African trypanosomiasis, the disease
2 occurs within a few years, and the latter causes acute infection with death within a few months (Steverding, 2008). N'dama cattle (Bos taurus) can limit parasitemia and disease symptoms caused by T.
Classification of trypanosomes
The life cycle and distribution of African trypanosomes
Once colonized, the salivary glands are the site of production of the MCF that is infectious to mammals (Sbicego et al., 1999). The slender forms replicate by asexual division and are susceptible to elimination by host proteases (Sbicego et al., 1999).
The genomic organisation of trypanosomes
However, in the PCF parasites, all ESs are silent, as the PCF parasites lack the VSG coat and are coated with PARPs and procyclins (Ersfeld, 2011). An ES is either switched off, another ES is activated, or an existing VSG gene in an active ES is replaced by another VSG gene via genetic recombination and results in the expression of a new variant of VSG (Horn and Cross, 1997).
Immune response to trypanosomal infection
The waves of parasitemia explain the constantly changing levels of circulating trypanosomal parasites in the bloodstream (Ross and Thomson, 1910) which in turn affects the limited sensitivity of parasitological detection methods in clinical practice (Chappuis et al., 2005a).
Control strategies for trypanosomiasis
Research has shown that treating livestock with insecticide has resulted in the same degree of tsetse control as can be achieved using scent bell traps, but this method has been less effective in areas where wild animals are the food source for the tsetse fly (Hargrove et al ., 2003).
The clinical features of human and animal African trypanosomiasis
The final stages of HAT infections are usually treated with melarsoprol (Schmid et al., 2004; Chappuis et al., 2005b), an arsenate that is toxic and known to cause reactive arsenic encephalopathy in patients (Pépin and Milord, 1994; WHO, 1998). Due to the ability to cross the blood-brain barrier, humoral parasite species can cause neurological symptoms during the course of the disease (Lonsdale-Eccles and Grab, 2002; Antoine-Moussiaux et al., 2008).
Techiques used for diagnosis of African trypanosomiasis
- Serological screening
- Parasitological confirmation
- Stage determination
- Other molecular diagnostic approaches to screening and confirmation of
The TL test is based on the complement-mediated lysis of trypanosomal parasites (Chappuis et al., 2005a). Based on the technique developed by Lanham and Godfrey (1970), in which the less negatively charged trypansomal parasites are separated from red blood cells by anion-exchange chromatography, the mini-anion-exchange centrifugation technique (mAECT) was introduced (Lumsden et al., 1979) .
Drug therapy for African trypanosomiasis
Isomethamide chloride (Samorin®) and homidium chloride (Novidium®) have prophylactic and therapeutic properties, while diminazene aceturate (Berenil®) has only therapeutic properties and are used to treat AAT (Geerts et al., 2001). The use of a sanative pair for AAT treatment consisting of isomethamide chloride and diminazene aceturate is unlikely to result in cross-resistance (Whitesand, 1960; Chitanga et al., 2011).
An anti-disease strategy for the control of African trypanosomiasis
Due to the extensive use of trypanocides, it is no surprise that drug resistance has emerged in 21 African countries (Geerts and Holmes, 1998) (Delespaux et al., 2008; Chitanga et al., 2011). Peptides of the trypanosomal parasite have been shown to be virulence factors and thus ideal chemotherapeutic targets and diagnostic candidates (Antoine-Moussiaux et al., 2009).
Protozoan peptidases
- Serine peptidases
- Oligopeptidase B
- Cysteine peptidases
- Cathepsin L- like peptidase
- Pyroglutamyl peptidase
- Metacaspases
Multiple sequence alignment of the CA clan, C1 family of cysteine peptidases generated using ClustalX (Larkin et al., 2007). Multiple sequence alignment of the CF clan, C15 family of cysteine peptidases generated using ClustalX ( Larkin et al., 2007 ).
Objectives of current study
The functions of these inactive peptidases are unknown, but may play a role in binding to natural peptidase inhibitors or may be substrates for endogenous enzymes that aid in the modulation of the activities of enzymatically active proteases (Mottram et al., 2003). The results of using the purified antigens in the ELISAs for inhibition and indirect antibody detection using infected and uninfected T.
Introduction
Trypanosomatid metacaspases are characterized by their domain composition as well as their gene copy number ( Alvarez et al., 2011 ). Knockdown of TbMCA4 by RNAi (RNAi) resulted in cell cycle arrest resulting in cell death ( Proto et al., 2011 ).
