Brucella Isolates Research Articles

A “One Health” surveillance and control of brucellosis in developing countries: Moving away from improvisation

Although a "One Health" approach has been successfully implemented for emerging infectious zoonotic diseases with pandemic potential, we still lack a conceptual framework to address enzootic diseases like brucellosis. The vast majority of published brucellosis studies in the developing world rely solely on serology. An important shortcoming of brucellosis serology is the impossibility to infer which (smooth) Brucella spp. induced antibodies in the host. In this respect, mixed farming and especially raising small ruminants along with cattle, a common practice in the developing world, is reported to be a risk factor and a central question that has to be answered is whether cattle are infected with B. melitensis or with B. abortus or with both Brucella species. Therefore the isolation, identification and molecular characterization of Brucella spp. in human and the different livestock species needs to be undertaken to define a sound conceptual framework, identify the source of infection and plan appropriate control measures.

Molecular genetic characterization of canine and rangiferine Brucella isolates from different regions of Russia

Comparative molecular-genetic characterization of Brucella isolates from dogs and reindeers in Russia by molecular-genetic typing methods. 19 canine and 2 rangiferine Brucella isolates were studied by molecular typing methods based on PCR for differential species and biovar specific molecular targets and MLVA (multiple locus variable number tandem repeats analysis) using primers to 12 known variable loci. Using PCR for differential molecular targets, canine Brucella isolates were characterized as B. canis and rangiferine isolates as B. suis biovar 4. MLVA revealed 5 identical and 7 variable MLVA loci. Using the dendrogram. all the isolates on the data of 12 loci were classified into the close related cluster. On the other hand, high discrimination power of MLVA with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.9842 was shown to reveal genetic diversity for the isolates of 17 MLVA genotypes. B. canis and B. suis isolates from different geographical regions in Russia were genetically close related, thereby confirming known genetic relationship between these species. Related MLVA genotypes of isolates were connected to certain regions of preliminary isolation in Russia. To improve the system ofbrucellosis surveillance in Russia MLVA typing of more canine and rangiferine Brucella isolates having epidemiological danger for humans is required to be studied.

PUNCTURE OF BOAR’S EPIDIDYMIS FOR DIAGNOSING PORCINE BRUCELLOSIS

In Serbia, mostly along the Danube, Morava and Mlava rivers, in animals reared under extensive conditions a disease named “rednja” may be detected. In the settlements where abortion is diagnosed all the sows mate with one boar that is infected with brucellosis. The Veterinary Program designs the control of Brucellosis twice a year, but the boars raised in extensive system are not controled and they are not registered. The causative agent was only recently isolated, confirmed and typed as Brucella suis biotype 2 (7). The owners have not reported the cases of abortion because they had thought that the reason are mechanical injuries that occur during feeding and drinking. After the visit of a vet a suspicion of brucellosis is notified. The symptoms of this disease are nonspecific. Only after abortion and if the swelling of boar scrotum occurs Brucellosis can be suspected. Brucellosis can be diagnosed in pigs by clinical examination, isolation, identification and typing of pathogens, serological and biological examination. Blood sampling is usually done in the case of abortion for serology examination, and also the placenta or aborted foetus is used for determining the cause. In addition to the above mentioned, the most important samples for bacteriological examination are the mandibular, retropharyngeal and supramamary lymph nodes, boar semen, i.e. testis after castration. All these samples cannot be taken when the reports on abortion are untimely. This means, only blood samples for serological examination can be taken. However, the literature does not mention epididymal puncture as a possible sample that is important for bacteriological examination, in addition to blood sampling from live animals. When the boars are fixed for blood sampling, the puncture of the epididymis can also be carried out for isolation of Brucella suis. After the disinfection, a sterile needle is stitched about 1 cm in depth and about 1-2 ml of the content is aspirated. Occassional sanies does not interfere the testing. The material can be frozen and examined when there the conditions are favourable and the laboratory available. Epididymal puncture is recommended as a safe and reliable way of sampling the diseased animals for the isolation and identification of Brucella suis biotype 2. During the bacteriological examination it was found that other bacterial species did not affect the growth of Brucella suis.

