Evaluation of dot-blot test for serological diagnosis of bovine brucellosis

Abstract The objective of this study was to standardize and validate the dot-blot test for the serological diagnosis of bovine brucellosis, compare the results with those found in the 2-mercaptoethanol (2-ME) and complement fixation test (CF), and estimate the relative sensitivity and specificity of the dot-blot compared to these tests. Fifty bovine blood serum samples were used for the test standardization, and 1315 samples were used for evaluation and comparison between the tests; the results were compared using the Kappa indicator. At the end of standardization, it was established as optimal for the antigen obtained from Brucella abortus B19 after passing through a microorganism rupture process, the blood serum samples diluted at 1:100, and the conjugate at 1:30,000. The comparison of the dot-blot results with 2-ME showed Kappa index of 0.9939, sensitivity of 99.48%, and specificity 99.91%, with CF, Kappa index of 0.8226, sensitivity 100% and specificity 95.32%. Using the combination of the test results 2-ME and CF to establish the true condition of the animal, the dot-blot showed relative sensitivity of 100%, and relative specificity of 99.91%. The evaluated test proved to be effective and reliable, besides being easy to handle and interpret the results.

Epidemiología de la brucelosis caprina y ovina en la provincia de Formosa, Argentina / Epidemiology of caprine and ovine brucellosis in Formosa province, Argentina

Se realizó un estudio epidemiológico de brucelosis en 516 majadas caprinas o mixtas (caprinos/ovinos) de las 3 regiones agroecológicas de la provincia de Formosa, Argentina. Mediante las pruebas de aglutinación en placa con antígeno tamponado y de fijación del complemento en suero se estudiaron un total de 25.401 caprinos y 2.453 ovinos. Además, se realizaron cultivos bacteriológicos y PCR en muestras de leche de cabras de 3 majadas con brucelosis y abortos recientes. Se detectó brucelosis en 4 de los 9 departamentos de la provincia, la prevalencia global fue del 2 % y la intrapredial varió entre el 1 y el 40%. La proporción de majadas positivas fue del 3,6, el 12 y el 36 % para las regiones este, centro y oeste, respectivamente. Se aisló Brucella melitensis bv. 1 de cabras por primera vez en la provincia. La PCR amplificó fragmentos esperados de 827 pb correspondiente al gen omp2ab (Brucella spp.) y de 731 pb correspondiente al inserto IS711 (B. melitensis). La detección de anticuerpos en ovinos que cohabitan con caprinos sugiere que las infecciones habrían sido causadas por B. melitensis, lo que constituye un riesgo adicional para la salud pública. Los programas de control y erradicación de la brucelosis deberían considerar las majadas mixtas como una sola unidad epidemiológica. Los resultados indican que la brucelosis por B. melitensis bv. 1 es altamente endémica en las regiones centro y oeste de la provincia de Formosa.

An epidemiological study of brucellosis was carried out in 516 goats and mixed flocks (goat/sheep) from the three agro-ecological regions of Formosa province, Argentina. Serum samples from a total of 25401 goats and 2453 sheeps were analyzed using buffered plate agglutination test (BPAT) and complement fixation test (CFT). Bacteriological and PCR analyses on milk samples from goats in three flocks with a history of brucellosis and recent abortions were also performed. Brucellosis was detected in four of the nine departments of the province with an overall prevalence of 2 % and an intra-flock prevalence ranging between 1 % and 40 %. The proportion of infected flocks was 3.6 %, 12 % and 36 % for the eastern, central and western regions, respectively. Brucella melitensis bv. 1 was isolated efrom goats for the first time in the province. The expected fragments of 827 bp from the omp2ab gene (Brucella spp.) and 731 bp from the insert IS711 (B. melitensis) were amplified by PCR. Detection of antibodies by BPAT and FCT in sheep cohabiting with goats suggests that infections could have been caused by B. melitensis, posing an additional risk to public health. Control and eradication programs for brucellosis should consider mixed flocks as a single epidemiological unit. The results indicate that brucellosis by B. melitensis bv1 is highly endemic in the central and western regions of Formosa province.

