ELISAs have historically been based on crude antigen with batch-to-batch variation, though standardisation has been improved with modifications such as a monoclonal capture antibody (Rebeski, Winger et al. 1999) and fractionation of crude antigen (Pathak, Arora et al. 1993; Reid and Copeman 2003).
Trypanosomes are known for their variable surface antigen coats and most antibodies produced by the hosts are directed against these variable surface antigens. Many
18 serological tests are therefore based on variable surface antigens produced by the trypanosomes. Although variable surface glycoprotein sequences are subject to change, it is possible that, in the case of T. evansi, which is non-cyclically transmitted and thus has limited genetic variation (Boid 1988), VSGs may be preserved. The VSG repertoire of a single stock of T. evansi is limited and thought to be relatively small (Yang, Suo et al. 2007) and there can be shared variable antigen types (VATs) between related stocks of T. evansi, with more closely related strains sharing more VATs (Yang, Suo et al. 2007). Infected hosts also produce antibodies to minor non-variable antigens (Shapiro and Murray 1982). It is possible that the host immune response to conserved antigens participates in control of the trypanosome infections in Cape buffaloes (Guirnalda, Murphy et al. 2007) and trypanotolerant N’Dama cattle (Authie, Muteti et al. 1993), possibly by neutralising toxic or pathogenic molecules produced by the parasite (Naessens, Teale et al. 2002).
2.2.3.1 RoTat 1.2
Rode Trypanozoon antigen type 1.2 (RoTat 1.2) is a variable antigen type (VAT) which was cloned from T. evansi isolated from an Indonesian water buffalo in 1982 (Bajyana Songa and Hamers 1988; Urakawa, Verloo et al. 2001). It is one of the predominant VATs and has been found early in the infection in the majority of T. evansi strains. Rabbits infected with stocks and clones from different parts of the world developed antibodies to RoTat 1.2 within 32 days of infection (Verloo, Magnus et al. 2001; Claes, Verloo et al. 2002). Tests using RoTat 1.2 do not cross react with antibodies to T.
theileri or other pathogenic trypanosomes, although they cannot distinguish between
infections with trypanosomes currently classified as T. evansi and T. equiperdum
(Claes, Verloo et al. 2002; Claes, Radwanska et al. 2004). The RoTat 1.2 gene is a
19 strains isolated from Kenya, which may limit its diagnostic utility (Claes, Radwanska et al. 2004; Ngaira, Olembo et al. 2005; Njiru, Constantine et al. 2006). It is not known whether type B strains exist in Asia.
2.2.3.2 GM6
GM6 is a minor non-variant trypanosome antigen. A partial sequence of GM6 was identified when a cDNA library from T. brucei gambiense was screened using sera from cattle soon after they were infected with T. brucei (Muller, Hemphill et al. 1992). GM6 is a large protein with a repetitive structure (units of 68 amino acids) that is located at the flagellum-cell body interface (Muller, Hemphill et al. 1992). It is a structural calpain-like protein (Ersfeld, Barraclough et al. 2005) that plays an essential role in the immune response because it is targeted by a number of micro-RNA genes in the trypanosome genome (Mallick, Ghosh et al. 2008). GM6 is well conserved between different species of salivarian trypanosomes, though there are slightly less similarities between the salivarian and the stercorarian species. Analogues are also present in some species of Leishmania (Muller, Hemphill et al. 1992).
2.2.3.3 Paraflagellar rod protein A
Each trypanosome has a single flagellum which exits from a flagellar pocket where endo/exocytosis takes place. Trypanosomes swim with the flagellum leading, dragging the cell behind it. Functions of the flagellum include motility, probably to reach and invade host cells (reviewed by (Bastin, McRae et al. 1999), to attach the parasite to host surfaces and as an environment sensor. The flagellum is attached to the cell body along its entire length at the flagellar attachment zone (FAZ). The flagellum contains an axoneme which is well conserved throughout eukaryotic evolution and consists of nine peripheral microtubule doublets plus two single central microtubules (Bastin, Pullen et
20 al. 2000). T. brucei forms a new flagellum whilst retaining the old one. The flagellum, flagellar pocket and pellicular membrane are covered by a variable surface glycoprotein coat, though glycoprotein on the pocket membrane appears to be a different to those on the other surfaces (Bastin, Pullen et al. 2000).
The trypanosome flagellum contains an extra-axonemal paraflagellar rod (Bastin, Pullen et al. 2000) which is vital for trypanosome motility (Bastin, Sherwin et al. 1998). The paraflagellar rod (PFR) is present from the flagellar exit at the flagellar pocket to the distal tip of the flagellum. The crescent-shaped paraflagellar rod is made up of fibres crossing at defined angles (de Souza and Souto-Padron 1980; Bastin, Matthews et al. 1996). It is 150nm in diameter and attached strongly to axoneme microtubules 4-7 via fibres that are very strong, and is difficult to remove (Bastin, Pullen et al. 2000). A paraflagellar rod has only been found in three groups of protists so far: kinetoplastids, euglenoids (Hyams 1982) and dinoflagellates and is not restricted to parasitic species. A morphologically similar structure is present in Giardia and Tritrichomonas sp., with further work to characterise similar structures in other protozoa ongoing (Bastin, Pullen et al. 2000). The paraflagellar rod is made up of PFR1 (C) and PFR2 (A) proteins. These proteins are found in all other organisms with a paraflagellar rod (Gallo and Schrevel 1985). PFR1 and PFR2 are well conserved between species and isolates, with only the ends showing significant diversity. The first monoclonal antibody to a paraflagellar rod protein from euglena recognized both PFR1 and PFR2 and cross reacted with all trypanosomes tested, including T. brucei (Gallo and Schrevel 1985), although the physical structure of the euglena PFR is different to that of trypanosomes (Hyams 1982). Anti-PFR protein antisera exclusively recognized the flagellum (Bastin, Pullen et al. 2000).
21 PFR is immunogenic and immunisation of mice with PFR elicits a strong T helper 1 response, which results in protection against trypanosomiasis. Alum preparations result in the strongest immunity by causing an increase in IFN-γ and IL-2 (Michailowsky, Luhrs et al. 2003). Although it is present in low copy numbers, PFRA has been targeted using a loop-mediated isothermal amplification (LAMP) test to diagnose trypanosomiasis (Kuboki, Inoue et al. 2003). This immunogenic non-variable antigen could be useful for detecting infection with pathogenic trypanosomes.