Genetic diversity of Neisseria meningitidis strains isolated in Rio de Janeiro, Brazil, evaluated by multilocus enzyme electrophoresis.

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Some current problems in the systematics of Trypanosomatids.

Trypanosomatids have been traditionally allocated to a number of genera that were described based on morphological features and host range. Recently molecular studies have provided new data that has allowed a reexamination of the genera. While in some cases the molecular data has been in agreement with the morphological characters they have also reinforced existing doubts about some current generic divisions as well as raising new concerns. A revision of the trypanosomatid genera is required. Suggested features of such a revision would include: (1) The possible division of Trypanosoma into new genera to reflect the wide genetic diversity of this group; (2) The inclusion of Leishmania, Sauroleishmania and Endotrypanum within a single genus given their high genetic affinity; (3) The complete revision of the monogenetic typanosomatid genera to reflect monophyletic groups; (4) A more precise redescription of Phytomonas so as to only include the monophyletic plant flagellates.

Differentiation of environmental and clinical isolates of Vibrio mimicus from Vibrio cholerae by multilocus enzyme electrophoresis.

In this study, we demonstrated that analyzed strains of Vibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V. mimicus and V. cholerae. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.

Molecular and biologic characterization of Leishmania parasites implicated in an epidemic outbreak in northwestern Argentina.

Leishmania (Viannia) braziliensis and its variants were implicated in the epidemic outbreak of mucocutaneous leishmaniasis that occurred in Salta, northwestern Argentina, in 1985. A total of 24 suspected, untreated cases were evaluated clinically and parasitologically. Four of five stable isolates were consistent with the reference strain of L. (V.) braziliensis as determined by monoclonal antibodies and indirect immunofluorescence or radioimmunobinding assays. Zymodeme analysis in agarose gels showed a close relationship with L. (V.) guyanensis and L. (V.) panamensis. All zymograms obtained with polyacrylamide gels belonged to the subgenus Viannia; the patterns were different from, but very closely related to, the reference strains of L. (V.) braziliensis as determined by dendrogram analysis. Hamsters infected with two isolates showed a pattern consistent with L. (V.) braziliensis. The pattern of development in the gut of Lutzomyia longipalpis was consistent with members of Viannia.

Speculations on the origin and evolution of the genus Leishmania.

Recently two hypotheses have been proposed for the evolution of Leishmania involving respectively a Neotropical or Paleartic origin for the species. Here an alternative proposal on the phylogeny of Leishmania based on the major divisions within the genus is presented. In this hypothesis a Neotropic origin is retained for L. (Viannia) and Paraleishmania, a recently described section within the genus Leishmania, while an African origin is proposed for L. (Leishmania) and possibly Sauroleishmania. The current distribution of Leishmania in the Neotropics is explained as the product of multiple introductions of Leishmania parasites into the New World. Problems with organismal identity in Sauroleishmania and the use of molecular sequence data in inferring phylogenies are also discussed.

Intraspecific heterogeneity in the mini-exon gene localization of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis from Colombia.

Intraspecific heterogeneity was demonstrated in the mini-exon gene localization from Leishmania (Viannia) panamensis and L. (Viannia) guyanensis. Different karyotypes were detected in human isolates circulating in endemic areas of Colombia. The presence of mini-exon gene sequences on chromosomes of different sizes, ranging from 370 to 800 kb in L. (V.) panamensis and from 500 to 800 kb in L. (V.) guyanensis, was observed and was neither strain nor species specific. In some cases, hybridization with 2 chromosomes in the same strain was observed. The variability of chromosomal localization of mini-exon gene sequences of these 2 species highlights the genetic variability of the Viannia subgenus and the potential utility of the mini-exon gene as a molecular epidemiologic marker.

Molecular population genetics of the primary neotropical malaria vector Anopheles darlingi using mtDNA.

Samples of the neotropical malaria vector Anopheles darlingi from Bolivia, Brazil, and Venezuela were analyzed to test for differences in mitochondrial haplotype frequencies. With the use of molecular variance components and F-statistics, significant genetic variability of An. darlingi was found apportioned primarily among populations within regions or within populations, with regions defined either as biomes (n = 5) or ecoregions (n = 2). The Mantel analysis resulted in a significant correlation [Prob (r) = 0.009] between genetic and geographic distances, evidence that these populations are genetically isolated by distance. Such isolation could reflect differences in phenotypes for factors affecting vector capacity.

