Ongoing quiescence in the Borborema Plateau Plague focus (Paraiba, Brazil).

Plague is a zoonosis caused by Yersinia pestis, whose cycle is based on a reservoir system composed of mammals and their fleas. Its transmission cycle presents long enzootic periods with undetected cases, sometimes misleading that the cycle is extinct. While surveillance activities in Brazil are being carried out only in some focal areas, the serologic results confirm the persistence of Y. pestis in all monitored areas. We studied the small mammal assembly and Y. pestis presencein the Borborema Plateau Focus within the state of Paraíba, which staged the last Brazilian plague outbreak (1986-1987), through aninventory and Y. pestis detection survey of small mammals in peridomestic and sylvatic areas from two municipalities in the state of Paraíba.The field sampling captured 45 specimens (27 marsupials, 18 rodents), of 10 species. Only two species (one marsupial, one rodent) were captured in both peridomestic and sylvatic ecotopes. The sylvatic ecotope had higher richness and abundance. No evidence of circulation of the pathogen was detected, however, this result does not discard the necessity of continuous epidemiological surveillance due to the risk of rekindling the foci after long dormant periods, especially given the current epidemiological transition occurring on a Global scale.

Plague reservoir species throughout the world.

Plague has been known since ancient times as a re-emerging infectious disease, causing considerable socioeconomic burden in regional hotspots. To better understand the epidemiological cycle of the causative agent of the plague, its potential occurrence, and possible future dispersion, one must carefully consider the taxonomy, distribution, and ecological requirements of reservoir-species in relation either to natural or human-driven changes (e.g. climate change or urbanization). In recent years, the depth of knowledge on species taxonomy and species composition in different landscapes has undergone a dramatic expansion, driven by modern taxonomic methods such as synthetic surveys that take into consideration morphology, genetics, and the ecological setting of captured animals to establish their species identities. Here, we consider the recent taxonomic changes of the rodent species in known plague reservoirs and detail their distribution across the world, with a particular focus on those rodents considered to be keystone host species. A complete checklist of all known plague-infectable vertebrates living in plague foci is provided as a Supporting Information table.

A new recombinant F1 antigen as a cost and time-effective tool for plague diagnosis.

The Yersinia pestis capsular antigen F1 is widely used in plague laboratory diagnosis. Here, we describe the production of an F1 recombinant protein within reduced time and biosafety requirements. Its evaluation in hemagglutination tests indicated that the recombinant F1 can replace the conventional F1 protein for plague diagnosis.

A single introduction of Yersinia pestis to Brazil during the 3rd plague pandemic.

Yersinia pestis was introduced to Brazil during the third plague pandemic and currently exists in several recognized foci. There is currently limited available phylogeographic data regarding Y. pestis in Brazil. We generated whole genome sequences for 411 Y. pestis strains from six Brazilian foci to investigate the phylogeography of Y. pestis in Brazil; these strains were isolated from 1966 to 1997. All 411 strains were assigned to a single monophyletic clade within the 1.ORI population, indicating a single Y. pestis introduction was responsible for the successful establishment of endemic foci in Brazil. There was a moderate level of genomic diversity but little population structure among the 411 Brazilian Y. pestis strains, consistent with a radial expansion wherein Y. pestis spread rapidly from the coast to the interior of Brazil and became ecologically established. Overall, there were no strong spatial or temporal patterns among the Brazilian strains. However, strains from the same focus tended to be more closely related and strains isolated from foci closer to the coast tended to fall in more basal positions in the whole genome phylogeny than strains from more interior foci. Overall, the patterns observed in Brazil are similar to other locations affected during the 3rd plague pandemic such as in North America and Madagascar.

Humoral and cellular immune response of mice challenged with Yersinia pestis antigenic preparations.

OBJECTIVES: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. METHODS: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40µg or 20µg for each preparation. Immunophenotyping was performed by flow cytometry. RESULTS: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40µg or 20µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. CONCLUSION: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40µg dose) favoured CD8+ T cell-mediated cellular immunity.

