No more cold-chain failures, using dehydrated reagents in ELISA antibody-detection against animal trypanosomes of African origin.

Animal trypanosomoses due to trypanosomes of African origin (ATAO), mainly caused by Trypanosoma congolense type Savannah (TCS), T. brucei brucei (TBB), T. vivax (TV), and T. evansi, are widespread diseases that affect domestic and wild mammals and have a huge economic impact. ATAO clinical suspicions are usually confirmed by parasitological and molecular methods, while sero-epidemiological surveys are generally carried out using the OIE-recommended ELISA method based on whole cell lysate soluble antigens (WCLSA) from purified trypanosomes; this reagent is usually stored frozen. With a view to expanding this ELISA test, we assessed, standardized, and validated the use of dehydrated rather than frozen WCLSA and serum samples. For the three ELISA assays (TV, TCS & TBB), a repeatability study revealed no significant difference between repeats. The results obtained using frozen rather than freeze-dried antigen and serum strongly correlated for Pearson's correlation values (>0.93) and Lin's measure ("very good" to "excellent"). Reproducibility was robust, with Pearson's correlation values >0.97 for inter technician effects, and 0.87 (TV) to 0.97 (TBB & TCS) for inter-laboratory tests; their combination was "very satisfactory" to "excellent" according to Lin's measure and there was no impact on qualitative test results. Dehydrated reagents offer the advantage of shipment at room temperature, allowing the secured provision of reagents to regional laboratories. Together with a compendium of standard diagnostic protocols for ATAO (/OIE), dehydrated reagents will enable the serological diagnosis of ATAO at regional level in endemic countries. This very welcome improvement in the context of the Progressive Control Pathway for trypanosomes, recently launched by African countries, will possibly be extended to Latin America in the near future.

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.

Association studies in QTL regions linked to bovine trypanotolerance in a West African crossbred population.

African animal trypanosomosis is a parasitic blood disease transmitted by tsetse flies and is widespread in sub-Saharan Africa. West African taurine breeds have the ability, known as trypanotolerance, to limit parasitaemia and anaemia and remain productive in enzootic areas. Several quantitative trait loci (QTL) underlying traits related to trypanotolerance have been identified in an experimentally infected F(2) population resulting from a cross between taurine and zebu cattle. Although this information is highly valuable, the QTL remain to be confirmed in populations subjected to natural conditions of infection, and the corresponding regions need to be refined. In our study, 360 West African cattle were phenotyped for the packed cell volume control under natural conditions of infection in south-western Burkina Faso. Phenotypes were assessed by analysing data from previous cattle monitored over 2 years in an area enzootic for trypanosomosis. We further genotyped for 64 microsatellite markers mapping within four previously reported QTL on BTA02, BTA04, BTA07 and BTA13. These data enabled us to estimate the heritability of the phenotype using the kinship matrix between individuals computed from genotyping data. Thus, depending on the estimators considered and the method used, the heritability of anaemia control ranged from 0.09 to 0.22. Finally, an analysis of association identified an allele of the MNB42 marker on BTA04 as being strongly associated with anaemia control, and a candidate gene, INHBA, as being close to that marker.

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.

Bovine trypanosomosis in the Upper West Region of Ghana: entomological, parasitological and serological cross-sectional surveys.

Baseline surveys were conducted in the Upper West Region of Ghana to assess the distribution and densities of tsetse species, as well as the prevalence of bovine trypanosomosis. The entomological survey was designed to cover the suitable tsetse habitats along the three main rivers in the study area (i.e. Black Volta, Kulpawn and Sissili). Results indicated the presence of Glossina tachinoides in all three river basins, whilst Glossina palpalis gambiensis was only found close to the southern limit of the study area. A random sampling of 1800 cattle of the West African Short Horn, Sanga and Zebu breeds from 36 randomly selected grid cells covering the study area showed substantial differences between parasitological and serological prevalences. The average parasitological prevalence was estimated at 2.5% (95% CI: 1.06-5.77) with the majority of the infections due to Trypanosoma vivax. Most of the infected cattle were found close to the major river systems. The serological prevalence, measured using Enzyme Linked Immunosorbent Assay (ELISA), test was 19% (95% CI: 14.03-25.35). Cattle with anti-trypanosomal antibodies were also found throughout the study area.

Experimental evaluation of xenodiagnosis to detect trypanosomes at low parasitaemia levels in infected hosts.

