Clinicopathological alterations in wild mammals from the reservoir system of Trypanosoma cruzi:a scoping review

Trypanosoma cruzi is the etiologic agent of Chagas disease.This flagellated protozoan is transmitted to humans as well as different species of domestic and wild animals via vectors from the Reduviidae family(known as"kissing bugs").Despite the fact that hundreds of species of wild mammals are part of the reservoir system,the morphologi-cal changes and clinical manifestations resulting from the pathogenesis of the infection have been largely neglected.The aim of this review is to systematically compile the available information regarding clinicopathological altera-tions in wild mammals due to natural infection by T.cruzi.Information was obtained from six online bibliographic data search platforms,resulting in the identification of 29 publications that met the inclusion criteria.Mortality was the most common clinical manifestation,cardiac damage was the main finding at necropsy,and lymphoplas-macytic inflammation was the most frequent microscopic injury.Thus,regardless of its role as a reservoir,T.cruzi has the potential to affect the health status of wild mammals,a situation that highlights the need for further research to analyze,measure,and compare its effects at both the individual and population levels.

Achillea fragrantissima,rich in flavonoids and tannins,potentiates the activity of diminazine aceturate against Trypanosoma evansi in rats

Objective:To evaluate activity of methanol extract of Achillea fragrantissima(meth)(A.fragrantissima) alone or in combination with diminazine aceturate(DA) against Trypanosoma evansi in experimentally infected rats.Methods:Sixty adult male Wister albino rats were divided equally into 6 groups(A-F).Rats in groups A-E were experimentally infected with T.evansi and those in group F were uninfected.The groups were treated respectively as follows:group A- with 3.5 mg/kg DA;group B- with 1 000 mg/kg meth,A.fragrantissima;group C-3.5mg/kg DA plus 500 mg/kg meth A.fragrantissima;group D-3.5 mg/kg DA plus 1 000 mg/kg meth A.fragrantissima.Group E was left untreated.Parasitaemia,survivability,packed cell volume,hemoglobin concentration,total leucocytes count,lymphocyte count,and serum malondialdehyde and reduced glutathione(GSH) levels were estimated.Phytochemical screening of meth A.fragrantissima was also performed.Results:The phytochemical analysis of the meth A.fragrantissima indicated a higher content from polyphenols tannins and non tannins and flavonoids.The efficacy percentage against trypanosomiasis in groups A to E was respectively as follows 80,40,90.100,0.The administration of meth-A.fragrantissima(1000)mg/kg b.wt.) produced a moderate efficacy against trypanosomiasis.Untreated rats in group E died between 25 and 30 d post infection.The rats given DA and meth A.fragrantissima combinations(C and D) showed faster and higher recovery rates than the uninfected control and groups A and B.The initial reduction in packed cell volume,hemoglobin,total leucocytes count,increases in serum malondialdehyde and decreases in GSH levels were reversed by the treatments.C onclusions:The administration of the methanol extracts of A.fragrantissima and DA combination therapy was more effective than each product alone in the treatment of rats infected with Trypanosoma evansi and further studies are required to isolate more active ingredients.

Hematological and serum biochemical aspects associated with a camel(Camelus dromedarius)naturally infected by Trypanosoma evansi with severe parasitemia in Semnan,Iran

Objective:To determine the presence of Trypanosoma eransi(T.evansi) and the effect of trypanosomosis on hemato-biochemical profile of dromedary camels in Semnan,Iran,which has not been reported yet.Methods:To perform this project,blood samples were collected by venipuncture into plain and EDTA-K2-containing vacutainer tubes from 21 dromedary camels(12 males and 9 females) aged3—18 years,from 4 different regions of Semnan.Results:Microscopic examination of stained thin blood smears revealed the presence of T.evansi in one of the samples.However,it should be noted that this sample showed a very high parasitemia(more than 5 trypomastigote were visible per microscopic field with MGG,1000×.This heavy parasitemia was associated with an 18-year-old female camel that showed symptoms of corneal opacity,intense emaciation and pale mucous membranes.Comparison of hematological and serum biochemical profiles between the camel infected by T.eransi and uninfected camels indicated anemia,leukocytosis,hyperproteinemia.hypoalbuminemia,hyperglobulinemia,reduction A/G ratio,increased a,,p and globulins and decreased of a,globulins and increased the concentration of gumma-glutamyl transferase enzyme.Conclusions:Results of the present study revealed that trypanosomosis was present in dromedary camels of Semnan,Iran(infection rate is 4.76%) and hemato-biochemical parameters were markedly affected by camel trypanosomosis.

