Alzira Maria Paiva de Almeida

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.

RAPD-PCR typing of Yersinia enterocolitica (Enterobacteriaceae) O:3 serotype strains isolated from pigs and humans

Sixteen strains of Yersinia enterocolitica serotype O:3, isolated from apparently healthy pigs collected in Rio de Janeiro, and four human strains of serotypes O:4, O:5, O:6 and O:13 were analyzed by RAPD-PCR. The strains were grouped into five genotypic profiles according to the amplification patterns obtained with three random primers. Fifteen of the 16 pig strains had identical amplification patterns, which was named genotypic profile 1. The one different profile was named genotypic profile 2. Genotypic profile 1 was also exhibited by the O:6 human serotype strain. The O:4 and O:13 human serotype strains showed similar amplification profiles with two primers. However, the third primer induced a distinct profile in each strain. Therefore, these two strains were placed into genotypic profile 3 and 4, respectively. Each primer produced a completely different amplification profile in the O:5 human serotype strain; therefore, it was named genotypic profile 5. The presence or absence of plasmids in the strains studied did not affect the amplification results. These results show that genetic variations can exist within a serotype, and strains of different serotypes can exhibit the same amplification profile when compared using other primers.

Development and Evaluation of a Single Tube Nested PCR Based Approach (STNPCR) for the Diagnosis of Plague

The performance of a single-tube nested-PCR (STNPCR) technique was evaluated for plague diagnosis in comparison to conventional (one step) and two step nested PCR (NPCR). Assays were carried out with primers targeting the gene caf1 that encodes the Yersinia pestis F1 antigen. For STNPCR inner primers were immobilized onto the inside of the microtube caps and after the first amplification they were eluted by inversion of the tube. This procedure avoids opening the tube, reducing the risks of false-positive results by cross-contamination. The immobilized primers are stable for several months at -20 degrees C, thus, the tubes can be prepared beforehand and stored until use. STNPCR was more sensitive than conventional PCR, and less sensitive than NPCR. This drawback is compensated by a lower risk of cross-contamination. The experiments with infected animals showed that NPCR and STNPCR were able to produce positive results in all samples tested, despite contamination with other organisms. In contrast, conventional PCR yielded positive results in a smaller number of samples. Three out of 62 culture-negative rodents from plague areas, were positive by STNPCR. In conclusion, the PCR approaches evaluated, particularly NPCR and STNPCR have potential to be used as alternative tools in epidemiological surveys of plague. Furthermore, as the results can be obtained quickly (less than 24 hour), these techniques could be useful in emergency situations in which the rapidity in diagnosis is essential for adoption of immediate measures of control.

Vigilância da peste no Estado do Ceará: 1990-1999

Serological surveillance activities regarding the foci of plague in Ceará State have detected a rising number of sentinel animals with antiplague antibodies in 1995, with a peak in 1997 demonstrating an increase in the plague bacteria activities throughout all the foci investigated. From a total of 110,725 serum samples collected from rodents (7,873) and domestic carnivores (102,852) analyzed by the Hemaglutination technique (HA) for antibodies against F1 antigen of Yersinia pestis 905 samples tested positive. In these samples there were 15 rodents (4 Rattus rattus and 11 Galea spp), 720 dogs and 170 cats. Of the 652 human suspected and contact cases investigated by HA, only two were positive. A third case had a positive hemoculture for Y. pestis. The isolate is highly pathogenic for laboratory animals and showed sensitivity to the antimicrobial drugs used for plague treatment.

