Effects of Allium Sativum Ethanolic Extract on Trypanosoma brucei brucei Parasites' Morphometric Parameters and Clinical Outcome in White Albino Laboratory Rats

BACKGROUND Trypanosomosis affects humans as well as wild and domestic vertebrates, yet has no successful prophylaxis, chemotherapy nor cure. OBJECTIVES The study was to investigate the effects of Allium sativum extract on Trypanosoma brucei brucei parasites' morphometric parameters, parasitemia and the clinical outcome in white infected Albino laboratory rats in order to determine its trypanocidal effects. METHODOLOGY The study was conducted at the department of Biological Sciences Laboratory of the Moi University Eldoret. Thirty two (32) mature rats randomly divided into four groups (M, N, P and Q) were kept in four (4) cages in a well ventilated room, with adequate light supply in the day. Sixteen (16) rats were infected with T. b. brucei (1.0 x 104 parasites per rat); eight (8) of which (Group N) were treated with the A. sativum ethanolic extract on day 5 and day 9 after infection, while the other eight (8)rats (Group Q) received saline treatment on the same days. Sixteen (16) non-infected rats (controls) were also divided into two groups of eight rats each (P and M) and treated as in group N and Q, respectively. The rats were obtained from University of Nairobi, Chiromo Campus. RESULTS All infected rats became parasitemic two days after infection and reached peak levels on day 4 and 5 post infection. Parasitemia in saline treated infected rats fluctuated between 4025.5 ± 0.05 - 5544.4 ± 0.05 parasites per 200WBC whereas in the extract treated rats parasitemia declined from 6976.6 ± 0.05 - 311.0 ± 0.05 parasites per 200WBC after the first treatment. Uninfected saline treated rats maintained normal Hb level (10.6g/L to 11.8g/L) as compared to the uninfected extract treated rats' whose Hb levels was at 13.41g/L to 14.36g/L. The haemoglobin level changed to 8.0g/L four days after the infection in the group N rats before rising to 10.2g/L on day 8 post-infection following the extract treatments. Group Q rats' Hb declined to 6.43g/L by the end of the study. RBC count of the infected saline treated rats declined to 3.38 x 106/µL as compared to 4.93-7.61 x 106/µL in the normal rats by 11 days postinfection.There was however no significant change in WBC, temperature and weight between the saline extract treated rats. The extract produced a shrinking effect on the parasite's body with some of the morphometric parameters appearing significantly (P<0.05) reduced as observed under a microscope with ocular and stage micrometer scale. The mean nucleus, posterior ends to nucleus centre, the nucleus centre to the anterior end and the body length were reduced from 2.41µm to 1.42µm(P=0.00), 4.42µm to 3.68µm(P=0.017) , 4.65µm to 4.18µm(P=0.001) and 8.58µm to 7.19µm(P=0.001) respectively. CONCLUSION In conclusion it was evident that, A. sativum ethanolic extract exhibited Trypanocidal effects that can be exploited to control clinical progression of Trypanosomosis in rats. In addition, the data presented demonstrates the plant extract had the potential to improve the red and white blood cell indices reducing parasitaemia following T. b. brucei infection. These findings suggest that, the garlic extract affected the plasma membrane of the parasites since shrinking was only possible with disrupted membrane biochemistry

Evidence for regulated expression of Telomeric Repeat-containing RNAs (TERRA) in parasitic trypanosomatids

The Telomeric Repeat-containing RNAs (TERRA) participate in the homeostasis of telomeres in higher eukaryotes. Here, we investigated the expression of TERRA in Leishmania spp. and Trypanosoma brucei and found evidences for its expression as a specific RNA class. The trypanosomatid TERRA are heterogeneous in size and partially polyadenylated. The levels of TERRA transcripts appear to be modulated through the life cycle in both trypanosomatids investigated, suggesting that TERRA play a stage-specific role in the life cycle of these early-branching eukaryotes.

