Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests.

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l'Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.

Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats.

The polymerase chain reaction (PCR) was used to diagnose goat brucellosis and compare its sensitivity against some of the most commonly used serological and bacteriological techniques. Twenty two female and one male out of 300 clinically healthy, mixed-breed goats were randomly chosen from a ranch located at Marín, Nuevo León, Mexico. Milk and blood samples were taken from each animal and used to obtain both microbiological cultures and DNA of the pathogen, and sera was tested against Rose Bengal antigen (RBT). Results showed that 86% of the blood samples were positive on the PCR test, while 60% were positive on the serological test. The pathogen was isolated from only one blood culture. Sixty four percent of the milk samples were positive on PCR tests, but failed to yield bacteria in culture. Biochemical and PCR specific assay demonstrated that Brucella abortus biovar 1 was associated with the infection. This study demonstrates the higher sensitivity of PCR over RBT and blood culture and its potential towards a rapid identification of Brucella strains.

Single-step PCR for detection of Brucella spp. from blood and milk of infected animals.

A versatile method for the extraction of Brucella DNA and PCR are presented as reliable tools for the detection of Brucella spp. from body fluids of infected animals. Two oligonucleotides homologous to regions of the gene encoding for an outer membrane protein (omp-2) were designed to detect the pathogen from milk and/or blood of infected goats, bovines, and human patients. The sensitivity of our test and its ability to detect the pathogen in samples from the field reveal a promising advance in the diagnosis of brucellosis in animals and humans.

[Haemophilus influenzae infections in 2 hospitals in the city of Puebla, Mexico]. / Infecciones por Haemophilus influenzae en dos hospitales de la ciudad de Puebla, México.

Haemophilus influenzae has been recognized as one of the most important pathogen in the pediatric population younger than 5 years old. We analized some characteristics of the infectious diseases by Haemophilus influenzae in the pediatric group from 1985 to 1990 in two hospitals of Puebla city. From 321 children studies cases included for this work, fifty of those had infectious diseases by H. influenzae. Forty six percent of the patients developed meningitis, 22% otitis media, 12% sepsis, 10% pneumonia and 10% other infectious diseases. The majority of cases occurred in males but the difference was not statistically significant (p > 0.05). Eighty percent of diseases by H. influenzae occurred in infants younger than 24 months. The serovar b was the most frequent in invasive infections and nontypable strains were frequent in otitis media. The biovars I and IV were isolated from invasive infections. During this study two children died with meningitis and 3 patients had severe neurological damage. This finding suggests that is necessary to vaccine this population to prevent the morbilethality of infectious diseases by Haemophilus influenzae.

[Survival of Listeria monocytogenes in milk fermented with a starter culture for making yogurt]. / Sobrevivencia de Listeria monocytogenes en leche fermentada con un cultivo iniciador para elaborar yogur.

The purpose of this research was to determine the survival of Listeria monocytogenes in sterile skim milk during the fermentation with a yogurt starter culture and during storage at refrigeration temperature. Sterile skim milk was inoculated with 10(3), 10(5) and 10(7) cfu/ml of L. monocytogenes and with 10(6) cfu of lactic acid bacteria. Inoculated milks were fermented for 8 h at 42 degrees C, followed by refrigeration at 4 degrees C. Samples were taken at 2 h intervals during fermentation and at 2 days intervals during storage. Acidity and pH were measured, as well as viable count of lactic acid bacteria and pathogen. L. monocytogenes survived 8h, 10 days and 32 days in the fermented milk, when the inocula were 10(3), 10(5) and 10(7) cfu/ml, respectively. Inhibition of the pathogen was associated with a decrease of pH below 4.0 and increase in acidity. It was demonstrated that this pathogen is able to survive several weeks in milk fermented with a starter culture, contrary to the general belief which considered it very difficult due to the low pH. Therefore fermented milks may play an important role in the transmission of these bacteria.

Cell response to a salt-extractable and sonicated Brucella melitensis 16M antigen in human brucellosis.

