Acute phase proteins: a potential approach for diagnosing chronic infection by Trypanosoma vivax

O objetivo do presente estudo foi verificar possíveis alterações nas proteínas de fase aguda em ovinos infectados experimentalmente com Trypanosoma vivax. Para tanto, foram utilizados oito ovinos machos, sendo quatro usados como controle e quatro infectados com 10(5) tripomastigotas de T. vivax. Colheram-se amostras de sangue em dois tempos antes da infecção e, posteriormente, aos 5, 7, 9, 11, 13, 15, 20, 30, 45, 60, 75, 90, 105 e 120 dias após a infecção (dpi); após centrifugação e aliquotização das amostras. As proteínas de fase aguda foram separadas por eletroforese em gel de acrilamida, contendo dodecil sulfato de sódio, e suas concentrações foram determinadas através de densitometria computadorizada. A dosagem de proteína total foi realizada pelo método colorimétrico do biureto. A contagem dos tripanossomas foi realizada diariamente, utilizando-se uma alíquota de 5 µL de sangue disperso em lâmina de microscopia, sob lamínula de 22 × 22 mm, contando-se os parasitos em 100 campos microscópicos, com objetiva de 40×, multiplicados pelo fator de correção do microscópio, e o resultado expresso em parasitos por mL de sangue. Para a análise estatística, empregou-se o teste de Wilcoxon a 5% de probabilidade. Foi observada a diminuição de diversas proteínas de fase aguda e aumento de antitripsina e transferrina que podem ser utilizadas para auxiliar no diagnóstico da infecção por T. vivax, principalmente na fase crônica da infecção.

Avaliação clínico-laboratorial de bovinos Nelore infectados experimentalmente com Trypanosoma vivax

Avaliaram-se as alterações clínico-laboratoriais de seis bezerros Nelore, de ambos os sexos, inoculados experimentalmente com 10(7) organismos viáveis de Trypanosoma vivax, isolados de bovinos da região de Poconé, Estado de Mato Grosso. Os animais foram observados diariamente, durante 30 dias, quanto aos parâmetros de temperatura retal, volume globular (VG), parasitemia, produção de anticorpos, coloração de mucosas, comportamento e apetite. Determinaram-se os níveis séricos de aspartato aminotransferase (AST), fosfatase alcalina (FA), gama glutamiltransferase (GGT), creatina kinase (CK), colesterol, uréia, creatinina, cálcio, fósforo e o perfil eletroforético das proteínas séricas aos 4, 8, 12, 16, 23 e 30 dias pós-inoculação (DPI). Durante os 6 meses seguintes, os animais foram observados semanalmente, avaliando-se a temperatura retal, o VG e a parasitemia. T. vivax foi evidenciado a partir do terceiro e quarto DPI em todos os bezerros e persistiu até o 30° DPI em cinco dos seis animais em estudo. Ocorreu um decréscimo significativo (p<0,05) do valor médio do VG (25%) aos dez DPI. Os animais não apresentaram qualquer alteração no quadro clínico, bem como na avaliação da bioquímica sérica durante o período experimental. A soroconversão ocorreu aos 6 e 8 DPI, permanecendo todos os animais soropositivos nos 30 dias experimentais. Bovinos nelores jovens, infectados experimentalmente com T. vivax, foram capazes de estabelecer um equilíbrio na relação hospedeiro-parasita.

First report of Trypanosoma vivax outbreak in dairy cattle in São Paulo state, Brazil

Esta é a primeira descrição de um surto de Trypanosomavivax ocorrido no Estado de São Paulo, no município de Lins. Animais acometidos apresentaram febre, icterícia, diminuição da produção de leite, perda de peso, diarreia profusa, abortos, anemia, leucocitose e hiperfibrigenemia. Foram registrados 31 óbitos de vacas e bezerros em 1.080 bovinos no total. Três vacas apresentaram sintomatologia nervosa, como dismetria, ataxia e fraqueza muscular, além de ptialismo, aumento de linfonodos e edema submandibular. Hemoparasitas flagelados foram observados em esfregaços sanguíneos, e a espécie de tripanossomo foi diagnosticada como T.vivax por PCR. A cepa de T. vivax mostrou ser resistente ao tratamento com aceturato de diaminozeno e a infecção disseminou rapidamente no rebanho. Pelo ELISA, observou-se que 98,36% (599) das amostras de soro colhidas apresentaram títulos positivos para IgG anti-T.vivax. O surto ocorreu em condições de baixa precipitação pluviométrica, fato que indica que outros fatores estavam envolvidos na ocorrência desse surto, como a ausência de tabanídeos e a grande presença de Haematobia irritans e Stomoxys calcitrans, cujo aumento populacional pode ser devido ao uso de resíduos de usinas de açúcar e álcool nos canaviais que circundavam a granja leiteira.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

Background: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion.Methods: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like.Results: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V-repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP.Conclusions: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

The genetic diversity of Plasmodium vivax: a review

The genetic diversity of Plasmodium vivax has been investigated in several malaria-endemic areas, including the Brazilian Amazon region, where this is currently the most prevalent species causing malaria in humans. This review summarizes current views on the use of molecular markers to examine P. vivax populations, with a focus on studies performed in Brazilian research laboratories. We emphasize the importance of phylogenetic studies on this parasite and discuss the perspectives created by our increasing understanding of genetic diversity and population structure of this parasite for the development of new control strategies, including vaccines, and more effective drugs for the treatment of P. vivax malaria.

