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.

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.

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.

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.

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.

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.

Cardiac Lesions in Naturally Infected Dogs with Trypanosoma cruzi

With the purpose to determine the frequency and type of cardiac lesions in naturally exposed dogs to Trypanosoma cruzi, ninety one stray dogs, capture by the Canine and Feline Control Center （dog pound） from the municipality of Merida, were studied. Before euthanasia, blood samples were taken to detect 72 cruzi antigens by indirect immunofluorescence antibody test and Western Blot and to detect the genome of parasite by Polymerase Chain Reaction. Immediately after euthanasia, hearts were macroscopically evaluated and a sample of the middle right atrial wall of each dog was taken for histopathological analyses. DNA was also obtained from paraffin blocks of seropositives animals with microscopic lesions to detect 72 cruzi genome. Of ninety one dogs, thirteen were seropositive. All seropositive dogs showed an association （P 〈 0.05） with lymphocytoplasmatic myocarditis. The presence of the 72 cruzi genome was also detected by PCR in cardiac septum tissue of seropositive dogs and in all the cases with microscopic lesions indicating the high pathogenicity of the local circulating strain. No association with macroscopic lesions was observed in seropositive dogs. Also, the presence of Dirofilaria immitis （D. immitis） was found in 6% of dogs evaluated. This study demonstrates a high tropism to cardiac tissue and virulence of the strains of 72 cruzi circulating in the studied dog population.

Synthesis of N1-Substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyra-zolethiocarboxamide as Novel Small Molecule Inhibitors of Cysteine Protease of T.cruzi

A series of N1-substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyrazolethiocarboxamide were prepared from the Mannich bases of aryl ketones in good yields. Some derivatives were found to be active against the cysteine protease of T.cruzi..

Behavioral parameters of six populations of/Wecavs phyllosomus longipennis(Heteroptera:Reduviidae)from areas with high and low prevalences of Trypanosoma cruzi human infection

Three behaviors of epidemiological importance,namely feeding latency,feeding duration and defecation latency,for six populations of Meccus phyllosomus longipennis(Usinger)from areas of central,western and north-central Mexico with high(HP)and low(LP)prevalence of Trypanosoma cruzi(Chagas)human infection were evaluated in this study.The median feeding latency(the time taken to begin feeding)was highly variable between instars.Within-instar comparisons showed that at least 65%of the LP populations(N3 to adult)started to feed significantly(P<0.05)later than the HP population,with N1 showing no difference,and N2 from LP populations feeding sooner than those from HP populations.The six populations had similar median feeding durations within instars.A higher(P<0.05)percentage of the instars from HP populations defecated faster than the respective instars from the three LP populations.Approximately 25%of the young nymphs(N1 to N3)and females in the HP populations defecated<2 min postfeeding,compared with 4%-6%of the young nymphs and 1.3%-3%of females in the LP populations.Moreover,17.7%-38.8%of the older nymphs(N4 to N5)in the HP populations and 6.8%-13.4%in the LP populations defecated during or immediately after feeding.Our results indicate that the HP populations have a greater potential than the LP populations to transmit T.cruzi infections,which may underlie the differences in the prevalence of T.cruzi infection in some areas where M p.longipennis is currently distributed.

Re-emerging threat of Trypanosoma cruzi vector transmission in El Salvador, update from 2018 to 2020