We compared the immunological responses of leukocytes taken from healthy negative controls, laboratory workers immunized with the phenol-insoluble French vaccine against brucellosis, patients acutely ill with brucellosis, and patients chronically infected with Brucella melitensis. A salt-extractable antigen (protein-rich but with traces of lipopolysaccharide) and a sonicated suspension from B. melitensis 16M were used as antigens for in vitro lymphocyte proliferation test. Quantitation of T cells showed that the ratio of CD4+/CD8+ cells decreased as the condition of the patient deteriorated. An assay to quantitate the cell-mediated immunity showed that the activities of mononuclear cells stimulated with concanavalin A increased as the disease progressed as well.

Molecular detection of Brucella spp.: rapid identification of B. abortus biovar I using PCR.

Brucellosis is an important zoonotic disease caused by several species of the genus Brucella. The most reliable diagnostic tests are based on microbiological analysis, bacterial growth, and biochemical reactions which are cumbersome and represent a risk of infection for technicians performing them. Recently, safe molecular genetic techniques have been incorporated in studies regarding taxonomy and evolution of Brucella. The polymerase chain reaction (PCR) was used here to analyze a duplicated gene (omp 2) encoding an outer membrane protein in members of the genus Brucella. The developed procedure detects each of the five species herein tested and their biovars: B. abortus, B. suis, B. melitensis, B. canis and B. ovis. It also permits the detection of an approximately 115 bp deletion in the omp 2a gene, consistently found in five different strains of B. abortus biovar 1: vaccine strain S19, rough mutant RB51, reference strains 544 and A99, and a strain isolated from naturally infected bovines.

[Seroepidemiology of brucellosis in Mexico]. / Seroepidemiología de la brucelosis en México.

Brucellosis is an important and widely distributed zoonosis in Mexican cattle which also affects an unknown proportion of the human population. This report presents the brucellosis antibody levels registered in the National Seroepidemiology Survey (NAS) in sera obtained from 66,982 healthy persons from one to 98 years of age and determined by the test of plaque microagglutination. Seroprevalences by states ranged from 0.24 per cent in Morelos to 13.5 per cent in the state of Mexico. The national mean was estimated to be 3.42 per cent. The analysis showed no statistical differences for brucellosis antibody levels by urban and rural residence and by density of family sleeping areas (three or more persons vs. one or two persons per bedroom). Adults between 20 and 39 years of age had greater seropositivity and children from one to nine years had the least. Women were most affected and had 48 per cent more seropositivity than men. According to the information obtained in the study, brucellosis in Mexico has the following characteristics: it is related to gender but not to occupation; affects persons in all age groups, social strata and is independent of size of the community of residence. Historically, brucellosis has been an endemic disease in Mexico. Recently an increasing incidence has been reported, and this is possibly due to a better national notification system.

Prueba rapida para el diagnostico de la oncocercosis. / Rapid test for the diagnosis of onchocerciasis

Se describe una aplicacion del metodo de fijacion en superficie y retencion del colorante para reconocer la presencia de anticuerpos y antigeno circulante en el suero de pacientes oncocercosos. La especificidad de la prueba se confirma por haber observadp reacciones negativas empleando sueros de diversas condiciones clinicas prevalentes en el Distrito Federal, quedando pendientes investigaciones con suero de pacientes no oncocercosos de las mismas zonas tropicales donde la filariasis a que nos referimos ha sido endemica

Immunization by an insoluble fraction extracted from Brucella melitensis: immunological and chemical characterization of the active substances.

A peptidoglycan-containing fraction called fraction P.I. (phenol insoluble), extracted from Brucella melitensis and previously described by some of us, had immunogenic and protective properties and did not produce any allergic reactions. Since it is well known that bacterial peptidoglycans studied so far have immunoadjuvant properties, the isolation of the active factor(s) of Brucella was undertaken. By successive enzymatic and chemical treatments, a new, much more purified fraction, called "4A" (approximately 5% of fraction P.I.), is obtained, retaining the same properties as P.I. and giving better protection against infection by Brucella. Immunogenicity, immunoadjuvant activity, allergizing capacity, and specific and nonspecific protective effects of fractions P.I. and 4A are compared. Chemically, fraction 4A is constituted by a lipoprotein covalently linked to peptidoglycan and by a few (lipo)proteins that could be solubilized by hot sodium dodecyl sulfate. Intrinsic properties of peptidoglycan could not be studied, but it does not seem to be essential for the activity. In conclusion, fractions P.I. and 4A are not agglutinogenic and, since fraction 4A induces better protection against infection by Brucella, it could advantageously replace fraction P.I. as a vaccine for humans.