Caracterização molecular de Sarcocystis spp. em amostras de carne / Molecular characterization of Sarcocystis spp. in samples of meat

A sarcocistose é uma doença distribuída mundialmente, podendo acometer aves, répteis e diversos mamíferos, incluindo o homem. O objetivo desse trabalho foi detectar a presença de Sarcocystis spp. e caracterizar as espécies encontradas em 375 amostras de produtos cárneos (filé mignon bovino, carne moída bovina e salame colonial). Para isso, foi realizada a detecção do parasita através da técnica de PCR para amplificação parcial do gene 18S rRNA e sua caracterização molecular utilizando o polimorfismo no comprimento do fragmento de restrição (RFLP) com as enzimas de restrição Bcl I, Rsa I e Alu I. A ocorrência de Sarcocystis spp. foi de 17% (64/375) do total de amostras testadas pelo PCR. Entre os produtos cárneos avaliados, 5,6% (7/125) das amostras de filé mignon, 12,8% (16/125) de carne moída e 32,8% (41/125) de embutido colonial, foram positivas para presença do DNA do Sarcocystis spp. Entre estas amostras positivas, as espécies caracterizadas foram Sarcocystis hirsuta e Sarcocystis hominis com prevalências de 93,7% (60/64) e 6,3% (4/64), respectivamente. Considerando à relevância da sarcocistose na área da saúde pública, a ocorrência de S. hominis encontrado neste estudo, pode ser um fator de risco para a contaminação humana. Porém, a presença do DNA deste protozoário não significa necessariamente potencial de infecção aos humanos, pois cuidados nos processos de fabricação podem reduzir a viabilidade dos cistos.(AU)

The sarcocystosis is a worldwide spread disease and can affect birds, reptiles and many mammals, including man. The aim of this study was to detect the presence of Sarcocystis spp. and characterize the species found in 375 samples of meat products (filet mignon, ground beef and colonial salami). For this, we carried out the detection of the parasite by PCR for the amplification of the partial 18S rRNA gene and molecular characterization using the restriction fragment length polymorphism (RFLP) with restriction enzymes Bcl I, Alu I and Rsa I. The occurrence of Sarcocystis spp. was 17% (64/375) of all samples. Among the meat products evaluated, the filet mignon samples were positive in 5.6% (7/125), the ground beef in 12.8% (16/125) and the colonial salami in 32.8% (41/125). Of the positive samples, Sarcocystis hirsuta and Sarcocystis hominis were detected, with prevalence of 93.7% (60/64) and 6.3% (4/64), respectively. Considering the relevance of sarcocystosis in public health, the occurrence of S. hominis found may be a risk factor to human contamination. However, the presence of DNA of this parasite does not necessarily mean potential of infection to humans, because good practices in the manufacturing processes can reduce the viability of the cysts.(AU)

Detecção de proteínas imunorreativas de Rickettsia sp. cepa Mata Atlântica / Detection of immunoreactive proteins of Rickettsia sp. Atlantic Forest strain

A Febre Maculosa Brasileira (FMB) é uma doença infecciosa, transmitida por carrapatos ao homem. Uma nova riquetsiose humana foi descrita como causadora de Febre Maculosa no Estado de São Paulo, sendo denominada de Rickettsia sp. cepa Mata Atlântica. O presente trabalho teve como objetivo detectar e identificar proteínas com potencial de estimular o sistema imune de hospedeiro mamífero, desta nova cepa descrita. Para tanto, foi realizado a extração proteica total de Rickettsia sp. cepa Mata Atlântica. As proteínas extraídas foram fracionadas por eletroforese. As bandas proteicas foram transferidas para membranas de nitrocelulose por migração elétrica e submetidas à técnica de Western-blot, para detecção proteica. Ao todo sete proteínas imunorreativas foram detectadas. Duas proteínas apresentaram maior abundancia, com peso molecular, de 200 e 130 kDa respectivamente. Através da comparação de mapas proteômicos existentes e pelo peso molecular que estas proteínas apresentaram, sugere-se que as duas proteínas detectadas representem rOmpA (200 kDa) e rOmpB (130 kDa). As demais proteínas detectadas apresentaram menor ocorrência e peso molecular inferior a 78 kDa, podendo representar membros da família de antígenos de superfície celular (Sca - Surface cell antigen). As proteínas detectadas poderão servir como base de estudo na elaboração de métodos diagnósticos sensíveis e específicos, no desenvolvimento de vacinas, além de possibilitarem novos estudos para terapias mais eficazes.(AU)