Respuesta humoral y consecuencias reproductivas en ovejas desafiadas con Brucella ovis al final de la gestación / Immune response and reproductive consequences in experimentally infected ewes with Brucella ovis during late pregnancy

La brucelosis ovina por Brucella ovis es una enfermedad de prevalencia alta en Argentina. Para evaluar la patogenicidad de B. ovis y la respuesta serológica durante el último mes de gestación, 6 ovejas se distribuyeron en dos grupos: G1, ovejas preñadas, n = 4 y G2, ovejas no preñadas, n = 2. Tres ovejas del G1 (15 días preparto) y una del G2 fueron inoculadas con B. ovis. Se analizaron muestras de suero mediante diferentes pruebas serológicas. Se realizó aislamiento y PCR a partir de mucus cérvico-vaginal (mcv), placenta y leche. En las muestras de placenta se realizó histopatología. Las hembras del G1 parieron corderos vivos; se detectaron anticuerpos en las ovejas desafiadas del G1 a partir de los 5 días posinoculación. El mcv de las ovejas desafiadas resultó negativo al aislamiento en ambos grupos. Las muestras de leche del G1 fueron positivas por cultivo y PCR a B. ovis. La técnica de PCR resultó positiva en las placentas de las ovejas desafiadas del G1. La histopatología reveló una placentitis necrótica supurativa en una de las ovejas desafiadas. El desafío con B. ovis preparto resultó en la invasión de la placenta y de la glándula mamaria, con la consecuente excreción de la bacteria por leche. La infección con B. ovis indujo una respuesta humoral temprana en las ovejas. La colonización de la placenta por B. ovis y la excreción de la bacteria por la leche sugieren un potencial riesgo de infección activa para los corderos y la posibilidad de que estos se comporten como portadores latentes de la infección.

Ovine brucellosis by Brucella ovis is a highly prevalent disease in Argentina. This study aimed to evaluate the pathogenicity of B. ovis and the serological response in ewes during late pregnancy and in their offspring. Six adult ewes were distributed in two groupsGI (pregnant females, n = 4) and G2 (nonpregnant females, n = 2). Three pregnant ewes at 15 days prepartum and one nonpregnant eve were inoculated with B. ovis. Sera of sheep and their offspring were analyzed by different serological tests. Samples of cervicovaginal mucus, placenta and milk were studied by bacteriology. A Brucella genus-specific PCR assay was carried out in placenta and milk samples. Placenta samples were hystopathologically processed. G1 females gave birth to live lambs, but one died hours postpartum. Serological techniques employed detected antibodies in serum of inoculated pregnant animal 5 days postchallenge. Sera of female controls G1 and G2 remained negative throughout the study. Cervicovaginal mucus of infected ewes in G1 and G2 yielded negative results to bacteriology, but B. ovis was isolated from milk. The PCR assay was positive for the placenta and milk from inoculated pregnant ewes. Histopathology revealed necrotic suppurative placentitis in one placenta. However, although results demonstrated that B. ovis can invade the placenta and mammary gland, this bacterium did not cause abortion when it was inoculated intravenously at 15 days prepartum. B. ovis infection induced an early humoral response in pregnant ewes, but their lambs remained seronegative, indicating that there was no transfer of antibodies in infancy. Placenta colonization and milk excretion of B. ovis involves a potential source of infection for lambs, which could play a role as latent carriers of infection.

Endocarditis por Brucella canis: primer caso documentado en un paciente adulto en Argentina / [Brucella canis endocarditis: first documented case in Argentina].

We herein present the case of an adult male patient who consulted for lower extremity edema, a 2- month history of fever and oppressive chest pain radiating to the left arm. He referred neither contact with breeding animals nor consumption of unpasteurized dairy products. A diagnosis of endocarditis was confirmed by cardiac studies. Since the empirical treatment with cephalotin, ampicillin and gentamicin failed, the patient underwent aortic valve replacement. A total of four blood cultures were positive with a gram-negative rod. Bacterial identification was performed using the API 20 NE technique (bioMÞrieux), the Phoenix automated method (BD) and conventional biochemical tests which were unable to classify the isolate as to genus and species. The strain was sent to the INEI-ANLIS "Dr. Carlos G. Malbrán" where it was identified as Brucella canis. The antimicrobial treatment was switched to doxycycline, rifampicin and trimethoprim-sulfamethoxazole with good evolution of the patient. The clinical significance of this case report lies in the possible failure of the empiric antibiotic therapy administered for endocarditis, since B. canis did not respond to the conventional antimicrobial treatment for this pathology.

Clinical spectrum of fever of unknown origin among adult Egyptian patients admitted to Ain Shams university hospitals: a hospital based study

This prospective study was designed to analyze the clinical spectrum of fever of unknown origin [FUO] among adult Egyptian patients admitted to Ain Shams University Hospitals during the period from May 2009 till the end of December 2010. All Egyptian patients fulfilling the criteria of FUO admitted during this period were followed up till reaching the diagnosis. 93 patients were included in the study. They were 48 [51.6%] females and 45 [48.4%] males, their ages ranged from 15 to 65 years [34.39 +/- 13.6]. Infections were the commonest cause of FUO[41.94%] followed by malignancies[30.11%]. While autoimmune diseases represented 15.05% and in 12.9% of patients the diagnosis was not established. Brucellosis and infective endocarditis were the commonest infections, while hematological malignancies were the commonest oncological diseases. Systemic lupus erythematosus [SLE] was the commonest auto-immune disease. Brucellosis, infective endocarditis, hematological malignancies and SLE must be considered in the differential diagnosis of adult FUO in Egypt

