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In the context of the Pan African Tsetse and Trypanosomiasis Eradication Campaign, the value of tsetse saliva antibodies as a biomarker of cattle exposure to tsetse flies was evaluated, as this could provide an alternative and complementary tool to conventional entomological methods. Serum immune reactivity to Glossina (G.) palpalis (p.)gambiensis, G. tachinoides and G. morsitans (m.) submorsitans whole saliva extracts (WSE) were monitored in cattle from both tsetse free and tsetse infested areas, and in cows experimentally exposed to tsetse flies and other hematophagous arthropods. In the tsetse infested area, cattle IgG responses to Glossina WSE were significantly higher during the dry season (p < 0.0001) when herds are most exposed to tsetse flies and in infected animals (p = 0.01) as expected in the case of a biomarker of exposure. Experimental studies further confirmed this as a quick rise of specific IgGs was observed in animals exposed to tsetse flies (within weeks), followed by a rapid clearance after exposure was stopped. In contrast to the two other tsetse species, G. m. submorsitans WSE enabled to detect exposure to all tsetse species and were associated with low level of cross-reactivity to other blood sucking arthropods. Finally, IgG responses to G. m. submorsitans salivary antigens enabled to distinguish different groups of cows according to exposure levels, thus indicating that tsetse saliva antibodies are not only indicators of tsetse exposure but also are correlated to the intensity of tsetse contacts (p = 0.0031). Implementation of this new sero-epidemiological marker of cattle exposure to tsetse flies in the framework of tsetse elimination campaigns is discussed.

African animal trypanosomosis (AAT) is considered the most important cattle disease in sub-Saharan Africa but its diagnosis in the field is difficult, resulting in inappropriate treatments, excessive delay in treatments and under-treatment. A field study in West Africa investigated the usefulness of anemia in the diagnosis of trypanosomosis. A total of 20,772 cattle blood samples were taken from 121 villages in 3 countries. The average packed cell volume (PCV) of trypanosomosis positive cattle was 23%, versus 28% for negative cattle. In a sub-set of animals, other causes of anemia were investigated showing most of the anemia burden was attributable to trypanosomosis. Anemia was a reasonably accurate indicator of trypanosomosis in the study area, with a sensitivity of 56% and a specificity of 80% and a diagnostic odds ratio of 4.2, the highest of all the signs evaluated (anemia, emaciation, staring coat, lymphadenopathy, fever, lacrimation and salivary or nasal discharge). Having confirmed the usefulness of anemia as a predictor of trypanosomosis, two potential pen-side tests for anemia were evaluated (the first reported trial of their use in cattle), firstly a color chart developed for anemia detection in sheep through visual inspection of conjunctival membranes (FAMACHA) and secondly the Hemoglobin Color Scale (HbCS) developed for assessing hemoglobin levels in human patients by comparing blood drops on filter paper with color standards. In a population of cattle suspected by their owners to be sick with trypanosomosis (n=898) the sensitivity of the HbCS test was 56% and the specificity was 77%, while the sensitivity of the FAMACHA test was 95% and the specificity was 22%. The higher sensitivity but lower specificity suggests the FAMACHA may be useful as a screening test and the HbCS as a confirmatory test. The two tests were also evaluated in cattle randomly selected from the village herd. Using cut-off points to optimize test performance, the HbCS test had a sensitivity of 81% and a specificity of 62% (n=505 cattle), while the FAMACHA had a sensitivity of 92% and a specificity of 30% (n=298 cattle). Recommendations are made for the appropriate use of these tests in the West African region.

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.

Trypanocidal drug resistance is unanimously recognized as a threat for livestock production in regions where the prevalence of trypanosomosis is high. To assess the impact of the disease and the effect of drug resistance on the health of small ruminants, twelve Trypanosoma vivax isolates collected in 6 villages in the vicinity of Bobo Dioulasso (Burkina Faso) were injected into 12 groups of 5 Sahelian goats, two being treated with 3.5 mg/kg body weight diminazene aceturate (DA), two with 0.5 mg/kg body weight isometamidium chloride (ISM) and one left untreated as control. A monitoring was performed every 5 days for 100 days to evaluate the parasitaemia by buffy coat examination, the hematocrit and the body weight. Among the 12 groups, 6 were additionally monitored using a trypanosome specific 18S-PCR–RFLP every 5 days from day 30 to day 100 to verify the complete clearance of the parasites from the blood of the hosts. In six groups of goats, trypanosomes disappeared completely after treatment, five groups showed relapses in at least one goat treated with ISM and one group showed relapses in one goat treated with DA and one with ISM. For the 6 groups that were screened both using microscopic examination and trypanosome specific 18S-PCR–RFLP, the following results were observed: for the groups treated with DA, no relapses by microscopic examination and 83.3% (10/12) using the 18S-PCR–RFLP. For the groups treated with ISM, 25% (3/12) relapses by microscopic examination and 83.3% with the 18S-PCR–RFLP (10/12). The evolution of the PCV and the weight during the observation period from relapsing (either by microscopical examination or by 18S-PCR–RFLP diagnosis) and non relapsing animals were compared. The relative average PCV in goats that relapsed microscopically, decreased significantly more than in non-relapsing goats. This difference was not significant when relapses were detected using the trypanosome specific 18S-PCR–RFLP. This indicates that only the animals with the highest parasitaemia suffered from the infection. Relapses after treatment where the host controls the parasitaemia to a level below the sensitivity of the microscopical examination do not affect body weight nor PCV.

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 (Debe, 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.

a Institute for Parasitology and Tropical Veterinary Medicine, Freie Universitat Berlin, Konigsweg 67, D-14163Berlin,Germany b International Animal Health, Freie Universitat Berlin, Konigsweg 67, D-14163Berlin,Germany c International Livestock Research Institute (ILRI), BP 320,Bamako,Mali d Laboratoire Central Veterinaire (LCV), BP 2295,Bamako,Mali e Centre International de Recherche-Developpement sur l’Elevage en Zone subhumide (CIRDES), BP 454,Bobo-Dioulasso,Burkina Faso f Pan African Tsetse and Trypanosomosis Eradication Programme (PATTEC) Burkina Faso, BP 454,Bobo-Dioulasso,Burkina Faso g OIE ad hoc Group on Non Tsetse Transmitted Animal Trypanosomoses (NTTAT) Diagnosis, 12, rue de Prony, F-75017Paris,France h Institute of Development and Agricultural Economics, Leibniz Universitat Hannover, Konigsworther Platz 1, D-30167Hannover,Germany i International Livestock Research Institute (ILRI), P.O. Box 30709,Nairobi 00100,Kenya

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 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 savannahtype 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.