Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report.

BACKGROUND: It is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus. CASE PRESENTATION: A 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI-TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence. CONCLUSION: Automated identification instruments and MALDI-TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI-TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.

Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

The Use of Flocked Swabs with a Protective Medium Increases the Recovery of Live Brucella spp. and DNA Detection.

Brucellosis is a worldwide zoonosis caused by bacteria from the genus Brucella. Once established, it is very hard to eradicate this disease, since it contaminates animals, the environment, and humans, causing problems for veterinary and public health as well as wildlife protection programs. Swabs are used for sampling in bacteriological and/or molecular diagnostics, from seropositive animals with disease symptoms, from genitalia or tissue lesions, as well as from contaminated environments. The aim of this study was to compare main of the commercially used swab types for sampling and diagnostics of Brucella spp. and determine the optimal storage conditions and time frame for testing. To achieve this, we tested bacterial and molecular methods for detection of Brucella abortus, Brucella melitensis, and Brucella suis using nine swab types, all with different tip materials, treated immediately after spiking, after 72 h at +4°C, and after 72 h at -20°C. Flocked swabs showed the highest capacity to preserve bacterial viability and DNA quality, regardless the storage conditions. Flocked swabs immersed in a protective medium provided the best conditions for Brucella survival in all three storage conditions. At the same time, the efficacy of quantitative PCR (qPCR) detection for all swabs, including the positive control, was above 50%, irrespective of the storage conditions, while bacterial survival was significantly lowered when swabs were kept at +4°C or -20°C for 72 h (48.2% and 27.5%, respectively). Compared to the positive control and other types, the flocked swabs maintained higher reproducibility regarding their capacity to preserve live bacteria in all three storage conditions. IMPORTANCE In order to protect public and veterinary health from highly zoonotic bacteria such as members of the genus Brucella and prevent their dissemination into the environment, direct diagnostics are of utmost importance. However, in addition to the highly specific diagnostic tests, the sampling methods, time necessary for specimens to reach the laboratories, and transport conditions are important factors to consider in order to increase the sensitivity of performed tests, especially bacterial culturing and qPCR. This paper shows how different swab types and storage conditions influence classical bacteriological diagnostics of the most prevalent Brucella species - B. melitensis, B. abortus, and B. suis - but have little impact on molecular methods. The presented results highlight (i) the choice of swab regarding the storage and transport conditions, (ii) the importance of immediate swab treatment upon sampling, and (iii) that molecular methods do not depend on storage conditions, unlike classical bacteriological isolation.

An Adolescent With Neurobrucellosis Caused by Brucella abortus Cattle Vaccine Strain RB51.

We present the case of an 18-year-old female with a 1-month history of fever, headache, and double vision, whose examination revealed papilledema and cranial nerve VI palsy. Blood cultures grew Brucella abortus cattle vaccine strain RB51, which is inherently resistant to rifampin. We discuss the management of the first known case of neurobrucellosis by this strain.

Seroprevalence of brucellosis and Q fever infections amongst pastoralists and their cattle herds in Sokoto State, Nigeria.

Brucellosis and Q fever are neglected zoonoses of global health importance, with unknown true prevalence in occupationally vulnerable settings, partly due to misdiagnosis for other febrile conditions and poor access to primary health care. We examined the seroprevalence of these diseases and associated factors amongst pastoralists and their cattle in Sokoto State, a hub of cattle and pastoral populations in Nigeria. Serum samples randomly collected from 137 pastoralists and 366 cattle from 27 herds in three selected Local Government Areas (LGAs) in the state were analysed for antibodies to Brucella abortus using Rose Bengal Plate Test (RBT) and competitive Enzyme-Linked Immunosorbent Assay (cELISA) as well as antibodies to Coxiella burnetti using indirect ELISA. Consenting pastoralists' knowledge, perception and practices about the diseases were assessed using a semi-structured questionnaire. Data were analysed using descriptive statistics and bivariate analysis at p ≤ 0.05 level of significance. Brucellosis adjusted individual seroprevalence were 0.83% (95%CI: 0.04-4.59%) and 0% among pastoralists; 2.28% (95%CI: 1.16-4.43%) and 5.70% (95%CI: 3.68-8.74%) in cattle by RBT and cELISA, respectively. Adjusted herd-level seroprevalence for brucellosis were 23.20% (95%CI: 11.07-42.54%) and 42.00% (95%CI: 25.27-61.11%) by RBT and cELISA, respectively. For Q fever, higher seroprevalence of 62.57% (95%CI: 54.04-70.46%) and 2.98% (95%CI: 1.57-5.58%) were recorded amongst the pastoralists and their cattle, respectively. with adjusted herd-level seroprevalence of 40.36% (95%CI: 22.57-63.17%). The LGAs of sampling were significantly (OR: 0.2; 95%CI: 0.02-1.00) associated with Q fever infection, though marginal. The majority of the pastoralists had poor knowledge, perception and practices towards the diseases. This is the first study establishing the presence of brucellosis and Q fever at the human-animal interface in Sokoto State, Nigeria. The pastoralists' poor knowledge, perception and practices about these diseases are worrisome and are important factors for consideration in disease control.

