Genotipificación y estudio de los genes pauA y sua de aislamientos de Streptococcus uberis de mastitis bovina / Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6 % concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8 % (134/137) and 94.9 % (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95 % and 100 % with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Este estudio pretendió determinar la relación clonal entre 137 aislamientos de S. uberis obtenidos de leche de bovinos con mastitis clínica o subclínica en la Argentina, como así también la prevalencia y la conservación de los genes sua y PauA entre dichos aislamientos. Esta información es crítica para el diseño racional de una vacuna que prevenga la mastitis bovina por S. uberis. Los aislamientos se tipificaron molecularmente por amplificación al azar del ADN polimórfico (RAPD) y mediante electroforesis de campos pulsados (PFGE). Los 137 aislamientos mostraron 61 pulsotipos mediante PFGE y 25 tipos de RAPD diferentes. Los índices de Simpson calculados fueron 0,983 por PFGE y 0,941 por RAPD; esto evidencia el elevado poder discriminatorio de ambas técnicas. El análisis de la relación entre pares de aislamientos mostró un 92,6 % de concordancia entre ambas técnicas, lo que indica que cualquier par de aislamientos que fue distinguido por un método tendió a ser distinguido por el otro. La prevalencia de los genes sua y puaA fue del 97,8 % (134/137) y 94,9 % (130/137), respectivamente. Las secuencias de nucleótidos y de aminoácidos codificados por los genes sua y pauA de los 20 aislamientos de S. uberis seleccionados sobre la base de su tipo de PFGE y RAPD y origen geográfico tuvieron un porcentaje de identidad de entre 95 % y 100 % con respecto a todas las secuencias de referencia registradas en GenBank. Estos resultados demuestran que, a pesar de la diversidad clonal de S. uberis, los genes sua y pauA son prevalentes y están altamente conservados y deberían ser incluidos en futuros estudios de vacunas para prevenir mastitis bovina causada por S. uberis.

OCD and Bacterial Infection: Is there a link.

The data which addresses the validity of the proposed unique subgroup of children with early and abrupt onset of obsessive-compulsive disorder (OCD) and/or tic disorder subsequent to streptococcal infection was reviewed. The aetiology of OCD and tic disorder is unknown, although it appears that both disorders may arise from a variety of genetic and environmental factors. Post-streptococcal autoimmunity has been postulated as one such mechanism. Although much research remains to be done, an increasing body of evidence provides support for the postulate that OCD may arise from post-streptococcal autoimmunity. The unique clinical characteristic of the PANDAS sub group, the presence of volumetric changes in basal ganglia and the dramatic response to immunomodulatory treatment suggest that symptoms arise from a combination of local, regional and systemic dysfunction and further research is required in order to provide novel strategies of prevention and treatment.

Indice CPOD, capacidad amortiguadora salival, niveles salivales de Streptococcus mutans y anticuerpos IgA, en escolares de la ciudad de México / DMFT Index, salivary buffer capacity, salivary Streptococcus mutans levels and IgA antibodies in schoolchildren form Mexico city

A 139 estudiantes de 12 a 15 años de edad, se les levantó el índice CPOD y en sus salivas se determinó: la capacidad amortiguadora, cantidad de colonias de Streptococcus mutans como un grado de infección, la respuesta de anticuerpos de tipo IgA salival contra los antígenos I/II de Streptococcus mutans mediante ELISA, y la especificidad de esta reacción se corroboró mediante inmunoelectrotransferencia (western blot). El índice CPOD presentó una mediana de 4, la capacidad amortiguadora de la saliva mostró una mediana de 2, que correspondió a una capacidad amortiguadora media, la prevalencia de la infección por el microorganismo mencionado fue de 94.2 por ciento, la mediana del grado de infección fue de 2, lo que indica una infección moderada en la población de estudio y el título de anticuerpos de tipo IgA salival contra antígenos I/II de S. mutans mostró una mediana de 0.170. Al realizar una correlación bivariada de Spearman entre la capacidad amortiguadora y título de anticuerpos IgA salivales contra Streptococcus mutans, se observó una correlación positiva (r = 0.126, P = 0.042). No se encontró correlación estadísticamente significativa entre el resto de variables. Conclusión: En la población estudiada el índice CPOD mostró una mediana de 4, con respecto a la saliva: se encontró una alta prevalencia de la presencia de S. mutans, observándose un moderado grado de infección, una capacidad amortiguadora media y se observó una correlación positiva estadísticamente significativa entre la capacidad amortiguadora y el título de anticuerpos IgA salivales contra antígenos I/II de Streptococcus mutans que resultó específica al determinarla mediante inmunoelectrotransferencia.

