6. Animal trypanosomiasis

(a) Survey and distribution

[See also 27: nos. 12661, 12690, 12696.]

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, T. theileri, T. congolense and T. vivax. Results showed that mixed infections are detectable at a minimum ratio of 2 percent:98 percent 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.

Indirect ELISAs using denatured antigen preparations of Trypanosoma (T.) congolense (TcAGd) and T. vivax (TvAGd) for detection of anti-trypanosome antibodies in bovine serum (I-TAB ELISAs) were adapted for serodiagnosis in goats. The diagnostic proficiency, the cross-reactivity with sera from heterologous trypanosome infections and the operational performance of the assays were evaluated on experimentally trypanosome-infected goats. The I-TAB ELISA (TcAGd) detected antibodies in all T. congolense infected goats (100 percent overall sensitivity) from 2 to 4 weeks post-infection (p.i.) until the end of the experiments. Specificity tested on 92 uninfected goats was 96.7 percent. Extensive cross-reactions of I-TAB ELISA (TcAGd) with sera from T. vivax or T. brucei infected goats were observed. The I-TAB ELISA (TvAGd) detected antibodies in 5 of the 6 T. vivax infected goats; specificity tested on uninfected goats was 100 percent. Cross-reactivity with sera from T. congolense or T. brucei infected goats remained limited. Infecting species identification based on the highest percent positivity (PP) in both systems, correctly identified all T. congolense infections, but misidentified on 2/19 occasions a T. vivax infection as a T. congolense infection. In the absence of T. brucei specific antigen coated plates, T. brucei infections were identified on, respectively, 7/9 and 2/9 occasions as T. congolense or T. vivax infections. Acceptable inter-plate repeatability was observed. The implications of results and technical requirements for ongoing applied research are discussed.

The clinical, parasitological and molecular diagnosis of bovine trypanosomosis were compared using samples from 250 Zebu cattle exposed to natural trypanosome challenge in Uganda. Clinical examination, molecular and parasitological diagnoses detected 184 (73.6 percent), 96 (38.4 percent) and 36 (14.4 percent) as diseased, respectively. The sensitivity and specificity of clinical examination were 87.5 percent and 35 percent, and 78 percent and 27 percent based on molecular and parasitological diagnoses, as gold standards, respectively. Of the cattle that had parasitologically-confirmed infections of T. brucei (33), T. congolense (3), T. vivax (13) or mixed infections (12), 78 percent, 33 percent, 84 percent and 100 percent respectively manifested clinical signs. Of the cattle that had infections detected by molecular diagnosis there were mixed infections (24), T. brucei (89), T. vivax (12), T. congolense forest-type (3), T. congolense Savannah-type (6) and T. congolense Tsavo-type (27); of these, 100 percent, 83 percent, 91 percent, 100 percent, 67 percent and 81 percent had clinical signs, respectively. In conclusion, treatment of cattle based on clinical examination may clear up to 87.5 percent or 78 percent of the cases that would be positive by either molecular or parasitological diagnosis, respectively. Under field conditions, in the absence of simple and portable diagnostic tools or access to laboratory facilities, veterinarians could rely on clinical diagnosis to screen and treat cases of bovine trypanosomosis presented by farmers before confirmatory diagnosis in diagnostic centres for the few unclear cases is sought.

A low incidence of trypanosomiasis (four cases) was observed in a sample of 100 cattle at abattoirs in the highland area of south-west Cameroon. The lowest packed cell volume and erythrocyte counts were found in two of these cases.

(b) Pathology and immunology

[See also 27: nos. 12696, 12739.]

Nitric oxide has been implicated as an effector cytotoxic molecule in trypanosomiasis. In this work, we investigated the presence of circulating antibodies directed against nitrosylated epitopes as biological indicators for nitric oxide (NO) production in the sera of trypanosome-infected mice. We tested these sera with synthetic antigens, such as S-nitrosated protein or nitrosylated conjugates of amino acids that possess a high affinity to NO, by an immunoenzymatic assay. We detected antibodies directed against nitroso epitopes in the sera of infected mice, as compared to non-infected control mice. The antibody response was linked to the IgM isotype. Our results indicate the production of NO and its derivatives in trypanosomiasis. This production may potentially induce the synthesis of nitroso epitopes in vivo and favour the development of a humoral immune response.

The first visible response in livestock to the bite of a trypanosome-infected tsetse fly is the formation of a localized skin reaction, also known as a chancre. This is an inflammatory response in the skin associated with swelling and an influx of cells. It is thought to be associated with an acquired immune response to the injected metacyclic trypanosomes. In this study, we examined the role of T lymphocytes in the development of the inflammatory response, by depleting cattle of T cell subpopulations and monitoring the development of chancres. Depletion of CD4 cells, but not CD8 cells, resulted in a significant reduction in chancre formation, confirming that an acquired response mediates the inflammatory response. In addition, it was established that the CD4 T cells mediate the generation of memory for immunity to a homologous re-challenge. The inflammatory response in the skin did not affect further progress of the infection.