In Human African Trypanosomosis (HAT) endemic areas, there are a number of subjects that are positive to serological tests but in whom trypanosomes are difficult to detect with the available parasitological tests. In most cases and particularly in West Africa, these subjects remain untreated, thus posing a fundamental problem both at the individual level (because of a possible lethal evolution of the disease) and at the epidemiological level (since they are potential reservoirs of trypanosomes). Xenodiagnosis may constitute an alternative for this type of cases. The objective of this study was to update the use of xenodiagnosis to detect trypanosomes in infected host characterized by low parasitaemia levels. This was carried out experimentally by infecting cattle and pigs with Trypanosoma congolense and T. brucei gambiense respectively, and by feeding tsetse flies (Glossina morsitans submorsitans and G. palpalis gambiensis, from the CIRDES colonies) on these animals at a time when the observed blood parasitaemia were low or undetectable by the classical microscopic parasitological tests used for the monitoring of infected animals. Our results showed that: i) the G. p. gambiensis colony at CIRDES could not be infected with the T. b. gambiense stocks used; ii) midgut infections of G. m. submorsitans were observed with both T. congolense and T. b. gambiense; iii) xenodiagnosis remains positive even at very low blood parasitaemia for both T. congolense and T. b. gambiense; and iv) to implement T. b. gambiense xenodiagnosis, batches of 20 G. m. submorsitans should be dissected two days after the infective meal. These results constitute a first step toward a possible implementation of xenodiagnosis to better characterize the parasitological status of seropositive individuals and the modalities of parasite transmission in HAT foci.

The prevalence of African animal trypanosomoses and tsetse presence in Western Senegal.

In 2005, the Government of Senegal initiated a tsetse eradication campaign in the Niayes and La Petite Côte aiming at the removal of African Animal Trypanosomosis (AAT), which is one of the main constraints to the development of more effective cattle production systems. The target area has particular meteorological and ecological characteristics that provide great potential for animal production, but it is unfortunately still infested by the riverine tsetse species Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae). The tsetse project in Senegal has adopted an area-wide integrated pest management (AW-IPM) approach that targets the entire tsetse population within a delimited area. During the first phase of the programme, a feasibility study was conducted that included the collection of entomological, veterinary, population genetics, environmental and socioeconomic baseline data. This paper presents the parasitological and serological prevalence data of AAT in cattle residing inside and outside the tsetse-infested areas of the target zone prior to the control effort. At the herd level, a mean parasitological prevalence of 2.4% was observed, whereas a serological prevalence of 28.7%, 4.4%, and 0.3% was obtained for Trypanosoma vivax, T. congolense and T. brucei brucei, respectively. The observed infection risk was 3 times higher for T. congolense and T. vivax in the tsetse-infested than in the assumed tsetse-free areas. Moreover, AAT prevalence decreased significantly with distance from the nearest tsetse captured which indicated that cyclical transmission of the parasites by tsetse was predominant over mechanical transmission by numerous other biting flies present. The importance of these results for the development of a control strategy for the planned AW-IPM campaign is discussed.

The application of PCR-ELISA to the detection of Trypanosoma congolense type savannah (TCS) in bovine blood samples.

PCR-ELISA was set up to detect strains of Trypanosoma congolense type savannah (TCS) in field samples of buffy coats. Results of PCR-ELISA and PCR were compared and the effectiveness of both techniques was also compared with the Murray's method for the detection of TCS in 257 bovine buffy coats. The PCR products were labelled with digoxigenin (DIG-dUTP) during amplification cycles of the repetitive satellite DNA. A biotinylated DNA capture probe was used to detect the PCR products by ELISA in streptavidin coated microplates. Both the PCR-ELISA and PCR were more sensitive and more specific than the Murray's method. Of the 257 buffy coats analysed by the three techniques, PCR-ELISA and PCR detected TCS in 98 and 97 buffy coats respectively, whereas the Murray's method detected only 39 samples. PCR-ELISA and PCR had almost the same sensitivity and specificity. PCR-ELISA and PCR respectively detected TCS in 39.2% and 38.6% in all the 334 samples analysed by both techniques in this study.

Calf morbidity, mortality and parasite prevalences in the cotton zone of Burkina Faso.