Análise de evidências de comunicação quorum sensing mediada pelo isoprenóide farnesol em Leishmania amazonensis / Analysis of evidence of quorum sensing communication mediated by isoprenoid farnesol in Leishmania amazonensis

Quorum sensing (QS) é um tipo de comunicação intercelular descrito em microorganismos. Ela é intermediada por moléculas sinalizadoras (QSMs) liberadas constitutivamente pelos espécimes e por meio delas os microorganismos percebem a densidade populacional. [...] Em fungos, trans,trans-farnesol (t,t-FOH) atua como QSM. Esse isoprenóide regula a virulência de Candida albicans modulando os processos de transição morfológica. O t, t- FOH é produzido por uma rota alternativa a partir do farnesilpirofosfato (FPP), um intermediário da via biossíntética de esteróis. Em protozoários, QS já foi descrita no tripanossomatídeo Trypanossoma brucei. Adicionalmente, o processo de metaciclogênese que ocorre em Leishmania possui características de regulação QS, devido sua relação com a densidade populacional. Interessantemente, fungos e tripanossomatídeos possuem maquinaria própria de síntese de esteróis. Essa similaridade e a descrição do t,t-FOH como QSM em fungos sugere que ele pode desempenhar atividade similar em tripanossomatídeos. Em nossa hipótese, t,t-FOH é uma QSM que causa uma redução da capacidade proliferativa de Leishmania amazonensis quando a cultura atinge um quorum. Porém, o parasito mantem-se viável, de modo que a cultura fica estacionária. Inicialmente constatamos que L. amazonensis libera t,t-FOH ou seus derivados no sobrenadante. [...] Essas observações estão de acordo com o perfil de uma possível QSM. Em seguida determinamos as condições de cultivo em que o t,t-FOH possivelmente atua como QSM nas culturas...

Quorum sensing (QS) is a type of intercellular communication described in microorganisms.It is mediated by signaling molecules (QSMs) constitutively released by specimens andthrough them, the microorganisms sense the population density. [...] Infungi, trans,trans-farnesol (t,t-FOH) acts as QSM. This isoprenoid regulates virulence ofCandida albicans interfering the morphological transitions. t,t-FOH is produced by analternative route from farnesylpyrophosphate (FPP), an intermediate of the sterolbiosynthetic pathway. In protozoa, QS has been described in the trypanosomatidTrypanosoma brucei. Additionally, the metacyclogenesis of Leishmania has caracteristics ofQS regulation, due to its relationship with population density. Interestingly, fungi andtrypanosomatids have their own machinery sterol synthesis. This similarity and thedescription of the t, t-FOH as QSM in fungi suggests that it may play similar activity intrypanosomatids. In our hypothesis, t,t-FOH is a QSM which causes a reduction in theproliferative capacity of Leishmania amazonensis when the culture reaches a quorum.However, the parasite keeps viable, so the culture enters in the stationary phase. Initially, wefound that L. amazonensis releases t,t-FOH or its derivatives in the supernatant. Theconcentration of these metabolites enhances with the increase in population density. Theseobservations are consistent with the profile of a possible QSM. Then, we determined thegrowing conditions in which t,t-FOH possibly acts as QSM. [...] We observed that the concentrations that inhibited the proliferation were also apparently toxic. Consideringthat the t,t-FOH is lipophilic, we decided to remove the lipophilic compounds of thesupernatant...

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

Anti-trypanosomal Activity of Potential Inhibitors of Trypanosoma brucei Glycolytic Pathway Enzymes Selected by Docking Studies

Human African trypanosomiasis (HAT); a potentially fatal protozoan infection caused by tsetse-fl mediated transmission of Trypanosoma brucei (T. Brucei); is largely recognized as a neglected disease. The repertoire of drugs that is effective against the infection is limited and all drugs have several drawbacks including high level of toxicity; difficult administration regimens; and the resurgence of resistance. At present the biology of the parasite is well studied and a number of technologies are now available which can aid in the identifiation of potential drug targets. This review identifis putative inhibitors of trypanosomal glycolytic enzymes

Effect of diminazene aceturate, levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei

OBJECTIVE@#To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei.@*METHODS@#Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability.@*RESULTS@#Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A.@*CONCLUSIONS@#Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei.

Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the a heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.