Background:Since the late twentieth century,Chagas disease gained global attention to suppress the vector burden as a main control strategy in endemic countries.In Central America,multi-national initiative successfully achieved significant reduction in the estimated disease prevalence as well as elimination of the region's principal vector species at the time in 2012.While the last decade has witnessed significant changes in ecosystem-such as urbanization and replacement of the main vector species-that can possibly affect the vector's habitation and residual transmission,the up-to-date vector burden in the region has not been evaluated thoroughly due to the cessation of active vector surveillance.The aim of this study was to update the risk of vector-borne Trypanosoma cruzi infection in El Salvador,the top Chagas disease-endemic country in Central America.Methods:A nationwide vector survey was conducted in the domestic environment of El Salvador from September 2018 to November 2020.The selection of the houses for inspection was based on expert purposeful sampling.Infection for T.cruzi was examined by microscopic observation of the insects'feces,followed by a species confrmation using PCR.The data were analyzed using R software version 4.1.3.Proportion estimates with 95%confidence intervals were inferred using the Jeffrey's method provided under the epiR package.Results:A total of 1529 Triatoma dimidiata was captured from 107 houses(infestation rate,34.4%;107/311)in all the fourteen departments of the country visited within the period;prevalence of T.cruzi infection was as high as 10%(153/1529).In the country,domestic T.dimidiata infestation was distributed ubiquitously,while T.cruzi infection rates varied across the departments.Five out of fourteen departments showed higher infection rates than the average,suggesting sporadic high-risk areas in the country.Conclusions:Our comprehensive study revealed substantial T.cruzi infection of T.dimidiata across the country,indicating potential active transmission of the disease.Therefore,strengthened surveillance for both vector and human infection is required to truly eliminate the risk of T.cruzi transmission in Central America.

Seropositivity for Trypanosoma cruzi in domestic dogs from Sonora,Mexico

Background:Chagas disease is an important health problem in Latin America due to its incapacitating effects and associated mortality.Studies on seropositivity for Trypanosoma cruzi in Mexican dogs have demonstrated a direct correlation between seropositivity in humans and dogs,which can act as sentinels for the disease in this region.The objective of this study was to determine the seropositivity for T.cruzi infection in dogs from Sonora,a northern borderstate of Mexico.Methods:Responsible pet owners were selected at random from an urban area of Empalme municipality,Sonora,Mexico,and from there,180 dog samples were collected.Anti-T.cruzi antibodies were determined using the enzymelinked immunosorbent assay(ELISA)method.Reactive ELISA sera were processed by indirect immunofluorescence to confirm the presence of anti-T.cruzi antibodies.For the statistical analysis,chi-square tests were conducted.Results:Dogs’sera showed a seropositivity rate of 4.44%.The rate of seropositivity was not associated with the dogs’age,sex,or socioeconomics pertaining to the geographical area.One sample(1/180,0.55%)showed the acute state of the disease.Conclusions:The study found a presence of anti-T.cruzi antibodies in dogs in this area,which suggests vector transmission.There is a need for active surveillance programs throughout the state of Sonora and vector control strategies should also be implemented in endemic regions.

ANTI-TRYPANOSOMA CRUZI ACTIVITY OF 20 MEDICINAL PLANTS USED IN NORTH EAST MEXICO

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi.This disease is also known as American trypanosomiasis and approximately 7-8 million people are currently infected(WHO,2014.Nifurtimox has been used for over 40 years to treat Chagas disease,however,this drug is only effective during the acute phase of infection,and certain protozoan strains have developed resistance

Challenges in the chemotherapy of Chagas disease: Looking for possibilities related to the differences and similarities between the parasite and host

Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countries. This short review highlights the enormous need for new studies aimed at the development of novel and more specific drugs to treat chagasic patients. The primary tool for facing this challenge is deep knowledge about the similarities and differences between the parasite and its human host.

美洲锥虫病——美洲新型艾滋病

美洲锥虫病,又称查加斯病,是由克氏锥虫引起的潜在致命的寄生虫性人兽共患病。除人外,在美洲200多种哺乳动物感染了克氏锥虫。人和动物感染的主要传播媒介为吸血昆虫——锥蝽,偶尔通过非吸血昆虫媒介机制传播,如输血、污染食品、母婴感染。该病的临床症状因感染阶段不同而异,早期急性期无症状或症状轻微,隐匿期亦无症状,慢性期的典型症状为渐进性慢性心脏和消化系统损害,持续终生,部分病例死亡。因克氏锥虫与人免疫缺陷病毒(HIV)的早期传播模式、漫长的潜伏期和难以治愈等方面极其相似,有专家将查加斯病称为"美洲的新艾滋病"。该病曾局限于美洲地区,但现已随人口迁移传播到欧洲等大陆,成为全球性疫病。在急性期,使用苄硝唑和硝呋替莫抗寄生虫药物治疗很有效;在慢性期,也可延迟或防止症状恶化,但仍有部分慢性病例最终发展成致命的心脏和消化道衰竭。查加斯病可通过病原鉴定、血清学和分子学技术进行诊断。目前尚无查加斯病疫苗,媒介昆虫控制是最有效的预防手段。论文详细介绍了美洲锥虫病的病原学、生活史、流行病学、临床症状、病理变化、诊断、治疗以及防控措施,以供参考。

