Characterization of Bordetella bronchiseptica strains using phenotypic and genotypic markers.

Thirty-five strains of Bordetella bronchiseptica, recovered primarily from pigs, rabbits, dogs, cats and humans, were characterized by phenotypic and genotypic markers. Biochemical typing only showed variation in the ability to reduce nitrate to nitrite. OMP profiles from virulent strains showed variations in the region of 85-95kDa, which lead us to describe five OMP-types alpha, beta, gamma, delta and epsilon. Genotypic markers included the presence of IS1001, and polymorphisms in the flagellin gene (flaA) and pertussis toxin (PT) promoter region. The IS1001 was detected in 16 isolates (2 from humans and 10 from pigs) but was absent in rabbit isolates. The restriction profiles of the flaA gene allowed us to differentiate the strains into types A-C. The PT types were characterized by an RFLP assay and could be typed through patterns III-V. There was no apparent association between the flaA or PT types and the origin of the isolates. Eleven groups of isolates were identified on the basis of specific combinations of the analyzed markers. The combination of phenotypic and genotypic tests used could be useful in characterizing isolates and differentiating between certain clonal types of B. bronchiseptica.

[Biotyping and molecular phenotypic characterization of Bordetella bronchiseptica]. / Biotipificación y caracterización fenotípica molecular de Bordetella bronchiseptica.

In the present study biochemical tests and outer membrane protein profile (OMP) capacity for typing Bordetella bronchiseptica field isolates were evaluated. The biochemical tests were performed by API 20NE system. OMP enriched fractions were obtained from cultures under virulent and modulated conditions and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have identified five patterns by differences in the bands in the 85-95 kDa region (alpha, beta, gamma, delta and epsilon) from virulent cultures; and three different patterns by the flagellin expressed isotype from modulated cultures (A: 40 kDa, B: 45 kDa, and C: 40 and 45 kD simultaneously). Isotypes alpha, beta and gamma were linked to isotpye A, isotype delta to B and C, and isotypes epsilon to B. There is no evident correlation between characterized isotypes and the origin of the isolate. Nitrate reduction was the unique variable biochemical characteristic, only observed in rabbit isolates. It was possible to differentiate seven groups with the traits included in the study. The capacity of discrimination of the traits analyzed using the Hunter and Gaston index was 0.829.

Biotipificación y caracterización fenotípica molecular de Bordetella bronchiseptica / [Biotyping and molecular phenotypic characterization of Bordetella bronchiseptica]

In the present study biochemical tests and outer membrane protein profile (OMP) capacity for typing Bordetella bronchiseptica field isolates were evaluated. The biochemical tests were performed by API 20NE system. OMP enriched fractions were obtained from cultures under virulent and modulated conditions and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have identified five patterns by differences in the bands in the 85-95 kDa region (alpha, beta, gamma, delta and epsilon) from virulent cultures; and three different patterns by the flagellin expressed isotype from modulated cultures (A: 40 kDa, B: 45 kDa, and C: 40 and 45 kD simultaneously). Isotypes alpha, beta and gamma were linked to isotpye A, isotype delta to B and C, and isotypes epsilon to B. There is no evident correlation between characterized isotypes and the origin of the isolate. Nitrate reduction was the unique variable biochemical characteristic, only observed in rabbit isolates. It was possible to differentiate seven groups with the traits included in the study. The capacity of discrimination of the traits analyzed using the Hunter and Gaston index was 0.829.

Biotipificación y caracterización fenotípica molecular de Bordetella bronchiseptica. / [Biotyping and molecular phenotypic characterization of Bordetella bronchiseptica]

In the present study biochemical tests and outer membrane protein profile (OMP) capacity for typing Bordetella bronchiseptica field isolates were evaluated. The biochemical tests were performed by API 20NE system. OMP enriched fractions were obtained from cultures under virulent and modulated conditions and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have identified five patterns by differences in the bands in the 85-95 kDa region (alpha, beta, gamma, delta and epsilon) from virulent cultures; and three different patterns by the flagellin expressed isotype from modulated cultures (A: 40 kDa, B: 45 kDa, and C: 40 and 45 kD simultaneously). Isotypes alpha, beta and gamma were linked to isotpye A, isotype delta to B and C, and isotypes epsilon to B. There is no evident correlation between characterized isotypes and the origin of the isolate. Nitrate reduction was the unique variable biochemical characteristic, only observed in rabbit isolates. It was possible to differentiate seven groups with the traits included in the study. The capacity of discrimination of the traits analyzed using the Hunter and Gaston index was 0.829.