Materials and methods
- Materials
- Molecular weight marker calibrations
- Protein quantititation
- Cloning of TcMCA5 into the pGEM ® -T cloning vector
- Subcloning of the TcMCA5 gene into the bacterial pET-28a expression
- Recombinant expression of TcMCA5
- Solubilisation, refolding and purification of recombinant TcMCA5
- Enzymatic characterisation of recombinant TcMAC5
- Activity assay
- Gelatin containing SDS-PAGE for recombinant TcMCA5 activity
- Antibody preparation and ELISA optimisation
- Preparation of immunogen, the immunisation of chickens and IgY
- ELISA evaluation of antibody production
- Culture of procyclic Trypanosoma congolense (strain IL 3000) parasites
A sample of the produced PCR mixture (2 μl) was added to an equal volume of GLB and subjected to electrophoresis on a 1% (w/v) agarose gel containing ethidium bromide (0.5 μg/ml) in TAE buffer. Plasmid DNA of positive clones was subjected to small volume (10 μl) restriction digestion with EcoRI in its unique buffer at 37 °C for 2 h followed by heat inactivation at 65 °C for 15 min. Restriction digestion products (10 µl) resulting from EcoRI and BamHI restriction digests were added to an equal volume of GLB and analyzed on a 1% (w/v) agarose gel containing ethidium bromide (0.5 µg/ml) in buffer TAE. to confirm the correct size of the TcMCA5 gene.
Samples of the supernatant and the pellet containing the solubilized protein lysate and insoluble fractions, respectively, were.
Results
- Cloning of TcMCA5 gene into the pGEM ® -T cloning vector
- Subcloning of the TcMCA5 gene into the bacterial pET-28a expression
- Recombinant expression, solubilisation, refolding and purification of TcMCA5
- Expression optimisation of recombinant TcMCA5
- Solubilisation, refolding and purification of recombinantly expressed
- Enzymatic characterisation of TcMCA5
- Activity assay
- Gelatin containing SDS-PAGE
- Autocatalytic processing of purified recombinant TcMCA5
- Evaluation of anti-TcMCA5 antibody production by ELISA
- Three dimensional structure prediction of TcMCA5
Ligation mixtures of the TcMCA5 insert with the different expression vectors were prepared, including. The presence of the two lower molecular weight proteins/cleavage products contributes to the fact that the expressed recombinant TcMCA5 may be in a partially native state and have retained some degree of activity. Some degree of native conformation of the recombinant TcMCA5 protein is present in the inclusion bodies, as is evident due to the products of autocatalytic cleavage/lower molecular weight proteins (Figs. 2.14 and 2.15).
2.16, solubilization and on-column refolding using the urea and sarkosyl methods respectively, it is clear that in the presence of urea, recombinant TcMCA5 has less activity as visualized by the low concentration of the two lower molecular weight products/cleavage products .
Discussion
Three strips are obtained; the one corresponding to the full length of LmMCA and the lower two bands thought to be the result of autocatalytic cleavage of LmMCA ( Gonzáles et al., 2007 ). Recombinant TcrMCA3 and -5 were solubilized using a chaotropic (urea) method according to Middleberg (2002) and successfully folded using IMAC purification ( Kosec et al., 2006b ). When incubated with calcium, recombinant TcMCA5 purified using the sarkosyl method underwent further processing as seen with TbMCA2 ( Moss et al., 2007 ).
It has been observed that in the presence of calcium, using far UV circular dichroism, reversible structural changes occurred in recombinant TbMCA2 (Machado et al., 2013).
Introduction
If TcrPGP is found to be expressed in the infectious BSF of mammals, it may be important for the pathogenesis of Chagas disease (Kosec et al., 2006a) (Fig. 1.14). It has since been shown that IgG antibodies against congopain can completely inhibit the enzymatic activity of congopain (Authié et al., 2001). Congopain is involved in immunosuppression and the development of anemia (Authié et al., 2001) and plays a key role in the development of trypanotolerance (Authié et al., 1993a).
The antibody detection ELISAs do have application in the surveillance of AT as well as in the initial phases of the development of lateral-flow assays (Chappuis et al., 2005a).
Materials and methods
- Materials
- Calibration of HiPrep™ 16/16 Sephacryl™ S200 HR and S300 HR Molecular
- Recombinant expression and purification of full length oligopeptidase B from
- Recombinant expression and purification of full length oligopeptidase B from
- Recombinant expression and purification of the catalytic domain of vivapain
- Antibody production and ELISA optimisation
- Coupling of the TcCATL N-terminal peptide to rabbit albumin via MBS
- Preparation of immunogen for the immunisation of chickens and IgY
- ELISA evaluation of antibody production
- Affinity purification of isolated IgY antibodies
- Coupling of TcCATL N-terminal peptide to SulfoLink ®
- Coupling of TcCATL FL to AminoLink ®
- Western blot of recombinantly purified proteins with their respective IgY
The elution volume (Ve) or retention volume of blue dextran was taken as the void volume (V0) of the column. Supernatant and pellet samples containing soluble and insoluble fractions were analyzed at 12.5%. Samples of unbound and eluted fractions were electrophoresed on a 12.5% reducing SDS-PAGE gel (Laemmli, 1970) and stained with Coomassie blue R-250 (according to Section 2.2.7).
Samples of the supernatant and pellet containing the soluble and insoluble fractions, respectively, were analyzed by 12.5% reducing SDS-PAGE (Laemmli, 1970) and stained with Coomassie blue R-250 (as in Section 2.2.6).