Vacinas em desenvolvimento: estreptococo do grupo B, herpes-zóster, HIV, malária e dengue / Vaccines under development: group B streptococcus, herpes-zoster, HIV, malaria and dengue

OBJETIVOS: As vacinas contra o estreptococo B, o herpes-zóster, o HIV, a malária e a dengue, selecionadas por critérios de comercialização iminente ou devido a problemas específicos para sua obtenção, foram objeto de uma revisão sobre o estado atual do seu desenvolvimento. FONTE DOS DADOS:Foi realizada revisão da literatura através da MEDLINE no período de 1996 a 2006, sobre a epidemiologia e imunologia das doenças, analisando tanto os maiores problemas para a obtenção de uma vacina como o estado atual dos estudos, com ênfase para os que estavam em fase mais adiantada. SíNTESE DOS DADOS: Cada uma das cinco doenças escolhidas apresenta problemas específicos para o desenvolvimento de uma vacina. No entanto, a maioria deles já foi ou está em vias de ser resolvido, permitindo prever que uma vacina - ou vacinas - eficaz e segura estará disponível em futuro próximo. CONCLUSÕES:Apesar dos problemas enfrentados para o desenvolvimento dessas vacinas, os avanços da biologia molecular e da imunologia permitiram superar a maioria deles, abrindo a perspectiva para a obtenção de novas vacinas.

Construction of recombinant polypeptides based on beta antigen C (Bac) protein & their usage for protection against group B streptococcal infection.

BACKGROUND & OBJECTIVES: immunocompromized adults. Approximately 50 per cent of the GBS strains carry and express the gene of BAC antigen which is capable to bind IgA. Gene encoding for the BAC antigen has been cloned and sequenced but actual IgA binding region on the protein has not been detected. The aim of the present work was to localize the region of IgA binding on Bac protein, to evaluate the role of one of the Bac protein regions MLKKIE in IgA binding, and to investigate the ability of Bac based recombinant proteins to generate protective antibodies against GBS infection. METHODS: Recombinant proteins based on beta antigen C were generated after PCR amplification of the fractions of bac gene with the following cloning of the PCR products into expression plasmids. Recombinant peptides were tested for IgA binding by immunoprecipitation and Western blot. One of the recombinant proteins expressing IgA binding was used as an antigen for immunization of mice and for GBS protection studies. RESULTS: Several bac gene constructs were generated. Their ability to bind IgA varied dramatically depending on the size of the construct and location of the fragment on the bac gene map. The smallest peptide expressing IgA binding was 14 kD in size. Amino acid substitutions in MLKKIE region facilitated IgA binding ability. Immunization of mice with recombinant Bac based peptide induced the appearance of anti-GBS antibody with high affinity level providing protection against GBS infection. INTERPRETATION & CONCLUSION: Size dependence of Bac based recombinant peptides proved that the effective IgA binding required specific folding of the protein binding IgA. Region MLKKIE could not be considered as region, responsible for IgA binding. Generation of antibodies against Bac based recombinant peptides with high titre and affinity makes these proteins a potent candidates for generating a vaccine against GBS infection.

Nasal associated lymphoid tissue & M cells, a window to persistent streptococcal infections.