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity.

AIMS: The present study shows that Congo red binding and urease activity assays are useful for selection of virulent (Bvg+) Bordetella bronchiseptica cultures. METHODS AND RESULTS: Congo red binding and urease activity of Bvg+ B. bronchiseptica cultures in different liquid media were compared with the expression of virulence markers such as filamentous haemagglutinin and some outer membrane proteins (OMP). The correlation with the reference virulence markers allowed the establishment of cut-off values for the proposed markers to assure the virulent phenotype (> or = 26 nmol ml-1 of CR and < or = 2.6 U). Using both assays, modulated cultures with avirulent phenotype (Stainer-Scholte broth, with MgSO4 20 mmol l-1 and brain heart infusion broth) and semi-modulated cultures with intermediate phenotypes (tryptose phosphate broth and 83% Stainer-Scholte with MgSO4 5 mmol l-1 cultures) could be distinguished. CONCLUSION: CR binding assay and urease activity are specific and sensitive enough to detect intermediate phenotypes that could only be detected by subtle changes in OMP profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of effective veterinary vaccines is hampered by reversible B. bronchiseptica antigenic modulation. The proposed assays are technically suitable for selection of virulent cultures to optimize vaccine production.

Use of Neo-melubrina, a banned antipyretic drug, in San Diego, California: a survey of patients and providers.

BACKGROUND: Dipyrone is an antipyretic drug that has been associated with agranulocytosis. It is banned in the United States but is available in Mexico under the name Neo-melubrina. OBJECTIVES: To define the use of Neo-melubrina in the Hispanic population of 2 San Diego, California, community clinics and to determine local physicians' and nurse practitioners' awareness of the drug and its risks. DESIGN: Patient survey and provider survey. PATIENTS: 200 parents of Hispanic pediatric patients. Providers: members of San Diego chapters of the American Academy of Pediatrics, the American Academy of Family Physicians, and the California Coalition of Nurse Practitioners. MAIN OUTCOME MEASURES: Self-reported use of Neo-melubrina by patients, and provider awareness of Neo-melubrina and its most significant side effects. RESULTS: Of the 200 patients, 76 (38.0%) reported a lifetime use of Neo-melubrina. Most (56%) used it for both pain and fever. Most providers were unable to correctly identify why Neo-melubrina might be used or its adverse effects. Physicians answered correctly more often than nurse practitioners and pediatric providers more often than family medicine providers. Providers who trained within 75 miles of the US-Mexico border, who reported a patient population of more than 50% Hispanic, and who were resident physicians at the time of the survey were most likely to answer correctly. CONCLUSIONS: Neo-melubrina has been used by a substantial percentage of Hispanic patients in the community clinics surveyed. Many San Diego health care providers are unaware of this medication and may, therefore, miss opportunities to educate patients about safer alternatives.

Identification of Bordetella pertussis virulence-associated outer membrane proteins.

Bordetella pertussis virulence-associated 30-, 32-, 90- and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30- and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.

Selección de variantes Bvg- de Bordetella pertussis / Selection of Bordetella pertussis Bvg- variants

