Prevalência de anticorpos contra Yersinia pestis em carnívoros domésticos nos focos pestosos, do Estado do Ceará

Analisou-se a prevalência de anticorpos contra Yersinia pestis em carnívoros domésticos (cães e gatos) nas áreas pestígenas do Estado do Ceará, visando estabelecer a importância do monitoramento desses animais na rotina do Programa de Controle da Peste. No decênio 1997-2006, analisaram-se 146.732 amostras de soros (95.883 cães e 50.849 gatos), das quais 2.629 (2.234 cães e 395 gatos) revelaram-se positivas. A prevalência entre os cães (85%) foi superior a dos gatos (15%) em todo o decênio e locais, exceto em Ibiapina, em 1998. O significado desses achados ainda não foi determinado. Os estudos sobre a zoonose no Brasil pautaram-se por paradigmas que não contemplavam todos os elementos envolvidos na zoonose, impossibilitando a devida elucidação do papel desses carnívoros. O monitoramento da atividade pestosa, realizado exclusivamente por inquéritos caninos, pode redundar no desconhecimento progressivo da situação epidemiológica da peste, caso não sejam desenvolvidas pesquisas interinstitucionais suplementares.

Infecção por Yersinia pestis, no Estado da Bahia: controle efetivo ou silêncio epidemiológico?

INTRODUÇÃO: A peste, doença infectocontagiosa milenar, continua sendo considerada da maior importância do ponto de vista epidemiológico devido ao alto potencial epidêmico, estando inclusive sujeita ao Regulamento Sanitário Internacional. Apesar da ausência de casos humanos da doença no Brasil, seu agente etiológico, a bactéria Yersinia pestis, permanece firmemente arraigado em seus focos naturais. A ocorrência de sorologia positiva em carnívoros domésticos de regiões pestígenas da Bahia, nos últimos anos, objetivou a realização deste estudo, que se propõe a verificar a existência de circulação do agente no estado, tendo em vista que fatores condicionantes para a doença são mantidos, oferecendo riscos à população. MÉTODOS: Trata-se de um estudo para verificação da presença de infecção por Y. pestis através do inquérito de soroprevalência em humanos, cães e roedores; e pesquisa da bactéria em roedores e pulgas. Utilizou-se de questionário estruturado para avaliação da associação existente entre fatores ambientais, sócioeconômicos e biológicos e a soroprevalência da infecção em humanos. RESULTADOS: Os 630 soros examinados (88 de humanos, 480 de cães, 62 de roedores) apresentaram-se não reagentes para peste e as análises bacteriológicas realizadas em 14 roedores e dois lotes de pulgas não identificaram a bactéria. CONCLUSÕES: Os resultados não configuram erradicação da doença no estado, pois sua natureza cíclica indica que pode passar longos períodos silente e depois ressurgir acometendo um grande número de pessoas. Portanto, a manutenção da vigilância ativa e permanente se faz necessária para a detecção precoce da doença e desenvolvimento oportuno das medidas de controle pertinentes.

Seroprevalence of hantavirus and Yersinia pestis antibodies in professionals from the Plague Control Program

Introduction Professionals who handle rodents in the field and in the laboratory are at risk of infection by the microorganisms harbored by these animals. Methods Serum samples from professionals involved in rodent and Yersinia pestis handling in field or laboratory work were analyzed to determine hantavirus and plague seroprevalence and to establish a relationship between these activities and reports of illnesses. Results Two individuals had antibodies against hantavirus, and two harbored antibodies against the plague; none of the individuals had experienced an illness related to their duties. Conclusions These results confirm the risks of hantavirus- and plague-related field and laboratory activities and the importance of protective measures for such work.

Patologia da infecção experimental de roedores domésticos com diferentes cepas de Yersinia pestis

Os autores estudaram 253 ratos domésticos (Rattus norvegicus e Rattus r. frugivorus), infectados experimentalmente (via subcutânea) com duas cepas de Yersinia pestis (cepas "PEXU-19" e "RANGEL"), isoladas de roedores silvestres capturados no nordeste brasileiro (Exú - Pernambuco) e na Venezuela, respectivamente, as quais foram mantidas em meio de cultura e subinoculadas periodicamente. A sobrevida dos animais infectados variou de dois a sete dias, sendo os sobreviventes sacrificados no 20º dia após a data da inoculação. Para cada uma das duas espécies de rato, foram empregadas cargas infectantes de intensidades diferentes (750 - 4.250; 7.500 - 41.250 e 75.000 - 750.000 bacilos), correspondentes a cada uma das duas cepas de Y. pestis utilizadas. A patologia foi estudada em relação ao fígado, baço, pulmão e linfonodo inguinal, órgãos onde as lesões se mostraram mais exuberantes. As lesões hepáticas foram essencialmente representadas por congestão, necrose coagulativa multifocal e infiltração inflamatória aguda portal e sinusoidal, com formação de microabscessos. No baço ocorreu atrofia acentuada dos folículos linfóides de Malpighi, congestão, hemorragia e esplemite aguda abscedada. No pulmão, foi achado freqüente a pneumonite aguda, ás vezes acompanhada de abscessos. De um modo geral, a cepa PEXU-19 revelou maior poder patogênico que a cepa RANGEL, embora, em relação à espécie de roedor infectado não tenha sido possível surpreender diferenças quanto á sua susceptibilidade frente ás duas cepas Y. pestis utilizadas no presente trabalho.