<i>Trypanosoma evansi</i>: A Qualitative and Quantitative Ultrastructural Analysis of the Spleen during Experimental Murine Infections

A murine model is used to study qualitatively and quantitatively the splenic ultrastructural changes induced by two Trypanosoma evansi strains derived from naturally infected local equine hosts (Equusasinus and E. caballus);T. evansi causes ultrastructural modifications in the spleen of the infected mice. The modifications include tissular disorganization, fibrosis, mitochondrial swelling, apoptosis and necrosis. The initial phases of the infection are quite similar, whereas the final phases differ qualitatively depending on the strain’s source. The ultrastructural quantitative changes were studied in the reticular splenocytes covering alterations in the area of the cytoplasm and nucleus. Analysis of the results shows the induction of various splenic alterations caused by local T. evansi strains. Also, it was documented that discriminative time modulation, as well as progressive tissular, cellular and subcellular changes, are more associated with derived infections from E. caballus strain.

Therapeutic Activity of Partially Purified Fractions of Emblica officinalis （Syn, Phyllanthus embfica） Dried Fruits against Trypanosoma evansi

Emblica officinalis （E. oJficinalis） dried fruits were evaluated for its antitrypanosomal activity and cytotoxic effects. Vero cell line maintained in DMEM （Dubecco＇s Modified Eagle Medium） and incubated with Trypanosoma evansi for more than 12 h. MPE was added to the Vero cell culture medium at different concentrations （250-1,000 μg/mL） with trypanosomes concentration （1 × 106 trypanosomes/mL in each ELISA plate well） and incubated at appropriate conditions for 72 h. In-vitro cytotoxieity of MPE of E. officinalis was determined on Vero cells at concentrations （（1.56-100 ~tg/mL）. Acute toxicity and in-vivo infectivity tests were done in mice. Obtained MPE ofE. officinalis underwent process of purification via column chromatography, preparative chromatography and HPLC （higher performance liquid chromatography） with bioassay at different strata on Alsever＇s medium. In-vivo assay for trypanocidal activity, MPE and PPFs （partially purified fractions） of E. officinalis with two sets of mice, each mouse was inoculated with 1 × 104/mL oftrypanosomes and treated （48 h post inoculation） at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 [tL per mouse via intraperitoneal route （in treating parassitemic mice） to different groups of mice, 6 mice per concentration. HPLC of partially purified fractions ofE. officinalis was carried out with mobile phase ofacetonitdle： water （40：60） in gradient mode. In vitro, MPE induced immobilization and killing of the parasites in concentration-time dependent manner. Significant reduction of trypanosomes counts from concentration of 250μg/mL and complete killing of trypanosomes at 5th hour of observation, which was statistically equivalent to 4th hour of Diminazine Aceturate （Berenil）, standard reference drug used. HPLC of the partially purified fractions revealed two major prominent peaks at retention time of 1-4 min. In vivo, both MPE and PPFs of test material did prolong lives of mice by 6-9 days but could not cure them. At concentration of 2,000 kg/kg body weight of MPE in acute test, all mice survived. For in-vivo infectivity test, mice injected with immobilized trypanosomes developed parasitemia and died while, the other group survived. MPE, PPFs and Diminazine Aceturate were toxic to Vero cells at all concentrations exception of 1.56, 1.56-3.13 and 1.56-6.25 μg/mL, respectively. From this report, PPFs ofE. officinalis dried fruits demonstrated potential pathway for a new development oftrypanocide in near future if additional investigations are put in place.