The effect of trypanosomosis on reaction time and semen characteristics of 12 Zebu (Bunaji) × Friesian crossbred bulls aged between 3 and 5 years was studied for a duration of 12 weeks. Four of the bulls were infected with Trypanosoma vivax, another four with Trypanosoma congolense and the remaining four bulls served as controls. Rectal temperatures and haematological parameters were monitored twice weekly. The pre-infection mean value of the rectal temperature was 38.3 °C, and this rose to a mean of between 40.5 and 41.1°C in the infected animals. Concurrently, the infected animals exhibited signs of anaemia shown by pale mucous membranes and decreased packed cell volume (PCV), weight loss, lethargy, weakness and dullness. The reaction time (ejaculation time) of semen collection significantly increased from a pre-infection mean value of 20.46-25.14 s to a mean of 290.33-301.15 s within 12 weeks post-infection. Semen characteristics deteriorated progressively within the same period in the infected bulls. There were highly significant and drastic decreases in sperm concentration and volume of semen and increases in sperm morphological defects. By the third week, all the infected bulls were unfit for breeding because of very poor semen characteristics. Deterioration, also characterized by oligospermia at 6 weeks post-infection in all bulls which later culminated in azoospermia in two bulls infected with T. vivax and two bulls infected with T. congolense, continued to the end of the investigation. The present results indicate that trypanosomosis due to T. vivax and T. congolense infections is very pathogenic and devastating in its effect on the reaction time (ejaculation time) and semen characteristics which resulted in very poor semen quality. The practical implication is infertility and sterility in Zebu × Friesian crossbred bulls in trypanosome endemic areas.

Detailed studies of sperm morphological abnormalities were carried out on 12 Zebu × Friesian crossbred bulls used in a study of the effects of trypanosomosis. Four bulls were infected with T. vivax, another four with T. congolense, while four served as controls. The infected bulls developed chronic trypanosomosis. All the bulls initially had very low sperm morphological abnormalities that were within acceptable limits for fertile animals. After infection there was a rapid and progressive increase in all sperm abnormalities. Spermatozoa of infected bulls were highly deformed with multiple morphological defects. Mean percentage pre-infection baseline values for acrosomal, spermhead, detached heads, proximal cytoplasmic droplets, distal cytoplasmic droplets, sperm-tail, midpiece and total sperm morphological defects ranged between 0. 1 ± 0. 1 for acrosomal and 8.3 ± 3.2 for total morphological abnormalities in the semen of the bulls. All the infected bulls developed sperm morphological abnormalities of more than a mean of 40.0 percent from the 4th week after infection until the end of the investigation and were considered unfit for breeding. At 7 weeks post-infection (PI) until the end of the study (12 weeks PI), the controls had a mean of less than 5 percent sperm morphological defects, while the infected bulls had 100 percent. Mean percentage values of sperm morphological defects throughout the duration of the investigation for control bulls were low and within the normal range for fertile bulls. These values differed significantly (p < 0.001) from the elevated values of the infected bulls. The results show that trypanosomosis due to T. vivax or T. congolense infection can render Zebu × Friesian crossbred bulls unfit for breeding within a very short time. The resultant infertility could be of economic importance in trypanosomosis-endemic sub-Saharan Africa where Zebu x Friesian crossbred bulls are kept.

(c) Trypanotolerance

[See also 27: nos. 12664, 12689, 12731.]

In Africa, trypanosomiasis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine (Bos taurus) breeds, such as the Longhorn (N’Dama) cattle are well known to control trypanosome infections. This gene-based ability called ‘trypanotolerance’ results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New postgenomic biotechnologies such as transcriptome analyses are efficient in characterizing the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from peripheral blood mononuclear cells of an N’Dama cow, two total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down-regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow the setting up of specific microarray sets for further metabolic and pharmacological studies and the design of field marker-assisted selection by introgression programmes.

Genetic parameters for growth traits in N’dama cattle under tsetse challenge were estimated using a univariate and bivariate animal model. Animals were born and weaned in a low to medium tsetse challenge area, and after weaning transported to a high tsetse challenge area until three years of age. Animals were weighed monthly, starting at birth. Heritabilities ranged from 0.11 for weight at 24 months (W24) to 0.4 for weight at 36 months (W36). Genetic correlations between birth weight (BW) and weight at 12 (W12) and 15 (W15) months were moderately high (0.51 and 0.6, respectively). It was concluded that additive genetic variance exists for the growth traits considered here, which, therefore, may be included in a breeding scheme. Based on the high genetic correlations between traits, not all the traits need to be included.