水牛枯氏住肉孢子虫在水牛与黄牛体内的发育史

11头5～8月龄公水牛犊和2头7日龄黑白花奶牛犊,分别人工感染80～2000万个水牛源枯氏住肉孢子虫孢子囊,2头同龄公水牛犊不感染作对照。感染后12、22和32天在大、中、小血管和毛细血管内出现第一代裂殖体,32.86×16.43(19.08～46.64×12.72～21.2)μm,含100～450个裂殖子;32～46天在毛细血管和小血管内发现大量的第二代裂殖体,18.21×9.87(10.60～29.68×6.36～13.78)μm,含30～110个裂殖子;28～40天,第二代裂殖子出现于血液中或单核细胞内;46天初级幼包囊形成,78天部分包囊成熟,98天(黄牛)包囊成熟。

N,N-双取代脲类克鲁斯氏锥体虫半胱氨酸蛋白酶小分子抑制剂的合成及其活性研究

目的 寻找克鲁斯氏锥体虫体半胱氨酸蛋白酶Cruzain的小分子抑制剂。方法 根据对Cruzain分子结构的计算机模拟设计结果 ,选用N ,N 双取代脲为先导结构 ,目标化合物的合成采用Curtius重排反应 ,测定了化合物的体外抑制Cruzain的IC50 值。结果 设计并合成了 2 1个未见文献报道的双取代脲衍生物 ,确证了它们的化学结构。结论 生物活性测定结果显示 ,所合成的化合物均有不同程度的抑制Cruzain的活性 ,其中化合物IV9和IV17的活性好于对照药tf 175。IV8的活性与对照药tf

In vitro-in vivo studies on anti-trypanosomal potentials of Zapoteca portoricensis

Objective:Aqueous extracts of Zapoteca portoricensis are used traditionally as antidiarrhea agent and in the treatment of diverse gastrointestinal disorders here in Nigeria specifically,the southern part.Similarly,the aqueous extract of the plant is also used traditionally as anticonvulsant,antispasmodic and in the treatment of tonsillitis.Recently too,the anti-inflammatory and antimicrobial activities of the methanol extracts of the root of Zapoteca portoricensis was reported.In this research,we are set to investigate the trypanocidal activity of Zapoteca portoricensis.Methods:The methanol extract of the root of Zapoteca portoricensis was investigated for both in vitro and in vivo trypanocidal activity following established models.In summary,phytochemical analysis was carried out on both the crude powdered root and on the methanol extract following standard procedures. The oral acute toxicity test(LD50 ) of the crude methanol extract was determined according to the method described by Lorke（1983）.Albino mice（17g-21g） of either sex were used.The methanol extract was suspended in 3%v/v tween 85 and administered orally at doses of 10 mg/kg,100 mg/kg and 1 000 mg/kg to three groups of mice（n = 3 ）.The animals were observed for 24 hours.Based on the result obtained in this initial test,doses of 4 mg/kg,6 mg/kg,and 8 mg/kg were administered to three different mice.The LD50 was calculated as the geometric mean of the lowest dose killing a mouse and the highest dose showing no death.The invivo /in-vitro antitrypanosomal evaluations were carried out in experimental animals and tissue cell culture respectively. Results:The result of the in vitro studies shows the inhibitive concentration-50（IC-50） against Trypanosoma brucei rhodesiense（T.b.rhodesiense） to be 0.372 mg/kg,while the control drug melarsoprol was 0.006 mg/kg.On Trypanosoma brucei brucei（T.cruzi）,the IC-50 is 6.42 mg/kg against 0.87 of the reference drug Benznidazole.The cytotoxicity on L-6 cells exhibited an IC-50 of 0.039 6 mg/kg against the reference drug,podophyllotoxin of 0.01 mg/kg.However,the in vivo study shows that the extract,at the administered doses,could not exhibit appreciable reduction of parasitemia and hence resulted to the death of test animals. Conclusion:The present data suggests that Zapoteca portoricensis could yield useful leads for the development of potentially potent antitrypanocides.

