Seroprevalence of bactericidal antibodies against serogroup B and C Meningococci in a University Hospital.

Meningococcus serogroup B (MenB), clonal complex 32 (cc 32), was the Brazilian epidemic strain of meningococcal disease (MD) in the 1990's. Currently, meningococcus serogroup C (MenC), cc 103, is responsible for most of the cases of the disease in Brazil. The aim of this study was to investigate the seroprevalence of bactericidal antibody (SBA) against representative epidemic strains of MenC, (N753/00 strain, C:23:P1.22,14-6, cc103) and MenB, (Cu385/83 strain, B:4,7:P1.15,19, cc32) in students and employees of a university hospital in the State of Rio Grande do Sul (RS, Brazil). A second MenC strain (N79/96, C:2b:P1.5-2,10, cc 8) was used as a prototype strain of Rio de Janeiro's outbreak that occurred in the 1990's. Our previous study showed a 9% rate of asymptomatic carriers in these same individuals. A second goal was to compare the SBA prevalence in meningococcal carriers and non-carriers. Fifty-nine percent of the studied population showed protective levels of SBA titers (log2≥2) against at least one of the three strains. About 40% of the individuals had protective levels of SBA against N753/00 and Cu385/83 strains. Nonetheless, only 22% of the individuals showed protective levels against N79/96 strain. Significantly higher antibody levels were seen in carriers compared to non-carriers (P≤0.009). This study showed that, similar to other States in Brazil, a MenC (23:P1.22,14-6, cc103) strain with epidemic potential is circulating in this hospital. Close control by the Epidemiological Surveillance Agency of RS of the number of cases of MD caused by MenC strains in the State is recommended to prevent a new disease outbreak.

Outbreak of Neisseria meningitidis C in workers at a large food-processing plant in Brazil: challenges of controlling disease spread to the larger community.

SUMMARYAn outbreak of meningococcal disease (MD) with severe morbidity and mortality was investigated in midwestern Brazil in order to identify control measures. A MD case was defined as isolation of Neisseria meningitidis, or detection of polysaccharide antigen in a sterile site, or presence of clinical purpura fulminans, or an epidemiological link with a laboratory-confirmed case-patient, between June and August 2008. In 8 out of 16 MD cases studied, serogroup C ST103 complex was identified. Five (31%) cases had neurological findings and five (31%) died. The attack rate was 12 cases/100 000 town residents and 60 cases/100 000 employees in a large local food-processing plant. We conducted a matched case-control study of eight primary laboratory-confirmed cases (1:4). Factors associated with illness in single variable analysis were work at the processing plant [matched odds ratio (mOR) 22, 95% confidence interval (CI) 2·3-207·7, P<0·01], and residing <1 year in Rio Verde (mOR 7, 95% CI 1·11-43·9, P<0·02). Mass vaccination (>10 000 plant employees) stopped propagation in the plant, but not in the larger community.

Induction of cell migration and activation in mice by the freshwater sponge Drulia uruguayensis Bonetto & Ezcurra de Drago, 1968 (Porifera: Metaniidae)

Freshwater sponges are abundant in the Amazon region and they have been known to cause dermatitis (acute inflammation) since the beginning of the 20th century. To determine whether additional constituents, besides their body spicules, cause dermatological reactions in humans, an experimental study was developed and carried out using mice and Drulia uruguayensis prepared in three different forms: intact sponges (IS), macerated sponges (MS) or isolated spicules - megascleres (ISM). The cells most commonly involved in inflammatory reactions (mast cells, eosinophils and neutrophils), as well as intraepithelial lymphocytes and degranulated mast cells, were counted so that they could be used as parameters to determine which of the sponge preparations induced the greatest reaction. The effects of the sponge on the skin were then determined by histological analysis. The results obtained showed that IS caused the greatest inflammatory reaction (p = 0.000005), activating mainly mast cells (p = 0.0018). The histopathological analysis revealed a slight loss of continuity of the epidermis when ISM or IS were applied. These findings allow us to conclude that a structurally intact sponge can cause a greater inflammatory reaction in the first contact because of its ability to perforate the skin and allow inflammatory agents to enter. Other proteins present in dried sponge bodies could induce allergic but not toxic responses (in contact with the entire sponge, a large number of pharmacologically inert proteins may be introduced, with a potential allergen).

Epidemiology of meningococcal disease in southern Brazil from 1995 to 2003, and molecular characterization of Neisseria meningitidis using multilocus sequence typing.