Endocarditis por Brucella abortus: Sobrevivir a los 74 años de edad / Brucella abortus endocarditis: Survival of a 74 year old patient

Brucellosis is not frequent in Chile but it may present with life threatening complications like endocarditis. The case reported refers to a 74 year old man admitted to the Infectious Diseases Hospital Dr. Lucio Córdova in Santiago. He had been febrile for 3 months with no specific symptoms. The trans-esophageal echocardiography confirmed múltiple large vegetations and important involvement of the aortic valve. Blood cultures yielded Brucella abortus. The patient required cardiac surgery, along with antibiotics, and he had a satisfactory outcome, being alive at the moment of this report???. Brucellosis can be the responsible for prolonged fever of unknown origin. It is necessary to take in mind brucellosis to obtain the specific laboratory tests. For a best prognosis an early treatment with associated antibiotics for at least 4 a 6 weeks is important. If endocarditis is present valve replacement is often necessary.

Brucelosis es una infección poco frecuente en Chile pero que puede presentar algunas complicaciones graves como endocarditis. Se presenta el caso de un paciente de 74 años que ingresa al Hospital de Enfermedades Infecciosas Dr Lucio Córdova (HLC) de Santiago, con un síndrome febril de 3 meses de evolución, sin síntomas específicos. El ecocardiograma trans-esofágico mostró múltiples vegetaciones en válvula aórtica y en los hemo-cultivos se desarrolló Brucella abortus. El paciente recibió tratamiento médico-quirúrgico en el Instituto de Enfermedades Respiratorias y Cirugía de Tórax (INERYCT), evolucionando satisfactoriamente. Ante un paciente con un síndrome febril prolongado debe tenerse presente la posibilidad diagnóstica de brucelosis de modo de solicitar los exámenes específicos en forma oportuna y, si se confirmase, iniciar precozmente el tratamiento. Este debe comprender un esquema asociado de antimicrobianos, que se mantendrá por al menos 4 a 6 semanas. En caso de endocarditis, con frecuencia se requerirá también de reemplazo valvular.

[Microbiological study and antimicrobial susceptibilities of brucella isolates in serologic diagnosed cases]

Brucellosis is a zoonotic disease with worldwide distribution that is endemic in Iran. Worldwide, brucellosis remains a major cause of morbidity in humans and domesticated animals. The disease has a wide spectrum of clinical manifestation and can affect a variety of organs and systems. This study focused on blood culture of serologic diagnosed brucellosis and antimicrobial susceptibility test. In this cross sectional study, microbiologic survey was done on a total of 30 serum samples with STA titer of 1:160 or greater and 2ME titer of 1:40 or greater, which were presumptive for brucellosis. Blood cultures were done by lysis centrifugation and antimicrobial susceptibility test, against 9 antimicrobial agents by disk method. The data was analyzed by stata V8.0 software. At the end this study, the blood culture isolation rate was 23.3% [7 cases out of 30 patients] and all of the isolates were brucella melitensis. Antimicrobial susceptibility tests showed high in vitro activity of ofloxacin, ciprofloxacin and doxycycline and also, low in vitro activity of streptomycin and cotrimoxazole. Brucellosis is endemic in Iran. Brucella melitensis was the most common strain of brucella in our patients. Except cotrimoxazole and streptomycin, high in vitro activity was found with other antibrucella agents, especially with ofloxacin, ciprofloxacin and doxycycline

Relapsed human brucellosis and related risk factors

To determine risk factors of relapse among outpatient treated brucellosis. It is a Cohort study carried out from 2004 to 2006, in an Infectious Diseases Clinic in Ahvaz, Iran. We studied 115 patients with brucellosis. The diagnostic criteria were the finding of > 1/80 [Wright] with a 2 mercaptoethanol [2 ME] >1/40, in association with compatible clinical findings [back pain, sweating and fever]. Treatment by standard drug regimen against brucellosis was established according to National Program against Brucellosis [NPB] in Iran. The patients were evaluated at the end of treatment and after two, four and six months by clinical and serological examinations. Patients with relapse and patients without relapse were placed separately in two groups. The data in the two groups were statistically compared with SPSS, 11.5 by chi square test. Of the 115 patients, 12 were excluded because of study's exclusion criteria. Of 93 studied patients 17 [18.3%] had relapse, results in whom are as fallow: 12 [70.6%] had Longer than three months duration of the symptoms prior to diagnosis [P=0.0001]. 100% of relapses were in male [P=0.003]. Aging was associated with increased risk of relapses [P=0.04]. Eleven patients [64.6%] had lymphopenia [P<0.0001]. Increased in ESR and CRP values were observed in 16[94.1%] and 17[100%], respectively [P0.05]. The present study showed that aging, gender, chronic infection and lymphopenia are risk factors for relapsing brucellosis

Infective endocarditis due to brucella.