Trypanosomatid comparative genomics: contributions to the study of parasite biology and different parasitic diseases

In 2005, draft sequences of the genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, also known as the Tri-Tryp genomes, were published. These protozoan parasites are the causative agents of three distinct insect-borne diseases, namely sleeping sickness, Chagas disease and leishmaniasis, all with a worldwide distribution. Despite the large estimated evolutionary distance among them, a conserved core of ~6,200 trypanosomatid genes was found among the Tri-Tryp genomes. Extensive analysis of these genomic sequences has greatly increased our understanding of the biology of these parasites and their host-parasite interactions. In this article, we review the recent advances in the comparative genomics of these three species. This analysis also includes data on additional sequences derived from other trypanosmatid species, as well as recent data on gene expression and functional genomics. In addition to facilitating the identification of key parasite molecules that may provide a better understanding of these complex diseases, genome studies offer a rich source of new information that can be used to define potential new drug targets and vaccine candidates for controlling these parasitic infections.

In vitro trypanocidal activity of antibodies to bacterially expressed Trypanosoma brucei tubulin

There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth. T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450micro g each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera. Low antisera dilutions [25%] from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions. Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes

Observations on estrus as monitored by receptivity to mating activity and pattern of vaginal exfoliates in trypanosoma brucei ­ infected WAD goat does synchronized with Estrumate® / Observaciones sobre el estro monitorizado por la receptividad a la actividad de apareamiento y el patrón de exfoliación vaginal en cabras WAD infectadas por trypanosoma brucei sincronizadas con Estrumate®

The effects of experimental Trypanosoma brucei infection on receptivity to mating activity and pattern of vaginal exfoliates were monitored using twenty-one adult WAD goats which were synchronized with double injection, seven days apart of Estrumate®. The twenty-one goats consisted of 3 bucks and 18 does. The does were randomly divided into control group 'A' having 10 does and test group 'B' with 8 does. The goats were fed with Elephant grass in the morning and commercial feed containing 15.23 percent CP at the rate of 0.25kg/head in the afternoons. Freshwater was provided ad libitum. Results showed that while all the control does were observed to stand to be mounted and mated, none of the infected does did. Also, the pattern of the mean percentage vaginal exfoliated cell types encountered between the control and infected doe groups were converse. While parasabal cells changed from 2.90±0.03 percent during proestrus through 3.05 +/- 0.46 percent during estrus to 2.42 +/-0.08 percent at diestrus in the control does, it changed from 22.07 +/- 0.56 percent during expected proestrus through 8.48 +/- 0.05 percent during expected estrus to 28.05 +/-1.09 percent respectively in the infected does. In like manner, intermediate cell changed from 11.10 +/- 0.03 percent during proestrus through 11.10 +/- 0.31 percent during estrus to 1.21 +/- 1.00 percent during diestrus in control does while it changed from 27.27 +/- 0.08 percent during expected proestrus through 42.37 +/- 2.39 percent during expected estrus to 40.24 +/- 1.06 percent during expected diestrus in infected does. Similarly, superficial cells changed from 56.25 +/- 0.75 percent during proestrus through 63.70 +/- 1.05 percent during estrus to 7.37 +/- 0.01 percent during diestrus while it changed from 0.00 percent during expected proestrus through 3.39 +/- 0.02 percent during expected estrus to 63.70 +/- 1.05 percent during estrus to 6.10 +/- 0.01 percent during expected diestrus. In the control does, the ...

Los efectos de la infección experimental por Trypanosoma brucei sobre la receptividad a la actividad de apareamiento y el patrón de exfoliación vaginal fueron monitoreados utilizando 21 cabras WAD adultas sincronizadas con doble inyección, a los siete días de diferencia de Estrumate®. De las 21 cabras utilizadas eran 3 machos y 18 hembras. Las hembras se dividieron al azar en grupo control "A" con 10 sujetos y un grupo de prueba "B" con 8. Las cabras fueron alimentadas con pasto y alimento comercial que contenía 15,23 por ciento de CP en tasa de 0,25kg/por cabeza en las tardes. Agua fresca fue proporcionada ad libitum. Los resultados mostraron que mientras todos las cabras del grupo control pudieron ser montadas y acopladas, ninguna de las infectadas pudo. Además, fue contradictorio el patrón de la media porcentual de los tipos de células vaginales exfoliadas encontradas entre los grupo control e infectadas. Mientras que las células parabasales cambiaron desde un 2,90 +/- 0,03 por ciento durante el proestro, al 3,05 +/- 0,46 por ciento durante el estro y 2,42 +/- 0,08 por ciento al diestro en el grupo control, el grupo infectado cambió desde un 22,07 +/- 0,56 por ciento durante el proestro, al 8,48 +/- 0,05 por ciento durante el estro y 28,05 +/- 1,09 por ciento al diestro. De la misma forma, las célula intermedias cambiaron de un 11,10 +/- 0,03 por ciento durante el proestro, al 11,10 +/- 0,31 por ciento durante el estro y al 1,21 +/- 1,00 por ciento durante el diestro en el grupo control, mientras que en el grupo infectado pasó del 27,27 +/- 0,08 por ciento durante el proestro, al 42,37 +/- 2,39 por ciento durante el estro y al 40,24 +/- 1,06 por ciento durante el diestro. Las células superficiales pasaron desde un 56,25 +/- 0,75 por ciento durante el proestro, 63,70 +/- 1,05 por ciento durante el estro, hasta un 7,37 +/- 0,01 por ciento durante el diestro, mientras en el grupo infectado pasaron de un 0.00 por ciento durante el proestro, al 3,9 +/- 0,02 p...

