Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso

Background Animal African Trypanosomosis (AAT) is a parasitic disease of livestock that has a major socio-economic impact in the affected areas. It is caused by several species of uniflagellate extracellular protists of the genus Trypanosoma mainly transmitted by tsetse flies: T. congolense, T. vivax and T. brucei brucei. In Burkina Faso, AAT hampers the proper economic development of the southwestern part of the country, which is yet the best watered area particularly conducive to agriculture and animal production. It was therefore important to investigate the extent of the infection in order to better control the disease. The objective of the present study was to assess the prevalence of trypanosome infections and collect data on the presence of tsetse flies. Methods Buffy coat, Trypanosoma species-specific PCR, Indirect ELISA Trypanosoma sp and trypanolysis techniques were used on 1898 samples collected. An entomological survey was also carried out. Results The parasitological prevalence of AAT was 1.1%, and all observed parasites were T. vivax. In contrast, the molecular prevalence was 23%, of which T. vivax was predominant (89%) followed by T. congolense (12.3%) and T. brucei s.l. (7.3%) with a sizable proportion as mixed infections (9.1%). T. brucei gambiense, responsible of sleeping sickness in humans, was not detected. The serological prevalence reached 49.7%. Once again T. vivax predominated (77.2%), but followed by T. brucei (14.7%) and T. congolense (8.1%). Seven samples, from six cattle and one pig, were found positive by trypanolysis. The density per trap of Glossina tachinoides and G. palpalis gambiensis was 1.2 flies. Conclusions/Significance Overall, our study showed a high prevalence of trypanosome infection in the area, pointing out an ongoing inadequacy of control measures.

Animal sampling 134 Sampling was carried out on all domestic animals proposed by the breeders (cattle, sheep, 135 goats and pigs). Thus in some farms the selection of animals to be sampled was not random but 136 would follow the farmer's choice. In some instance, the selection was skewed towards animals 137 with poor body conditions, or on the contrary towards valuable animals such as drought oxen, 138 introducing an occasional bias. Dogs, usually included in such surveys, are rare in the area and 139 were not sampled in our study. 140 Biological data for each animal were recorded on a questionnaire-type sheet. The 141 recorded information were: sampling localization, name of farm, species, age classes, sex, and physical condition of the animals noted from 1 to 5 (1-recumbent animal, 2-animal in very 143 poor condition, 3-animal in fair condition, 4-animal in good condition, and 5-animal in very 144 good condition). The packed cell volume (PCV) values and field diagnostic test results were 145 also noted. 146 A 5 mL blood sample was taken with a heparinized Vacutainer™ tube from the jugular 147 vein for cattle, goats, and sheep, and vena cava for pigs. Tubes were kept on ice all through the 148 various procedures. 149 Animal field survey 150 Two heparinized microcapillaries for each animal were filled with the previously 151 collected blood and centrifuged at 3000 rpm for 5 min. The PCV was measured in duplicate 152 using a microhematocrit reading plate and recorded. Animal with PCV value below 24% were 153 considered to be anemic. For each of the two microcapillaries, the buffy coats (BC) were 154 subsequently collected through breaking off the capillary just under the white blood cells layer 155 and spread between slide and cover slip, and examined under an optical microscope with a 40 156 X magnitude objective to search for the presence of trypanosomes (19). The Vacutainer™ tubes 157 were centrifuged at 3000 rpm on site for 5 min. Plasmas were first collected with a 1000 µL 158 micropipette and transferred to individual 1.5 mL Eppendorf™ tubes. The BC were collected 159 separately by pipetting the white layer of leucocytes (500 µL), avoiding as far as possible to 160 pipette red blood cells. Samples were stored at -20°C in a portable freezer until brought back to 161 the lab and moved to a -20°C freezer. 163 For DNA extraction the 500 µL BC samples were added to a sterile 1.5 mL Eppendorf™ 164 tube containing 500 µL of 5% w/v suspension of Chelex ® 100. The mixture was incubated first in a 56°C water bath for 1 h and then at 95°C for 30 min. The tubes were centrifuged at 13,000 166 rpm for 3 min, the supernatant recovered in new 1.5 mL Eppendorf™ tubes and stored at -20°C. 167 Determination of trypanosome species was done first using the TRYP1(R & S) primers, 168 which amplify the internal transcribed spacer 1 (ITS1) of the ribosomal DNA of most 169 trypanosome species, and display size polymorphism. Positive samples were subsequently 170 submitted to species-specific primers pairs (Table 1) (TRYP1R & S, TBR1/TBR2, TCF1/TCF2, TCS1/TCS2 and TVW1/TVW2) started   177 with an initial denaturation step at 95°C for 3 min followed by 40 cycles composed of a 178 denaturation step at 95°C for 30 s, a hybridization step at 55°C for 1 min 30 s and an extension 179 step at 72°C for 1 min, and a last final extension at 72°C for 5 min. The TgsGP PCR was 180 performed using a program consisting of an initial denaturation step at 94°C for 15    available. An antiseptic was applied after each sampling.

Parasitological investigations
The BCT was positive for 20 animals, leading to an overall prevalence of 1.1% (Table   258   4   (Z= -4.71, P= 2.5.10-6) and pig (Z= -3.41, P= 6.5.10-3) species tended to decrease it. In  (Table 6). and Sud-Ouest respectively. There is no statistical difference between these prevalence (P˃ 323 0.05). This test also shows that the cattle were the most affected.

324
The Venn diagram shows the levels of concordance of detection of the tests used. All

325
BCT positive cases were also PCR positive, however only 16/20 cases were ELISA positive.

326
The concordance of cases identified by ELISA and PCR was 22.1% for 248/1124 cases 327 identified ( Figure 2).  showed a molecular prevalence of 11.6%, lower than in the present study (30). Similar

384
The sero-prevalence of 49.2% that we found in our survey is higher than that of there is no significant impact of sex and age on prevalence. However, there could be a difference 436 due to age as farmers often do not take young animals to pasture, which limits their contact with 437 tsetse flies and therefore adults are most exposed (18).

438
The diagnostic tests that we used in the survey have different levels of sensitivity and 439 specificity. BCT often lacks sensitivity for the diagnosis of trypanosomes when the parasitemia 440 is low (< 300 trypanosomes per mL), but is species specific (29 The apparent densities of flies per trap observed in relation to the season are low.