OBJECTIVE: To describe the epidemiology of meningococcal disease (MD) in southern Brazil. METHODS: Retrospective cohort study among 2215 MD cases reported from 1995 to 2003 in Rio Grande do Sul (RS) State. RESULTS: The overall incidence fell by 50%; the case-fatality rate during this period was 22%. Even so, the incidence of MD remained high after the epidemic period ended in 1999. Together, the age groups of 1-4 years and infants accounted for 54.1% of reported cases with incidences of 11.3/100 000 and 31.3/100 000, respectively; 69.8% of cases were caused by Neisseria meningitidis serogroup B, which increased significantly. There was a significant decrease in serogroup C cases in the whole period. The phenotypes B:4,7:P1.19,15, B:15:P1.7,16 and B:NT:P1.3 caused almost 50% of all serotyped cases. Fifty-six isolates obtained from RS patients during the first non-epidemic year 2000 plus 20 isolates from other southern Brazilian states (Santa Catarina and Paraná), Denmark and France were typed by multilocus sequence typing. Twenty sequence types (STs) were identified, eight of them found only in RS. ST-33 (27%) and ST-259 (18%) were the most frequent; both belong to the ST-32/ET-5 complex. ST-259 cases showed a trend towards higher risk of fatal outcome. ST-259 isolates were not detected among geographic controls or in other studies in Brazil. CONCLUSION: Our data suggest that ST-33 and ST-259 clones and the emergence of the ST-103 isolates contributed to the continued high incidence of MD in RS.

Immune response to native NadA from Neisseria meningitidis and its expression in clinical isolates in Brazil

A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60% of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+).

The plasma membrane (Ca(2+) + Mg(2+))ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca(2+) ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca(2+) strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca(2+)-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca(2+) is related to opposite long-term hydration effects on the substrate binding domain and the Ca(2+) binding domain.

Serosubtypes and PorA types of Neisseria meningitidis serogroup B isolated in Brazil during 1997--1998: overview and implications for vaccine development.

Meningococcal disease caused by N. meningitidis serogroup B (MenB) has been endemic in Brazil since 1997. In this study, we determined the prevalence of serosubtypes of MenB isolated in 10 Brazilian states and the Federal District during 1997 and 1998 and investigated the extent of PorA VR sequence variation among the most prevalent serosubtypes to evaluate the possible use of an outer membrane vesicle (OMV)-, PorA-based vaccine to prevent meningococcal disease in Brazil. During this period, a total of 8,932 cases of meningococcal disease were reported. Only 42% (n = 3,751) of the reported cases were laboratory confirmed, and about 60% (n = 2,255) of those were identified as MenB. Among 1,297 MenB strains selected for this study, the most prevalent serosubtypes were P1.19,15 (66%), P1.7,1 (11%), and P1.7,16 (4%). PorA VR typing showed that 91% of the P1.19,15 strains analyzed had VR1 and VR2 sequences identical to those of the prototype strain. No sequence variation was detected among the 40 strains representing all isolated MenB P1.7,16 strains in the three southern states, where this serosubtype accounts for 75% of the serosubtypes identified. Similarly, all P1.7,1 strains were identified by PorA typing as P1.7-1,1. Although further improvements in the reporting of cases and collection of strains in Brazil are needed, our data suggest that a trivalent OMV-based vaccine prepared with PorA types P1.19,15, P1.7-1,1, and P1.7,16 may be appropriate to control serogroup B meningococcal disease in most of the Brazilian states.

Genetic relationships among serogroup B: serotype 4 Neisseria meningitidis strains.

We compared the results obtained by serotyping of PorB epitopes using an expanded panel of monoclonal antibodies (mAb) including mAb 7 and mAb 10, with results obtained by RFLP of rRNA genes (ribotyping). The purpose of this study was to assess the correlation between phenotypic- and genotypic- methods for typing N. meningitidis. The ribotypes obtained using ClaI or EcoRV endonucleases grouped the strains in seven and two different patterns, respectively. This additional characterization of PorB epitopes improved the correlation between these two methods of typing N. meningitidis.

A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence.

The plasma membrane of cells contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using living cells. In this work we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (5.39 +/- 0.71 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 30.75 +/- 2.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.21 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.98 mM and free Mg(2+) did not increase the ecto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells were viable, the acid phosphatase activity decreased, while the Mg(2+)-dependent ATPase activity increased. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. A comparison between the Mg(2+)-dependent ATPase activity of virulent and avirulent promastigotes showed that avirulent promastigotes were less efficient than the virulent promastigotes in hydrolyzing ATP.

Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase.

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 M, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20%) inhibiting the ATPase activity, also protect the F0F1-ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 M), and in the presence of three polyols, the Ki for urea shift from 0.7 M to 1.2 M. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 M, 2 M or 3 M urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1-ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.

Proposed standardization of Neisseria meningitidis PorA variable-region typing nomenclature.