The objective of this study is to describe morbidity and mortality in calves <1-year-old and to associate the impact of gastrointestinal and haemoparasitoses on morbidity and mortality by comparing these parameters in ill and well calves in the cotton zone of Burkina Faso. Fifty-nine herds selected in two villages were involved. Calves born between February 1997 and February 1999 were monitored up to 1 year of age or until the end of the study in April 1999. Blood and faecal samples were taken from ill calves and matched with samples taken from well calves (as control) for analysis of haemoparasites and gastrointestinal worms. Infected ill calves were treated with a trypanocidal drug and/or an anthelmintic. Dead calves were necropsied; adult-worm burdens were determined and brain smears taken to detect the presence of Cowdria ruminantium. Diarrhoea was the main clinical observation and most calves shed worm eggs. The EPG for gastrointestinal parasites was higher neither in ill calves nor in diarrhoeal calves. Infections by trypanosome species were observed in ill calves only (prevalences of 8% and 15% in Daboura and Kourouma, respectively). Average PCV in infected calves (28%) was lower than that in non-infected calves (37%). Most ill calves (86%) recovered 2 weeks after the treatment with anthelmintic and/or trypanocide. The post-mortem worm counting in 12 calves revealed that half of necropsied calves had a burden ranging from 2040 to 20,072 helminths. Infection by Babesia bigemina was found in the blood smear of one ill calf and the presence of C. ruminantium was noted in the brain smear of one calf.

Comparative pathogenicity of three genetically distinct types of Trypanosoma congolense in cattle: clinical observations and haematological changes.

The pathology of African bovine trypanosomosis was compared in Zebu cattle subcutaneously inoculated with three clones of trypanosomes corresponding to the three genetically distinct types of Trypanosoma congolense; savannah-type, west African riverine/forest-type and kilifi-type. All inoculated animals became parasitaemic between 7 and 11 days post-infection (dpi). The savannah-type showed consistently higher levels of parasitaemia and lower packed red cell volume percentages and leukocyte counts than the other two types. The syndrome was also more severe in the savannah-type and led inexorably to death between 29 and 54 dpi while animals with the forest or the kilifi-types recovered from earlier symptoms and haematological alterations after 3 months of infection. By the end of the experiment, the animals self-cured from the forest-type infection and the kilifi-type passed under control. The results of the present study indicated clear difference in pathogenicity between the three types of T. congolense; the savannah-type was virulent while the forest-type was of low pathogenicity and the kilifi-type was non-pathogenic.

Comparative pathogenicity of three genetically distinct Trypanosoma congolense-types in inbred Balb/c mice.

Inbred Balb/c mice were infected with three clones of Trypanosoma congolense (Sam.28.1, Dind.3.1 and K60.1A) corresponding, respectively, to the three genetically distinct types (savannah, forest and kilifi) defined within this species, for the purpose of comparing their pathogenicity for a better understanding of the epidemiology of African trypanosomosis. Another clone of savannah type, IL 3000, was also tested simultaneously to study a probable strain variation. Both the clones of savannah type were found of extreme virulence with loss of appetite, rough hair, rapid respiration, lethargy, and all mice died within a week. Parasitaemias evolved rapidly to the first peak by day 3-5 post-inoculation without any remission and the course of disease was correlated positively with the prepatent period. The clones of the forest type and the kilifi type were of low virulence with chronic infection and symptoms progressively less patent throughout the infection; only one mouse died in each experimental group.

The analysis of the cross-reactions occurring in antibody-ELISA for the detection of trypanosomes can improve identification of the parasite species involved.

In Africa, the main pathogenic trypanosomes of livestock are Trypanosoma vivax, T. congolense and T. brucei. The geographical distributions and hosts of these three species are very similar. As they differ markedly in pathogenicity and epidemiology, however, a species-specific serological test for infection would be very useful for epidemiological studies. The antibody-ELISA (Ab-ELISA) that have been developed for detecting the Trypanosoma spp. most commonly infecting livestock give satisfactory sensitivity and genus specificity. Unfortunately, they are not species-specific because of strong cross-reactions between the pathogenic Trypanosoma spp. In the present study, carried out in Burkina Faso, the results of standardized Ab-ELISA for T. vivax, T. brucei or T. congolense were compared using 1288 plasma samples from sheep experimentally infected with T. vivax, T. evansi and/or T. congolense. If the results were interpreted, as usual, only using a positivity threshold (PT), the strong cross-reactions observed led to a mean species-specificity of < 30%. However, analysis of the reactions observed in the three types of Ab-ELISA revealed that the homologous reactions were stronger than the heterologous for almost all of the single and mixed infections (98.3% and 99.0%, respectively). In monospecific infections exceeding the PT study of the positivity score produced in each of the three types of Ab-ELISA increased species-specificity to > 96%. It therefore appears that comparison of the strengths of the reactions seen in Ab-ELISA could greatly improve sero-epidemiological surveys of trypanosome infections in domestic ruminants, although the technique remains to be evaluated in experimentally infected cattle.

