Trypanosoma congolense is a major causative agent of the bovine disease
trypanosomosis which has a considerable economic impact on sub-Saharan Africa.
Current control methods for trypanosomosis are unsatisfactory and vaccine development
has been hampered by antigenic variation.
An anti-disease vaccine is based on the idea that disease is caused by the pathogenic
factors released by the parasite, rather than by the parasite itself. Therefore, if these
pathogenic factors could be neutralised by antibodies produced by vaccination, the
disease could be circumvented. The method used here for identification of novel
pathogenic factors is based on the concept that trypanotolerant cattle are able to mitigate
the disease by generating a specific immune response against a few key antigens
(pathogenic factors).
Two immuno-affinity columns were therefore prepared: one containing IgG from noninfected
sera and a second column containing IgG from trypanotolerant N’Dama cattle
serially infected with T. congolense. The differential binding of antigens to the two
columns allowed identification of antigens specifically recognised by the immune system
of a trypanotolerant animal, i.e. potential pathogenic factors. The most promising antigens
identified included several variant cathepsin L-like cysteine peptidases (CPs) and the
Family M1 Clan MA aminopeptidases (APs). For the CPs, a study of the genetic
organisation was conducted in order to further understand the variability present in this
gene family.
To this end, two different mini-libraries of cathepsin L-like genes were prepared: one in
which genes as different as possible from congopain (the major CP of T. congolense)
were selected, and a second which contained all possible genes present in the congopain
array. Analysis of the sequences obtained in these two mini-libraries showed that there
was significant variability of the genes within the congopain array. Two variants of CPs,
chosen for differences in their catalytic triads, were cloned for expression. The
recombinantly expressed CP variants differed in substrate preferences from one another
and from C2 (the recombinant truncated form of congopain), and surprisingly, all enzymes
were active at physiological pH. The two APs were cloned and expressed as insoluble inclusion bodies in an E. coli
system, and subsequently refolded. The refolded APs showed a substrate preference for
H-Ala-AMC, an optimum pH of 8.0, localisation to the cytoplasm and inhibition by
puromycin. The two APs were not developmentally regulated and present in procyclic,
metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi
resulted in a slightly reduced growth rate in procyclic parasites in vitro. Immunisation of
BALB/c mice with the APs did not provide protection when challenged with T. congolense.
For an anti-disease vaccine to be protective, it would possibly have to include all
pathogenic factors, including the two APs and at least one CP described in the present
study. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10942 |
| Date | January 2010 |
| Creators | Pillay, Davita. |
| Contributors | Coetzer, Theresa Helen Taillefer. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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