Las cepas virulentas (Bvg+) de Bordetella pertussis expresan numerosos factores de virulencia. Estos factores están regulados por el locus bvg en respuesta a estímulos ambientales, a través del proceso reversible de modulación antigénica. A su vez, mutaciones en el locus bvg originan variantes avirulentas (Bvg-) que no expresan factores de virulencia en ninguna condición de cultivo. En este trabajo hemos seleccionado variantes espontáneas Bvg- de B. pertussis Tohama I y 10536, potencialmente útiles para el estudio de marcadores de virulencia, utilizando como medios selectivos Stainer-Scholte agarizado suplementado con 1 o/o de Casamino-acids (SSA-CAS) y Jones-Kendrick con 0,20 microgramos/ml de eritromicina (JK-Ery). Paralelamente hemos estudiado la eficiencia de recuperación de células de B. pertussis Tohama 1, 10536 y 40103 (cepas Bvg+) y de la cepa Bvg- 347 (control del fenotipo avirulento) en SSA-CAS y en Steiner-Scholte agarizado (SSA), y analizado el fenotipo de las células recuperadas a partir de ellos. Para la caracterización fenotípica se utilizaron los siguientes marcadores de fase virulenta: producción de hemólisis, producción de hemaglutininas y perfiles de proteínas de fracciones enriquecidas en membrana externa. Las tres cepas Bvg+ ensayadas mostraron diferente comportamiento en estos medios. B. pertussis Tohama 1 y 10646 no crecieron en SSA, mientras que la eficiencia de recuperación en el medio SSA-CAS fue inferior al 0,001 o/o, obteniéndose variantes Bvg- estables de la cepa Tohama 1, en cambio la cepa 10536 sufrió el fenómeno de modulación, ya que recuperó el fenotipo virulento al ser subcultivada en Bordel-Gengou. El medio JK-Ery permitió seleccionar variantes Bvg- estables de B. pertussis Tohama 1 y 10536. B. pertussis 40103 mostró alta eficiencia de recuperación en SSA y SSA-CAS y retuvo el fenotipo virulento en todos los medios ensayados. Por otra parte, B. pertussis 347, a pesar de ser avirulenta, presento una eficiencia de recuperación pobre en SSA y un crecimiento escaso en JK-Ery, corroborando que no todas las cepas Bv- adquieren la capacidad de crecer en medios que resultan inhibitorios para muchas cepas virulentas

Selección de variantes Bvg- de Bordetella pertussis / Selection of Bordetella pertussis Bvg- variants

Las cepas virulentas (Bvg+) de Bordetella pertussis expresan numerosos factores de virulencia. Estos factores están regulados por el locus bvg en respuesta a estímulos ambientales, a través del proceso reversible de modulación antigénica. A su vez, mutaciones en el locus bvg originan variantes avirulentas (Bvg-) que no expresan factores de virulencia en ninguna condición de cultivo. En este trabajo hemos seleccionado variantes espontáneas Bvg- de B. pertussis Tohama I y 10536, potencialmente útiles para el estudio de marcadores de virulencia, utilizando como medios selectivos Stainer-Scholte agarizado suplementado con 1 o/o de Casamino-acids (SSA-CAS) y Jones-Kendrick con 0,20 microgramos/ml de eritromicina (JK-Ery). Paralelamente hemos estudiado la eficiencia de recuperación de células de B. pertussis Tohama 1, 10536 y 40103 (cepas Bvg+) y de la cepa Bvg- 347 (control del fenotipo avirulento) en SSA-CAS y en Steiner-Scholte agarizado (SSA), y analizado el fenotipo de las células recuperadas a partir de ellos. Para la caracterización fenotípica se utilizaron los siguientes marcadores de fase virulenta: producción de hemólisis, producción de hemaglutininas y perfiles de proteínas de fracciones enriquecidas en membrana externa. Las tres cepas Bvg+ ensayadas mostraron diferente comportamiento en estos medios. B. pertussis Tohama 1 y 10646 no crecieron en SSA, mientras que la eficiencia de recuperación en el medio SSA-CAS fue inferior al 0,001 o/o, obteniéndose variantes Bvg- estables de la cepa Tohama 1, en cambio la cepa 10536 sufrió el fenómeno de modulación, ya que recuperó el fenotipo virulento al ser subcultivada en Bordel-Gengou. El medio JK-Ery permitió seleccionar variantes Bvg- estables de B. pertussis Tohama 1 y 10536. B. pertussis 40103 mostró alta eficiencia de recuperación en SSA y SSA-CAS y retuvo el fenotipo virulento en todos los medios ensayados. Por otra parte, B. pertussis 347, a pesar de ser avirulenta, presento una eficiencia de recuperación pobre en SSA y un crecimiento escaso en JK-Ery, corroborando que no todas las cepas Bv- adquieren la capacidad de crecer en medios que resultan inhibitorios para muchas cepas virulentas (AU)

Flagellin, a major protein present in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from Bordetella bronchiseptica Bvg- or modulated Bvg+ strains.