伊氏锥虫感染的昆明小鼠血常规变化和病理组织学观察

为了解伊氏锥虫感染昆明小鼠的血常规和病理组织学变化,人工感染昆明小鼠、制作血液涂片、检测血常规、分子生物学(PCR)鉴定、病理组织切片,结果显示伊氏锥虫对小鼠具有较高的致病性,随着感染天数的增加,白细胞(WBC)、中性粒细胞计数(NEU)、红细胞压积(HCT)、平均红细胞血红蛋白量(MCH)、血小板计数(PLT)值逐渐降低,淋巴细胞百分数(LYM%)、红细胞平均体积(MCV)、红细胞分布宽度的标准差(RDWR-SD)、血小板平均容积(MPV)值在死亡时明显升高,健康小鼠血常规正常。对比试验和对照小鼠各器官大小,感染小鼠脾脏、肝脏,心脏均明显变大,肺脏及肾脏变化较小。PCR鉴定在799 bp处出现目的条带,与预期片段大小相符。病理组织学观察,试验小鼠脾脏淤血,髓质间可见大量红细胞,出现较多形态改变的淋巴滤泡;肝脏中央静脉、小叶间静脉淤血,肝细胞肿胀,肝窦间隙变窄;肺脏肺泡肿胀较明显,肺泡壁增宽,支气管出现纤维化,间质可见炎症细胞;心脏中心肌纤维稍肿胀,颗粒变性,颜色加深,并伴有较多的伊氏锥虫虫体;肾小囊间隙变窄,肾小管上皮细胞肿胀,管腔变窄;对照组小鼠均未出现病理变化。研究结果可为伊氏锥虫病的诊断与预防提供理论依据。

新疆部分养马区域马匹血源性和虻源性伊氏锥虫PCR检测

为了解新疆部分养马区域马伊氏锥虫感染情况,保障当地马伊氏锥虫病的综合防控和马产业的健康发展,论文采集了不同区域马体表吸血虻(n=157)、疑似血液样本(n=238)进行PCR检测,并将结果分析及防疫建议提供给马场。通过对虻的形态学鉴定及其特异性基因(COI)和携带病原ISG 65基因扩增、测序、同源性比对,鉴定和检测了采样区域放牧马体表吸血虻及其携带病原情况。结果显示,伊犁、阿勒泰、塔城等地马血源性伊氏锥虫PCR阳性率分别为29.6%(16/54)、12.8%(17/133)、19.6%(10/51);马虻鉴定为突额瘤虻,其虻源性伊氏锥虫PCR携带率为35%。研究结果表明马锥虫具有一定的感染率,对该病进行病原监测和综合防控十分重要。

伊犁地区马媾疫的诊断与治疗效果的观察

【目的】为研究新疆伊犁地区马媾疫的发病情况和药物治疗效果,【方法】对疑似有马媾疫的马匹,通过ELISA方法检测马匹血清中马媾疫抗体,采用PCR方法检测马匹生殖道黏膜拭子中马媾疫锥虫,伊氏锥虫PCR检测排除假阳性,对确诊马媾疫的马匹使用抗锥虫药物治疗并监测治疗效果。【结果】21匹临床表现疑似马媾疫的马匹,ELISA抗体检测结果显示阳性率为19.05%(4/21);用PCR检测出3匹马呈阳性,同时该3匹马均为抗体检测呈阳性马匹,PCR与ELISA检测阳性符合率75%;抗体阳性而PCR阴性马匹使用伊氏锥虫PCR检测呈阴性,结合流行病学分析该马匹也为马媾疫。4匹阳性马经抗锥虫药物治疗后,2匹马痊愈,1匹联合抗菌药物巩固治疗后痊愈,1匹死亡。【结论】马媾疫ELISA抗体检测联合PCR分子检测可提高马媾疫的检出率,诊断时需排除伊氏锥虫感染。早期发现马媾疫通过药物治疗后易痊愈,疾病进展至后期难以治愈成功。本研究为新疆伊犁地区马媾疫的诊断和治疗提供科学依据。

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 aceturale(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 5rats each were used.They were either infected with 1×10~a 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.S mg/kg DA and 100 mg/kg vitamin C;and group D-3.S mg/kg DA and 7.S mg/kg levamisole and 100 mg/kg vitamin C.Croup 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 between25 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.

