A real-time quantitative PCR based on molecular beacon for detecting Brucella infection / PCR quantitativo em tempo real, baseado em guia molecular para detecção de infeção por Brucella

O objetivo deste comunicado é desenvolver um método quantitativo PCR em tempo real, baseado em guia molecular (MB) (MB-qPCR) para detecção de infecção por espécies de Brucella, e avaliar seu potencial de utilização clínica. Os primers e as sondas MB foram desenhados para amplificação específica e determinação de sequência conservada do código do gene para os primeiros 58-aa da proteína de membrana externa OMP-2a, que é compartilhada em cinco espécies de Brucella epidêmicas. A avaliação metodológica foi realizada por análise de sensibilidade, especificidade, coeficiente de variação intra e inter, e a linearidade do qPCR. O potencial diagnóstico foi avaliado comparando-se o método qPCR desenvolvido com ensaios de exames bacteriológicos convencionais, incluindo os testes de soroaglutinação convencionais (SATs) e os testes do Rosa Bengala (RBPTs). O método exibiu alta sensibilidade (tão baixo quanto 50 cópias) e grande faixa de linearidade (102-108 cópias). Nenhuma reação cruzada foi encontrada com bactéria clínica comum. A sensibilidade diagnóstica foi superior ao exame bacteriológico, e a especificidade diagnóstica foi superior ao SAT ou ao RBPT. Um método MB-qPCR altamente sensível e específico para DNA de Brucella foi estabelecido com sucesso, provando ser uma ferramenta útil no diagnóstico molecular de brucelose.(AU)

Preliminary Comparative Assessment of Brucellergene Skin Test for Diagnosis of Brucellosis in Dromedary Camels (Camelus dromedarius).

This study was conducted to evaluate the use of Brucellergene skin test (BST) for the diagnosis of Brucellosis in camels (Camelus dromedarius) in comparison with Rose Bengal test (RBT) and competitive enzyme-linked immunosorbent assay (c-ELISA). A total of 68 apparently healthy adult dromedary camels of either gender from three different geographical locations of Abu Dhabi Emirate, United Arab Emirates (UAE), were included in the study. The skin test was applied on two shaved areas at the middle of the neck: one for the test and the other area was injected with normal saline as a control. Reading was done 72 h postinjection. Results were subjected to Bayesian analysis to assess the test performances in camels. The model estimated the following sensitivity and specificity median values: BST: Se = 70.72%, Sp = 98.82%; RBT: Se = 93.27%, Sp = 97.79%; and c-ELISA: Se = 94.78%, Sp = 98.48%. As the BST investigated in this study proved to be a highly specific test, we propose using it as a confirmatory test in camels particularly when the serological tests give doubtful results on individual animals.

Brucella melitensis B115-based ELISA to unravel false positive serologic reactions in bovine brucellosis: a field study.

BACKGROUND: Brucellosis is a zoonosis whose incidence is not declining worldwide despite the global effort to control the disease. Accurate and precise diagnosis is a crucial step in any prophylaxis program but single tests to unequivocally detect animals infected with Brucella spp. are currently unavailable. In Italy, serological diagnosis of bovine brucellosis is performed with two official tests: a rapid agglutination test (i.e., Rose Bengal Plate test, RBPT) and a complement fixation test (CFT) that detect antibodies directed mainly to the smooth lipopolysaccharide (S-LPS). Neither of the two tests is able to avoid the detection of false positive serological reactions (FPSRs) caused by bacteria sharing S-LPS components with Brucella spp. and responsible for the single reactors (SR) phenomenon. A B. melitensis R strain-based ELISA showed a good diagnostic performance in unravelling FP animals; however, since a limited number of animals were analyzed in that study, a large field study was conducted here to discriminate between Brucella-infected from FP animals, with the final aim of reducing the unnecessary slaughter of the latter. An ELISA based on a R strain of Brucella, i.e., Brucella melitensis B115, was employed to measure specific IgG responses in a collection of bovine sera (n = 648). Sera were obtained from 180 farms (either officially brucellosis-free or not brucellosis-free) recruited during an extended period of time (2014-2018) and were preliminarily assayed with the official tests by the Italian Reference Centers and then subjected to the ELISA. RESULTS: Negative sera, when subjected to the ELISA, gave O.D. values below the cutoff; SR sera, i.e. RBPT positive and CFT negative, as well as double positive (DP) sera, i.e. RBPT and CFT positive, gave O.D. values that were below the cutoff. All positive sera, i.e. from Brucella-infected animals, were RBPT positive and CFT positive (ICFTU ranging from 20 to 1280) and gave ELISA O.D. values above the cutoff. CONCLUSIONS: The B. melitensis B115-based ELISA systematically unravelled all false positive (FP) sera while confirming the diagnosis in Brucella-infected animals. Thus, the test employed in the present study may complement the official assays to avoid the costly slaughter of FP animals.

