Identifikacija podtipova protozoona Trypanosoma vivax izdvojenih iz goveda i koza pomoću mikrosatelitskih markera.

Microsatellite DNA polymorphisms can be utilised to assess intra-specific genetic diversity and hence are useful for characterisation of species and strains of trypanosomes. Here, we present four new microsatellite markers specific for T. vivax isolated from Ugandan cattle and goats. The GeneDB partial shotgun 5x coverage sequence of T. vivax available as of 1st August 2005 was used and targeted the genomic sequence of T. vivax that has no cross amplification with other livestock-infective trypanosomes. Only di-; tri-; tetra;- and pentanucleotide microsatellites not less than five units were selected. Although pentanucleotide repeats on screening appeared to have the desired variability, they gave poorer PCR products compared to di-, tri- and tetranucleotide repeats. Mononucleotide repeats presented difficulty in detecting visible bands on agarose gels from their amplification and were omitted from this study. Clear length polymorphism was obtained with guanine, thymine and adenine repeated 16 times (GTA)16 while cytosine, adenine, cytosine and thymine (CACT)15 gave size and length variability. Bands of similar size were obtained from thymine and adenine (TTA)24 microsatellite, approximately 150 base pairs long and 180-200 base pairs from the cytosine and adenine (CA)26 microsatellite. These findings suggest that different subtypes of T. vivax exist in Uganda; the polymorphic forms derived from microsatellite band size differences may suggest this parasite exhibits virulence differences as has been shown in T. Congolense subtypes.Polimorfizam mikrosatelitske DNA može se rabiti za procjenu unutarvrsne genetske raznolikosti pa tako i za karakterizaciju vrsta i sojeva tripanosoma. Prikazana su četiri nova mikrosatelitska markera specifična za vrstu T. vivax izdvojenu iz goveda i koza u Ugandi. GenDB kratka i specifična sekvencija T. vivax dostupna nakon 1. kolovoza 2005. bila je ciljano rabljena za određivanje genomskoga slijeda za protozoon T. vivax koji nije pokazivao križne reakcije s drugim tripanosomama zaraznima za stoku. Izabrani su bili samo di-, tri-, tetrai pentanukleotidni mikrosateliti. Premda se činilo da pentanukleotidne ponavljajuće sekvencije u probirnom testu imaju potrebnu varijabilnost, one su dale lošije PCR proizvode u odnosu na di-, tri- i tetranukleotidne ponavljajuće sekvencije. Mononukleotidne ponavljajuće sekvencije nisu dale jasno vidljive trake na agaroznom gelu pa nisu bile dalje istražene. Jasan polimorfizam postignut je upotrebom gvanina, timina i adenina sa šesnaesterostrukim ponavljanjem (GTA)16 dok je sekvencija citozin, adenin, citozin i timin (CACT)15 bila varijabilna u odnosu na veličinu i dužinu. Sekvencije slične veličine bile su dobivene od mikrosatelita koji su sadržavali timin i adenin (TTA)24, a one od 150 parova baza te 180 - 200 parova baza od mikrosatelita citozina i adenina (CA)26. Ovi nalazi govore u prilog postojanju različitih podtipova protozoona T. vivax u Ugandi, koji bi se mogli odlikovati i različitom virulencijom kao što je dokazano za podtipove T. congolense

A Potential Malaria Vaccine Candidate Identified Using an Insilico Approach

The search for an effective malaria vaccine has yielded no success yet. Unfortunately, resistance to post-infection treatments is on the increase hence the need to develop an efficient vaccine. The aim of many reverse vaccinology studies is to identify novel proteins found exposed on the surface. Many malaria vaccine candidates can be effective tools against malaria but gross allelic polymorphism is a major hinderance which could be overcome by using highly conserved proteins. Also peptide based vaccines can be of great importance in fighting malaria however this is limited by HLA restriction which can be maneuvered  by using promiscuous peptides. In the current work, our objective was to computationally identify conserved hypothetical, antigenic, surface proteins in pathogenic plasmodium falciparum parasite. So in this study, we employed an in silico approach to screen the proteins on the basis of surface localization, non-homology with host proteome, and MHC class I and II binding promiscuity. The analyses reported XP_001351004.1 an uncharacterized protein as a novel vaccine candidate. Generation of the 3D model of the protein was done using RaptorX server. Furthermore, the B cell and T cell epitopes were also predicted. B cell epitopes were predicted using ABCpred and Kolanskar and Tongaonkar antigenicity method while Tcell epitopes were predicted using CTLpred.Five peptides were selected based on their hydrophobicity. Results from this study could be extended to in-vivo and in-vitro experiments for future vaccine development

Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci

Theileria parva is a tick‐transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti‐transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south‐western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen‐encoding genes that are targets of CD8+T‐cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south‐western Uganda. Cattle‐derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved

Anti-Paraflagellar Rodc Antibodies Inhibit the In-Vitro Growth of Trypanosoma Brucei Brucei

Paraflagellar rod (PFR), a conserved structure expressed in all lifecycle stages of the order kinetoplasida except in the amastigotes is vital for the parasites survival. In T.b.brucei, the PFR protein has two major components, PFRc and PFRa with molecular mass 73kDa and 68kDa respectively. Experimental evidences implicate the PFR protein as a highly immunogenic and protective antigen. However, its immunogenic properties underlying its suitability as vaccine candidate has not been adequately investigated in-vitro. This study aimed to demonstrate the growth inhibitory potential of PFR protein against T.b.brucei parasites in–vitro. Antibodies against a recombinant form of the PFRc protein were produced and used to generate immune response. A deoxyribonucleotide (DNA) segment of approximate 672bp encoding the PFRc protein component was amplified using polymerase chain reaction (PCR), cloned and expressed in E.coli (BL21) cells. A 200 µg portion of the purified PFRc protein mixed with 100µl Freund's complete adjuvant (FCA) was used to immunize rabbits. An antibody titre of 2.5 x 104 reciprocal dilutions was obtained following three immunisation boosts, spaced two weeks apart. Western blot analysis showed that rabbit anti-PFRc antibodies recognised specifically a 25kDa protein corresponding to the estimated size of the expressed PFRc protein. 25% of purified anti-rabbit IgG antibodies were able to inhibit ~70% T.b.brucei parasite in vitro. This confirmed that the PFRc protein is immunogenic in rabbits and can elicit specific growth inhibitory antibodies. However, we recommend invivo studies in humans and domestic animals infected by trypanosomes to ascertain the vaccine potential of this candidate protein for trypanosomiasis

A Single-Blind randomized controlled trial to evaluate the effect of extended counseling on uptake of pre-antiretroviral care in eastern uganda

<p>Abstract</p> <p>Background</p> <p>Many newly screened people living with HIV (PLHIV) in Sub-Saharan Africa do not understand the importance of regular pre-antiretroviral (ARV) care because most of them have been counseled by staff who lack basic counseling skills. This results in low uptake of pre-ARV care and late treatment initiation in resource-poor settings. The effect of providing post-test counseling by staff equipped with basic counseling skills, combined with home visits by community support agents on uptake of pre-ARV care for newly diagnosed PLHIV was evaluated through a randomized intervention trial in Uganda.</p> <p>Methods</p> <p>An intervention trial was performed consisting of post-test counseling by trained counselors, combined with monthly home visits by community support agents for continued counseling to newly screened PLHIV in Iganga district, Uganda between July 2009 and June 2010, Participants (N = 400) from three public recruitment centres were randomized to receive either the intervention, or the standard care (the existing post-test counseling by ARV clinic staff who lack basic training in counseling skills), the control arm. The outcome measure was the proportion of newly screened and counseled PLHIV in either arm who had been to their nearest health center for clinical check-up in the subsequent three months +2 months. Treatment was randomly assigned using computer-generated random numbers. The statistical significance of differences between the two study arms was assessed using chi-square and t-tests for categorical and quantitative data respectively. Risk ratios and 95% confidence intervals were used to assess the effect of the intervention.</p> <p>Results</p> <p>Participants in the intervention arm were 80% more likely to accept (take up) pre-ARV care compared to those in the control arm (RR 1.8, 95% CI 1.4-2.1). No adverse events were reported.</p> <p>Conclusions</p> <p>Provision of post-test counseling by staff trained in basic counseling skills, combined with home visits by community support agents had a significant effect on uptake of pre-ARV care and appears to be a cost-effective way to increase the prerequisites for timely ARV initiation.</p> <p>Trial registration</p> <p>The trial was registered by Current Controlled Trials Ltd C/OBioMed Central Ltd as <a href="http://www.controlled-trials.com/ISRCTN94133652">ISRCTN94133652</a> and received financial support from Sida and logistical support from the European Commission.</p