Validation of a polymerase chain reaction assay for monitoring the therapeutic efficacy of diminazene aceturate in trypanosome-infected sheep.

The diagnostic performance of a polymerase chain reaction assay (PCR) for monitoring the effectiveness of aceturate diminazene treatment was compared with those of an antibody-detection ELISA test and the buffy-coat technique using sheep experimentally infected with either savannah-type or forest-type Trypanosoma congolense or T. vivax. Within the period of infection, the PCR using specific savannah-type T. congolense primers showed a significant higher diagnostic sensitivity (p<0.05) than the buffy-coat technique. Both techniques gave closed results for detecting forest-type T. congolense or T. vivax infections. Following trypanocidal treatment, the PCR showed that specific product disappeared definitively 1 or 2 days later in animals in which a decrease of the antibody level and a significant improvement of the red packed cell volume were observed. The occurrence of relapse infection was detected by the PCR in one animal infected by T. vivax on day 19 post-treatment and confirmed by the persistence and increasing antibody level whereas the buffy-coat technique detected parasites 42 days later. Then, the PCR signals remained positive on several occasions while parasitaemia was detected only two times.The application of PCR combined with the antibody detection appeared to provide a useful tool as compared to the buffy-coat technique for monitoring the effectiveness of trypanocidal treatment.

Review on the molecular tools for the understanding of the epidemiology of animal trypanosomosis in West Africa.

The epidemiology of animal trypanosomosis around Bobo-Dioulasso (Burkina Faso, West Africa) benefited a lot in the last years from the progress of molecular tools. The two most used molecular techniques were the polymerase chain reaction for the diagnosis of the disease in cattle and the characterization of the trypanosomes in the host and the vector on one hand, and the microsatellite DNA polymorphism in tsetse flies to study the intraspecific genetic variability of the vector on the other hand. The results obtained in the Sideradougou area during a recent two year survey with these techniques, associated with many other georeferenced informations concerning vector and cattle distribution, natural environment, landuse, ground occupation, livestock management, were combined in a Geographical Information System. This new approach of a complex pathogenic system led to a better evaluation of the risk of trypanosome transmission.

New epidemiological features on animal trypanosomiasis by molecular analysis in the pastoral zone of Sideradougou, Burkina Faso.

A multidisciplinary work was undertaken in the agropastoral zone of Sidéradougou, Burkina Faso to try to elucidate the key factors determining the presence of tsetse flies. In this study the PCR was used to characterize trypanosomes infecting the vector (Glossina tachinoides and Glossina palpalis gambiensis) and the host, i.e. cattle. A 2-year survey involved dissecting 2211 tsetse of the two Glossina species. A total of 298 parasitologically infected tsetse were analysed by PCR. Trypanosoma vivax was the most frequently identified trypanosome followed by the savannah type of T. congolense and, to a lesser extent, the riverine forest type of T. congolense, and by T brucei. No cases of T. simiae were found. From the 107 identified infections in cattle, the taxa were the same, but T. congolense savannah type was more frequent, whereas T. vivax and T. congolense riverine forest types were found less frequently. A correlation was found between midgut infection rates of tsetse, nonidentified infections and reptile bloodmeals. These rates were higher in G.p. gambiensis, and in the western part of the study area. T. vivax infections were related to cattle bloodmeals, and were more frequent in G. tachinoides and in the eastern study area. The PCR results combined with bloodmeal analysis helped us to establish the relationships between the vector and the host, to assess the trypanosome challenge in the two parts of the area, to elucidate the differences between the two types of T. congolense, and to suspect that most midgut infections were originating from reptilian trypanosomes.

PCR analysis and spatial repartition of trypanosomes infecting tsetse flies in Sidéradougou area of Burkina Faso.