Induced proteins profile in the haemolymph of desert locust, schistocerca gregaria, following trypanosoma brucei brucei challenge

Innate immunity plays a key role in the control of microbial infections in both vertebrates and invertebrates. Haemolymph samples from Shistocerca gregaria, obtained after Trypanosoma brucei brucei challenge were analyzed for their protein profiles using SDS and 2D-PAGE and also evaluated for antitrypanosomal activity in vitro. Protein induction was found to increase with time, peaking at about 18 hours. In SDS-PAGE, the intensity levels of five polypeptides were found to vary from prechallenge levels. Further analysis of the polypeptides on 2D-PAGE showed variations in their induction pattern with some being induced, upregulated or suppressed with time of induction. Samples collected from insects challenged with parasites followed by sugars, D-glucosamine had the highest inhibitory effect on the level of protein induction while D-galactose had the least effect. When screened for trypanolytic activity against T. brucei brucei, the samples had pronounced antitrypanosomal activity which peaked with the 18 hour sample. Antibodies raised against Glossina proteolytic lectin [Gpl], showed no cross-reactivity to Shistocerca gregaria induced haemolymph proteins in Western blots. Antitrypanosoma proteins induced during vector-parasite interaction have the potential of being used to modulate tsetse fly vectorial capacity

In vivo antitrypanosomal effects of some ethnomedicinal plants from Nupeland of North Central Nigeria

Abstract: Four medicinal plants Acacia nilotica, Bombax buonopozense, Terminalia avicennioides and Zanthoxylum zanthoxyloides traditionally used for treatment of sleeping sickness in Nupeland were investigated for in vivo antitrypanosomal activity. Methanol extracts of different parts of each plant (stem barks and fruits) were obtained and evaluated for their in vivo antitrypanosomal activities against Trypanosoma brucei brucei. Phytochemical screening of the methanol extracts of each plant were performed by standard procedures. Methanol extracts of A. nilotica (stem bark), B. buonopozense (stem bark), T. avicennioides (round fruit) and Z. zanthoxyloides (stem bark) were effective on trypanosomes. The extracts of A. nilotica and B. buonopozense exhibited antitrypanosomal effects at 200 and 300 mg/kg body weight respectively. Doses were able to clear the parasites from circulation within 6 and 7 days of treatment respectively with prolonging survival period of up to 30 days. While the extracts of T. avicennioides and Z. zanthoxyloides showed trypanostatic effects and could not clear the parasites completely. The methanol extracts of these plants contain metabolites that are associated with antitrypanosomal effects; therefore, these medicinal plants may be sources of new compounds that may be active against T. b. brucei. This study has also justified the claim that some medicinal plants of Nupeland possess antitrypanosomal activity and could be useful in the management of trypanosomiasis

Reconstrução in silico das vias de processamento da informação genética nos Tritryps (Tripanosoma cruzi, Trypanosoma brucei e Leishmania major): busca por análogos funcionais / Reconstruction of roads in silico processing of genetic information in Tritryps (Trypanosoma cruzi, Trypanosoma brucei and Leishmania major): the search for functional analogues

