PCR-based diagnosis of surra-targeting VSG gene: experimental studies in small laboratory rodents and buffalo.
Trypanosoma evansi, the causative organism of 'surra' expresses its variable surface glycoprotein (VSG) at early, middle and late stages of infection in animals. The variable antigenic nature of VSG caused by switching its expression type favours evasion from the host immune response and leads to chronic and persistent infection. Developing a polymerase chain reaction (PCR)-based diagnostic tool targeting the VSG gene is expected to be highly specific and sensitive for diagnosis of surra. Hence, in the present study, we have designed EXP3F/4R primer pair and amplified the 1.4 kb of VSG gene of T. evansi and studied the phylogenetic relationship by in silico analysis. The PCR method was standardised using another set of primer, DITRYF/R, and 400 bp was amplified from blood and tissue samples of experimentally infected animals. Applying the PCR method, we were able to detect as low as 0.15 trypanosomeml(-1). Considering the number of parasite-to-DNA concentration, the PCR method has a sensitivity of 0.015 pg ml(-1). The PCR could detect the presence of the parasite as early as 24h post-infection (p.i.) and 72 h p.i., respectively, in experimentally infected rats and buffalo. No amplification was observed with DNA of Babesia bigemina and Theileria annulata, indicating the primers are specific for T. evansi. The PCR method could detect the dog, lion and leopard isolates of T. evansi. Similarly, amplifying the DNA from the experimentally infected tissues was also found to be sensitive. Thus, the findings of this study favour the application of PCR over the parasitological methods for the detection of the early and/or chronic stage of surra in domestic and wild animals.
Sengupta
P P
PP
Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore 560024, Karnataka, India. pinakiprasad_s@rediffmail.com
Balumahendiran
M
M
Suryanaryana
V V S
VV
Raghavendra
A G
AG
Shome
B R
BR
Gajendragad
M R
MR
Prabhudas
K
K
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes / Q000469:parasitology*; D002353:Carrier State / Q000175:diagnosis / Q000469:parasitology*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*
D016054:DNA, Protozoan; D008562:Membrane Glycoproteins