A parasitological and entomological survey was conducted in the Sideradougou area (south of Bobo Dioulasso, Burkina Faso) in order to identify transmission factors of African trypanosomosis. A total of 3600 tsetse flies (Glossina tachinoides, Glossina palpalis gambiensis) were captured along 120 km of linear gallery forest and half of them were dissected. PCR analysis was undertaken on parasitologically positive flies (161 G. tachinoides, 92 G. palpalis gambiensis) to characterize the different trypanosomes. All the results were integrated in a GIS (Geographical Information System). Spatial repartition of the characterized trypanosomes enabled to recognize different areas with specific patterns of infection. Relations with environmental factors are discussed.

[Use of Trypanosoma antigen detection ELISA during an epidemiological follow-up in the Sideradougou, Burkina Faso]. / Utilisation du test ELISA de détection des antigènes circulants de trypanosomes dans le cadre d'un suivi épidémiologique dans la zone de Sidéradougou, Burkina Faso.

Plasmas taken during a parasitological survey on bovine trypanosomosis were tested a posteriori with an antigen detection ELISA test. The objective was to evaluate the value of this test for epidemiological monitoring. The results were compared to those obtained using the usual parasitological technique (buffy coat examination). Of the 297 zebu cattle heads studied, 216 came from four villages in the pastoral zone of Sideradougou. A large eradication programme had led to the disappearance of tsetse in the area. Blood samples were taken during the rainy saison (September 1986), at the beginning (January 1987) and at the end (May 1987) of the dry season. The parasitological diagnosis was carried out in the field and plasmas were stored at -20 degrees C. The serological test was performed in August 1993. The incidence rates of bovine trypanosomosis obtained with antigen detection ELISA were low in the center of the zone. These rates decreased proportionally to the distance between the blood sampling area and the limit of the infested area. The results of the parasitological diagnosis were similar except for a village located on the border of the treated zone. The use of antigen detection ELISA confirmed the low incidence of trypanosomosis in the center of the area of Sideradougou following the eradication campaign. It also enables to obtain finer parasitological results pointing out the trypanosome persistence in peripheric zones, more exposed to reinfestations.

Evaluation of an antigen detection-ELISA test for the diagnosis of trypanosomiasis in naturally infected cattle.

The sensitivity and the specificity of the antigen detection ELISA proposed by Nantulya and Lindqvist (1989) for the diagnosis of African Animal Trypanosomiasis have been assessed in naturally-occurring infections. 1633 cattle were sampled in trypanosomiasis endemic area and examined for trypanosomes by darkground/phase contrast buffy-coat method described by Murray et al. (1977) and for circulating antigen by ELISA. Fifty sera from Markoye, a tsetse free area in north of Burkina Faso, and 49 sera from Germany were also tested. In trypanosomiasis infested area, BCT detected 144 (8.8%) positive animals while Ag-ELISA revealed 65.8% of positive. Out of the 144 BCT-parasite-positive, Ag-ELISA enable to detect 75% of positive. The predominant trypanosomes identified by BCT was Trypanosoma vivax followed by T. congolense while Ag-ELISA indicated T. congolense followed by T. brucei. Ag-ELISA detected 76.5% out of the 51 T. congolense-BCT-positive and only 17% of all T. vivax BCT-positive. Cattle carring mixed infection involving two or three trypanosomes, particularly those with T. brucei and T. congolense are the most frequent. In tsetse free area, Ag-ELISA detected one positive cattle carring T. brucei and T. congolense and showed an apparent specificity of 98%. No serum from Germany was detected positive. This study suggests the joint use of Ag-ELISA and BCT for the diagnosis of trypanosomiasis particularly in epidemiological study in endemic area.

[Absence of interaction between Trypanosoma theileri infections with the diagnosis of animal trypanosomiasis by detection of circulating antigens]. / Absence d'interaction des infections à Trypanosoma theileri avec le diagnostic des trypanosomoses animales par détection des antigènes circulants.

This work presents data gathered at the CIRDES (Centre international de Recherche-Développement sur l'Elevage en Zone subhumide) during epidemiological monitoring. The prevalence levels of Trypanosoma vivax, Trypanosoma congolense and Trypansoma brucei obtained using antigen-detection ELISA were compared in non-infected animals and in animals infected with Trypanosoma theileri. The aim was to investigate whether there were any serological cross-reactions between T. theileri and the pathogenic trypanosomes. The results show that there was no interaction by Trypanosoma theileri with the diagnosis of the pathogenic trypanosomes using antigen-detection ELISA.