Leishmania major, Trypanosoma brucei e Trypanosoma cruzi (Tritryps) são protozoários unicelulares que causam a leishmaniose, a doença do sono e a doença de Chagas, respectivamente. Essas doenças causam ônus econômicos principalmente em regiões subtropicais e tropicais. Atualmente não existem vacinas comercialmente disponíveis e não há tratamento eficaz para tais doenças. Isso se deve ao fato dos fármacos disponíveis apresentarem muitos efeitos colaterais e estarem propensas ao desenvolvimento de resistência. A maioria desses fármacos foi descoberta através da seleção de um grande numero de compostos contra parasitas íntegros. Porém, nos últimos anos, uma nova abordagem vem ganhando espaço sob o termo de “desenho racional de fármacos” Este termo representa a busca por compostos contra alvos moleculares específicos, visando diferenças bioquímicas e fisiológicas entre o parasita e o hospedeiro. A era pós-genômica gerou uma grande quantidade de informações que permitem a identificação ótima de novos alvos. Neste contexto, a partir de dados públicos dos genomas de Tritryps, reconstruímos as vias de processamento da informação genética (com ênfase nas vias de replicação e reparo, transcrição e tradução) nesses organismos, para adquirir uma melhor representação das enzimas envolvidas nestes processos. Estas análises permitiram estudos comparativos para identificar candidatos a novos alvos terapêuticos. Em nossa metodologia utilizamos a ferramenta AnEnPi (http://bioinfo.pdtis.fiocruz.br/AnEnPi/) para buscar nas seqüências genômicas por enzimas análogas. Utilizando os dados provindos do KEGG, primeiro houve uma etapa de clusterização das estruturas primárias de todas as enzimas desse banco de dados anotadas com o mesmo EC. Para isso, utilizou-se uma pontuação (score) de similaridade no Blastp de 120, como parâmetros de corte. Encontramos 830 grupos de ECs com mais de um cluster e 1430 com um único cluster. Após isso, foi realizado um passo de reanotação. Para isto, foi rodado um novo Blastp, assumindo como ponto de corte um e-value de 10e-20, entre todas as proteínas preditas nos genomas de cada Tritryp contra todos os clusters. Desses dados geramos mapas de vias de interesse para esses organismos e os comparamos aos mapas que o KEGG disponibiliza como padrão. Identificamos alguns casos de analogia nestas vias entre seres humanos e Tritryps que podem vir a ser utilizados como novos alvos terapêuticos para o desenvolvimento de fármacos contra esses parasitas. Foi feita a modelagem por homologia de um análogo (6.1.1.-, de T. brucei), utilizando a ferramente MHOLline. Além disso, buscamos no banco de alvos terapêuticos para doenças negligenciadas, TDRTARGETS (http://tdrtargets.org/), pelos ECs identificados como possíveis novos alvos, e não encontramos nenhuma ocorrência. Tal fato pode indicar que com a metodologia aplicada conseguimos identificar novos candidatos a alvos terapêuticos contra estes parasitas. Em análises futuras, vamos testar e-values mais restritivos na etapa de reanotação, para assim, testar o potencial de reanotação da ferramenta.

Cloning, expression, purification and activity assay of Trypanosoma brucei phenylalanyl-tRNA synthetase in Escherichia coli / 生物工程学报

Phenylalany--tRNA synthetase is a key enzyme for protein synthesis in Trypanosoma. Its validation as an inhibition. target will enable the development of a new generation of anti-Trypanosoma drugs. However, little is known about the isolation of the Trypanosoma Phenylalanyl-tRNA synthetase. Here we report the cloning, expression, purification, and activity assay of Phenylalanyl-tRNA synthetase from Trypanosoma brucei in Escherichia coli host. We co-cloned the alpha-subunit and beta-subunit of Phenylalanyl-tRNA synthetase from Trypanosoma brucei genomic DNA into the co-expression vector pCOLADuet. We successfully expressed the Trypanosoma brucei Phenylalanyl-tRNA synthetase in E. coli host, purified the whole enzyme by Ni-Hind affinity column and verified it by Western blotting. In addition, we tested its enzymatic activity by isotope labeling. The whole work laid a solid foundation for in vitro the screening and optimization of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitors.

