Authors

Discussion

Species of Trypanosoma transmitted by the tsetse fly (Glossina) vector are responsible for important medical and veterinary diseases across sub-Saharan Africa. Although advances have been made in the control of human African trypanosomiasis (HAT), animal African trypanosomiasis (AAT) remains a disease of significant economic burden and livestock mortality in sub-Saharan Africa. Current AAT surveillance tools suffer from poor sensitivity and specificity, with serological methods also requiring animal restraint and blood collection by trained personnel. Faecal sampling is an attractive potential option for more accessible sample collection and screening. This study set out to determine whether it is possible to detect DNA of AAT aetiological agents (T. brucei and T. congolense savannah) in the faeces of experimentally-infected cattle. 

Five male Holstein-Friesian calves of post-weaning age were inoculated with T. brucei AnTat 1.1 and the infection course was followed for a total of 68 days. A total of 146 faecal samples (12 pre-inoculation, 134 post-inoculation) and 148 blood samples (10 pre-inoculation, 138 post-inoculation) were collected. In a parallel study, six male Holstein-Friesian calves were inoculated with T. congolense savannah IL3000 and infection course followed for 66 days. A total of 151 faecal samples (6 pre-inoculation, 145 post-inoculation) and 167 blood samples (12 pre-inoculation, 155 post-inoculation) were collected. All samples were screened using Typanosoma species-specific PCR assays and novel probe-based qPCR assays targeting Trypanozoon-specific and T. congolense savannah-specific repeat regions in kinetoplast minicircle DNA respectively.

T. brucei target DNA was successfully detected in 85% (n=114) of post-inoculation faecal samples by qPCR and 50% (n=67) by PCR. T. brucei target DNA was detected in faecal samples collected between four days post-inoculation (dpi) to 66 dpi by both qPCR and PCR. Amplification of target DNA was confirmed by Sanger sequencing of PCR products, which revealed significant homology to the target sequence. T. brucei DNA was detected in 100% (n=138) of post-inoculation blood samples by qPCR. Linear regression analysis revealed a weak yet statistically significant positive relationship (p=0.0354, R²=0.06) between Cq values obtained from matched T. brucei blood and faecal samples. However, T. congolense target DNA was detected in just 3.4% (n=5) of post-inoculation faecal samples by qPCR and none by PCR. T. congolense DNA was detected in 100% (n=155) of post-inoculation blood samples.

These results confirm, for the first time, the ability to consistently detect T. brucei DNA from the faeces of infected cattle. By contrast, T. congolense DNA could not be reliably detected in faeces, despite the respective qPCR assays having the same approximate limit of detection. This finding may be explained by the differences in Trypanosoma species tissue distribution; with T. brucei capable of tissue invasion whilst T. congolense remains largely restricted to the blood circulatory system. Whilst these findings show the potential of using faeces as an easily-accessible sample to screen for active T. brucei infection, blood sampling is still required to reliably detect T. congolense in cattle. Future research should focus on refining this novel diagnostic method in field and wildlife samples to broaden T. brucei surveillance.