Characterisation of Brucella species and biovars in South Africa between 2008 and 2018 using laboratory diagnostic data.

BACKGROUND: Brucellosis is an infectious zoonotic bacterial disease of humans and other animals. In the Republic of South Africa (RSA), animal brucellosis is widespread and the current available data on the prevalence of this disease rely solely on serological testing. The primary limitation of brucellosis serology is the lack of discriminatory powers to differentiate between Brucella species and biovars as well as the cross-reactivity observed with other Gram-negative bacteria. AIM: The aim of this study was to conduct a retrospective laboratory-based survey on Brucella species and biovars isolated from various animal species in SA between 2008 and 2018. MATERIAL AND METHODS: The isolation of Brucella species and biovar typing was performed using conventional microbiological techniques. RESULTS AND DISCUSSION: A total of 963 strains of Brucella species were included in this study with a frequency of detection for B. abortus (n = 883; 91.6%) followed by B. melitensis (n = 42; 4.4%), B. ovis (n = 29; 3.0%) and B. canis (n = 9; 0.9%). Of the 883 strains of B. abortus, 90.1% were typed as B. abortus biovar-1 while 5.7% as B. abortus biovar-2, and 3.3% and 0.5% were B. abortus S19 and B. abortus RB51 vaccine strains, respectively. Among the 42 B. melitensis strains, 71.4% were reported as B. melitensis biovar-1 and 26.2% as B. melitensis biovar-3 while 2.4% was B. melitensis biovar-2. CONCLUSION: A retrospective study, such as this one, provides useful information that can be critical in formulating policies and strategies for the control and eradication of brucellosis in animal populations in RSA.

Determining Antibody Retention in Hemolyzed, Bacterially Contaminated, and Nobuto Filter Paper-Derived Serum Utilizing Two Brucella abortus Fluorescence Polarization Assays.

We evaluated hemolyzed, bacterially contaminated, and Nobuto filter paper-derived serum, collected from 50 Rocky Mountain elk (Cervus elaphus nelson) in 2017 and 2019, divided into eight treatments to determine antibody retention. Serum was analyzed on Brucella abortus-specific fluorescence polarization assay utilizing plates and tubes. Reference titers and serostatus were compared to serum held at 22 C for 4, 8, 12, and 16 d; frozen clotted blood; blood with 2% and 10% elk rumen content (held for 8 d at 22 C); and serum eluted from Nobuto filter paper. Using Cohen's kappa test of agreement, plate assay serostatus agreement was substantial or outstanding in all treatments. Serostatus agreement was outstanding in all treatments utilizing tubes. The mean change in score (treatment minus reference) showed significant negative bias in serosuspect or seropositive animals in the frozen, 2% rumen, and 10% rumen treatments on the plate assay, and the day 16 and 10% rumen treatments on the tube assay, that could ultimately result in an animal being misclassified into a serosuspect or seronegative category. Serum eluted from Nobuto filter paper produced inconsistent results and is not recommended as an alternative to serum derived from blood. Although the potential for misclassification of animals with low titers exists, analyzing hemolyzed and bacterially contaminated serum from Brucella abortus nonendemic areas can increase sample size and the potential to detect seropositive animals.

