Trypanosoma

The cdentcfccatcon of trypanosomes was performed by PCR amplcfccatcon of ITS 1. Inctcally, trypanosome specces were prelcmcnarcly asscgned accordcng to the band scze of the product ycelded. As descrcbed cn the lcterature [24], sczes cn the range between 550 and 700 bp, 400 – 450 bp and 198 – 250 bp were conscdered to come from T. congolense kclcfc and T. congolense forest/savannah, T. brucei ssp., and T. vivax, respectcvely. Also, unexpected band sczes of about 320 and 100 bp were amplcfced (Fcg. 2b, Addctconal fcle 1: Tables S1-S 3).

To further check the cdentcfccatcon, samples were subjected to PCR wcth speccfcc prcmers where avaclable. Fcnally, representatcve PCR products were subcloned and sequenced for fcnal confcrmatcon or more detacled

a Trypanosoma Species ITS 1 ampl -

icon size

T. brucei rhodesiense

T. brucei gambiense 426

T. brucei brucei

T. congolense savannah 645

T. congolense kilifi 562

T. congolense

662

riverine/forest

T. vivax 198

T. grayi 317

T. theileri 349

b

M 1 2 3 4 5 6 7 8 9 10 C M

500 bp

250 bp

50 bp

Fig. 2 ITS 1 amplcconm of Trypanosoma mpeccem. a Amplccon mczem expected fol amplcfccatcon wcth genelcc plcmelm ITS 1-InF and ITS 1-InR wele calculated flom avaclable databame mequencem and clommchecked wcth mequencem flom mcleened mamplem. b PCR wam pelfolmed wcth leplementatcve mamplem contacncng DNA of the cndccated olcgcn and genelcc (a) ol mpeccfcc plcmelm (Table 1). Lane M: Malkel GeneRulel

50 bp Laddel (Thelmo Sccentcfcc); Lane 1: NI 100 (genelcc plcmelm); Lane 2: Bodoncd (genelcc plcmelm); Lane 3: T. vivax (genelcc plcmelm); Lane 4: T. grayi (genelcc plcmelm); Lane 5: T. theileri (genelcc plcmelm); Lane 6: T. brucei mmp. (genelcc plcmelm); Lane 7: T. grayi (mpeccfcc plcmelm); Lane 8: T. congolense (mpeccfcc plcmelm); Lane 9: T. congolense folemt (mpeccfcc plcmelm); Lane 10: T. congolense folemt and T. congolense kclcfc (mpeccfcc plcmelm); Lane C: contlol wcthout DNA template

cdentcfccatcon. The fragment sczes and respectcve Trypanosoma specces are lcsted cn Fcg. 2 and Addctconal fcle 1: Tables S1-S 4).