The isolation of Brucella spp. from sheep and goat farms in Sicily

Brucellosis is a worldwide zoonotic disease. The role of sheep and goats is particularly important as they are considered reservoir animals. Although it has been eradicated in some countries, it continues to be an important disease in many farming areas of the Mediterranean region. The study reports the results of field and microbiological examinations carried out at the Istituto Zooprofilattico Sperimentale della Sicilia over the three year period 2007–2009.

Management of Brucella endocarditis: results of the Gulhane study

Brucella endocarditis (BE) is a rare but life-threatening complication of human brucellosis. The aim of this study was to investigate the course of BE along with the therapeutic interrelations. A total of 53 patients with BE hospitalised in 19 health institutions between 2006 and 2011 were included in the Gulhane study. Diagnosis of brucellosis was established by either isolation of Brucella sp. or the presence of antibodies, and the definition of endocarditis was made according to Duke's criteria. There were four treatment groups: ceftriaxone combined with oral antibiotics (Group 1); aminoglycosides combined with oral antibiotics (Group 2); oral antibiotic combinations (Group 3); and aminoglycoside plus ceftriaxone combined with an oral antibiotic (Group 4). Involvement rates of the aortic, mitral and tricuspid valves were 49.1%, 43.4% and 5.7%, respectively. Thirty-two patients (60.4%) had an underlying cardiac valvular problem, including previous prosthetic valve replacement (n=18). Medical treatment was provided to 32 patients (60.4%), whilst concordant medical and surgical approaches were provided to 21 patients (39.6%). Mortality in Group 1 was 15% (3/20), whilst in Group 2 it was 5.3% (1/19). In Group 3, 25.0% (3/12) of the cases died, whereas none of the cases in Group 4 died. In conclusion, mortality increased 47-fold with pericardial effusion and 25-fold due to congestive heart failure that developed after BE. Although mortality was lower in the aminoglycoside-containing arm (Groups 2 and 4), statistical analysis could not be performed owing to the small number of patients.

Construction of nutrient media for L-form brucella

Dry nutrient media on the basis of hepatic infusion and Siberian roach hydrolyzate are constructed. Experimental data demonstrate that these media possess high sensitivity, completely inhibit growth of S-form Brucella, do not require рН adjustment, filtration and autoclaving. They can be used for isolation, cultivation and accumulation of L-form Brucella for bacteriological diagnostics of brucellosis. As these media are accessible for transportation, they are applicable for stationary and field conditions.

Characterization of Brucella species in Mexico by Bruce-Ladder polymerase chain reaction (PCR)

The typing of 153 Brucella strains isolated from different regions of Mexico was performed by multiplex PCR Bruce-Ladder. Results showed that microbiological typing and multiplex Bruce-ladder amplification were identical for all Brucella isolates tested. Because of its speed, Bruce-Ladder PCR is a useful method in the typing of Brucella species isolated from animal and humans. Key words: Brucella, brucellosis, typing, Mexico, Bruce-ladder polymerase chain reaction (PCR).

Serial Kinetics of the Antibody Response against the Complete Brucella melitensis ORFeome in Focal Vertebral Brucellosis

Human brucellosis is a common zoonosis worldwide. Here we present a case of focal vertebral brucellosis in a 71-year-old Mexican-American woman who contracted infection from unpasteurized goat milk. Standard agglutination serology was negative; the diagnosis was established by the isolation of Brucella melitensis from abscess fluid. A B. melitensis protein microarray comprised of nearly all proteins encoded by the bacterial genome was used to determine the kinetics of this patient's antibody responses to the complete collection of open reading frames existing in the genome (ORFeome). Three patterns of antibody responses against B. melitensis antigens were seen for serum samples obtained on days 0 (pretreatment), 14, 49, 100, and 180: (i) stable titers over time, (ii) a steady fall in titers, and (iii) an initial rise in titers followed by declining titers. Sera from this patient with chronic brucellosis recognized some of the same B. melitensis proteins as those recognized by sera from acute/subacute, blood culture-positive brucellosis patients but also recognized a distinct set of proteins. This study is the first to determine the kinetics of the human antibody responses to the complete repertoire of proteins encoded by a bacterial genome and demonstrates fundamentally different immunopathogenetic mechanisms between acute human brucellosis and chronic human brucellosis. While an extension of these findings to a larger patient population is necessary, these findings have important clinical and diagnostic implications and lead toward new insights into the fundamental immunopathogenesis of brucellosis.

