LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Seguimiento clínico, serológico y mediante la reacción de polimerasa en cadena de una familia con brucelosis / Clinical, serological and polymerase chain reaction follow-up of a family with brucellosis

Introduction: Human brucellosis diagnosis is based on isolation of Brucella spp. from blood or tissue cultures with a positivity rate of 40-70% and serology techniques are used as complementary tools; recently molecular biology diagnostic techniques have been developed intending to optimize the etiological confirmation. Aim: The main objective of this work was to compare the polymerase chain reaction (PCR), against serological diagnostic tests during the clinical follow-up of a family presenting brucellosis. Methods: Seven family members who lived in the urban area of Mexico City, were monitored using the Rose Bengal test, the agglutination test as well as agglutination with 2 mecapto ethanol, blood cultures and serum PCR for a period of 27 months. The suspected source of infection was fresh goat cheese from a known endemic zone. Results: Brucella melitensis was isolated from the blood cultures of two patients. All of the patients were positive in serological and PCR tests at the beginning of this follow-up. At the end of the study, three patients responded well to the treatment and showed negative results in the serological and PCR tests. While two patients with diabetes mellitus type 2, showed positive results in the serological and PCR tests as well as persistent symptoms. Conclusion: Clinical follow-up of patients with brucellosis is of great importance, to properly evaluate the given treatment. In this sense the PCR is a great supporting tool in diagnostic testing.

Introducción: El diagnóstico de brucelosis humana es difícil pues los cultivos de sangre y tejidos tienen un rendimiento limitado (40-70%) y usualmente se recurre a la serología como recurso complementario; últimamente se han desarrollado técnicas de biología molecular que intentan optimizar la confirmación etiológica. Objetivo: Comparar la reacción de la polimerasa en cadena (RPC) con las pruebas de diagnóstico serológicas en el seguimiento clínico de una familia con brucelosis. Métodos: Siete integrantes de una familia con brucelosis que habitaban la zona urbana de Ciudad de México fueron monitoreados mediante aglutinación con antígeno Rosa de Bengala, prueba de aglutinación, aglutinación en presencia de 2 mercapto-etanol, hemocultivos y RPC en suero durante 27 meses. La probable fuente de infección de los pacientes fue el consumo de queso fresco de cabra originario de una zona endémica. Resultados: Brucella melitensis se obtuvo del hemocultivo de dos pacientes. Todos los pacientes fueron positivos a las pruebas serológicas y al RPC al inicio del seguimiento. Tres pacientes respondieron bien al tratamiento y mostraron resultados negativos en serología y RPC al final del estudio. Mientras que en dos pacientes con diabetes mellitus tipo 2 la sintomatología fue persistente, serología positiva y RPC positivos al finalizar el estudio. Conclusión: El seguimiento clínico de pacientes con brucelosis es muy importante para valorar el tratamiento, en este sentido la RPC es una herramienta que puede apoyar a otras pruebas de diagnóstico.

Efficacy evaluation of some antibiotics against syrian brucella spp isolates, in vitro

Brucellosis is an endemic zoonosis in Syria, affecting large numbers of animals and there are an increasing number of cases in humans. The aim of this study is to investigate the in vitro efficacy of various traditional and new antibiotics against89 Brucella isolates (isolated from domestic animals) collected from different Syrian regions. Minimum inhibitory concentrations (MICs) of seventeen antibiotics were determined. Ciprofloxacin and ofloxacin were the most effective antibiotics, whereas sparfloxacin, levofloxacin, doxycycline and tetracycline had a moderate activity. In contrast, moxifloxacin and rifampicin had a low activity, while streptomycin, spiramycin and cephalosporines were ineffective. As a result, we come to the conclusion that a combination between one effective quinolone and doxycycline has a good efficacy against Brucella. Further in vivo studies are necessary to support this suggestion.

Brucellosis in India - a review.

Brucellosis is an important re-emerging zoonosis with a worldwide distribution. It is still an uncontrolled serious public health problem in many developing countries including India. Brucellosis in India is yet a very common but often neglected disease. Currently, Brucella melitensis accounts for most recorded cases globally with cattle emerging as a important reservoir with the few cases of B. suis. Isolated cases of non-terrestrial brucellosis and continuing transmission from wild animals have raised important epidemiological issues. Routine serological surveillance along with high clinical suspicion and screening of family members of index cases would be essential in delineating the real magnitude of human brucellosis in endemic countries. Increased business and leisure travel to endemic countries have led to diagnostic challenge in non-endemic areas. Laboratory testing is indispensable for diagnosis. Advances in newer rapid, sensitive, and specific testing methodologies and alternate treatment strategies are urgently needed. A safe and effective vaccine in human is not yet available. Prevention is dependent upon increasing public awareness through health education programmes and safe livestock practices. Active co-operation between health and veterinary services should be promoted. This review collates world literature and its impact to the discovery, isolation and diagnosis and epidemiology along with the control measures adapted in the Indian scenario.