Brucella species circulating in rural and periurban dairy cattle farms: a comparative study in an endemic area.

Brucellosis is among the most important zoonotic infectious diseases worldwide affecting both humans and domestic animals. The present study aimed to determine and compare the seroprevalence of brucellosis among rural and periurban dairy cattle farms of four Iranian provinces from 2017 to 2019. We applied different serological tests, including RBT, SAT, and iELISA to evaluate the brucellosis prevalence among 2808 dairy cattle. Species-specific multiplex PCR and biotyping tests were also used to further identify the implicated Brucella species. Serological screening using RBT, SAT, and iELISA led to 157 (5.6%), 112 (3.9%), and 139 (4.9%) positive results among tested cattle, respectively. Brucella abortus biovars 1 (2 cases) and biovars 3 (42 cases) were identified by biotyping experiments and multiplex PCR in all 44 tested lymph node samples. Further, Cohen's kappa statistical analysis revealed that the best degree of agreement was seen between RBT and iELISA (99.4%), followed by SAT/iELISA (98.5%) and finally RBT/SAT (98.4%). Our results also showed a significantly lower seroprevalence of brucellosis in periurban dairy cattle when compared to rural dairy cattle population (p value= 0.01). These results reflect the need for better vaccine coverage using RB51 combined with an appropriate test-and-slaughter program in the rural dairy cattle population.

Identification, geographic distribution and risk factors of Brucella abortus and Brucella melitensis infection in cattle in Algeria.

Brucellosis is an infectious disease of several terrestrial and marine animals and humans caused by bacteria of the genus Brucella. This study aimed to identify Brucella species and biovars circulating in cattle and to analyze their geographic distribution across Algeria. Two hundred ninety eight milk and lymph node samples from 161 seropositive cattle of different local and foreign breeds were collected from 97 dairy farms in 56 towns of 13 wilayas (states/ provinces) of the central, eastern, western and southern regions. The samples were cultured on selective media and the obtained isolates were identified using bacteriological and molecular tests. Eighty-five Brucella isolates (72 B. abortus and 13 B. melitensis) were recovered from 63 animals in 37 dairy farms. In total, 71 (83.5 %) B. abortus bv 3, 11 (12.9 %) B. melitensis bv 2, 2 (2.4 %) B. melitensis bv 3 and 1 (1.2 %) unidentified B. abortus biovar were detected. The identification of B. abortus biovar 3 and B. melitensis biovar 2 is a new finding for Algeria and the Maghreb, respectively. B. abortus (84.7 %) was the main etiological agent of brucellosis. B. abortus showed a scattered distribution across Algeria. The fact that 60 % of the seropositive cattle showed no clinical signs, but 36 % were culture positive is an alarming observation. These data will rise awareness for the current epidemiological situation of bovine brucellosis in Algeria. To the best of our knowledge, this is the first representative countrywide bacteriological investigation of Brucella species and biovars in cattle across Algeria, which is a developing country where resources might be limited and the working conditions might not be very friendly.

Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines.

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

Brucella melitensis and Brucella abortus genotyping via real-time PCR targeting 21 variable genome loci.

Brucella melitensis and Brucella abortus account for almost all cases of brucellosis in Turkish population. We developed a fourplex quantitative real-time PCR (qPCR) assay for the electrophoresis-free, rapid and cost-effective differentiation of B. abortus and B. melitensis from the other Brucella spp. The 4-plex species differentiation assay was combined with a qPCR assay targeting 17 different single nucleotide polymorphism (SNP) loci in Brucella genomes. This combination resulted in a 21 Variable Genome Loci (21-VGL) qPCR assay for high resolution genotyping of B. abortus and B. melitensis. A total of 486 Brucella was analyzed using the qPCR assay to create a 21-VGL profile database. The database contained the profiles of 55 B. abortus, 352 B. melitensis, 3 B. ceti, 6 B. neotomae, 7 B. ovis, 6 B. pinnipedialis, 44 B. suis and 13 B. canis strains. The 21-VGL Brucella genotyping clearly distinguished B. abortus, B. melitensis, B. neotomae and B. ovis. The 21-VGL approach could not distinguish B. pinnipedialis from B. ceti and some B. suis genotypes from B. canis. The results revealed that more than 99% of the Brucella isolates in Turkey were B. melitensis and 21-VGL genotyping can be reduced to 8-VGL B. melitensis genotyping without any loss of genotyping resolution. To our knowledge, we introduced the fastest and the lowest-cost B. abortus and B. melitensis genotyping and species differentiation methodology in the literature.