In vitro antibiotic susceptibility testing of Brucella isolates from Egypt between 1999 and 2007 and evidence of probable rifampin resistance

BackgroundBrucellosis poses a significant public health problem in Mediterranean countries, including Egypt. Treatment of this disease is often empirical due to limited information on the antibiotic susceptibility profiles of Brucella spp. in this region of the world. The aim of this study was to determine the antibiotic susceptibility profiles of Brucella blood isolates in Egypt, a country endemic for brucellosis.MethodsBrucella spp. isolates were identified from the blood cultures of acute febrile illness (AFI) patients presenting to a network of infectious disease hospitals from 1999–2007. Minimum inhibitory concentrations were determined for tetracycline, gentamicin, doxycycline, trimethoprim-sulfamethoxazole, streptomycin, ceftriaxone, ciprofloxacin and rifampin using the E-test. Interpretations were made according to Clinical and Laboratory Standards Institute (CLSI) guidelines.ResultsA total of 355 Brucella spp. isolates were analyzed. All were susceptible to tetracycline, doxycycline, trimethoprim-sulfamethoxazole, streptomycin and ciprofloxacin; probable resistance to rifampin and ceftriaxone was observed among 277 (64%) and 7 (2%) of the isolates, respectively. Percentages of isolates showing probable resistance to rifampin were significantly lower before 2001 than in the following years (7% vs. >81%, p < 0.01).ConclusionsDespite the high burden of brucellosis in Egypt and frequent empirical treatment, isolates have remained susceptible to the majority of tested antibiotics. However, this is the first report of high rates of probable resistance to rifampin among Brucella isolates from Egypt. Patients should be closely monitored while following standard treatment regimens. Continued surveillance, drug susceptibility studies and updated CLSI interpretive criteria are needed to monitor and update antibiotic prescribing policies for brucellosis.

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 against 89 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.

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

BackgroundThe genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays.ResultsIn this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences.ConclusionsMALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

The First Detection of Brucella canis in Cattle in the Republic of Korea

Twenty mammary lymph node samples were collected from cattle on a farm in the Republic of Korea. These cattle were serologically negative for Brucella by tube agglutination test (≤ 1:50) and serum agglutination test (≤ 1:50). Out of 20 lymph node samples, two samples were positive for Brucella growth on Brucella agar as well as blood agar. Tests for urease, hydrogen sulphide and reactions against monospecific sera A and M indicated that these two isolates (No. 15 and 16) belong to the genus Brucella. Genus specific, AMOS (abortus, melitensis, ovis, suis) and Bruce-ladder multiplex polymerase chain reaction (PCR) assays confirmed the Brucella isolates as either a B. abortus or a B. canis strain. This is the first report of the occurrence of a B. canis infection in cattle in Korea. More survey data are needed to determine whether B. canis is a significant aetiology in the cases of cattle brucellosis in Korea.

Epidemiological survey of rifampicin resistance in clinic isolates of Brucella melitensis obtained from all regions of Turkey

The aim of the present study was to assess the antimicrobial susceptibility of Brucella melitensis isolates to rifampicin (RIF) depending on time and regional differences. A total of 94 human Brucella isolates collected in an 8-year period from the beginning of 2002 to the end of 2009 throughout Turkey were investigated. The isolates were identified at species and biovar levels by conventional methods, and minimum inhibitory concentrations (MIC) of RIF was determined by using the E test method. All isolates were identified as B. melitensis (93 isolates, biovar 3; 1, biovar 1), and MIC(50) and MIC(90) values of RIF were 1 and 1.5 μg/ml, respectively (MIC range, 0.25-1.5 μg/ml). All isolates were sensitive to RIF except 2 isolates, which had intermediate susceptibility to RIF. These findings indicated that B. melitensis biovar 3 may be the most frequently agent responsible for human brucellosis in Turkey. None of the isolates in our region was resistant to RIF.