Trypanosoma brucei Infection in Asymptomatic Greater Kudus (Tragelaphus strepsiceros) on a Game Ranch in Zambia

Trypomastogotes of Trypanosoma brucei were detected from 4 asymptomatic kudus (Tragelaphus strepsiceros) on a game ranch located approximately 45 km north east of Lusaka, Zambia. Blood smears examined from 14 wildlife species comprising of the impala (Aepyceros melampus), Kafue lechwe (kobus leche kafuensis), sable antelope (Hippotragus niger), tsessebe (Damaliscus lunatus), warthog (Phacochoerus aethiopicus), puku (Kobus vardoni), zebra (Equus burchelli), waterbuck (Kobus ellipsiprymnus), bushbuck (Tragelaphus scriptus), reedbuck (Redunca arundinum), wilderbeest (Connochaetes taurinus), hartebeest (Alcephelus lichtensteini), African buffalo (Syncerus caffer), and kudu (Tragelaphus strepsiceros) showed that only the kudu had T. brucei. Although game ranching has emerged to be a successful ex-situ conservation strategy aimed at saving the declining wildlife population in the National Parks, our findings suggest that it has the potential of aiding the re-distribution of animal diseases. Hence, there is a need for augmenting wildlife conservation with disease control strategies aimed at reducing the risk of disease transmission between wildlife and domestic animals.

Trypanosome Glycosylphosphatidylinositol Biosynthesis

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.

Trypanosome Glycosylphosphatidylinositol Biosynthesis

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90 percent of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

Characterisation of the Trypanosoma brucei rhodesiense isolates from Tanzania using serum resistance associated gene as molecular marker

Serum resistance associated (SRA) gene has been found to confer resistance to the innate trypanolytic factor (TLF) found in normal human serum; thus allowing Trypanosoma brucei brucei to survive exposure to normal human serum. This study was carried out to examine the presence of SRA gene and identify the origin of T. b. rhodesiense isolates from three districts in Tanzania, namely Kibondo, Kasulu and Urambo. Twenty-six T. b. rhodesiense isolates and two references T. b. rhodesiense isolates from Kenya were examined for SRA gene using simple Polymerase Chain Reaction technique. The gene was found to be present in all 26 T. b. rhodesiense isolates including the two references isolates from Kenya. The SRA gene was confirmed to be specific to T. b. rhodesiense since it could not be amplified from all other Trypanozoon including T. b. gambiense; and gave an amplified fragment of the expected size (3.9kb), confirming that all these isolates were T. b. rhodesiense of the northern variant. Although the geographic distributions of T. b. gambiense and T. b. rhodesiense are clearly localized to west/central Africa and eastern Africa, respectively, natural movement of people and recent influx of large number of refugees into Tanzania from the Democratic Republic of Congo, could have brought T. b. gambiense in western Tanzania. The overlap in distribution of both of these pathogenic sub-species could result in erroneous diagnoses since both trypanosome sub-species are morphologically identical, and currently serologic methods have low specificity. Both the susceptible and resistant T.b. rhodesiense isolates possessed the SRA gene suggesting that there is no correlation between drug resistance and presence of SRA gene. The use of SRA gene helps to confirm the identity and diversity of some of the isolates resistant to various drugs.

A comparative study on the susceptibility of male and female albino mice to Trypanosoma brucei brucei.

BACKGROUND & OBJECTIVES: Trypanosomiasis has remained a major set-back in the development of livestock farming in tropical Africa. Thus the need for ascertaining the trypanotolerant levels of domestic animal breeds and possible improvement on them cannot be over-emphasised. METHODS: Level of trypanotolerance in animals was compared between sexes using albino mice infected with a Nigerian strain of Trypanosoma brucei brucei at a 50% mouse lethal dose (MLD50). RESULTS: The male mice showed unrestrained parasite growth with a prepatent period (PP) of two days and a mean survival period (MSP) of six days corresponding to a gradual decrease in packed cell volume (PCV), body weight, diet response and white blood cells (WBC) count to the time of death. Their female counterparts showed a PP of three days and MSP often days with a similar PCV gradient but a refractory WBC count. There was no significant difference in the differential leucocytes count in both sexes. However, the eosinophils count was significantly higher in the infected animals. It was found that female albino mice exercised more parasite restraint than their male counterparts. INTERPRETATION & CONCLUSION: The result suggests that the female animals may be more trypanotolerant hence may be more useful in protein production in trypanosomiasis endemic areas. However, further research using large domestic breeds like goats and sheep may be required to confirm the hypothesis.