Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Serum Levels of Interferon Gamma in Patients with Brucellosis in a Saudi Hospital.

Background: Brucellosis is a major zoonotic disease that is endemic in Saudi Arabia and it remains a major health problem that has not been eradicated in the country yet. Place and Duration of Study: This retrospective study was conducted in a Saudi Hospital at Al Madinah city during the period of 1 November, 2010 to 31 October, 2011. Methodology: All sera of patients suspected to have brucellosis (n= 65) and 18 healthy subjects were tested for brucella antibody using slide latex agglutination (SAT) and ELISA. Quantitation of IFN-ɣ was also done using ELISA. Results: Brucellosis was detected in all age groups but the incidence was higher and reached 33.3% in age group (40- <50) years with average of 43.9±2.53 years. Male to female ratio in infected patients was 2:1 by using SAT. The incidence of seropositive cases was high (80.1%) in the three months (April, May and June), with the highest peak in May (46.7%). Drinking raw milk was the most encountered risk factor with a prevalence of 66.1% followed by consumption of milk products (11.9%). The most prevalent species among the examined cases was B. melitensis (93.3%). Among the studied cases, 60 cases (92.3%) were serologically positive for brucellosis by SAT. Among the 60 cases yielding significant titers against brucella, 14 sera (23.3%) had agglutinin levels of 1:80, 34 sera (56.7%) had titers of 1:160 and 12 sera (20%) had titers of 1:320. By estimating IgM and IgG levels in the sera of examined cases using ELISA, 52 cases (80%) had brucellaIgM while 42 cases (64.6%) had brucella IgG. Sensitivities of SAT, IgM ELISA and IgG ELISA were 91.5%, 88.1% and 71.2%, respectively compared with combined ELISA. Mean IFN-ɣ levels ± SD in the subacute phase was 136.7±70.07pg/ml, 120.2±54.25pg/ml in the acute phase, and 121.3±51.09 pg/ml in the chronic phase of brucellosis. Conclusion: The sensitivity and specificity of ELISA to diagnose human brucellosis was higher when combined ELISA (IgM/IgG or both) was used. Mean IFN-ɣ levels were lower, but not significantly, in the chronic phase of the disease than in the sub acute phase and healthy subjects.

Diseño y evaluación de una prueba de PCR para detección de Brucella spp. y Brucella abortus / Design and evaluation of a PCR test for detection of Brucella spp. and Brucella abortus / Desenho e avaliação de um teste de PCR para a detecção de Brucella spp. e Brucella abortus

Resumen Problema: el diagnóstico oportuno de la brucelosis, zoonosis que afecta ganado y trabajadores pecuarios, es difícil. Las pruebas serológicas Rosa de Bengala y ELISA, el hemocultivo y mielocultivo, tienen sensibilidades entre 15-70% dependiendo del estadío de la infección. El desarrollo de métodos diagnósticos rápidos, sensibles y específicos utilizando técnicas moleculares como la PCR, permite diagnosticar y tratar oportunamente esta enfermedad. Objetivo: Desarrollar y evaluar dos pruebas de PCR para detección de Brucella spp. y B. abortus. Materiales y métodos: se diseñaron y evaluaron iniciadores para detectar el gen ugpA utilizando Primer3, y otros reportados en la literatura. Se calculó sensibilidad y especificidad, usando 3 grupos (G) de muestras humanas y bovinas utilizando el teorema de Bayes: G1:30 muestras de suero y 30 de sangre de humanos sanos y 30 de suero y 30 de sangre de bovinos sanos, inoculadas con Brucella abortus; G2: 30 muestras de suero y 30 de sangre de humanos sanos y 30 de suero y de 30 sangre de bovinos sanos inoculadas con Salmonella Typhi, Escherichia coli, Klebsiella sp., Psudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae y Enterococcus sp. G3: 60 muestras de suero y 60 sangre de bovinos y 60 muestras de suero y 60 sangre de humanos asintomáticos. Resultados: se obtuvo una sensibilidad y especificidad de detección de Brucella sp. y Brucella abortus del 100% en muestras humanas y bovinas. Conclusiones: por la alta sensibilidad y especificidad encontradas en las pruebas de PCR estudiadas, se recomienda continuar un estudio clínico de aplicación para evaluar su comportamiento en muestras de pacientes y animales infectados.