Pesquisa de bactérias patogênicas em leite pasteurizado tipo C comercializado na cidade do Recife, Pernambuco, Brasil

In order to improve information about the microbiological quality of the milk commercially available in the city of Recife, 250 samples of pasteurized type-C milk and 50 samples of raw milk were analyzed for Yersinia enterocolitica and Listeria monocytogenes and verify the possible occurrence of Yersinia enterocolitica and Listeria monocytogenes. These bacteria can develop in refrigeration temperatures and are responsible for food-born diseases. Neither Y. enterocolitica nor L. monocytogenes were found in the samples analyzed. However, the presence of Y. intermedia and Y. frederiksenii was detected, these environmental species behave as opportunist pathogens. Through the methodology used for Listeria isolation, one isolate of Salmonella Montevideo was obtained from a sample of pasteurized milk and another isolated from one sample of raw milk. Besides these, several other bacteria species were found. It is likely that the large microbiota present in the samples and the procedures employed to destroy it could have hindered the isolation of Y. enterocolitica and L. monocytogenes.

Pesquisa da infecção natural por Yersinia pestis, em pulicídeos provenientes de focos pestosos do nordeste do Brasil

Three different containment transport processes of fleas were evaluated as an approach to the bacteriologic isolation of Yersinia pestis. The three methods employed were: live fleas in glass tubes containing pieces of wrapped filter paper; dead fleas in saline solution; and maceratedfleas in Cary-Blair culture medium. The two latter methods were almost equal and superior to the first method. A total of 29512 flea pools, from plague foci in Northeast Brazil collected during 1966 to 1982 were evaluated by the three methods. Among these samples, 236 (0.80%) flea pools were positive with regard to bacteriological cultivation and/or infection of susceptible animals.

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) synthesized 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 was studied. 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 be positive with the A13 probe, indicating that the locus was lost after subculture in 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, EcoRV or AvaII, indicating that 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.

[Prevalence of antibodies against Yersinia pestis in domestic carnivores, in plague foci in the State of Ceará].

The prevalence of antibodies against Yersinia pestis in domestic carnivores (dogs and cats), in plague areas in the State of Ceará, was analyzed to establish the importance of monitoring these animals within the routine practice of the plague control program. Over the decade 1997-2006, 146,732 serum samples were examined (95,883 from dogs and 50,849 from cats), of which 2,629 (2,234 from dogs and 395 from cats) proved to be positive. The prevalence among dogs (85%) was higher than among cats (15%) throughout the decade and in all places, except in Ibiapina in 1998. The significance of these findings has not yet been determined. Studies on this zoonosis in Brazil have been based on paradigms that did not cover all the elements involved in the zoonosis, thus making it impossible to properly understand the role of these carnivores. Monitoring of plague foci conducted exclusively by means of dog surveys may result in progressive lack of knowledge of the epidemiological situation of plague, if supplementary inter-institutional research is not developed.

Biossegurança, Proteção Ambiental e Saúde: compondo o mosaico

This work reflects on the development of the field of Biosafety. The scope of this field is presented, as well as its complex themes and its interdisciplinary perspective. The scope of this field is to propose actions capable of preventing and controlling the risk of worsening human and environmental health. This is done in order to provide alternatives to the theoretical and practical challenges posed by the rapid changes in the world, resulting from human intervention in nature and mediated by scientific and technological advances. We address questions that place Biosafety as a tool in the search for a model of sustainable development, establishing the relationship between environmental degradation, precarious health conditions, and control of the emergence and reemergence of diseases in populations.

Plague in Brazil: From Now and Then

In Brazil, plague is a greatly neglected disease. It received some attention when it was first introduced in 1899 and again during the first decades of the twenty century, when it spread to important cities. Plague was forgotten as soon as it became restricted to isolated and poor areas, but it received renewed attention in the 1960s, when the lack of control resulted in increased plague-related morbidity and mortality. Records of this zoonosis are lacking, and the biotic and abiotic factors in the epidemiological chain are virtually unknown by the public health services and universities. However, the systematic detection of Yersinia pestis antibodies in sentinel animals has provided evidence of its continued presence and the possibility of its reemergence. In this paper, some aspects of plague epidemiology and plague control from 1899 to 2011 are described and analyzed. This information could support new studies of the natural history of plague in Brazil.