Bleach-concentrated direct sputum smear microscopy procedure for diagnosis of pulmonary tuberculosis in poverty areas / Microscopia direta de amostra de escarro concentrado em água sanitária para diagnóstico de tuberculose pulmonar

ABSTRACT INTRODUCTION: Pulmonary tuberculosis caused by Mycobacterium tuberculosis is a serious public health problem affecting millions of people worldwide. The development of easy and low-cost diagnostic methods is crucial for disease control in rural remote and poverty areas and among vulnerable groups. OBJECTIVE: To evaluate the accuracy of laboratory methods for the diagnosis of Pulmonary tuberculosis. MATERIAL AND METHODS: Sputum samples from patients with clinical signs and symptoms were analyzed by microscopy after chemical treatment and spontaneous sedimentation and compared with methods employed routinely: direct sputum smear microscopy, culture, and GeneXpert®MTB/RIF. RESULTS: From the samples analyzed, 16% were positive by microscopy in the processed samples, 18% by both culture and Xpert®MTB/RIF, while 13% in the direct microscopy. The processed samples showed a 31% increase in positivity (57 samples) compared to conventional direct microscopy. In the analysis of the accuracy of the evaluated methods, all the results were statistically significant proving that they were not randomly positive or negative and confirming that there is a tendency for these results. CONCLUSION: Chemical treatment and spontaneous sedimentation of the sputum samples procedure represent an effective diagnostic tool in situations where more advanced technologies are not feasible. Besides the higher accuracy and greater detection of positive cases regarding the direct smear, the procedure strengthens biosafety by decreasing the risks of aerial contamination by Mycobacterium tuberculosis for laboratory professionals.

RESUMEN INTRODUCCIÓN: La tuberculosis pulmonar causada por Mycobacterium tuberculosis es un grave problema de salud pública que afecta millones de individuos en el mundo. El desarrollo de métodos de diagnóstico fáciles y de bajo costo es esencial para el control de la enfermedad en zonas rurales remotas y pobres y entre los grupos vulnerables. OBJETIVO: Evaluar la exactitud de métodos de laboratorio para el diagnóstico de tuberculosis pulmonar. MATERIAL Y MÉTODO: Las muestras del esputo de pacientes con signos y síntomas clínicos fueron analizadas por microscopía luego de tratamiento químico y sedimentación espontánea y comparados con métodos empleados ordinariamente: baciloscopía directa de esputo, cultivo y GeneXpert® MTB/RIF. RESULTADOS: Entre las muestras analizadas, 16% fueron positivas por microscopía en las muestras procesadas; 18% por cultivo y Xpert® MTB/RIF; y 13% por microscopía directa. Las muestras procesadas presentaran un aumento de 31% de positividad (57 muestras) con respecto a la microscopía directa convencional. En el análisis de los métodos, todos los resultados fueron estadísticamente significativos, comprobando que no eran aleatoriamente positivos o negativos y confirmando que hay una tendencia para esos resultados. CONCLUSIÓN: El tratamiento químico y la sedimentación espontánea de las muestras de esputo representan una herramienta diagnóstica eficaz en las situaciones en las cuales tecnologías más avanzadas no son viables. Además de la mayor precisión y mayor detección de casos positivos de lo que hace el frotis directo, el procedimiento fortalece la bioseguridad, disminuyendo los riesgos de contaminación del aire por Mycobacterium tuberculosis para el personal de laboratorio.