Anti-trypanosomal effect of Peristrophe bicalyculata extract on Trypanosoma brucei brucei-infected rats

Objective:To investigate thein vitroandin vivoeffect of whole plant extracts ofPeristrophe bicalyculataonTrypanosoma brucei brucei-infected rats.Methods:The experiment wasdivided into two phases:In the first phase,the anti-trypanosomal activity of the hot water,cold water,methanol and butanol extracts of the whole plant were determined by incubatingwithTrypanosoma brucei brucei.The cold water extract was partially-purified and the anti-trypanosomal activity of the fractions determined.In the second phase,Trypanosoma brucei brucei-infected rats were treated with fraction 2c for nine days.Packed cell volume(PCV),highdensity lipoprotein(HDL),low density lipoprotein(LDL),total cholesterol(TC),triacylglycerol(TAG),aspartate aminotransferase,alanine aminotransferases(ALT),alkaline phosphatase(ALP),total and direct bilirubin levels were determined at the end of the experiment.Results:Cold water extract immobilized 90%of the parasites after 60 min of incubation,and fraction 2ccompletely immobilized the parasites after 35 min.It significantly increased PCV inTrypanosoma brucei brucei-infected rats.Decreased TC,TAG,HDL and LDL levels of infected rats increasedsignificantly when rats were treated with the fraction,while elevated levels of total bilirubinand ALT also decreased.The difference in urea,direct bilirubin and ALP was not significantwhen infected rats were compared to rats in other groups.Conclusions:The ability of the plantto ameliorate the infection-induced biochemical changes calls for detailed investigation of thepotentials of the plant for antitrypanosomiasis drug delivery.

Unlocking the in vitro anti-Trypanosoma cruzi activity of halophyte plants from the southern Portugal

Objective:To evaluate the in vitro anti-Trypanosoma cruzi（T.cruzi） activity of organic extracts prepared from halophyte species collected in the southern coast of Portugal（Algarve）,and chemically characterize the most active samples.Methods:Acetone,dichloromethane and methanol extracts were prepared from 31 halophyte species and tested in vitro against trypomastigotes and intracellular amastigotes of the Tulahuen strain of T.cruzi.The most active extract was fractionated by preparative HPLC-DAD,affording 11 fractions.The most selective fraction was fully characterized by 1H-NMR.Results:From 94 samples tested,one was active,namely the root dichloromethane extract of Juncus acutus(IC50 < 20 μg/mL).This extract was fractionated by HPLC,affording 11 fractions,one of them containing only a pure compound（juncunol）,and tested for anti-parasitic activity.Fraction 8(IC50 = 4.1 μg/mL) was the most active,and was further characterized by 1H-NMR.The major compounds were phenanthrenes,9,10-dihydrophenanthrenes and benzocoumarins.Conclusion:Our results suggest that the compounds identified in fraction 8 are likely responsible for the observed anti parasitic activity.Further research is in progress aiming to isolate and identify the specific active molecules.To the best of our knowledge,this is the first report on the in vitro anti T.cruzi activity of halophyte species.

Trypanosoma rangeli:growth in mammalian cells in vitro and action of a repositioned drug(17-AAG)and a natural extract(Artemisia sp.essential oil)

Trypanosoma rangeli and T.cruzi are both parasitic unicellular species that infect humans.Unlike T.cruzi,the causative agent of Chagas disease,T.rangeli is an infective and non-pathogenic parasite for humans,but pathogenic for vectors from the Rhodnius genus.Because both species can coexist in different hosts and overlap their infective cycles but very little is known about the infection of T.rangeli in mammalian cells,we decided to characterize both the development of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action on it.We found that T.rangeli exhibits a cycle of infection in Vero cells similar to that for T.cruzi and that the repurposed drug,17-AAG,and the natural extract Artemisia sp.essential oil produce a toxic effect on epimastigotes showing a trypanocidal action from the fifth day of culture.Both treatments also affected the infection of trypomastigotes and reduced the capacity of replication of amastigotes of T.rangeli.Since T.cruzi/T.rangeli coinfection cases have been reported,the finding of drugs with potential activity against both species could be significant in the future.Furthermore,studies of susceptibility of both species to drugs could also help to know the different mechanisms of pathogenicity in humans displayed by T.cruzi that are absent in T.rangeli.

