and increased liveweight gain compared to lambs on the basal diet. The loss of appetite following infection was less in lambs fed the urea-supplemented diet. The observed effect of urea supplementation was seemingly due to greater food consumption as well as the better diet.

The use of enzyme linked immunosorbent assays to investigate the prevalence of Trypanosoma equiperdum in Ethiopian horses.

A field study involving 309 horses was undertaken in the provinces of Arsi and Bale in the Ethiopian highlands to investigate the prevalence of Trypanosoma equiperdum infections using enzyme linked immunosorbent assays (ELISAs) for the detection of both trypanosomal antigen and antibody. Adult horses of both sexes were examined for clinical signs of T. equiperdum infection and serum samples were collected for the assays. One hundred and one horses showed the presence of trypanosomal antibodies in their serum and 70 animals showed typical clinical signs of dourine. Nineteen horses showed the presence of trypanosomal antigen. Eight horses were positive for both T. equiperdum antibody and antigen. Blood and genital washes from seven antigenaemic horses were inoculated into mice and rabbits in an attempt to isolate trypanosomes but none became infected. Statistical analysis of the results of antibody assays indicated that there were significant differences in the distribution of serologically positive horses in the different clinical groupings, with seropositivity increasing with the severity of the observed clinical signs (P < 0.001). There was also a positive correlation between the presence of circulating trypanosomal antigen and clinical evidence of infection. Although it was not possible to obtain direct parasitological evidence of infection, the results of the serological assays, together with the clinical signs of disease observed in many of the horses, provide strong circumstantial evidence that T. equiperdum occurs in Arsi and Bale provinces of Ethiopia. Furthermore, in view of the large number of horses in Ethiopia and the unrestricted movement of animals throughout the country it is likely that dourine may be more widespread in Ethiopia than is currently realised. The assays used show potential for diagnosis of dourine, but to be widely applied in field situations for the diagnosis and control of dourine in Africa they require validation of their specificity and sensitivity.

Effect of experimental infection with Trypanosoma congolense and scrotal insulation on Leydig cell steroidogenesis in the ram.

Testicular steroid content and Leydig cell steroidogenesis in vitro were investigated in rams on Days 28 and 58 after Trypanosoma congolense infection and were compared with those of rams in which testicular temperature had been raised artificially by insulation of the scrotum for 58 d. Testicular testosterone content increased significantly on Day 28 after infection but was lower than that of controls on Day 58 while it increased in scrotal-insulated rams compared with that of controls by Day 58. Testicular progesterone was undetectable in the control and trypanosome-infected groups throughout the experiment, but it increased in the insulated rams by day 58. Basal (unstimulated) Leydig cell testosterone production in the infected rams was similar to that of control rams on Day 28 but was significantly lower on Day 58. Stimulation of Leydig cell testosterone production with hCG or 22R-hydroxycholesterol (22ROHC) significantly reduced in infected rams at both 28 and 58 d after infection as well as in scrotal-insulated rams on Day 58. It is concluded that the increase in testicular testosterone content in the infected and scrotal-insulated rams on Days 28 and 58, respectively, was induced by elevation of testicular temperature by trypanosome infection, perhaps through an effect on testicular blood flow. Reduced testosterone production by Leydig cells from infected and scrotal-insulated rams in response to hCG and 22ROHC suggests that trypanosome-induced pyrexia might be involved in reducing Leydig cell steroidogenesis and subsequent plasma testosterone levels, possibly by affecting enzymes involved in steroid biosynthesis.

New approaches to the integrated control of trypanosomosis.

Trypanosomosis is one of the most devastating diseases of animals and man in Sub-Saharan Africa. Over the past century numerous methods of control have been developed yet the disease has proved very difficult to eradicate. Current methods to control the parasite, in the absence of a vaccine, have to rely on the use of trypanocidal drugs and trypanotolerant breeds of livestock. Vector control previously depended on ground and aerial spraying of insecticide but now depends on the use of traps, targets and bait technology. The application of insecticides to cattle is currently of particular interest. Unfortunately all of the current methods of control have disadvantages and none has proved to be sustainable. There is growing interest in integrated control which can be at three levels; integration with rural development, integration with other disease control measures and integration of various tsetse and trypanosomosis control measures. It is anticipated that distinct benefits can be achieved by an integrated approach which will improve the effectiveness of control and enhance the prospects of sustainability.

The influence of supplementation with cotton seed cake on the resistance of Ugandan goats to primary and secondary challenges with Trypanosoma congolense and on their response to treatment.