Scoping review of brucellosis in Cameroon: Where do we stand, and where are we going?

Brucellosis is a zoonotic disease known to be endemic to parts of western and sub-Saharan Africa. However, the epidemiology for humans and animals remains largely unknown in many of these countries with Cameroon being a typical example. Despite common knowledge that brucellosis affects livestock, the actual number of infected animals remains unknown. Through a scoping review, the current known status of the disease is described. The aim is to ascertain relevant and publicly accessible research and knowledge of human and animal brucellosis in the country, and to provide an overview of the factors associated with its known persistence. Seroprevalence has been estimated and published in 12 separate instances (1 human; 9 cattle; 1 human and cattle; and 1 that includes cattle, pigs, and small ruminants), between 1982 and 2020, in 9 of the country's 10 geopolitical regions. In 1983, Brucella abortus and B. melitensis were isolated in cattle, but no further bacterial isolation has been published since. The seroprevalence from 196 total humans has ranged between 5.6% and 28.1%, and between 3.0% and 30.8% for 14,044 total cattle. As there is no ongoing surveillance program, it is not currently possible to identify the specific Brucella spp. that are endemic to the country and its regions. There are sufficient agricultural systems of cattle, pigs, goats, and sheep to sustain the presence of multiple Brucella spp. Surveillance information is the cornerstone of epidemiologic decision making, and is needed to direct policy makers, public health authorities, and veterinary services to appropriate actions. A combination of serological and molecular based diagnostics for surveillance is necessary to identify, quantify, and direct the appropriate public health interventions. Cameroon has an opportunity to build public and animal health infrastructure, leading the way for central Africa in the management and future eradication of brucellosis.

First identification of a Brucella abortus biovar 4 strain from yak in Tibet, China.

Brucellosis is one of the major zoonotic diseases in the world. In China, understanding on its causative agent Brucella is still limited. Recently, we isolated a Brucella strain XZ19-1 from yak in Lhasa, Tibet. Phenotypical characterization proved that it belongs to B. abortus biovar 4, a biotype that has never been reported in China. MLVA-16 genotyping revealed a novel profile (4-5-3-12-2-2-3-3-8-32-8-5-4-3-3-3) in this strain, while MLST sequence typing demonstrated that it belongs to ST 71. Furthermore, the whole genome of XZ19-1 strain was sequenced. Subsequent phylogenetic analysis demonstrated that XZ19-1was genetically more closely related to B. abortus strains originated from European countries rather than to those collected from China previously. Isolation and identification of XZ19-1 strain may thus indicate a unique Brucella lineage existing in Qing-Tibet plateau. These findings will help to improve the diagnosis and epidemiological studies of brucellosis in animals and human in this part of China.

Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk.

Brucellosis and tuberculosis are diseases of great economic impact in cattle herds and are controlled by governmental programs in many countries. The validation of a diagnostic technique is fundamental for its application in official control programs of these diseases. The aim of the present study was to validate a polymerase chain reaction in real time (qPCR) for detection of Mycobacterium bovis and Brucella abortus in samples of artificially contaminated raw milk. The technique was evaluated using tests of analytical sensitivity and specificity, repeatability, internal reproducibility, and robustness. Initially, five DNA extraction methodologies were tested, and the DNeasy Mericon Food Kit-Qiagen and the Maxwell® 16 Tissue DNA Purification Kit-Promega presented the best analytical specificity of all the commercial kits tested and were used exclusively in subsequent tests. The lowest limits of detection obtained in the qPCR were 2.3 pg for M. bovis DNA and 20.7 fg for B. abortus DNA. The repeatability and reproducibility associated with the robustness indicate that the evaluated methods are applicable as rapid tools for the official in vivo diagnosis of bovine tuberculosis and brucellosis in raw milk from dairy herds in Brazil.

Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis.

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10-100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 102 to 108 cells mL-1 and limit of detection at 103 cells mL-1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL-1) and mice IgG (detection antibody, 50 µg mL-1) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 102 cells mL-1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (KD) and Maximum Binding Capacity (Bmax) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

Persistence of Brucella abortus lineages revealed by genomic characterization and phylodynamic analysis.

Brucellosis, caused by Brucella abortus, is a major disease of cattle and humans worldwide distributed. Eradication and control of the disease has been difficult in Central and South America, Central Asia, the Mediterranean and the Middle East. Epidemiological strategies combined with phylogenetic methods provide the high-resolution power needed to study relationships between surveillance data and pathogen population dynamics, using genetic diversity and spatiotemporal distributions. This information is crucial for prevention and control of disease spreading at a local and worldwide level. In Costa Rica (CR), the disease was first reported at the beginning of the 20th century and has not been controlled despite many efforts. We characterized 188 B. abortus isolates from CR recovered from cattle, humans and water buffalo, from 2003 to 2018, and whole genome sequencing (WGS) was performed in 95 of them. They were also assessed based on geographic origin, date of introduction, and phylogenetic associations in a worldwide and national context. Our results show circulation of five B. abortus lineages (I to V) in CR, phylogenetically related to isolates from the United States, United Kingdom, and South America. Lineage I was dominant and probably introduced at the end of the 19th century. Lineage II, represented by a single isolate from a water buffalo, clustered with a Colombian sample, and was likely introduced after 1845. Lineages III and IV were likely introduced during the early 2000s. Fourteen isolates from humans were found within the same lineage (lineage I) regardless of their geographic origin within the country. The main CR lineages, introduced more than 100 years ago, are widely spread throughout the country, in contrast to new introductions that seemed to be more geographically restricted. Following the brucellosis prevalence and the farming practices of several middle- and low-income countries, similar scenarios could be found in other regions worldwide.

Prevalence and speciation of brucellosis in febrile patients from a pastoralist community of Tanzania.

Brucellosis is an endemic zoonosis in sub-Saharan Africa. Pastoralists are at high risk of infection but data on brucellosis from these communities are scarce. The study objectives were to: estimate the prevalence of human brucellosis, identify the Brucella spp. causing illness, describe non-Brucella bloodstream infections, and identify risk factors for brucellosis in febrile patients from a pastoralist community of Tanzania. Fourteen (6.1%) of 230 participants enrolled between August 2016 and October 2017 met study criteria for confirmed (febrile illness and culture positivity or ≥four-fold rise in SAT titre) or probable (febrile illness and single SAT titre ≥160) brucellosis. Brucella spp. was the most common bloodstream infection, with B. melitensis isolated from seven participants and B. abortus from one. Enterococcus spp., Escherichia coli, Salmonella enterica, Staphylococcus aureus and Streptococcus pneumoniae were also isolated. Risk factors identified for brucellosis included age and herding, with a greater probability of brucellosis in individuals with lower age and who herded cattle, sheep or goats in the previous 12 months. Disease prevention activities targeting young herders have potential to reduce the impacts of human brucellosis in Tanzania. Livestock vaccination strategies for the region should include both B. melitensis and B. abortus.

Comparative evaluation of fluorescence polarization assay and competitive ELISA for the diagnosis of bovine brucellosis vis-a-vis sero-monitoring.