Molecular typing of Brucella species isolates from livestock and human

Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus-melitensis-ovis-suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.

Comparison of direct culture versus PCR for the detection of Brucella in aborted fetuses of cattle and sheep in Turkey

The aim of this study was to detect Brucella in samples from aborted fetuses of sheep and cattle in Turkey using PCR and bacteriological analysis, and to determine the sensitivity...

Association between Brucella melitensis DNA and Brucella sp. Antibodies

Brucella sp. antibodies, despite falling to low levels, can remain measurable after recovery from acute brucellosis (1). Recently, several studies have shown the persistence of Brucella sp. DNA in both chronic brucellosis patients and asymptomatic subjects with a history of brucellosis (2–4, 6). However, to our knowledge, the association between serum antibodies and Brucella sp. DNA has not been investigated. We screened a cohort of 38 subjects with a well-documented history of brucellosis for the presence of Brucella melitensis DNA and Brucella antibodies. For that purpose, we tested both a quantitative real-time PCR (qPCR) assay (2) and an immunocapture-agglutination test (Brucellacapt; Vircell SL, Granada, Spain) that was performed as specified by the manufacturer. The Brucellacapt test has been described as offering results comparable to those of the Coombs anti-Brucella test, the most often used technique for the diagnosis of chronic brucellosis (5). Twenty-seven (71%) subjects were men, and 11 (29%) were women. The mean age was 49 ± 14 years (range, 26 to 83 years). The diagnosis of acute brucellosis was made between 3 and 33 years previously, according to one or both of the following criteria: isolation of Brucella spp. from blood or any sample of body fluid or tissue and the presence of a compatible clinical picture together with the demonstration of specific antibodies at significant titers (Wright test titer of ≥1:160 or Coombs anti-Brucella test titer of ≥1:320) or seroconversion. According to their clinical course after the initial episode, subjects were divided into three groups. Group A consisted of 10 (26%) focal disease subjects. Group B comprised 8 (21%) nonfocal disease subjects complaining of nonspecific symptoms, such as fatigue, malaise, arthralgia, and/or myalgia. The remaining 20 (53%) subjects were asymptomatic (group C). Chronic brucellosis patients included all patients diagnosed with focal disease and those whose symptoms had persisted for more than 1 year after the initial episode. Results are expressed as means ± standard deviations. P values less than 0.05 were considered statistically significant. We found an association between being B. melitensis DNA positive and being antibody positive. Among the 22 subjects with detectable B. melitensis DNA, 19 (86%) subjects had Brucella antibodies, while among the 16 subjects without B. melitensis DNA, Brucella antibodies were detected in 7 (44%) (P = 0.005; chi-square test). In the case of the asymptomatic subject group, the DNA-antibody concordance was not statistically significant (P = 0.264; two-tailed Fisher's exact test). The distribution of DNA-antibody results by group is shown in Table 1. Table 1. Distribution of the qPCR and Brucellacapt results from 18 patients with chronic brucellosis and 20 asymptomatic subjects The chronic brucellosis patients harboring B. melitensis DNA are more likely to show a seropositive sample than the remaining subjects. These findings suggest that after the initial infection, either the viable Brucella or its antigenic and structural components persist in the host and may have diagnostic and pathogenic implications.