BACKGROUND & OBJECTIVES: Intranasal infection of mice has been used as a model of streptococcal pharyngitis, as nasal associated lymphoid tissue (NALT) in this animal is structurally and functionally analogous to human tonsils. The present study was carried out to determine whether group A streptococci preferentially colonized or invaded NALT. METHODS: Lux(+) strain Sp3 was created and exponential phase bacteria were introduced intranasally into BALB/C female mice, total photon emission from selected areas was quantified, sections of NALT tissues were used for immunofluorescent staining and M cell staining. RESULTS: Intranasal infection of mice with bioluminescent group A streptococci or unlabeled streptococci demonstrated that NALT was a primary target of this pathogen. Streptococci readily gained access to the blood stream from this site of infection. Immunofluorescence microscopy studies showed that M cells, dispersed along the mucosal epithelium adjacent to NALT, were preferentially infected and likely to provide the window through which streptococci reached the underlying tissue. INTERPRETATION & CONCLUSION: The present study suggests that this upper respiratory Gram positive pathogen uses a mechanism similar to that of enteric pathogens in the intestine to gain access to underlying tissue, lymphatics and blood.

Human immunogenicity studies on group A streptococcal C5a peptidase (SCPA) as a potential vaccine against group A streptococcal infections.

BACKGROUND & OBJECTIVES: Group A streptococcal C5a peptidase (SCPA) is a major virulence surface factor. Its highly conserved nature among all tested serotypes of group A streptococci (GAS) as well as animal protection studies make SCPA a prime vaccine candidate. The present study was undertaken to explore the human immunogenicity to SCPA using an indirect enzyme-linked immunosorbent assay. METHODS: Children (n=72) who had signs and symptoms of acute pharyngitis and had GAS isolated from the throat at initial visit were included. Acute and convalescent sera were collected 4 weeks apart. ELISA was performed using recombinant SCPA peptide as antigen. RESULTS: The mean convalescent anti-SCPA level was twice the level of mean acute anti-SCPA and the difference was statistically significant (P < 0.0001). There was a rise in convalescent anti-SCPA in all children aged 2-12 yr. INTERPRETATION & CONCLUSION: Our observations confirmed that SCPA was highly immunogenic in children infected with group A streptococcal pharyngitis. Further studies need to be done to characterize the immune response including antibody subclass.

Diversidad clínica de las infecciones invasivas por streptococcus pyogenes y asociación con el superantíngeno SpeA / Clinical diversity of invasive infections by streptococcus pyogenes and SpeA superantigen association

Se presenta una serie de 5 casos con infecciones invasivas por S. pyogenes observados durante 1999, con el objetivo de ilustrar la diversidad clínica de estas infecciones. Todos los pacientes presentaron bacteremia y tenían condiciones mórbidas asociadas. Tres pacientes presentaron shock, dos de ellos de tipo tóxico y el mismo número de pacientes presentó manifestaciones cutáneas. Dos pacientes tuvieron alteraciones de coagulación. Todos los casos tenían leucocitosis (valor promedio 16800 por mm3) y aumento de la proteína C reactiva (300 mg/L en promedio). Los valores de desviación a izquierda y eritrosedimentación presentaron una amplia variación en sus resultados, observando pacientes con cifras normales. Cuatro pacientes recibieron tratamiento quirúrgico y 3 pacientes gamaglobulina endovenosa. Tres pacientes fallecieron (dos de ellos a las 72 horas). Los pacientes que sobrevivieron tuvieron una hospitalización prolongada que superó el mes de duración. Todos los aislamientos bacterianos portaban el gen que codifica por el superantígeno SpeA

Humoral immune response in a sample of Bangladeshi children with rheumatic fever.