Brazilian Spotted Fever (BSF) is an infectious disease transmitted by ticks to humans. A new human rickettsial infection was reported to cause spotted fever in the State of São Paulo and was named Rickettsia sp. Strain Atlantic Forest. This study aimed to detect and identify proteins with potential to stimulate the immune system of mammalian host of this new strain described. Therefore, we performed total protein extraction Rickettsia sp. Strain Atlantic Forest. The extracted proteins were fractionated by electrophoresis. The protein bands were transferred to nitrocellulose membrane by electrical migration and subjected to Western blot for protein detection. In all, seven immunoreactive proteins were detected. Two proteins showed higher abundance, with molecular weight of 200 and 130 kDa respectively. By comparing existing proteomic maps and the molecular weight of these proteins showed that, it is suggested that the two proteins detected representing rOmpA (200 kDa) and rOmpB (130 kDa). The other detected proteins had lower occurrence and molecular weight less than 78 kDa, which may represent members of the cell surface antigens Family (Sca - Surface cell antigen). The detected proteins may serve as a study based on the development of sensitive and specific diagnostic methods in the development of vaccines and they enable further studies to more effective therapies.(AU)

Ecology of a tick-borne spotted fever in southern Brazil.

Rio Grande do Sul is the southernmost state of Brazil, bordering Uruguay. Clinical cases of spotted fever group (SFG) rickettsiosis were recently reported in Rio Grande do Sul. None of these cases was lethal, and all were confirmed by seroconversion to R. rickettsii antigens. Because serological cross-reactions are well known to occur between different SFG agents, the SFG agent responsible for the clinical cases remains unknown in Rio Grande do Sul, where no rickettsial agent is known to infect ticks. During 2013-2014, ticks and blood sera samples were collected from domestic dogs and wild small mammals, and from the vegetation in a SFG-endemic area of Rio Grande do Sul. Dogs were infested by Amblyomma ovale adult ticks, whereas small mammals were infested by immature stages of A. ovale, Ixodes loricatus, and adults of I. loricatus. Ticks collected on vegetation were adults of A. ovale, and immature stages of A. ovale, Amblyomma dubitatum, and Amblyomma longirostre. Three Rickettsia species were detected: Rickettsia bellii in I. loricatus, Rickettsia amblyommii in A. longirostre, and a Rickettsia parkeri-like agent (Rickettsia sp. strain Atlantic rainforest) in A. ovale. Seroreactivity to SFG antigens were detected in 19.7 % (27/137) canine and 37.5 % (15/40) small mammal sera, with highest titers to R. parkeri. Results indicate that the R. parkeri-like agent, strain Atlantic rainforest, is circulating between A. ovale ticks, dogs and small mammals in the study area, suggesting that this SFG pathogen could be one of the etiological agents of SFG clinical cases in Rio Grande do Sul.

Amblyomma yucumense n. sp. (Acari: Ixodidae), a Parasite of Wild Mammals in Southern Brazil.

During 2013-2014, adult ticks were collected on the vegetation and subadult ticks were collected from small mammals [Didelphis aurita Wied-Neuwied, Sooretamys angouya (Fischer), Euryoryzomys russatus (Wagner), Akodon montensis Thomas, Oxymycterus judex Thomas] in an Atlantic rainforest reserve in southern Brazil. Analyses of the external morphology of the adult ticks revealed that they represent a new species, Amblyomma yucumense n. sp. Partial 16S rRNA sequences generated from males, females, and nymphs were identical to each other and closest (95% identity) to corresponding sequences of Amblyomma dubitatum Neumann. A. yucumense is morphologically and genetically closest related to A. dubitatum. Dorsally, male of these species can be separated by major longitudinal pale orange stripes associated with a pseudoscutum indicated by a pale stripe in A. yucumense, in contrast to pale creamy longitudinal stripes and absence of pseudoscutum in A. dubitatum. Ventrally, male coxal I spurs are separated by a space narrower than external spur width in A. yucumense, and wider than external spur width in A. dubitatum. Females of the two species can be separated by coxal I spurs, longer in A. yucumense than in A. dubitatum. In addition, the adult capitulum and ventral idiosoma of A. yucumense are generally dark brown colored, while A. dubitatum is yellowish or light brown colored. The nymph of A. yucumense differs from A. dubitatum by the scutal cervical groove length, slightly shorter in the former species. Currently, A. yucumense is restricted to southern Brazil.

HR-MAS NMR metabolomics of 'Swingle' citrumelo rootstock genetically modified to overproduce proline.

The accumulation of proline is a typical physiological response to abiotic stresses in higher plants. 'Swingle' citrumelo, an important rootstock for citrus production, has been modified with a mutated Δ(1)-pyrroline-5-carboxylate synthetase gene (VaP5CSF129A) linked to the cauliflower mosaic virus 35S promoter to induce the overproduction of free proline. This paper presents a comparative metabolomic study of nontransgenic versus transgenic 'Swingle' citrumelo plants with high endogenous proline. (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and multivariate analysis showed significant differences in some metabolites between the nontransgenic and transgenic leaves and roots. The overproduction of proline has reduced the sucrose content in transgenic leaves, revealing a metabolic cost for these plants. In roots, the high level of free proline acts for the adjustment of cation-anion balance, causing the reduction of acetic acid content. The same sucrose level in roots indicates that they can be considered as sucrose sink. Similar behavior may be waited for fruits produced on transgenic rootstock.