One hundred and eighty two F₂ trypanotolerant N’Dama Bos taurus × trypanosusceptible improved Kenyan Boran B. indicus, were challenged with Trypanosoma congolense and then monitored weekly for 22 weeks for body weight, anaemia and parasitaemia level. A genome-wide scan based on 477 markers revealed ten trypanotolerance quantitative trait loci (QTL) on nine chromosomes using the threshold of a false discovery rate (FDR) of 10 percent. Estimates of QTL effects indicate that the trypanotolerant allele comes from the trypanotolerant N’Dama parent on Bta 2, 7, 16 (position 18.0/19.3 cM), 26, 27 and from the susceptible Boran parent on Bta 4, 13, 16 (position 1.8 cM) 17 and 20. These results suggest that a synthetic breed from a cross of N’Dama and improved Kenyan Boran could achieve higher tolerance than currently exists in either parent.

Marker-assisted introgression (MAI) was conducted to transfer trypanotolerance QTL (quantitative trait loci) from a donor mouse strain, C57BL/6, into a recipient mouse strain, A/J. The objective was to assess the effectiveness of the introgression using genetic markers and to evaluate its effect on survival time. A backcross (BC) strategy was used with two lines, each carrying two of the donor QTL alleles through the BC phase. At the fourth BC generation, single carrier animals were selected to proceed with the intercross phase. Given the genotype on chromosomes 1, 5 and 17, ten groups of mice plus two control groups (A/J and C57BL/6) were constituted at the end of the intercross phase and challenged with Trypanosoma congolense. Introgressed mice showed better survival time to challenge than the recipient mice, but none of these mice reached the survival level of the donor strain.

Three QTL for trypanosomosis resistance in mice were previously identified using stringent genome-wide significance thresholds in separate analyses of two F₂ data sets. The two data sets were combined and reanalysed using the False Discovery Rate (FDR) to control for multiple hypothesis testing. Analysis of this data set revealed six additional chromosomes that potentially contained QTL of smaller effect. Three of these were confirmed as putative QTL by additional markers in an F₆ population. Use of standard controls on multiple hypothesis testing (Bonferroni correction, genome-wide significance thresholds) would have resulted in these regions being missed in the F₂ reanalysis.

Inbred strains of mice differ in their susceptibility to trypanosomosis. Trypanotolerance quantitative trait loci (QTL) designated as Tir1, Tir2 and Tir3 have been mapped to chromosomes 17, 5 and 1 respectively. Only Tir1 has been fine mapped to an interval less than 1 cM. In order to fine map Tir2 and Tir3, F₁₂ C57BL/6J × A/J advanced intercross lines (AIL) fixed for the susceptible and resistance alleles at Tir1 locus and the parental controls were generated and challenged with Trypanosoma congolense. Mice from the two survival extremes of the population fixed for the susceptible alleles were genotyped with a panel of microsatellite markers across the previously mapped region. QTL analysis results showed that chromosome 1 has three significant loci, with a 95 percent confidence interval (CI) between 3-6 cM, while chromosome 5 has two loci with a 95 percent CI of 1 and 2 cM. The confidence range can now facilitate positional cloning of trypanotolerance genes.

The aim of this study was to assess the effects of trypanosome infection on the work performance of trypanotolerant cattle in the subhumid zone of Senegal. The study was conducted on N’dama cattle with a mean live weight of 288 kg and a mean age of six years. The animals were trained individually (single ox). The experimental design was comprised of two phases of four weeks each. In the first phase, the animals worked five hours a day for five days a week, drafting the equivalent of 12 percent of their body weight. A three-week rest period followed. For the second phase, animals were infected intradermally with a strain of Trypanosoma congolense (infective dose: 10⁵ trypanosomes per ml) and subjected to the same work. The trypanosome infection had a significant effect on work power (P<0.001), work speed (P<0.001), distance travelled (P<0.001), volume of pellets obtained by centrifugation (P<0.05) and Andropogon gayanus intake (P<0.01). However, the infection had no significant effect on the animals’ live weights. Results showed that the work performance of infection-free animals was better than that of trypanosome-infected cattle. Health care and prophylactic measures against trypanosomes are proposed to improve the work performance of trypanotolerant N’dama cattle used for draft work. Cattle should be treated against trypanosomosis with diminazene aceturate (Berenil; 7 mg/kg body weight) at the end of the dry season, and with isometamidium at 0.5-1 mg/kg body weight during the rainy season in order to protect them during the land preparation period.