FILTER-PAPER BLOOD SAMPLES FOR ELISA DETECTION OF BRUCELLA ANTIBODIES IN CARIBOU

We evaluated blood collected on Nobuto filter-paper (FP) strips for use in detecting Brucella spp. antibodies in caribou. Whole blood (for serum) and blood-saturated FP strips were obtained from 185 killed arctic caribou (Rangifer tarandus groenlandicus). Sample pairs (serum and FP eluates) were simultaneously tested in duplicate using competitive enzyme-linked immunosorbent assay (c-ELISA) and indirect ELISA (i-ELISA) for Brucella spp. Prior work based on isolation of Brucella spp. revealed sensitivity (SE) and specificity (SP) of 100% and 99%, respectively, for both these serum assays in caribou. Infection status of the animals in the current study was unknown but recent sampling had revealed clinical brucellosis and >40% Brucella antibody prevalence in the herd. To assess the performance of FP relative to serum in these assays, serum was used as the putative gold standard. On both assays, the findings for duplicate runs (A and B) were similar. For c-ELISA run A, the FP Brucella prevalence (47%) was lower than serum prevalence (52%), with SE 89% (95% confidence interval [CI]: 82-95%) and SP 99% (97-100%). For i-ELISA run A, serum and FP Brucella prevalence rates were identical (43%), and the SE and SP of FP testing were 100% and 99% (97-100%), respectively. The findings suggest better FP test performance with i-ELISA than with c-ELISA; however, i-ELISA does not distinguish cross-reacting antibodies induced by Brucella vaccination or exposure to certain other Gram-negative pathogens. Results for duplicate FP eluates (prepared using separate FP strips from each animal) were strongly correlated for both protocols (r=0.996 and 0.999 for c-ELISA and i-ELISA, respectively), indicating minimal variability among FPs from any individual caribou. Dried caribou FP blood samples stored for 2 mo at room temperature are comparable with serum for use in Brucella spp. c-ELISA and i-ELISA. Hunter-based FP sampling can facilitate detection of disease exposure in remote regions and under adverse conditions, and can expand wildlife disease surveillance across temporospatial scales.

Antimicrobial susceptibilities of Brucella isolates from various clinical specimens.

Brucellosis is a worldwide zoonotic disease and still constitutes a major public health problem. In the study we claimed to identify Brucella species from clinical samples of patients with active brucellosis from Van region of Eastern Anatolia and to determine in vitro antimicrobial susceptibilities of these strains to commonly used anti-Brucella agents and a possible new alternative tigecycline. A total of 56 Brucella isolates were enrolled the study and the identification of the isolates were based on conventional methods. In vitro activities of antimicrobials were evaluated by the E test method. All isolates were identified as B. melitensis. MIC(90) values of doxycycline, streptomycin, rifampin, trimethoprim-sulfamethoxazole and tigecycline were 0.064 mg/L, 1 mg/L, 2 mg/L, 0.125 mg/L and 0.094 mg/L, respectively. Tigecycline had low MIC(50) and MIC(90) values against all B. melitensis strains; the highest MIC observed was 0.25 μg/mL. Our data suggest that tigecycline can be a therapeutic alternative option for the treatment of brucellosis.

Rapid Multiplex PCR Assay for the Simultaneous Detection of the Brucella Genus, B. abortus, B. melitensis, and B. suis

The routine identification and differentiation of Brucella species is a time-consuming and labor-intensive process, which frequently places personnel at risk of laboratoryacquired infection. Here, we describe the development of a rapid multiplex PCR assay for the confirmation of presumptive Brucella isolates. The assay was able to identify and differentiate major human pathogens, namely B. abortus, B. melitensis, and B. suis, in a single test of less than an hour and a half.

Study on species identification of brucella with small fragments of PCR

Objective To identify the species of brucella rapidly with small fragments of PCR products. Methods The primers were designed according to Brucella spp. repeated insertion sequence IS711 in addition to the specific gene sequences of Brucella melitensis, Brucella abortus and Brucella suis. The small fragments of 3 standard strains and 13 clinical isolates of Brucella were amplified by PCR. Then PCR products were T-A cloned and sequenced. The DNA of standard strains were diluted for sensitivity and stability testing. Results Four specific PCR products were obtained by PCR (63, 67, 81 and 83 bp). The results of T-A cloning and sequencing were in accord with the target gene fragment test. The detection limit of DNA was 1 μg/L. The expected results were achieved with different primers. Conclusion Species identification of Brucella with small fragments of PCR production is a method to identify Brucella strains with great specificity,sensitivity and stability. Key words: Brucella; Polymerase chain reaction