The use of molecular technology to investigate trypanosome infections in tsetse flies at Liwonde Wild Life Reserve

Abstract

Background: Trypanosomes are protozoan flagellates that cause human African trypanosomiasis (HAT) and African animal trypanosomiasis (AAT). HAT is caused by Trypanosoma brucei rhodesiense in East and Central Africa and T.b. gambiense in West Africa, whereas AAT is caused by a number of trypanosome species, including T. brucei brucei, T. evansi, T. vivax, T. congolense, T. godfreyi and T. simiae. The aim of this study was to establish if tsetse flies at Liwonde Wild Life Reserve (LWLR) are infected with these trypanosomes and thus pose a risk to both humans and animals within and surrounding the LWLR.

Methods: A total of 150 tsetse flies were caught. Of these, 82 remained alive after capture and were dissected such that the mid-gut could be examined microscopically for trypanosomes. DNA extractions were performed from both mid-guts and the 68 dead flies using a Qiagen Kit. Amplification techniques involved the Internal Transcriber Spacer 1 (ITS 1) conventional polymerase chain reaction (PCR) with primers designed to identify trypanosome species, and Repetitive Insertion Mobile Element - Loop Mediated Isothermal Amplification (RIME LAMP), a sequence specific to T. brucei.

Results: Analysis showed that 79/82 (96.3%) of the mid-guts examined microscopically were positive for trypanosomes and that 75/150 (50%) of the DNA extracts (from the mid-gut, and tsetse fly carcasses) were positive for T. brucei, as determined by the RIME LAMP method. ITS1 PCR further showed that 87/150 (58.0%) flies were positive for trypanosomes, of which 56/87 (64.4%) were T. brucei, 9/87 (10.3%) were T. vivax; 7/87 (8.1%) were T. simiae; 6/87 (6.9%) were T. congolense, and 6/87 (6.9%) were T. godfreyi. Ten samples had a mixture of infections.

Conclusion: Our analysis demonstrated a mixture of infections from trypanosome species in tsetse flies at LWLR, and that T. brucei, the species that causes HAT, was the most common. Our study successfully used molecular techniques to demonstrate the presence of T. b. rhodesiense at LWLR, a species that causes HAT in both East and Central Africa.

Keywords: African Trypanosomiasis; DNA; Malawi; Polymerase Chain Reaction; Southern Africa; Tsetse Flies.

Figure 1

Brightfield microscopy images showing trypanosomes (arrows)

Figure 2

Trypanosome species identified by Internal Transcriber Spacer 1 polymerase chain reaction (ITS 1 PCR).

Figure 3

Image of an agarose gel showing samples S61–S68: M is a 1000 bp marker; PC1 and PC2 were positive controls for T. congolense and T. simiae species; S61, S62, S64 are infections by T. brucei; S65 & S67 are mixed infections by T. brucei and T. vivax; S66 is a mixed infection by T. brucei and T. godfreyi; and S68 is a mixed infection by T. brucei, T. godfreyi and T. vivax.

Figure 4

RIME LAMP results: NC 1 is a negative control (nuclease free water); NC2 is a negative control (prepared master mix); PC is a positive control for a known T. brucei sample from Liwonde; S1, S2, S4, S5, S7–S9 are positives; and S3, S6 are negatives.