The bvg or vir locus positively regulates the expression of many Bordetella virulence-associated determinants (encoded by vag genes), including cell envelope proteins, in response to environmental stimuli. On the other hand, several genes named vrg genes are negatively controlled by the bvg regulon (Knapp and Mekalanos, 1988). Flagellin is encoded by a vrg gene, which is expressed when the principal virulence factors are eliminated during antigenic modulation or in phase variants (Akerley et al., 1992). We have previously analyzed SDS-PAGE profiles of Sarkosyl-outer membrane protein (OMP)-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains and reported a major band associated with the avirulent phenotype (Passerini de Rossi et al., 1995). In order to characterize this band we have purified flagellar filaments from Bvg- and modulated Bvg+ strains, and analyzed them by SDS-PAGE. These profiles revealed a single major band of 40 or 45 kDa depending on the strain. The N-terminal amino acid sequence of the putative flagellin expressed by BB7200a was identical over the first 21 residues analyzed to that of the flagellin from the modulated strain BB7865 reported by Akerley et al. (1992). Comparison of the SDS-PAGE profile of flagellar filaments with that of the OMP-enriched fraction of the corresponding strain showed that the flagellum-associated polypeptide had the same electrophoretic mobility as that of the characteristic band of the avirulent phenotype. Furthermore, this band was absent in the OMP-enriched fraction profile from a Bvg- strain subjected to a treatment that removes flagella. Our results indicate that the major protein observed in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains corresponds to flagellin.

[Selection of Bvg- variants of Bordetella pertussis]. / Selección de variantes Bvg- de Bordetella pertussis.

Bordetella pertussis virulent strains (Bvg+) produce a wide array of virulence factors. The production of these factors is coordinately regulated by the locus bvg in response to environmental signals in a process known as antigenic modulation. Mutations in the bvg locus originate avirulent variants (Bvg-) that are unable to express virulence factors regardless of growth conditions. In this paper we have obtained spontaneous variants Bvg- of B. pertussis Tohama I and 10536, which would be useful for studying virulence markers, using the selective media Stainer-Scholte agar supplemented with 1% Casamino-acids (SSA-CAS) and Jones-Kendrick with 0.20 microgram of erythromycin per ml (JK-Ery). We have also studied the efficiency of plating of B. pertussis Tohama I, 10536 and 40103 cells (Bvg+ strains) and of Bvg- strain 347 (control of the avirulent phenotype) on SSA-CAS and Stainer-Scholte agar (SSA), and we have analyzed the phenotype of the cells recovered from these media. The bacterial phenotype was characterized by using the following virulence markers: hemolysis production, hemagglutinin production, and outer membrane protein (OMP) enriched profiles. The three Bvg+ strains showed different behaviour in these selective media. B. pertussis Tohama I and 10536 could not be recovered on SSA, whereas on SSA-CAS the efficiency of plating was poor, less than 0.001%, but nevertheless allowed the selection of stable Bvg- variants of Tohama I since the OMP profile of this stain did not change after subculture in Cyclodextrin liquid medium. By contrast, strain 10536 grown on SSA-CAS suffered the process of antigenic modulation since this strain recovered the virulent phenotype when it was subcultured in Bordet-Gengou. Stable Bvg- variants from Tohama I and 10536 were obtained on JK-Ery. On the other hand, Bvg+ strain 40103 showed a high efficiency of plating on SSA and SSA-CAS and retained the virulent phenotype in all the selective media. Bvg- strain 347, in spite of being an avirulen variant, showed a poor efficiency of plating in SSA and a scant growth in JK-Ery, in agreement with the findings of other investigators which suggest that not all avirulent strains posses the ability to grow on media that inhibit most of virulent strains.

Increased prefrontal sulcal prominence in relatively young patients with unipolar major depression.