Neisseria meningitidis isolates are conventionally classified by serosubtyping, which characterizes the reactivities of the PorA outer membrane protein variable-region (VR) epitopes with monoclonal antibodies (MAbs). A newer method (PorA VR typing) uses predicted amino acid sequences derived from DNA sequence analysis. The resulting classification schemes are not standardized, offering conflicting and sometimes irreconcilable data from the two methods. In this paper, we propose a standardization of the PorA VR typing nomenclature that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences. We performed a comprehensive literature and database search, generating a collection of strains and DNA sequences that reflects the diversity within PorA that exists to date. We have arranged this information in a comprehensive logical model that includes both serosubtype and PorA VR type assignments. Our data demonstrate that the current panel of serosubtype-defining MAbs underestimates PorA VR variability by at least 50%. Our proposal for VR typing is informative because amino acid sequence and serologic information, when serosubtype-defining MAbs are available, can be deduced simultaneously from the PorA VR designation. This scheme will be useful in future classification and applied epidemiologic studies of N. meningitidis, being a systematic way of selecting PorA vaccine candidates and analyzing vaccine coverage and failure.

Meningococcal disease caused by Neisseria meningitidis serogroup B serotype 4 in São Paulo, Brazil, 1990 to 1996.

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988. A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

The use of oligonucleotide probes for meningococcal serotype characterization.

In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs). Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs) are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A) and 615U (VR3-B) used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

Correlation between serological and sequencing analyses of the PorB outer membrane protein in the Neisseria meningitidis serotyping system.

The current serological typing scheme for Neisseria meningitidis is not comprehensive; a proportion of isolates are not serotypeable. DNA sequence analysis and predicted amino acid sequences were used to characterize the structures of variable-region (VR) epitopes on N. meningitidis PorB proteins (PorB VR typing). Twenty-six porB gene sequences were obtained from GenBank and aligned with 41 new sequences. Primary amino acid structures predicted from those genes were grouped into 30 VR families of related variants that displayed at least 60% similarity. We correlated VR families with monoclonal antibody (MAb) reactivities, establishing a relationship between VR families and epitope locations for 15 serotype-defining MAbs. The current panel of serotype-defining MAbs underestimates by at least 50% the PorB VR variability because reagents for several major VR families are lacking or because a number of VR variants within some families are not recognized by serotype-defining MAbs. These difficulties, also reported for serosubtyping based on the PorA protein, are shown as inconsistent results between serological and sequence analyses, leading to inaccurate strain identification and incomplete epidemiological data. The information from this study enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities. Use of the expanded serotype panel enabled us to improve the sensitivity of serotyping by resolving a number of formerly nonserotypeable strains. In most cases, this information can be used to predict the VR family placement of unknown PorB proteins without sequencing the entire porB gene. PorB VR typing complements serotyping, and a combination of both techniques may be used for full characterization of meningococcal strains. The present work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date.

Carbohydrate protection of enzyme structure and function against guanidinium chloride treatment depends on the nature of carbohydrate and enzyme.

Baker's yeast cells accumulate osmolytes as a response to several stress conditions such as high-temperature and low-temperature shifts, dehydration, or osmotic stress. One of the major osmolytes that accumulates is trehalose, which plays an essential role affecting the survival of yeast at the time of stress. In this report, we show that trehalose efficiently protects the function and the structure of two yeast cytosolic enzymes against chemical denaturation by guanidinium chloride. Other sugars tested also protected yeast pyrophosphatase and glucose-6-phosphate dehydrogenase structure against guanidinium chloride effects, but were not as efficient at protecting enzyme activity. The thermostable pyrophosphatase from Bacillus stearothermophilus was also protected by several sugars against the chaotropic properties of guanidinium chloride, but was only protected by trehalose against functional inactivation. The function of the membrane-embedded H+-ATPase from yeast could not be protected by any of the tested sugars, although all of the sugars protected its structure from guanidinium-chloride-induced unfolding. The results presented in this study suggest that several sugars are able to prevent protein unfolding induced by a chaotropic compound. However, prevention of functional inactivation depends on the nature of the sugar. Trehalose was the most efficient, being able to protect many cytosolic enzymes against guanidinium chloride. The efficiency of protection also depended on the nature of the protein tested. This might explain why trehalose is one of the osmolytes accumulated in yeast and also why it is not the only osmolyte to accumulate.

Ultrastructural and biochemical analysis of the relationship of Crithidia deanei with its endosymbiont.