Saponins-rich fraction of Calotropis procera leaves elicit no antitrypanosomal activityin a rat model

Objective:To examine thein vitroandin vivoanti-Trypanosoma evansi(T.evansi)activity ofsaponins-rich fraction ofCalotropis procera(cpsf)leaves as well as the effect of the fraction onthe parasite-induced anemia.Methods:A 60-minutes time course experiment was conductedwith various concentrations of the fraction using a 96-well microtiter plate technique,andsubsequently used to treat experimentallyT.evansiinfected rats at 100 and 200 mg/kg bodyweight.Index of anemia was analyzed in all animals during the experiment.Results:The cpsfdid not demonstrate anin vitroantitrypanosomal activity.Further,the cpsf treatments did notsignificantly(P>0.05)keep the parasites lower than the infected untreated groups.At the end ofthe experiment,allT.evansiinfected rats developed anemia whose severity was not significantly(P>0.05)ameliorated by the cpsf treatment.Conclusions:It was concluded that saponins derivedfromCalotropis proceraleaves could not elicitin vitroandin vivoactivities againstT.evansi.

Experimental Trypanosoma brucei infection at immediate post partum period:effects on dam and the offspring

Objective:To investigate the effects of immediate post-partum infection with Trypanosoma brucei（T.brucei） on dam and offspring.Methods:Sixty female Albino rats（Rattus norvegicus） weighing between 130-170 g were used as animal model.The animals were divided as follows: 25 infected between 1-5 days post partum;10 infected unbred as positive controls;and 25 uninfected as negative controls.The following parameters were evaluated:packed cell volume （PCV）,level of parasitaemia,survival time,litter size and litter weight at birth and on days 7, 14 and 21 post delivery,using conventional methods.Possible trans-mammary transmission of infection to litter through milk was also assessed.Results:The results showed a comparatively （P【0.05） higher mean PCV value for the uninfected negative control on the 8 day post infection compared with the infected groups which corresponded with the increasing level of parasitaemia in the two infected groups.Mean litter size and litter weights were higher（P【0.05） in the uninfected controls on the 21st day.Survival time in the infected groups were similar.No evidence of trans-mammary transfer of infection was recorded.Conclusion:T.brucei infection during immediate post partum period is detrimental to the dam and impairs growth of the offspring.

Canova medication changes TNF-α and IL-10 serum levels in mice infected with Trypanosoma cruzi Y strain

Objective:To identify whether Canova medication changes TNF-α and IL-10 serum levels in mice infected with Trypanosoma cruzi Y strain.Methods:Animals were divided into five groups:non-treated infected animals(I); benznidazole-treated infected animals(Bz; 100 mg/kg body weight,single daily dose by gavage); Canova medication(CM) treated infected animals(CM;0.2 mL/animal,single daily dose by gavage); benznidazole- and Canova medication–treated infected animals with the above-mentioned dose(Bz+CM);and non-infected animals(C).TNF-α and IL-10 levels were determined in serum aliquots after 4,7,10,13,and 29 days of infection.An ELISA technique was employed with R&D System Inc.antibody pairs.Results:A high increase in TNF-α and IL-10 levels occurred in the infected and CM-treated groups within the treatment employed on the 10 th day after infection,coupled with a IL-10 decrease on the 13 th day after infection when compared with the other experimental groups.Conclusions:CM may change the balance between plasma cytokine levels(TNF-α and IL-10) in mice infected with Y strain T.cruzi,with important consequences leading towards a more severe infection.