Several researchers have investigated cellular response, and antibody response to different antigens of group A beta-haemolytic streptococci, cardiac tissues and B cell alloantigen in rheumatic fever (RF). However, immunoglobulins (Ig) which may indicate general immune status did not receive attention. This study was done in the outpatient clinic of the National Center for Control of Rheumatic Fever and Heart Diseases, Dhaka, to compare Ig levels in subjects with and without RF who have had recent group A beta-haemolytic streptococcal infections. We have recruited 44 RF cases aged 5 to 20 years, and 44 subjects without RF were randomly matched for age and sex as controls. Convalescent blood samples were used to measure antistreptolysin O and IgG, IgM, and IgA levels. The cases, as compared with the controls, had significantly higher levels of antistreptolysin O (mean 399 versus 321 IU/ml), IgG (mean 2386 versus 1885 mg/dl), IgM (mean 286 versus 222 mg/dl) and IgA (mean 258 versus 184 mg/dl). It is not clearly known why the immune response is higher in the RF cases. We have to elucidate factors responsible for higher immune response in children with RF.

Recognition of Streptococcus pyogenes and skin autoantigens in Guttate psoriasis

Background. Guttate psoriasis is associated with infections by Streptococcus pyogenes and cross-reaction between skin and streptococcal antigens have been reported, suggesting an autoimmune component in the disease. Methods. In this work, the authors looked for antibodies against S. pyogenes M-5 antigens by immunoblot in 52 sera of psoriasis patients and in 52 sera of normal individuals. Histological and immunohistochemical analysis in skin biopsies from lesions of another group of 16 clinically diagnosed guttate psoriasis patients and four healthy controls were also carried out. Results. All guttate psoariasis patients studied (11) had IgG antibodies that intensively recognized three different proteins of 70,60 and 14 kDa, as compared to sera from patients with other forms of psoriasis or from healthy controls. The diagnosis of psoariasis was confirmed in 14 of the patients by hematoxylin-eosin staining. Of the other two patients, one was diagnosed as parapsoriasis and the other as liquen. By indirect immunofluorescence (IFI), all 14 psoriatic patients had autoantibodies against their own lesional skin that did not recognized normal skin from control subjects or from the two non-psoriatic patients. The parapsoriatic and the liquen patients did not have autoantibodies. A rabbit immune serum against S. pyogenes antigens reacted with lesional skin from the 14 guttate psoriatic patients, but not with normal skin from controls or with lesional skin from the 2 non-psoriatic patients. Conclusions. The recognition by immunoblot of streptococcal antigens by serum of guttate psoriasis patients, the presence of autoantibodies against their own skin, and recognition of the same skin antigens by anti-streptococcal rabbit antibodies confirm the participation of the immune system and of streptococcal infection in guttate psoriasis

Streptococcus pyogenes: vigilancia de su resistencia a los antibióticos en un hospital pediátrico / Streptococcus pyogenes: surveillance of resistance to antimicrobial at a pediatric hospital

En varios países del mundo comenzaron a aislarse en forma creciente cepas de Streptococcus pyogenes resistentes a eritromicina. Este trabajo tuvo por objeto verificar la sensibilidad a penicilina, lincosamidas y macrólidos de los estreptococos del grupo A aislados en nuestro hospital entre 1989 y 1994. Se estudió un total de 373 cepas aisladas principalmente de exudados faríngeos por el método de dilución en medio sólido. Ninguna cepa resultó resistente a penicilina y sólo en 1989 se aislaron cepas resistentes a eritromicina (1,6 por ciento para ese año). Las dos cepas resistentes presentaron un patrón de sensibilidad compatible con el mecanismo MLS inducible. En conclusión, S. pyogenes continúa siendo uniformemente sensible a penicilina mientras que tanto la eritromicina como los nuevos macrólidos y las lincosamidas mantienen su efectividad in vitro como para seguir siendo considerados como alternativas para el tratamiento de infecciones estreptocócicas en nuestro medio

Caracterización de tolerancia a penicilina frente a Streptococcus equisimilis (S. ß-hemolítico grupo C) y Streptococcus canis (S. ß-hemolítico grupo G) / Penicillin tolerance of Streptococcus equisimilus and streptococcus canis