Evaluation of the Naturally Acquired Antibody Immune Response to the Pv200L N-terminal Fragment of Plasmodium vivax Merozoite Surface Protein-1 in Four Areas of the Amazon Region of Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Duffy blood group gene polymorphisms among malaria vivax patients in four areas of the Brazilian Amazon region

Background: Duffy blood group polymorphisms are important in areas where Plasmodium vivax predominates, because this molecule acts as a receptor for this protozoan. In the present study, Duffy blood group genotyping in P. vivax malaria patients from four different Brazilian endemic areas is reported, exploring significant associations between blood group variants and susceptibility or resistance to malaria.Methods: the P. vivax identification was determined by non-genotypic and genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP in 330 blood donors and 312 malaria patients from four Brazilian Amazon areas. In order to assess the variables significance and to obtain independence among the proportions, the Fisher's exact test was used.Results: the data show a high frequency of the FYA/FYB genotype, followed by FYB/FYB, FYA/FYA, FYA/FYB-33 and FYB/FYB-33. Low frequencies were detected for the FYA/FY(X), FYB/FY(X), FYX/FY(X) and FYB-33/FYB-33 genotypes. Negative Duffy genotype (FYB-33/FYB-33) was found in both groups: individuals infected and non-infected (blood donors). No individual carried the FY(X)/FYB-33 genotype. Some of the Duffy genotypes frequencies showed significant differences between donors and malaria patients.Conclusion: the obtained data suggest that individuals with the FYA/FYB genotype have higher susceptibility to malaria. The presence of the FYB-33 allele may be a selective advantage in the population, reducing the rate of infection by P. vivax in this region. Additional efforts may contribute to better elucidate the physiopathologic differences in this parasite/host relationship in regions endemic for P. vivax malaria, in particular the Brazilian Amazon region.

Plasmodium vivax infection among Duffy antigen-negative individuals from the Brazilian Amazon region: an exception?

We present evidence for Plasmodium vivax infection among Duffy blood group-negative inhabitants of Brazil. The P. vivax identification was determined by both genotypic and non-genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP and phenotyped using a microtyping kit. We detected two homozygous FY*B-33 carriers infected by P vivax, whose circumsporozoite protein genotypes were VK210 and/or P. vivax-like. Additional efforts are necessary in order to clarify the evidence that P. vivax is being transmitted among Duffy blood group-negative patients from the Brazilian Amazon region. (C) 2007 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine and Hygiene.

A new polymerase chain reaction/restriction fragment length polymorphism protocol for Plasmodium vivax circumsporozoite protein genotype (VK210, VK247, and P-vivax-like) determination

For the molecular diagnosis of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) using DNA amplification procedures in the laboratory, the choice of rapid and inexpensive identification products of the 3 different genotypes is an important prerequisite. We report here the standardization of a new polymerase chain reaction/restriction fragment length polymorphism technique to identify the 3 described P. vivax circumsporozoite protein (CSP) variants using amplification of the central immunodominant region of the CSP gene of this protozoan. The simplicity, specificity, and sensitivity of the system described here is important to determine the prevalence and the distribution of infection with these P. vivax genotypes in endemic and nonendemic malaria areas, enabling a better understanding of their phylogeny. (c) 2007 Published by Elsevier B.V.

Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed. © 2006 Cambridge University Press.

No evidence for association of the CD40, CD40L and BLYS polymorphisms, B-cell co-stimulatory molecules, with Brazilian endemic Plasmodium vivax malaria

Background: Plasmodium vivax is the most prevalent malaria species in Brazil. The parasite-host coevolutionary process can be viewed as an 'arms race', in which adaptive genetic changes in one are eventually matched by alterations in the other. Methods: Following the candidate gene approach we analyzed the CD40, CD40L and BLYS genes that participate in B-cell co-stimulation, for associations with P. vivax malaria. The study sample included 97 patients and 103 controls. We extracted DNA using the extraction and purification commercial kit and identified the following SNPs: 21C.T in the CD40 gene, 2726T.C in the CD40L gene and the 2871C.T in the BLyS gene using PCR-RFLP. We analyzed the genotype and allele frequencies by direct counting. We also compared the observed with the expected genotype frequencies using the Hardy-Weinberg equilibrium. Results: The allele and genotype frequencies for these SNPs did not differ statistically between patient and control groups. Gene-gene interactions were not observed between the CD40 and BLYS and between the CD40L and BLYS genes. Overall, the genes were in Hardy-Weinberg equilibrium. Significant differences were not observed among the frequencies of antibody responses against P. vivax sporozoite and erythrocytic antigens and the CD40 and BLYS genotypes. Conclusions: The results of this study show that, although the investigated CD40, CD40L and BLYS alleles differ functionally, this variation does not alter the functionality of the molecules in a way that would interfere in susceptibility to the disease. The variants of these genes may influence the clinical course rather than simply increase or decrease susceptibility. © Royal Society of Tropical Medicine and Hygiene 2013. All rights reserved.

Resposta imune-humoral e proteinogramas séricos de bovinos naturalmente infectados pelo Trypanosoma vivax

Pós-graduação em Medicina Veterinária - FCAV

Um novo protocolo de PCR/RFLP para a determinação dos genótipos da CSP de Plasmodium vivax (VK210, VK247 e P. vivax-like)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)