Some protozoa of the Trypanosomatidae family harbor in their cytoplasm bacterial endosymbionts that provide essential nutrients to and induce morphological alterations in the protozoa. In the present study, a close association between endosymbionts and glycosomes, a peroxisome-like organelle where most of the enzymes of the glycolytic pathway are compartmentalized, was identified by conventional transmission electron microscopy in Crithidia deanei. Such an association was further supported by the cytochemical localization of catalase in the glycosome and also confirmed by 3-D reconstruction of the protozoan. The enzymes cytochrome oxidase and succinate dehydrogenase were detected by ultrastructural cytochemistry. A positive reaction was observed in the protozoan mitochondrion but not in the endosymbiont envelope. Enzymatic assays for succinate cytochrome c reductase reinforced these results, as a low enzymatic activity was detected in an endosymbiont-enriched fraction, while high activity was observed in a purified protozoan mitochondrion fraction. We also demonstrated that a purified symbiont fraction was able to hydrolyze ATP. This activity was Mg+2 dependent, since it was highly stimulated by the presence of physiological concentrations of this ion. Taken together, these observations suggest that no electron transporting system is active in the symbionts of Crithidia deanei and that they might obtain energetic molecules derivated from the protozoan glycosomes.

Antibody response after immunization of Brazilian children with serogroup C meningococcal polysaccharide noncovalently complexed with outer membrane proteins.

We have studied the antibody response of Brazilian vaccinees to C meningococcal polysaccharide (C-PS) after one or two doses of a vaccine composed of C-PS, outer membrane proteins of B meningococci and aluminum hydroxide. Total IgG, IgG1 and IgG2 as well as bactericidal activity mediated by complement were measured in serum samples from children 3 to 83 months of age (post-vaccination IgG, IgG1 and IgG2 levels of 2.4 to 13.4 micrograms/ml; less than 18 to 67.8 U/ml and less than 18 to 106.8 U/ml, respectively) and from individuals 10 to 14 years of age (post-vaccination IgG, IgG1 and IgG2 levels of 14.6 micrograms/ml, 23.7 U/ml and 112.0 U/ml, respectively). The antibody response, measured as IgG levels, was age-dependent. Although high antibody levels were demonstrable by enzyme-linked immunosorbent assay (ELISA), bactericidal activity was not demonstrable (less than 1:4) in serum from children aged less than 24 months. A significant bactericidal activity was detected in serum of children older than 49 months of age and in individuals 10 to 14 years of age. A predominance of IgG2 was observed in post-vaccination serum samples from children belonging to those two age groups. The antibody concentration sufficient to confer protection as well as the possible causes of the poor correlation observed between ELISA and bactericidal activity results are discussed.

Antibody response after immunization of Brazilian children with serogroup C meningococcal polysaccharide noncovalently complexed with outer membrane proteins

We have studied the antibody response of Brazilian vaccines to C meningococcal polysaccharide (C-PS) after one or two doses of a vaccine composed of C-PS, outer membrane proteins of B meningococci and aluminum hydroxide. Total IgG, IgG1 and IgG2 as well as bactericidal activity mediated by complement were measured in serum samples from children 3 to 83 months of age (post-vaccination IgG, IgG1 and IgG2 levels of 2.4 to 13.4 µg/ml; less than 18 to 67.8 U/ml and less than 8 to 106.8U/ml, respectively) and from individuals 10 to 14 years of age (post-vaccination IgG, IgG1 and IgG2 levels of 14.6 µg/ml, 23,7 U/ml and 112.0 U/ml, respectively). The antibody response, measured as IgG levels, was age-dependent. Although high antibody levels were demonstrableby enzyme-linked immunosorbent assay (ELISA), bactericidal activity was not demonstrable (less than 1:4) in serum from children aged less than 24 months. A significant bactericidal activity was detected in serum of children older than 49 months of age and in individuals 10 to 14 years of age. A predominance of IgG2 was observed in post-vaccination serum samples from children belonging to those two age groups. The antibody concentration sufficient to confer protection as well as the possible causes of the poor correlation observed between ELISA and bactericidal activity results are discussed

Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes.

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

Considerations on the use of Neisseria meningitidis class 5 proteins as meningococcal BC vaccine components.

This investigation was carried out to evaluate the importance of individual meningococcal surface class 5 protein with respect to antibody induction and its functional activity. Two groups of mice were immunized with two vaccine preparations differing in the presence or absence of class 5 protein. The ELISA results show that both vaccines were immunogenic and elicited mainly IgG antibodies against the major classes of meningococcal surface proteins, and the absence of class 5 protein in the vaccine produced a significant change in the overall units ml-1 of antibodies against the homologous strain. The infant rat model and the bactericidal assay were used to evaluate the functional antibody activity. Our results showed that (1) even using two different challenge doses (10(6) and 10(7) bacteria/animal), mortality could not be detected when followed up at 48 h; (2) there was protection as determined by the infant rat model and bactericidal activity using sera from both vaccinated groups; (3) there were no differences in the bactericidal titres between these groups; (4) in the infant rat model there were no differences in the index of bacteraemia among the infected animals (counts ml-1 of blood); and (5) there were differences in the incidence of bacteraemia. This is the first evidence that some immunological differences in the vaccine response could be attributed to the absence of class 5 protein by using infant rat model but not by using the bactericidal assay.