The present study investigated the influence of supplementation with cotton seed cake on the resistance of the Small East African breed of goats to primary and secondary challenges with Trypanosoma congolense and on their response to chemotherapy with diminazene aceturate. The supplemented group received 300 g of cotton seed cake per day in addition to about 500 g of fresh napier grass which was available to the unsupplemented group. It was observed that the supplemented infected (SI) group tended to sustain higher intensities of parasitaemia than the unsupplemented infected (USI) group particularly during the primary challenge and both groups showed longer prepatent periods to secondary challenge than to primary challenge. Infection caused a significant reduction in the rate of live body weight gain in the USI group compared with the unsupplemented control (USC) group whilst the SI group grew at the same rate as the supplemented control (SC) group. This effect was observed both during primary and secondary challenges. Following primary challenge, both infected groups developed similar degrees of anaemia, but the packed red cell volume (PCV) levels in the SI group improved towards the end of the first challenge and were also significantly higher than those of the USI group during the second challenge. After treatments at 56 and 126 days after infection (DAI), the greatest response was observed in PCV values. The response of the SI group was superior to that of the USI group and by 4 weeks after treatment the PCV values of the SI and SC groups were similar while those of the USI group were significantly lower than those of the USC group. It is concluded that supplementation with cotton seed cake plays an important role in the rate of live weight gain, and rate of recovery from anaemia produced by trypanosome infection in goats.

Prophylactic effects of isometamidium- and ethidium-sustained release devices against Trypanosoma congolense in cattle.

Two successive experiments were carried out in which three cows were treated by intramuscular injection of either 0.5 mg/kg isometamidium or 1 mg/kg ethidium and compared with another group of three cows which received a subcutaneously implanted sustained release device (SRD) containing the same dose of drug. The prophylactic effect of both drug formulations was evaluated by exposing the animals at monthly intervals to Glossina morsitans morsitans infected with Trypanosoma congolense. The average protection period using the isometamidium- and the ethidium-SRD was extended by a factor of 3.2 and 2.8, respectively in comparison with the intramuscular injection of the drugs. In the analysis of isometamidium concentrations in the serum of the animals using a competitive drug-ELISA the drugs remained present for much longer periods in the sera of the implanted animals than in those of the intramuscularly treated cattle. The animals were still protected, however, a long time after the disappearance of detectable drug levels in the serum. No difference in drug sensitivity could be observed, when breakthrough isolates were compared from animals which received the ethidium-SRD and those treated intramuscularly, although a slight loss sensitivity occurred in the breakthrough isolates as compared to the parent trypanosome population.

Concentrations of isometamidium in the sera of cattle challenged with drug-resistant Trypanosoma congolense.

The relationship between serum concentrations of the prophylactic trypanocidal drug isometamidium chloride and protection against tsetse challenge with two populations of Trypanosoma congolense was investigated in Boran (Bos indicus) cattle, using an isometamidium-ELISA. Isometamidium chloride (Samorin) was administered to cattle at a dose rate of 1.0 mg/kg body weight by deep intramuscular injection. Thereafter, the animals were challenged at monthly intervals with either a drug-sensitive clone (T. congolense IL 1180) or a clone expressing a moderate level of resistance to isometamidium (T. congolense IL 3343). Untreated control cattle were used to confirm the infectivity of each challenge. Of ten drug-treated cattle that were challenged with T. congolense IL 3343, all were refractory to infection at the first challenge. 1 month after drug administration. However, all ten animals succumbed to infection at either the second (seven cattle) or third (three cattle) monthly challenges. By contrast, all five drug-treated cattle challenged with T. congolense IL 1180 resisted four monthly challenges. The mean isometamidium concentration at the time of the first, 1 month, challenge was 5.6 +/- 2.8 ng/ml. At the time of the second monthly challenge the mean concentration was 2.0 +/- 0.86 ng/ml: at this time, concentrations were not significantly different between those cattle refractory to challenge with T. congolense IL 3343 and those cattle that were not. Thus, differences in susceptibility to challenge at this time would appear to be due to differences in the drug sensitivity of the parasite challenge. Finally, the mean isometamidium concentration in uninfected cattle at the time of the fourth monthly challenge was 0.4 +/- 0.18 ng/ml. These results indicate that when T. congolense infection occurs in cattle under isometamidium prophylaxis, the parasites may be considered at least moderately drug resistant if the concentration of isometamidium in serum is 2.0 ng/ml. At concentrations between 0.4 and 2.0 ng/ml a low level of drug resistance may be inferred. Below 0.4 ng/ml, however, no inference regarding drug resistance should be made.

Susceptibility of three breeds of Ugandan goats to experimental infection with Trypanosoma congolense.

This study has indicated that differences in susceptibility to Trypanosoma congolense infection exist among the 3 main breeds of goats in Uganda namely, Kigezi, Mubende and Small East African (SEA). The Kigezi goats appeared to be the most susceptible suffering more severe anaemia, greater retardation of growth and more deaths than the other 2 breeds following experimental infection with Try-panosoma congolense. The Small East African goats appeared to be least susceptible. Following treatment after 84 days of infection, the SEA goats responded much better than the other 2 breeds. By 4 weeks after treatment with diminazene aceturate, the packed red cell volumes of the treated SEA goats were similar to those of control SEA goats while those of the Mubende and Kigezi goats were still much lower than those of control animals.