Se seleccionaron al azar 18 cepas de S. canis (S. ß-hemolítico grupo G colonia grande) y 13 de S. equisimilis, aisladas de materiales clínicos de pacientes pediátricos. Se estudió, en este grupo, los fenómenos de tolerancia y actividad bactericida, por medio de curvas de letalidad, frente a penicilina (CIM50: 0,007 mg/l, CIM90: 0,015 mg/l) a concentraciones de 0,5 µg/ml y 8 µg/ml y tiempo de exposición de 5 hs y 24 hs. El porcentaje de cepas tolerantes a la penicilina de S. canis fue 28 por ciento (5/18) y 17 por ciento (3/18) a 0,5 µg/ml y 8 µg/ml respectivamente. Sólo se observó actividad bactericida con 0,5 g/ml de penicilina, en el 17 por ciento de las cepas de S. canis (3/18) a las 5 hs de exposición al antibiótico; esta cifra se elevó al 50 por ciento cuando se utilizó 8 µg/ml. No se obtuvo tolerancia en ninguna de las 13 cepas de S. equisimilis; sin embargo, en 5 de estas cepas no fue posible alcanzar actividad bactericida a las 5 hs con 0,5 µg/ml de penicilina. Este trabajo sugiere que la actividad bactericida de la penicilina en S. canis y S. equisimilis es concentración dependiente frente a algunas cepas, mejorando con el incremento de las mismas y con el tiempo de exposición al antibiótico (24 hs). Consideramos importante la realización de estudios clínicos, tendientes a establecer la trascendencia de este fenómeno en las infecciones producidas por estos microorganismos

Acute rheumatic fever another scope at immunological profile

Sero-immunological study was carried out on 111 patients with acute rheumatic fever [ARF] in comparison with patients' controls and normal group. Using streptococcal antibodies tests [antistreptolysin O [ASO], antistreptokinase [ASK] and antideoxyribonucleotidase B [ADNAseB tests]. Seropositivity for the antecedent streptococcal infection was raised from about 79.2% using the conventional antistreptolysin O [ASO] test to as high as 96.3% when two additional streptococcal antibody test was measured. In chorea patients ADNAse B was proved to be the only marker for the detectipn of the antecedent streptococcal infection. Mean serum ASO, ASK and ADNAse B titers in individual manifestation of ARF patients were studied

El futuro de una vacuna antiestreptocócica / The future of a antistreptococcus vaccine

Para poder comprender los esfuerzos recientes encaminados a la elaboración de una vacuna antiestreptocócica es necesario revisar con mayor detenimiento la estructura molecular de la proteína M. Esta proteína sobresale de la célula estreptocócica, su extremo más alejado del punto de inserción en el estreptococo es el extremo N-terminal, mientras que el más cercano a la superficie celular corresponde al C-terminal. Este extremo tiene 19 aminoácidos hidrofóbicos que actúan como ancla de la proteína M en la membrana del germen. La región N-terminal tiene los epítopos variables responsables de la especificidad de tipo. Un factor importante a considerar en la elaboración de una vacuna tipo específica basada en epítopos, es el potencial del estreptococo para generar nuevos serotipos M, al modificar la porción N-terminal. A medida que los bloques de aminoácidos, designados A, B, C, y D (a partir del punto más alejado de la célula estreptocócica al más cercano), se aproximan más a la superficie, se vuelven más homogéneos. La región de aminoácidos del bloque C del estreptococo M-6, es la que se repite más consistentemente entre los diferentes serotipos distintos al M-6. Todo lo anterior es importante para elaborar una vacuna sintética basada en los epítopos inmunodeterminantes: es necesario identificar los epítopos con variación antigénica mínima, deben evitarse los epítopos de la proteína M que producen anticuerpos que reaccionan cruzadadamente con tejido cardíaco y debe incluír a los serotipos prevalentes en la comunidad, asociados a la fiebre reumática y a otras complicaciones supurativas y no supurativas