One of the complications of brucellosis is infective endocarditis, which carries a high mortality rate if undiagnosed or misdiagnosed. We report a case of Brucella infective endocarditis, which was diagnosed serologically and by polymerase chain reaction. After Brucella specific treatment, patient showed dramatic improvement clinically, as evident by echocardiogram findings and other investigations.

35 year old man with fever, weight loss and splenomegaly

A 35 year - old man was referred to an infectious disease specialist because of two weeks; fever, fatigue, 4kg weight loss and an obscure pain of whole body. He had otherwise been well until 1 5 days before. 10 days before; treatment with Procaine Penicillin [800,000 U per 12 hours] and Amoxicillin by a G.P was begun. 5 days later a physician sent him to a lab for routine blood examination and febrile agglutination tests. His physician on physical examination of patient found a palpable spleen and his temperature was 38c, CRP+ + and ESR was 45 mm per h our. In auscultation, heart and lungs were normal. A urine culture was negative.Blood sugar, Creatinine, Cholesterol and Triglycerides were normal. Hematologic lab values were: Hct%40, MCV 82, WBC [Per mm3] 9 100, dif count N=35, L=55, E=O, M=8, B=2, PLT250 000, Blood *chemical *values Creatinine 0.9, Sugar 95, Calcium normal, Liver enzymes; ALT=50, AST=48. And Tuberculin Test positive with 12 mm indurations. The patients' weight had dropped from 75 to 71 Kg. The temperature was 38 c.The pulse rate was 75, respiration rate was 22. The blood pressure was 1 10/70. The urine was positive [++] for protein, Urea Nitrogen 33 mg/dl, Creatinine 1.2mg/dI. Na 139 mmol; K, 4.5mmol/lit; Glucose 119 mg/dl; Bilirubin total 1.7 and direct 0.6; and normal liver enzymes, EKG showed sinusal tachycardia; the other findings were otherwise normal. Chest X-Ray showed no abnormality. Sample of blood was obtained for culture. Temperature was controlled each day and it was maximum 38.3 centigrade. On the next clinical visit of patient,three days later, serum sample obtained on the seventh day of illness was negative for Salmonellosis and Brucellosis and first report of blood culture was negative too. A CT-Scan of lumbo-saccreal portion and Chest x Ray gave no diagnostic information A diagnostic procedure was performed

Seroepidemiological and microbiological study of brucellosis in Kuwait

The primary objective of the study was to determine the prevalence of brucellosis and the antimicrobial susceptibility pattern of local Brucellae isolates in the Infectious Diseases Hospital, Kuwait. Subjects and A single serum sample was collected from each of 1,836 patients of different nationalities from January 2000 to December 2001. Any patient with a provisional diagnosis of fever or brucellosis had a standard tube agglutination [STA] test for the quantitation of Brucella antibodies. Blood cultures were done in 166 of 455 patients with significant STA titers, using the Bactec system. Antimicrobial susceptibility testing of 123 isolates of Brucella spp. was done against 8 antimicrobial agents. A total of 455 serum samples [24.8%] having an STA titer of >/= 1:160 were presumptively diagnosed as cases of brucellosis. The peak isolation was in April and May. Brucella spp. were isolated from 123 blood cultures [74.1%]. The blood culture isolation rate was significantly higher in patients with an STA titer of >/= 1:1,280 than in those with an STA titer of

Estudio comparativo de medios de cultivo para aislar Brucella ovis / Comparative study of culture media for isolating Brucella ovis

Se compararon los medios de cultivo agar Thayer-Martin modificado adicionado con 0,01 *g/mlde timetropina y 100 UI de nistatina (TMM) y Agar Skirrow (SK) para el aislamiento de Brucella ovis. Se utilizaron 11 cepas de B. ovis y se evaluaron mediante la técnica de recuento de viables comparando los resultados con agar base Columbia con sangre bovina 7% (ASC). También se cultivaron en los mismos medios 94 muestras de semen pertenecientes a 33 carneros de una majada con antecedentes de infección. El crecimiento de las cepas de B. ovis fue similar en los 3 médios, excepto una cepa que no desarrolló en TMM. Los resultados de las siembras de semen fueron similares para los medios TMM y SK, aislándose B. ovis del 27% de las muestras. Los resultados indican que los medios TMM y SK ofrecen excelentes alternativas para aislar B. ovis del semen de carneros en condiciones de campo