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Diagnosis plays a central role in the control of human African trypanosomiasis (HAT) whose mainstay in disease control is chemotherapy. However, accurate diagnosis is hampered by the absence of sensitive techniques for parasite detection. Without concentrating the blood, detection thresholds can be as high as 10,000 trypanosomes per milliliter of blood. The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) are promising molecular diagnostics that generally yield high sensitivity and could improve case detection. Recently, these two tests were coupled to oligochromatography (OC) for simplified and standardized detection of amplified products, eliminating the need for electrophoresis. In this study, we evaluated the diagnostic accuracy of these two novel tests on blood specimens from HAT patients and healthy endemic controls from D.R. Congo and Uganda. Both tests exhibited good sensitivity and specificity compared to the current diagnostic tests and may be valuable tools for sensitive and specific parasite detection in clinical specimens. These standardized molecular test formats open avenues for improved case detection, particularly in epidemiological studies and in disease diagnosis at reference centres

Benzimidazole (BZ) resistance in Haemonchus controtus : specific interactions of BZs with tubulin

The mechanism of benzimidazole (BZ) anthelmintic resistance in Haemonchus contortus was investigated. The total binding (TB), low-affinity binding (LAB) and high-affinity (specific) binding (HAB) of ($sp3$H) BZs (mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ) and oxfendazole (OFZ)) in supernatants derived from BZ-susceptible (S) and BZ-resistant (R) strains were examined and compared. The TB of all ($sp3$H) BZs was reduced for the R strain. The TB of OBZ, MBZ and ABZ was separated into LAB and HAB. However, OFZ bound with low-affinity. The binding affinity, K$sb{ rm a},$ and maximum binding, B$sb{ rm max},$ for the HAB of OBZ and MBZ were calculated using computer programs. Compared with the S strain, the B$sb{ rm max}$ of the R strain was reduced but the K$sb{ rm a}$ was not affected. LAB to parasite preparations devoid of tubulin was observed but HAB occurred to preparations containing tubulin only. The HAB per mg protein decreased from egg through larva to adult stage. It was shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA) analysis that the tubulin content per mg protein decreased from egg, through larva to adult worm. The ability of various BZs--OBZ, MBZ, ABZ, OFZ, fenbendazole (FBZ), albendazole sulphoxide (ABZSO), albendazole sulphone (ABZSO$sb2),$ and thiabendazole (TBZ)--to bind tubulin was compared by displacement analysis and their IC$sb{50}$ ( (BZ) required to inhibit 50% of the ($sp3$H) BZ binding) and K$sb{ rm a}$ values were determined. The IC$sb{50}$ and K$sb{ rm a}$ values approximately correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZs except for OFZ and ABZSO. Tubulin bound BZs at 4$sp circ$C with lower K$sb{ rm a}$ than at 37$sp circ$C. Western blot of tubulin separated by 2-dimensional electrophoresis showed that the $beta$-tubulin isoform pattern of the S and R strains were dissimilar whi

Trypanosoma brucei prenylated-protein carboxyl methyltransferase prefers farnesylated substrates.

Carboxyl methylation of the C-terminal prenylated cysteine, which occurs in most farnesylated and geranylgeranylated proteins, is a reversible step and is implicated in the regulation of membrane binding and cellular functions of prenylated proteins such as GTPases. The gene coding for prenylated-protein carboxyl methyltransferase (PPMT) of the protozoan parasite Trypanosoma brucei has been cloned and expressed in the baculovirus/Sf9 cell system. The protein of 245 amino acids has 24-28% sequence identity to the orthologues from other species including human and Saccharomyces cerevisiae. Methyltransferase activity was detected in the membrane fraction from Sf9 cells infected with the recombinant baculovirus using N -acetyl- S -farnesylcysteine (AFC) and S -adenosyl[ methyl -(3)H]methionine ([(3)H]AdoMet) as substrates. Recombinant T. brucei PPMT prefers AFC to N -acetyl- S -geranylgeranylcysteine (AGGC) by 10-50-fold based on the V (max)/ K (m) values. Native PPMT activity detected in the membrane fraction from T. brucei procyclics displays similar substrate specificity ( approximately 40-fold preference for AFC over AGGC). In contrast, mouse liver PPMT utilizes both AFC and AGGC as substrates with similar catalytic efficiencies. Several cellular proteins of the T. brucei bloodstream form were shown to be carboxyl methylated in a cell-free system. Incorporation of [(3)H]methyl group from [(3)H]AdoMet into most of the proteins was significantly inhibited by AFC but not AGGC at 20 microM, suggesting that T. brucei PPMT acts on farnesylated proteins in the cell. Cells of the T. brucei bloodstream form show higher sensitivity to AFC and AGGC (EC(50)=70-80 microM) compared with mouse 3T3 cells (EC(50)>150 microM)