Although several studies have reported ventricular enlargement and sulcal prominence in mixed samples of patients with affective disorders (unipolar and bipolar subtypes), it is not established if these findings extend to a homogeneous sample of relatively young patients with unipolar major depression ventricular:brain ratio (VBR) and prefrontal sulcal prominence (PSP). In the present study, measures of ventricle-brain ratio (VBR) and prefrontal sulcal prominence (PSP) were compared in patients with affective disorders (n = 24, mean age = 39), medical control subjects (n = 40), patients with schizophrenia (n = 101) on ventricular : brain ratio (VBR) and prefrontal sulcal prominence (PSP). No statistically significant differences were noted in VBR in the three groups. Both patient groups had significantly greater PSP than the medical control subjects but did not differ significantly from each other. The results of the present study extend the finding of prefrontal sulcal prominence, but not ventricular enlargement, to relatively young patients with unipolar depression. Furthermore, the results of the present study suggest that patients with schizophrenia and patients with affective disorders differ only slightly or not at all in brain morphology, at the level of resolution studied.

Meta-analyses of studies of ventricular enlargement and cortical sulcal prominence in mood disorders. Comparisons with controls or patients with schizophrenia.

BACKGROUND: The findings of ventricular enlargement and increased sulcal prominence are well documented in schizophrenia, but the consistency of similar findings in mood disorders is less well appreciated. Reliable documentation of the presence of these structural abnormalities in mood disorders would require a reassessment of their significance for both schizophrenia and mood disorders. In this article, we meta-analytically review the literature on ventricular enlargement and cortical sulcal prominence in patients with mood disorders compared with controls and patients with schizophrenia. METHODS: Four meta-analytic reviews were conducted, two comparing patients with mood disorders with normal controls on ventricular enlargement (meta-analysis 1) or sulcal prominence (meta-analysis 2) and two comparing patients with mood disorders with schizophrenic patients on these same measures (meta-analyses 3 and 4). RESULTS: Meta-analyses 1 and 2 revealed statistically significant (P < .001) moderate composite effect sizes (d) for the comparisons of patients with mood disorders with controls on both ventricular enlargement (d = 0.44) and sulcal prominence (d = 0.42). Meta-analysis 3 further revealed that patients with schizophrenia have significantly greater ventricular enlargement than patients with mood disorders (P = .002), but the effect size was small (d = -0.20). There were too few studies comparing these patient groups on sulcal prominence to support a quantitative meta-analysis. CONCLUSIONS: This review documents the presence of ventricular enlargement and increased sulcal prominence in mood disorders. Patients with mood disorders have less ventricular enlargement than patients with schizophrenia, but this effect is small. These results reinforce previous suggestions of the nonspecificity of structural brain changes in schizophrenia and mood disorders.

Oral rehydration of infants in a large urban U.S. medical center.

A prospective randomized study of 100 well-nourished infants with acute gastroenteritis resulting in dehydration and acidosis was carried out at the Jackson Memorial Hospital, Miami from 1981 to 1983. Patients were randomly assigned to receive either standard intravenous therapy or oral rehydration. Infants in the latter group first received solution A containing 75 mEq/L sodium, 30 mEq/L potassium, 75 mEq/L chloride [corrected], 30 mEq/L bicarbonate, and 2 gm/dL glucose [corrected]. After ad libitum feeding for six hours, solution B containing 50 mEq/L sodium, 30 mEq/L potassium, 50 mEq/L chlorine, 30 mEq/L bicarbonate, and 3 gm/dL [corrected] glucose was given. With three exceptions (6%), oral rehydration was comparable to the intravenous regimen in clinical estimates of improvement, although the oral group had more stools in the first day. The oral group had faster correction of acidosis and a sustained rise in serum potassium concentration, whereas in the intravenous group the potassium concentration showed first a drop with a later increase, but levels were at all times below those in the oral group. Although potassium was given from the beginning of oral rehydration, and at a higher concentration than recommended by the World Health Organization, no hyperkalemia occurred. We concluded that oral therapy is safe, less expensive for patients, and more convenient for the medical and nursing staffs.