Brucellosis is an important zoonosis that constitutes a serious public health hazard which is caused by a bacterium belonging to the genus Brucella. In the present study, two highly specific serological tests for brucellosis diagnosis, fluorescence polarization assay (FPA) and competitive ELISA (cELISA) were standardized in the laboratory, evaluated and compared with rose bengal plate test (RBPT), indirect ELISA (iELISA) and commercial cELISA kit. For test evaluation, 1386 serum samples [apparently healthy animals (n = 260), samples from Brucella infected farms (n = 701) and B. abortus S19 vaccinated animals (n = 425)] were analyzed to assess suitable diagnostic test in B. abortus S19 post vaccinated bovine population. In apparently healthy brucellosis free farms, RBPT, iELISA, in-house FPA and cELISA were found to be highly specific than commercial cELISA. Commercial cELISA kit was comparatively more sensitive than other serological tests in samples collected from infected farms. The FPA showed sensitivity nearly equal to RBPT and in-house cELISA showed greater sensitivity than RBPT in infected farms. In animals with persistent vaccinal antibodies, only in-house FPA and cELISA recorded higher specificity of 87.64 and 90.27%, respectively. The other tests, RBPT and iELISA displayed similar reactivity with vaccine antibodies to that of infection antibodies whereas commercial cELISA kit showed an intermediate specificity of 47.69%. With these findings, RBPT, iELISA and cELISA are suggested for screening infected herds, and in-house developed FPA and cELISA tests with a proven specificity can be used for confirmatory diagnosis of brucellosis in B. abortus S19 post vaccinated animal populations.

Comparative proteomic and genomic analyses of Brucella abortus biofilm and planktonic cells.

The present study aimed to explore the differences in protein and gene expression of Brucella abortus cultured under biofilm and planktonic conditions. The proteins unique to biofilms and planktonic B. abortus were separated by two­dimensional (2­D) electrophoresis and then identified by matrix­assisted laser desorption/ionization­tandem time of flight­mass spectrometry (MALDI­TOF/TOF­MS). High­throughput sequencing and bioinformatic analyses were performed to identify differentially expressed genes between B. abortus cultured under biofilm and planktonic conditions. The proteins and genes identified by proteomic and genomic analyses were further evaluated via western blot and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analyses. 2­D electrophoresis identified 20 differentially expressed protein spots between biofilms and planktonic cells, which corresponded to 18 individual proteins (12 downregulated and 6 upregulated) after MALDI­TOF/TOF­MS analysis, including elongation factor Tu and enolase. RT­qPCR analysis revealed that all of the 18 genes were downregulated in biofilms compared with planktonic cells. Western blot analysis identified 9 downregulated and 3 upregulated proteins. High­throughput sequencing and bioinformatic analyses identified 14 function and pathway­associated genes (e.g., BAbS19_I14970). RT­qPCR analysis of the 14 genes showed that they were upregulated in biofilm compared with in planktonic state. In conclusion, these differentially expressed genes may play important roles in bacterial defense, colonization, invasion, and virulence.

Survey of Leptospira spp. and Brucella abortus in Free-Ranging Armadillos from Pantanal, Brazil.

Out of the 20 recognized species of armadillos in the world, 11 are found in Brazil, and five of them are found in Pantanal, one of the world's largest wetlands. Beef cattle (Bos taurus) farming is the main economic activity in this region, which promotes intense wildlife-livestock contact and increases the likelihood of pathogen exposure, including to agents with zoonotic and economic relevance. Previous studies demonstrated that several wildlife species in Pantanal have been exposed to Brucella abortus and Leptospira spp.; however, little is known regarding the exposure and/or prevalence of zoonotic pathogens in armadillos. We used conventional PCR, the rose Bengal test (RBT), and the microscopic agglutination test (MAT) to investigate the exposure to and infection by Brucella spp. and Leptospira spp. using blood samples from four species of armadillos: nine-banded armadillo (Dasypus novemcinctus, n=2), southern naked-tailed armadillo (Cabassous unicinctus, n=8), yellow armadillo (Euphractus sexcinctus, n=16), and giant armadillo (Priodontes maximus, n=22), captured in Nhecolândia, Pantanal, Brazil. Samples were PCR- and RBT-negative for Brucella spp. infection and exposure. However, MAT revealed a Leptospira spp. seroprevalence of 31% (5/16; 95% confidence interval [CI]=0.11-0.58) in yellow armadillo and 18% (4/22; 95% CI=0.05-0.40) in giant armadillo specimens to serogroups Autumnalis, Cynopteri, and Pomona, with titers ranging from 200 to 1,600. Our results contribute to the understanding of zoonotic pathogens in armadillos in Pantanal and reinforce the importance of wildlife health surveillance in this area.