RESUMO INTRODUÇÃO: A tuberculose pulmonar causada por Mycobacterium tuberculosis é um grave problema de saúde pública que afeta mundialmente milhões de indivíduos. O desenvolvimento de métodos de diagnóstico fáceis e de baixo custo é essencial para o controle da doença nas áreas rurais remotas e de pobreza e entre os grupos vulneráveis. OBJETIVO: Avaliar a acurácia dos métodos laboratoriais para o diagnóstico de tuberculose pulmonar. MATERIAL E MÉTODOS: As amostras de escarro de pacientes com sinais e sintomas clínicos foram analisadas por microscopia após tratamento químico e sedimentação espontânea e comparadas com métodos empregados rotineiramente: baciloscopia direta do escarro, cultura e GeneXpert® MTB/RIF. RESULTADOS: Das amostras analisadas, 16% foram positivas por microscopia nas amostras processadas; 18%, por cultura e Xpert® MTB/RIF; e 13%, por microscopia direta. As amostras processadas apresentaram um aumento de 31% de positividade (57 amostras) em relação à microscopia direta convencional. Na análise dos métodos avaliados, todos os resultados foram estatisticamente significativos, comprovando que não eram positivos ou negativos aleatoriamente e confirmando que há uma tendência para esses resultados. CONCLUSÃO: O tratamento químico e a sedimentação espontânea das amostras de escarro representam uma ferramenta diagnóstica eficaz nas situações em que tecnologias mais avançadas não são viáveis. Além da maior precisão e maior detecção de casos positivos em relação ao esfregaço direto, o procedimento reforça a biossegurança, diminuindo os riscos de contaminação aérea por Mycobacterium tuberculosis para profissionais de laboratório

Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis.

The primary goal of clinical microbiology is the accurate identification of the causative agent of the disease. Here, we describe a method for differentiation between Yersinia species using PCR-HRMA. The results revealed species-specific melting profiles. The herein developed assay can be used as an effective method to differentiate Yersinia species.

A Taxonomic Update of Small Mammal Plague Reservoirs in South America.

Plague is a disease of epidemic potential that may emerge with discontinuous outbreaks. In South America, 50 wild rodent species have been identified as plague reservoirs, in addition to one lagomorph and two marsupials. To review the nomenclature of plague reservoirs, we examined specimens collected in plague foci, carried out new surveys in Brazilian plague regions, and re-evaluated the nomenclature of South American reservoirs on the basis of the current literature. Five of the 15 species involved with plague in Argentina, three of 10 species involved with plague in Bolivia, three of the seven species involved with plague in Peru, five of the nine species involved with plague in Ecuador, and six of the nine species involved with plague in Brazil have undergone taxonomic changes. In the last 20 years, plague cases were recorded in Bolivia, Brazil, Ecuador, and Peru. These four countries have a high rodent species richness in plague foci, a fact that may be decisive for the maintenance of plague in the wild.

Molecular analysis of clinical isolates of Providencia alcalifaciens.

In an attempt to elucidate the virulence factors and the pathogenic mechanisms of Providencia alcalifaciens, 36 isolates identified in 1994-1995 in Recife city, Brazil were analysed by PCR to investigate the presence of DNA sequences homologous to virulence genes described in other invasive enterobacteria, as well as their ability to invade HeLa cells, their plasmid profiles and antibiotic resistance patterns. The genetic diversity of the isolates was also analysed by RAPD-PCR. No homologous sequences of virulence genes were observed with any of the P. alcalifaciens isolates studied. Ten isolates had no plasmid and 26 harboured one-to-five plasmids of 147-<6.9 kb. Invasion of HeLa cells was observed in only 10 isolates. No correlation between the plasmid content of the strains, their invasion of HeLa cells or their resistance to antimicrobial drugs could be established. The isolates could be distributed into 10 genotypic groups by RAPD-PCR. Considering the genotypic profile and ability to invade HeLa cells, 7 of the 10 invasive isolates belonged to the same genotypic group. The presence of invasive isolates in the same or a related genotypic group suggests the existence of a clonal lineage responsible for the invasiveness.

Humoral and cellular immune response of mice challenged with Yersinia pestis antigenic preparations

ABSTRACT Objectives: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. Methods: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40 µg or 20 µg for each preparation. Immunophenotyping was performed by flow cytometry. Results: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40 µg or 20 µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. Conclusion: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40 µg dose) favoured CD8+ T cell-mediated cellular immunity.