Some West African Bos taurus cattle breeds such as the N’Dama and a number of West African Shorthorns are resistant to tsetse fly-transmitted trypanosomosis. The trait is termed trypanotolerance, for which changes in packed red cell volume percent (PCV) and growth rate following infection are generally considered to be indicators. An F₂ population was created with the N’Dama and the Kenyan Boran (a trypanosusceptible breed). Two hundred and fourteen F₂ N’Dama × Boran cattle were infected by the bites of tsetse flies infected with the cloned parasite Trypanosoma congolense IL1180. Body weight, PCV, and parasite counts were recorded on a weekly basis for 150 days post infection. Seventeen derived traits were defined based on the data recorded; seven derived from bodyweight, seven from PCV, two from parasite counts recordings and survival. Average values among the F₂ animals were intermediate between those of N’Dama and Boran control animals for all traits. The highest and lowest responders in the F₂ group, when selected on maximum decrease in PCV or on maximum decrease in body weight, were equal to the averages of the Boran and N’Dama animals, respectively. The most trypanotolerant F₂ animals followed similar courses to N’Dama controls and the most susceptible animals followed similar courses to the Borans. There were moderate to low phenotypic correlations (0.00-0.32) between average log(parasite count) or number of times an animal was detected parasitaemic, and the PCV and body weight derived traits. There were low to moderate phenotypic correlations (0.02-0.74) between and within (0.01-0.96) PCV and body weight derived traits. Most of the traits defined in this study were heritable. Heritabilities ranged from 0.01 for PCV recovery, to 0.88 for initial PCV. Some F₂ animals seemed to be able to control anaemia and have a higher average body weight and body weight gain than the pure-bred N’Dama. Overall, in this study body weight change following infection seems an appropriate and easy-to-measure indicator of trypanotolerance.

(d) Treatment

[See also 27: nos. 12723, 12779.]

Smallholders’ access to pour-on-type products was rendered difficult after the price increase of imported products, a consequence of the 1994 devaluation of the CFA franc. They thus turned to less reliable methods for the control not only of ticks, but also of African animal trypanosomoses (AAT) transmitted by tsetse flies. To address this problem, the Animal Trypanomosis Control Project in Togo investigated the use of a new application method that combined advantages of the pour-on method and a lower cost: Electrodyn^TM (Zeneca). This method is based on the electrodynamic spraying of an insecticide formulation (Karate 2.5 ED®) containing 1 percent lambda-cyhalothrin. The trial was conducted on 170 head of cattle in the village of Skriback in the north of Togo (304 head at the end of the trial). All the animals were treated between March 1996 and March 1997, and only half of them were treated between July 1997 and July 1998. Before starting the first treatment, preliminary surveys were carried over a one-year period (from February 1995 to February 1996) to obtain precise data on the area tsetse challenge, for before-and-after-treatment comparisons. Results showed that the system was very efficient within the study set-up. It helped reduce tsetse fly densities by 99.55 percent, and reduce AAT prevalence from 17 to 2 percent when used in combination with a trypanocide treatment. It also helped improve the herd mean packed cell volume from 27 to 32.5 percent, when used in combination with a regular anthelmintic treatment. The Electrodyn method is also cost-effective, its cost being a third of the traditional pour-on method. Furthermore, huge additional advantages can be obtained through the joint use of equipment in areas of intensive phytosanitary control. Handling of the applicator (fixed length) and the relatively high cost of batteries are the constraints that may affect acceptability of the technique.

A household survey was conducted in November 1998 in 21 villages of Kwale district, Kenya, to assess farmers’ trypanocidal drug use characteristics for treatment of bovine trypanosomosis and their relationship to drug effectiveness. Descriptive statistical tools were used to summarize the farmers' drug use patterns. The c² test was carried out to establish the relationship between proper drug use and recovery. The results indicated that the farmers had considerable knowledge about trypanocidal drugs with 82 percent (n = 65) having used these drugs within six months preceding the survey. Cases of incorrect drug use were reported. This study established that there was no significant relationship between correct drug use and recovery of the treated animals, suggesting the presence of drug resistance in Kwale district.

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 percent prevalence of bovine trypanosomosis during an earlier survey. A longitudinal study was conducted in the same three districts over a one-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 percent of infections caused by Trypanosoma congolense. The remainder was caused by Trypanosoma vivax (2 percent) and Trypanosoma brucei (2 percent). Tests in mice showed that of the stabilates collected, 24 (34 percent) were resistant to only isometamidium chloride, 8 (11.3 percent) were resistant to only diminazene aceturate, 1 (1.4 percent) was resistant to both drugs, and 38 (53.5 percent) 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 percent) of 1526 serum samples from cattle in the study. Only 6 (2.8 percent) 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.