Abstract Problem: brucelosis is a zoonosis that affects livestock and livestock workers. This disease is characterized by the difficulties for its early diagnosis. Sensitivity of marrow and blood cultures as well as serological tests (Rose Bengal and ELISA) ranges between 15 and 70%, depending on the stage of infection. Development of rapid, sensitive, and specific diagnostic methods using molecular techniques such as PCR would allow timely diagnose and treatment of the disease. Objective: To develop and evaluate two PCR tests for detection of Brucella spp. and B. abortus. Materials and methods: primers to detect ugpA gene were designed and evaluated using Primer3 and others reported in the literature. Sensitivity and specificity were calculated for 3 groups (G) of human and bovine samples using Bayes' theorem. G1 consisted of healthy human and healthy bovine samples (30 serum and 30 blood samples of each species); bovine blood samples were inoculated with Brucella abortus. G2 consisted of healthy human and healthy bovine samples (30 serum and 30 blood samples of each); bovine blood samples were inoculated with Salmonella typhi, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Enterococcus sp. G3 consisted of 60 serum and 60 blood samples from asymptomatic bovines, as well as 60 serum and 60 blood samples from asymptomatic humans. Results: sensitivity and specificity of the PCR test to detect Brucella sp. and Brucella abortus reached 100% in human and bovine samples. Conclusiones: considering the high sensitivity and specificity observed in the PCR tests studied, we recommend doing a follow up of the test in a clinical trial to evaluate its performance in infected humans and animals.

Resumo Problema: é difícil o diagnostico oportuno da brucelose, zoonose que afeta o gado e os trabalhadores pecuários. Os testes serológicos Rosa de Bengala e ELISA, e a hemocultura e mielocultura têm sensibilidades entre 15 e 70% dependendo do estádio da infecção. O desenvolvimento de métodos diagnósticos rápidos, sensíveis e específicos utilizando técnicas moleculares como a PCR, permite diagnosticar e tratar oportunamente esta doença. Objetivo: desenvolver e avaliar dois testes de PCR para a detecção de Brucella spp. e B. abortus. Materiais e métodos. Desenharam-se e avaliaram-se iniciadores utilizando o software Primer3 para detectar o gene ugpA, também se avaliaram outros iniciadores reportados previamente na literatura. Calculou-se a sensibilidade e especificidade do teste por PCR usando três grupos (G) de amostras humanas e bovinas utilizando o teorema de Bayes: G1: 30 amostras de soro e 30 de sangue de humanos sãos e 30 de soro e 30 de sangue de bovinos sãos, inoculadas com Brucella abortus; G2: 30 amostras de soro e 30 de sangue de humanos sãos e 30 de soro e de 30 sangue de bovinos sãos inoculadas com Salmonella Typhi, Escherichia coli, Klebsiella sp., Psudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae e Enterococcus sp. G3: 60 amostras de soro e 60 de sangue de bovinos e 60 amostras de soro e 60 sangue de humanos assintomáticos. Resultados. Obteve-se uma sensibilidade e especificidade de detecção da Brucella spp. e Brucella abortus de 100% em amostras humanas e bovinas. Conclusões: pela alta sensibilidade e especificidade encontrada com o teste de PCR estudado, recomenda-se continuar um estudo clínico de aplicação para avaliar seu comportamento em amostras de pacientes e animais infetados.

Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen / Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin

Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus.

Diagnostic Experience in the 3 Human Brucellosis Cases by the Microbiologic, Serologic and Gene Tests / 대한임상미생물학회지

Brucellosis is a zoonosis caused by Brucella species. B. melitensis, B. suis, B. abortus and B. canis can infect humans. Recently, as the cases of bovine brucellosis have increased every year in Korea, the cases of human brucellosis have also increased among livestock workers and veterinarians in rural areas, since the first human case was reported in 2003. Because clinical manifestations of the disease are nonspecific and may be very atypical, clinicians and laboratory persons need to be active in using diagnostic tools including polymerase chain reaction in addition to the ordinary culture and serologic tests, and taking an appropriate measure to prevent intralaboratory infection. We report herein our experience in three human brucellosis cases diagnosed by cultures, serologic tests and gene detection.