Evaluación de la reacción en cadena de la polimerasa para el diagnóstico de la brucelosis en un rebaño lechero infectado con Brucellaspp / Assessment of polymerase chain reaction (PCR) to diagnose brucellosis in a Brucella infected herd

Para el diagnóstico de la brucelosis bovina en muestras de sangre y/o leche, se comparó la reacción en cadena de la polimerasa (PCR) con el aislamiento in vitro de Brucella abortus, las pruebas serológicas defijación del complemento (FC) e inmunoenzimáticas de competición (ELISA-C) en suero e indirecto (ELISA-I) en leche. Se analizaron muestras de vacas lecheras de un rebaño infectado “A”, vacunadas con B. abortus cepa 19 antes de los 8 meses de edad y revacunadas con B. abortus cepa RB51 como adultas (n= 99) y de otro “B”, libre de brucelosis (n=100), como control. En A, la PCR identificó 14 vacas infectadas con B. abortus: nueve con cepa silvestre y cinco con cepa silvestre y RB51. No se identificó B. abortus cepa 19. El biotipo 1 se aisló en un caso. Las 14 vacas infectadas con la cepa silvestre resultaron positivas en las tres pruebas serológicas. En B, por PCR no se identificó Brucella. Las pruebas serológicas mostraron una sensibilidad del 100% respecto de PCR. La especificidad para FC, ELISA-C y ELISA-I fue del 100%, 99% y 95%, respectivamente. Se concluye que la PCR sería útil como complemento de las pruebas serológicas o cuando no hay un resultado concluyente.

The diagnosis of bovine brucellosis using PCR in blood and milk samples from two dairy herds were compared to in vitro isolation, complement fixation test (CF), competitive ELISA (C-ELISA) in serum, and indirect ELISA (I-ELISA) in milk. Samples were obtained from 99 cows vaccinated with Brucella abortus strain 19, from a naturally infected herd (A), whose cows were also vaccinated with B. abortus strain RB51 as adults, and 100 from brucellosis free herd (B). In herd A, PCR identified 14 B. abortus infected cows: nine infected with wild type, and five with wild type and RB51, B. abortus S 19 was not identified. B. abortus biotype 1 was isolated from one cow. All cows infected with a wild strain of B. abortus were positive in serologic tests. Brucella was not found in herd B using PCR. Serological test showed 100% sensitivity related to PCR. The specificity for CF, C-ELISA and I-ELISA was 100%, 99% and 95% respectively. PCR could be useful to identify Brucella biotypes and to complement serologic tests.

On serch of bacterial species definition

The bacterial species concept was examined within the framework of plant and animal associated alpha-2 proteobacteria, taking into consideration the phylogenetic, taxonomic and biological approaches as well as the microbiologists' perception. The virtue of the phylogenetic approach is that it gives an evolutionary perspective of the bacterial lineage; however the methods used possess low resolution for defining species located at the terminal branches of the phylogenetic trees. The merit of the taxonomic approach is that species are defined on the basis of multiple characteristics allowing high resolution at the terminal branches of dendograms; its disadvantage is the inaccuracy in the earlier nodes. On an individual level, the qualitative biological characteristics used for the definition of species frequently reveal shortcomings because many of these properties are the result of coevolution, parallel evolution or the horizontal transfer of genes. Nevertheless, when considered together with the phylogenetic and taxonomic approaches, important uncertainties are discovered: these must be weighed if a practical definition of bacterial species is conceived. The microbiologists' perception is the criterion expressed by a group of sponsors who, based on scientific and practical grounds, propose a new bacterial species. The success of this new proposal is measured by its widespread acceptance and its permanence. A difficult problem concerned with defining bacterial species is how to distinguish if they are independent evolutionary units or if they are reticulate evolutionary units. In the first case the inherence is vertically transmitted as a result of binary fission and clonal expansion. This may be the case of some animal cell associated bacteria in which recombination appears to be precluded or exceptional. In the second case adaptive changes occurring within an individual can be horizontally transferred to many or all group members. This seems to be the condition of many intestinal and plant associated bacteria. Genetic drift and specialization in clonal bacteria will depend almost exclusively on mutation and internal genetic rearrangement processes, whereas specialization in reticulate bacteria will depend not only on these processes but in their genetic interactions with other bacterial strains. This uncertainty, which corresponds to the evolutionary process, is at the same time one of the key factors in defining a bacterial species