RIL—2对急性克氏锥虫感染小鼠的抗SRBC直接溶血空斑反应及抗虫保护性免疫影响的初步研究

为研究ＩＬ—２在克氏锥虫感染过程中出现的免疫抑制机制中的作用，８只克氏锥虫感染的Ｃ３Ｈ／ＨｅＪｎ小鼠每日皮下注射两次，每日总量５０００μ重组人ｒＩＬ－２；对照组相同数量小鼠皮下注射等量的无钙、镁离子的ＰＢＳ。实验结果表明ｒＩＬ－２注射组小鼠脾细胞对羊红细胞的反应能力由ＰＢＳ注射鼠的１５。２％提高到９３。５％（Ｐ＜０．０５）。相当接近于正常的未感染小鼠的空斑形成细胞数。ｒＩＬ－２注射组小鼠仅表现轻度降低的寄生虫血症，其动态变化在两组间未显示出差异。除ＰＢＳ组的１只小鼠在感染后第３６ｄ死亡外，ｒＩＬ－２以及ＰＢＳ注射组的其它动物都在克氏锥虫急性感染期存活。尽管ｒＩＬ－２注射并未明显地提高克氏锥虫急性感染期Ｃ３Ｈ／ＨｅＪｎ小鼠的抗虫保护性免疫，本实验提示ＩＬ－２不足是克氏锥虫急性感染期Ｃ３Ｈ／ＨｅＪｎ小鼠非特异性免疫抑制的原因之一。

Modulation of immune response in experimental Chagas disease

Trypanosoma cruzi(T. cruzi), the etiological agent of Chagas disease, affects nearly 18 million people in Latin America and 90 million are at risk of infection. The parasite presents two stages of medical importance in the host, the amastigote, intracellular replicating form, and the extracellular trypomastigote, the infective form. Thus infection by T. cruzi induces a complex immune response that involves effectors and regulatory mechanisms. That is why control of the infection requires a strong humoral and cellular immune response; hence, the outcome of host-parasite interaction in the early stages of infection is extremely important. A critical event during this period of the infection is innate immune response, in which the macrophage's role is vital. Thus, after being phagocytized, the parasite is able to develop intracellularly; however, during later periods, these cells induce its elimination by means of toxic metabolites. In turn, as the infection progresses, adaptive immune response mechanisms are triggered through the TH1 and TH2 responses. Finally, T. cruzi, like other protozoa such as Leishmania and Toxoplasma, have numerous evasive mechanisms to the immune response that make it possible to spread around the host. In our Laboratory we have developed a vaccination model in mice with Trypanosoma rangeli, nonpathogenic to humans, which modulates the immune response to infection by T. cruzi, thus protecting them. Vaccinated animals showed an important innate response(modulation of NO and other metabolites, cytokines, activation of macrophages), a strong adaptive cellular response and significant increase in specific antibodies. The modulation caused early elimination of the parasites, low parasitaemia, the absence of histological lesions and high survival rates. Even though progress has been made in the knowledge of some of these mechanisms, new studies must be conducted which could target further prophylactic and therapeutic trials against T. cruzi infection.

Interferon-γ and other inflammatory mediators in cardiomyocyte signaling during Chagas disease cardiomyopathy

Chagas disease cardiomyopathy(CCC), the main consequence of Trypanosoma cruzi(T.cruzi) infection, is an inflammatory cardiomyopathy that develops in up to 30% of infected individuals. The heart inflammation in CCC patients is characterized by a Th1 T cell-rich myocarditis with increased production of interferon(IFN)-γ, produced by the CCC myocardial infiltrate and detected at high levels in the periphery. IFN-γ has a central role in the cardiomyocyte signaling during both acute and chronic phases of T.cruzi infection. In this review, we have chosen to focus in its pleiotropic mode of action during CCC, which may ultimately be the strongest driver towards pathological remodeling and heart failure. We describe here the antiparasitic protective and pathogenic dual role of IFN-γ in Chagas disease.