Histopatologia da infecção por Yersinia pestis em roedores de focos de peste do Nordeste brasileiro

O presente trabalho mostra a histopatologia da infecção pela Yersinia pestis, entre as diferentes espécies de roedores silvestres e comensais (cricetídeos, equimídeos, murídeos e cavídeos) que ocorrem na zona endêmica de peste do Nordeste do Brasil. Estes roedores foram encontrados naturalmente infectados nos campos ou inoculados experimentalmente no laboratório (vias percutânea, subcutânea ou picada de pulgas) com cepas locais e/ou estrangeiras de Yersiniapestis. Quase todos os animais, exceto alguns dos cavídeos, desenvolveram a forma bubosepticêmica da peste. Entre as lesões encontradas, a necrose coagulativa multifocal do fígado, a pneumonite intersticial aguda difusa e a atrofia linfoide do baço, podem, por sua constância, ser consideradas como os principais indicadores histológicos da infecção pestosa, embora estas lesões não sejam exclusivas da peste. A diversidade e a intensidade das lesões entre os Zygodontomys lasiurus pixuna, podem explicar a mortalidade elevada desta espécie e a disseminação da peste nos focos naturais do Nordeste brasileiro. Cricetídeos e murídeos mostraram alterações histopatológicas qualitativamente semelhantes. A resistência dos cavídeos à infecção pestosa foi evidenciada pela sobrevida desses roedores à fase aguda da infecção e pelo desenvolvimento de uma reação histiocitária interna, delimitando as áreas abscedadas. è possível que estas lesões crônicas abriguem bacilos virulentos, que permitirão a reinfecção periódica das pulgas e conseqüente reativação do processo epizoótico.

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

Survey of the irp2 gene among Yersinia pestis strains isolated during several plague outbreaks in northeast Brazil

The irp2 gene codes for a 190 kDa protein (HMWP2) synthesidez when highly pathogenic Yersinia are grown under conditions of iron starvation. In this work, the presence of irp2 in strains of Y. pestis isolated from different hosts during several plague outbreaks in the foci of Northeast Brazil wasstudied. For this purpose, 53 strains were spotted onto nylon filters and their DNA was hybridized with the A13 probe which is a 1 kb fragment of the irp2 coding sequence. All strains except two hybridized with the probe. However, when the initial stock culture of these two strains were analyzed, they both proved to bepositive with the A13 probe, indicating that the locus was lost after subculturein vitro but was always present in vivo. To examine the degree of conservation of the chromosomal fragment carrying irp2 among Brazilian strains, the hybridization profiles of 15 strains from different outbreaks, different hosts and different foci were compared. The hybridization profiles of these strains were all identical when their DNA was digested with either EcoRI, EcorRV or AvaII, indicatingthat the restriction sites surrounding the irp2 locus are very well conserved among Northeast Brazilian strains of Y. pestis. Altogether, these results suggest that the irp2 chromosomal region should be of prime importance for the bacteria during their multiplication in the host.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

Evaluation of three serological tests for the detection of human plague in northeast Brazil

The passive haemagglutination (PHA) test, enzyme-linked immunosorbent assay (ELISA) and the dot enzyme-immunosorbent assay (DOT-ELISA) were used to detect the levels of IgG antibodies against the Fraction 1 (F1) antigen of Yersinia pestis in sera of plague-infected patients from Northeast Brazil. Twenty three selected PHA-positive sera of subjects with bacteriological confirmation of plague were also positive in the DOT-ELISA but only 19 were detected by the conventional ELISA technique. Another group of 186 serum samples from subjects diagnosed as plague-infected by clinical and epidemiological parameters, but PHA-negative, were screened with DOT-ELISA and 11 gave positive results. The specificity of the assays on the serological detection of plague was confirmed in inhibition tests using purified F1 antigen. These results suggest that DOT-ELISA can be an useful, simple and more sensitive alternative for the serodiagnosis of plague in Northeast Brazil.

The use of filter paper plasticized with polyvinyl alcohol-glutaraldehyde in ELISA

F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3% w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.

Detection of Cryptococcus neoformans capsular polysaccharide antigen in asymptomatic HIV-infected patients

Serum samples from 242 HIV-positive persons were studied for the detection of capsular polysaccha-ride antigen of Cryptococcus neoformans; 193 of these patients presented less than 300 CD4+ cells/µl of blood and 49 patients had more than 300 CD4+ cells/µl. None of them had symptoms or signs characteristic of cryptococcosis. The capsular antigen of C. neofarmans was detected by latex agglutination technique with pronase pre-treatment (IMMY, Crypto-Latex Antigen Detection System, Immunomycologics Inc., OK, USA); in 61% of the samples, ELISA technique was also used (Premier, Cryptococcal Antigen, Meridian Diagnostic Inc., Cincinatti, Oh, USA). The comparative study of both methods showed that the results obtained were similar in 96.9% of the cases. The capsular antigen was detected in 13 out of 193 (6.7%) patients with less than 300 CD4+ cells/µl. Cryptococcosis was confirmed mycologically in 3 of these 13 cases (23%) by the isolation of C. neoformans in CSF or blood cultures. Three patients, who had presented negative results of both tests for capsular antigen, suffered disseminated cryptococcosis 4 to 8 months later. The predictive diagnostic value of capsular antigen detection of C. neoformans seems tobe low and we believe that it should not be done routinely in asymptomatic HIV-positive persons.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.