Results
- Recombinant expression and purification of full length oligopeptidase B from
- Recombinant expression and purification of full length oligopeptidase B from
- Full length oligopeptidase B from T. vivax, (TvOPB)
- Pyroglutamyl peptidase from T. congolense, (TcPGP)
- Catalytic domain of vivapain from T. vivax, (TvCATL)
- Full length congopain from T. congolense, (TcCATL FL )
- Catalytic domain of congopain from T. congolense, (TcCATL)
- Antibody preparation and ELISA optimisation for TcOPB
- Antibody preparation and ELISA optimisation for TvOPB
- Antibody preparation and ELISA optimisation for TcPGP
- Antibody preparation and ELISA optimisation for TvCATL
- Antibody preparation and ELISA optimisation for TcCATL FL
- Antibody preparation and ELISA optimisation for TcCATL
- Antibody preparation and ELISA optimisation for TcCATL N-terminal peptide . 119
To optimize TcOPB coating and anti-TcOPB IgY concentration, we used a checkerboard ELISA as described by Crowther (2000). 3.25 shows that there was significant production of anti-TcPGP IgY antibodies from weeks 3 to 16, except for week 12 in chicken 1, with absorbance values at 405 nm above those of the preimmune IgY antibody. Five affinity purifications of anti-TcCATL N-terminal peptide IgY antibodies from different weeks were performed and those with similarly high absorbance values at 280 nm were grouped into three separate groups.
Each of the affinity-purified N-terminal peptide IgY pools of anti-TcCATL was used in a checkerboard ELISA against TcCATL.
Introduction
Commercial ELISA kits for the diagnosis of HAT use the same antigens as LATEX/T. Antigen detection ELISAs use monoclonal antibodies which are used to detect trypanosomal antigens in serum samples and are available for the diagnosis of HAT and T. The use of ELISA as a diagnostic tool may be the step to develop flow lateral. the tests.
Due to the need for the development of AAT diagnostics, the use of multiple antigens in an antibody detection ELISA was investigated.
Materials and methods
- Materials
- Inhibition antibody detection ELISA
- Sensitivity and specificity calculations for the inhibition antibody
- Indirect antibody detection ELISA
- Sensitivity and specificity calculations for the indirect antibody detection
- Receiver-operating characteristic (ROC) analysis of the diagnostic potential
The cutoffs for each of the antigens were calculated by adding one, two, and three SDs to the mean percent inhibition from the panel of known negative sera. The sensitivity and specificity of the inhibition ELISA for each antigen was determined at each cutoff. These values were divided by the average of the triplicate absorbance values at 405 nm of the known positive control serum.
The sensitivity and specificity of the indirect ELISA for each antigen were determined at each cutoff.
Results
- Optimisation of inhibition and indirect TcOPB ELISA with Trypanosoma
- Inhibition TcOPB ELISA
- Indirect TcOPB ELISA
- Optimisation of inhibition and indirect TcPGP ELISA with Trypanosoma
- Inhibition TcPGP ELISA
- Indirect TcPGP ELISA
- Inhibition TcCATL FL ELISA
- Indirect TcCATL FL ELISA
- Optimisation of inhibition and indirect TcCATL ELISA with Trypanosoma
- Inhibition TcCATL ELISA
- Indirect TcCATL ELISA
- Optimisation of indirect TvCATL ELISA with Trypanosoma vivax infected and
- Optimisation of indirect TvOPB ELISA with Trypanosoma vivax infected and
- Trypanosoma congolense blinded serum panel using the inhibition and
- Trypanosoma vivax blinded serum panel using the indirect ELISA format with
Corrected absorbance values at 405 nm for the non-infected serum samples were found to be slightly higher than the values for the infected serum samples (Appendix C, Fig. C.2). The corrected absorbance values at 405 nm were slightly higher for the uninfected serum samples than for the infected serum samples (Appendix C, Fig. C.5). 4.15, the corrected absorbance values at 405 nm for the uninfected serum samples were higher than those for the infected serum samples at each of the conditions tested.
The ability of the TcCATLFL antigen in this ELISA format was able to distinguish between infected and uninfected serum samples.
Discussion
Both TcCATLFL and TcCATL antigens performed better in the indirect ELISA format than in the inhibition format. Although the indirect ELISA requires one less step than the inhibition format, detection of IgG in the test serum and diagnosis requires comparison of the results with that of a known strong positive control serum that was included in the test (Rebeski et al. . , 1999c). On the other hand, inhibition ELISA requires the generation of antibodies against each of the antigens, which is time-consuming.
The main advantage of the inhibition ELISA format is that the anti-trypansomal antibodies of a variety of livestock species are detectable using one set of ELISA reagents, whereas in the indirect ELISA format species-specific antibodies are required.
During the course of AT infection, disruption of the mammalian host's endocrine system is common (Tizard et al., 1978; Brandenberger et al., 1996). It was found that in the serum of infected rats, the degradation of peptide hormones followed an unusual pattern (Tetaert et al., 1993). The Tc38630 antigen was found to be able to detect antibodies earlier than the parasite lysate (Mochabo et al., 2013).
Due to the favorable performance in the antigen detection ELISA, it was concluded that the Tc38630 protein was a promising diagnostic antigen for the diagnosis of AAT (Mochabo et al., 2013).