Genotypic diversity among Brevibacillus laterosporus strains.

In comparison with other entomopathogenic Bacillus species, the genome of Brevibacillus laterosporus is poorly characterized. The aim of this study was to examine genetic variability in B. laterosporus by using a range of typing methodologies. Strains of B. laterosporus were examined for variation in 13 chromosomal genes encoding enzymes by multilocus enzyme electrophoresis. Optimal conditions of pulsed-field gel electrophoresis and randomly amplified polymorphic DNA were established that allowed analysis of the genome of B. laterosporus. None of these techniques allowed the identification of a convenient molecular marker for entomopathogenic strains, although one specific primer amplified only DNA from almost all mosquitocidal strains.

Genetic relationships among the different phenotypes of Streptococcus dysgalactiae strains.

The species Streptococcus dysgalactiae was proposed to accommodate a heterogeneous group of streptococci associated with infections in animals and human beings. This taxon is now considered to include animal isolates of alpha-haemolytic group C streptococci, previously called S. dysgalactiae; animal and human isolates of beta-haemolytic group C streptococci, previously called 'S. equisimilis'; beta-haemolytic group L strains associated with infections in animals and, rarely, in humans; and beta-haemolytic group G strains isolated from humans. DNA-DNA reassociation experiments (hydroxyapatite method) and multilocus enzyme electrophoresis (MEE) were performed on reference strains and clinical isolates to determine the genetic relationships among these different phenotypic categories. DNA-DNA hybridization tests showed that they were related at the species level, despite the phenotypic and host heterogeneity. Both genotypic and phenotypic characterization indicated that S. dysgalactiae could be separated into two major sub-groups. The first sub-group contained alpha-haemolytic strains that showed levels of DNA relatedness with the type strain of S. dysgalactiae ranging from 84 to 90% and from 82 to 88% under optimal (55 degrees C) and stringent (70 degrees C) conditions, respectively. The second sub-group contained beta-haemolytic strains showing levels of relatedness ranging from 71 to 79% (55 degrees C) and from 62 to 73% (70 degrees C). Percentage divergence varied from 0.5 to 1.0% (alpha-haemolytic group) and from 2.0 to 3.5% (beta-haemolytic group). A dendrogram based on phenotypic similarity between the enzyme bands produced by MEE showed a Jaccard similarity coefficient of 0.45 between the subclusters formed by the two sub-groups. The results of phenotypic and genotypic characterization were consistent with a published proposal to divide S. dysgalactiae into two subspecies, S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis, with a few modifications.

Genetic diversity in natural populations of New World Leishmania.

Our results have shown the wide diversity of parasites within New World Leishmania. Biochemical and molecular characterization of species within the genus has revealed that much of the population heterogeneity has a genetic basis. The source of genetic diversity among Leishmania appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction can not be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localized to a particular endemic focus. Epidemiological studies of leishmaniasis has been directed to the ecology and dynamics of transmission of Leishmania species/variants, particularly in localized areas. Future research using molecular techniques should aim to identify and follow Leishmania types in nature and correlate genetic typing with important clinical characteristics such as virulence, pathogenicity, drug resistance and antigenic variation. The epidemiological significance of such variation not only has important implications for the control of the leishmaniases, but would also help to elucidate the evolutionary biology of the causative agents.

Serotype H5a5b is a major clone within mosquito-pathogenic strains of Bacillus sphaericus.

Seventy six mosquito pathogenic strains of Bacillus sphaericus and 10 non-pathogens were examined by pulsed field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA. Non-pathogenic strains were clearly distinguished from the entomopathogenic types which were assigned to 21 groups (SmaI restriction patterns; SRPs). Some agreement between SRP based on PFGE and serotyping was noted, in particular all 39 strains of serotype 5a5b examined revealed identical SRPs indicating total conservation of the SmaI restriction site in these bacteria. Serotype 5a5b (SRP 12) strains comprise a widely distributed and abundant clonal lineage. Most serotypes, however, were divided into several SRPs. Seven strains from serotype 2a2b were covered in five SRPs in which toxin synthesis was correlated with chromosomal structure. Similarly, toxicity correlated with SRP in strains from serotypes 3 and 6.