Staphylococcal toxin genes in strains isolated from cows with subclinical mastitis / Detecção de genes de toxinas em linhagens de estafilococos isolados de vacas com mastite subclínica

The present study was carried out in 11 dairy herds in four municipal districts of the rural area of the State of Pernambuco, Brazil. Out of 984 quarter milk (246 cows), 10 (1.0%) were positive for clinical mastitis, 562 (57.1%) for subclinical mastitis and 412 (41.9%) were negative. A total of 81 Staphylococcus spp. isolates were obtained from milk samples from the cows diagnosed with subclinical mastitis. From these, 53 (65.0%) were S. aureus, 16 (20.0%) coagulase-positive staphylococci (CPS) and 12 (15.0%) coagulase-negative staphylococci (CNS). The isolates were further investigated for the presence of toxin genes by multiplex and uniplex PCR. The main gene observed was seg followed by seh, sei and sej. The distribution of these observed genes among the isolates obtained from different areas showed a regional pattern for the SEs. The presence of toxin genes in the strains isolated from bovine milk demonstrates a potential problem for public health.(AU)

O presente estudo foi realizado em 11 rebanhos leiteiros de quatro municípios da área rural do estado de Pernambuco, Brasil. Dos 984 quartos mamários examinados (246 vacas), 10 (1,0%) foram positivos para a mastite clínica, 562 (57,1%) para a mastite subclínica e 412 (41,9%) foram negativos para mastite. Foram isoladas 81 linhagens de Staphylococcus spp. do leite de vacas com mastite subclínica. Destes, 53 (65,0%) foram S. aureus, 16 (20,0%) estafilococos coagulase-positivo (SCP) e 12 (15,0%) estafilococos coagulase-negativo (SCN). O principal gene observado nos estafilococos foi o seg seguido pelo seh, sei e sej. Foi constatada distribuição regional dos genes dos estafilococos isolados dos animais nos municípios estudados. A presença dos genes das toxinas nas linhagens isoladas do leite de vacas representa risco potencial para a Saúde Pública.(AU)

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Marcadores de patogenicidade em Yersinia enterocolitica O: 3 isoladas de suínos do Rio de Janeiro / Genetic markers of pathogenicity in Yersinia enterocolitica O: 3 isolated from healthy pigs from Rio de Janeiro

Foi realizada a caracterização genotípica e fenotípica de fatores de patogenicidade em 16 amostras de Yersinia enterocolitica O:3 isoladas de suínos sadios do Rio de Janeiro. Foi observado que apenas 6 cepas possuíam o plasmídio de virulência, pYV (+ 70 kb) e apresentavam dependência ao cálcio no meio MOX a 37C. Um plasmídio críptico de cerca de 8,6 kb foi encontrado em uma cepa. Doze cepas revelaram sensibilidade à pesticina enquanto que apenas três se revelaram capazes de hidrolisar a esculina. Através de PCR com "primers" específicos, foi constatada a presença dos genes ail em 14 cepas, irp2, em 1 cepa e a ausência de psaA em todas as cepas analisadas. Quanto aos quimioterápicos, a quase totalidade das cepas mostrou-se ao mesmo tempo resistente à ampicilina e carbenicilina e sensível ao sulfazotrin e à cefoxitina. As respostas foram variadas frente ao cloranfenicol, tetraciclina, kanamicina, gentamicina e ácido nalidíxo

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

Standardization of the dot enzyme-lynked immunosorbent assay (dot-ELISA) for experimental plague

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against FIA protein obtained from Yersina pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of FIA protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate

The use of dacron plates for DOT enzyme-linked immunosorbent assay (DOT-ELISA)

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Triration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. Howwvwe, both procedures showed to have a significant better performance in comparasion with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4 graus Centígrados, 28 graus centígrados and -20 graus centígrados for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4 graus centígrados. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate

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 1.3µg 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 per cent 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.