Performance of an Immunochromatographic Test (ICT) in Comparison to Some Commonly Used Serological Tests for the Diagnosis of Brucellosis in Dromedary Camels (Camelus dromedarius).

Serological tests may represent an essential tool for the diagnosis of camel brucellosis; however, concerns arise in the scientific community regarding the direct transposition from cattle and small ruminants without adequate validation. The present study was made to compare four serological tests for the diagnosis of brucellosis in dromedary camels (Camelus dromedarius). In terms of sensitivity, our results show that the Immunochromatographic Test (ICT) shows the higher value of sensitivity, 98.67% (95% Confidence Level (C.L): 94.36%-99.99%), followed by the Fluorescence Polarization Assay (FPA) with 95.05% (95% C.L: 88.23%-99.51%), then the Competitive Enzyme-Linked Immunosorbent Assay (c-ELISA) with 94.94% (95% C.L: 88.25%-99.45%) and, finally, the Rose Bengal Test (RBT) with 68.95% (95% C.L: 56.55%-80.69%), which is the only test showing a significantly lower sensitivity compared to the others. On the other hand, our study revealed no significant difference in terms of specificity between all the tests under study, with a range from 99.06% (95% C.L: 98.34%-99.64%) for the ICT to 99.92% (95% C.L: 99.64%-100%) for the RBT. The ICT was found to be comparable in terms of sensitivity and specificity with the most commonly used tests for camel brucellosis. The results of the present study are of paramount importance for designing surveillance and control measures for brucellosis in camel populations.

Brucelosis canina en perros de la ciudad de Buenos Aires / Canine brucellosis in dogs in the city of Buenos Aires

La brucelosis canina, causada por Brucella canis, provoca epididimitis, atrofia testicular y esterilidad en los perros, mientras que en las hembras el síntoma principal es el aborto. La transmisión al hombre puede ser por contacto con el semen, orina, y/o fetos abortados de animales infectados. El presente estudio de tipo observacional de corte transversal, se realizó en caninos de barrios y asentamientos con alto índice de necesidades básicas insatisfechas (NBI) en 8 áreas de la ciudad de Buenos Aires. Se estudiaron 219 perros, 184 hembras y 35 machos, que fueron negativos a la prueba de aglutinación con antígeno tamponado (BPAT), que descartó la infección con especies lisas del género Brucella. Sedetectaron anticuerpos anti-B. canis en 16 perros (7.3%), 9 hembras y 7 machos, este último dato es relevante ya que la orina de los machos es considerada uno de los medios de diseminación de la infección. Aunque sólo se pudieron tomar hemocultivos a 175 animales, en 3 (2 hembras y un macho) se aislaron B. canis. Sólo 3 de los dueños de los perros positivos accedieron al diagnóstico serológico y dos resultaron positivos. Destacamos que la prueba de inmunodifusión en gel de agar (IGDA) ha demostrado ser poco sensible, detectó sólo uno de los 16 casos positivos y ninguno de los tres confirmados por aislamiento. Concluimos que en las áreas estudiadas el hallazgo de perros serológicamente positivos y el aislamiento de B. canis en 3 casos, son indicadores del riesgo en el que se encuentra la salud de la población expuesta.

Canine brucellosis, caused by Brucella canis, provokes epidydimitis, testicular atrophy and sterility in male dogs, while in females the major symptom is miscarriage. Transmission to humans may be through contact with semen, urine and/or aborted fetuses of infected animals. Our study, observational and cross-sectional, focused on dogs in lower class neighborhoods and slums with a high rate of unmet basic needs (UBN) in 8 areas of the city of Buenos Aires. We studied 219 dogs: 184 females and 35 males, that tested negative to the buffered plate antigen test (BPAT), which ruled out infection with smooth species of Brucella. We detected anti-B. canis antibodies in 16 dogs (7.3%): 9 females and 7 males, relevant data since the urine of males is considered one of the vectors for the spread of the infection. Although we could run blood cultures on only 175 animals, we isolated B. canis in 3 (2 females and 1 male). Only 3 of the owners of dogs that tested positive consented to a serological diagnosis and two of them were positive. We highlight that the agar gel immunodiffusion test (IGID) proved to have low sensitivity, having detected only one of the 16 positive cases and none of the three confirmed by isolation. We conclude that in the areas studied, the detection of serologically positive dogs and the isolation of B. canis in 3 cases are indicators of the health hazard for the population exposed to it.