Detection of Trypanosoma spp. in Bandicota indica from the Thai-Myanmar border area, Mae Sot District Tak Province, Thailand

Objective:To investigate the prevalence of trypanosome infection and their phylogeny in Bandicota indica rats from the cadmium-contaminated area of Mae Sot and the Myanmar border.Methods:Blood samples were taken from 100 animals,and parasite infection was examined by light microscopy observation and polymerase chain reaction(PCR)studies.Results:Trypanosoma spp.infection was found in 20%of the thin blood smear samples.PCR showed positive 623 bp DNA bands in 21 samples(21%).The sequencing analysis showed that all of the samples(100%)had the Trypanasoma lewisi 18 S ribosomal RNA gene.Phylogenetic analysis confirmed that these 16 isolates of Trypanosoma spp.were closely related to Trypanasoma lewisi.Conclusions:Molecular detection using PCR is as effective as conventional light microscopy analysis.This study confirms that trypanosomal infection in rodents is still high;therefore,fleas as their vectors need to be controlled in order to prevent transmission to humans.

Specific primers design based on the superoxide dismutase b gene for Trypanosoma cruzi as a screening tool:Validation method using strains from Colombia classified according to their discrete typing unit

Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained from Flowers of Anthemis Tinctoria, Against Trypanosoma Cruzi

In Brazil and several other Latin American countries, Chagas' disease still constitutes a serious medical and social problem, and there is a need to develop new, more-potent drugs with fewer side effects to effectively treat this disease. We investigated the antitrypanosomal effect of a crude extract, fractions, and a semi-purified subfraction rich in a mix- ture of isomeric labdane sesquiterpenes, obtained from flowers of Anthemis tinctoria, against Trypanosoma cruzi. In epimastigote forms, the aqueous crude extract, dichloromethane fraction, and semi-purified subfraction showed a dose-dependent inhibitory activity, with IC50 of 2.3 μg/ml, 1.8 μg/ml, and 0.2 μg/ml, respectively. In the interaction in- dex, the semi-purified subfraction showed a reduction in both the percentage of infected LLCMK2 cells and the mean number of amastigotes per infected cell. The cytotoxicity evaluation demonstrated that the cytotoxic concentrations of the semi-purified subfraction were higher for LLCMK2 cells than for the protozoans, with a selectivity index of 35.0. Epimastigote forms treated with the semi-purified subfraction showed ultrastructural and morphological alterations such as rounding of the cells and bleb formation in the flagellum and cytoplasmic membrane. These results show that the flowers from A. tinctoria may be a source of new drugs with antiprotozoal activity. However, additional in vitro and in vivo studies are needed to validate the use of A. tinctoria in the treatment of Chagas’ disease.

Regulation of RNA binding proteins in trypanosomatid protozoan parasites

Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins(RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of m RNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

Antibody delivery into viable epimastigotes of <i>Trypanosoma cruzi</i>as a tool to study the parasite biology

American trypanosomiasis is a zoonosis of worldwide medical importance and currently there is no effective treatment in chronic patients, hence the importance of the study of protein function of the parasite with the objective of finding new drug targets and to know better the biology of the agent causal (Trypano-soma cruzi). T. cruzi is an RNAi-negative parasite, therefore the silencing genes strategies by RNAi is not possible;for that reason, antibodies may be taken as a tool for studying the parasite proteins function by blocking these molecules with specific antibodies. The aim of this work was to establish a methodology for antibody delivery (antibody transfection) into viable parasites. We used anti-cyclin-A antibody (human origin) in western blot assay with epimastigote of T. cruzi proteins and this recognized a ~55 kDa polypeptide. Several methods for antibody transfection (electroporation, saponin permeabilization and a lipid-based formulation) were tested. The first two methods were unsuccessful. In electroporation was impossible to visualize the antibody inside parasites and with saponin permeabilization, antibodies were successfully introduced, but with loss of parasites viability. The lipid-based formulation method forms noncovalent complexes with antibodies. These complexes are internalized by cells and antibodies are released into the cytoplasm. With this method, a successful antibody delivery was achieved. Anti-cyclin antibodies were visualized in the cytoplasm from fixed transfected parasites (immunofluorescence assays). At 24 h post-transfection, parasites maintained their viability (90%) and were able to arrest the cell cycle in G0/G1-phase of cultured epimastigotes (cell population increased in G0/G1-phase from 50.5% to 66.2% and decreased in S-phase from 47.2% to 26%). It was also observed that anti-cyclin-A antibodies inhibit the parasite population doubling (p T. cruzi, with a simple and cheap technique, which will allows carrying out further studies of this protozoan.