If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

Diagnostic utility of LAMP PCR targeting bcsp-31 gene for human brucellosis infection.

PURPOSE: Human brucellosis is a neglected zoonotic disease of significant public health concern. Molecular diagnosis of brucella remains challenging in low resource settings, due to the high infrastructure and cost involved. Loop-mediated isothermal amplification (LAMP) is a rapid point of care polymerase chain reaction (PCR) with the utility of on-field molecular diagnosis and offers a convenient alternative to conventional PCR. In the present study, we developed and evaluated the diagnostic utility of in house LAMP PCR targeting the Brucella genus-specific bcsp-31 gene in patients having febrile illness. MATERIALS AND METHODS: The analytical sensitivity and specificity of bcsp-31 LAMP PCR was first evaluated using brucella (n â€‹= â€‹8) and non-brucella cultures (n â€‹= â€‹5), along with spiked clinical samples. The overall diagnostic utility of developed LAMP PCR was then further evaluated in 393 human samples suspected of brucellosis. RESULTS: The developed LAMP PCR could detect as low as 8 â€‹fg of DNA by visual detection within 35min. We report sensitivity and specificity of the developed LAMP PCR as 90.91% and 99.37%.The accuracy of the developed test assay was found to be 98.60%. In clinical samples, LAMP gave positivity of 20% with the concordance of 89% with conventional PCR. CONCLUSION: To conclude, a rapid, efficacious, sensitive LAMP PCR targeting the bcsp 31 gene was developed. The existing LAMP PCR can be used as a point of care screening test in various low resource endemic setting in lieu of conventional PCR for estimation of prevalence data, diagnosis and treatment of brucellosis.

Characterization of Brucella spp. and other abortigenic pathogens from aborted tissues of cattle and goats in Rwanda.

BACKGROUND: Abortions cause tremendous economic losses in food-producing animals and may lead to food insecurity. OBJECTIVES: This study aimed to characterize Brucella spp. and other abortigenic pathogens from aborted tissues of cattle. METHODS: For cattle, aborted tissues (n = 19) were cultured, and Brucella spp. were detected using the genus-specific 16S-23S ribosomal DNA interspacer region (ITS) assay and speciated using Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR assays. Brucella negative samples were screened using the eight abortigenic pathogens PCR panel. Samples from an abortion outbreak that occurred within a goat tribe were included in this investigation. Sera of females (n = 8) and males (n = 2) were analyzed using the Rose Bengal Test (RBT) and indirect enzyme-linked immunosorbent assay (i-ELISA), while vaginal swabs (n = 3) and aborted tissues (n = 1) were cultured and characterized. RESULTS: The ITS-PCR detected Brucella DNA in cultures from two aborted tissues of cattle (10.5%, [2/19]), which were identified as B. melitensis (n = 1), and B. abortus (n = 1) using AMOS and Bruce-ladder PCR assays. Campylobacter fetus (n = 7) and Leptospira spp. (n = 4) including co-infections (n = 2) of C. fetus and Leptospira spp. were identified from the Brucella negative samples of cattle. Goats (100.0%, 10/10) were brucellosis seropositive on RBT and i-ELISA. Mixed infections caused by B. melitensis and B. abortus were isolated from the vaginal swabs (n = 3) and aborted tissues (n = 1). DISCUSSION AND CONCLUSIONS: This is the first identification of abortion-associated pathogens in aborted cattle indicating the enormous financial losses and a threat to public health. It is therefore essential to include these identified pathogens in the surveillance scheme of veterinary and human services.

Herd-level contamination of Neospora caninum, Toxoplasma gondii and Brucella in milk of Iranian dairy farms.

The bulk milk examination is a reliable screening tool for monitoring the quality of milk in the farms. The infection to Neospora caninum, Toxoplasma gondii and Brucella sp. Was evaluated in bulk milk samples of dairy farms in Hamedan province, West part of Iran. All the dairy farms (n = 149) were examined for N. caninum, T. gondii and Brucella infections using milk ring test (MRT), microbiology, serology (Enzyme-linked Immunosorbent Assay), and molecular techniques. Based on molecular methods, Brucella-infection was negative in all farms; while, 55 %, 5.4 % and 2.7 % of samples were positive for N. caninum, T. gondii and mix infection, respectively. The highest Neospora-infection was detected in the farms with history of abortion in fall and winter. There was significant association between Neospora-infection and the presence of dogs and rodents in the farms, herd size, and age of the animals. Also, a significant association was seen between Toxoplasma-infection and the presence of cats and rodents in the farms, as well as age of the animals. Average total bacterial count (TBC) was calculated 1.14 × 106±1.1 × 106. The highest TBC was in the farms from Central locations of studied area (5.7 × 106±2.24 × 106), farms with more than 120 animals (7.9 × 106±2.8 × 106), and farms with ≥50-months age (1.74 × 106±6.3 × 105) in spring and summer (6.9 × 106±3.7 × 106). The number of somatic cells was estimated between 1 × 104 and 2 × 106 (Average = 4.2 × 105±3.39 × 105). The current study was a comprehensive evaluation of Neospora, Toxoplasma and Brucella infections in milk samples of Iranian dairy farms for the first time. Neospora-infection is responsible for economic losses in the region. Health education and milk pasteurization are so helpful for inhibiting the milk borne diseases. To reduce the risk factors, predict and design the appropriate schemes like redundant of heterogeneous animals are recommended.

Genomics of Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis) reveals a new blaOXA subgroup.

Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis) is an emerging pathogen in immunodeficient and immunocompetent patients. Most documented cases associated with Ochrobactrum are frequently catheter-related and exhibit wide-spectrum ß-lactam resistance. Misidentification of this pathogen using commercial bacterial identification kits is common. We identified a case of O. pseudogrignonense infection associated with cholelithiasis. The O. pseudogrignonense genome was sequenced and reconstructed using a Nanopore and Illumina hybrid strategy. A novel blaOXA-919 divergent from existing OXA members was identified and subsequent analysis revealed its existence in all available O. pseudogrignonense genomes, which forms a new phylogenetic subgroup distinct from other OXA clusters. Further analysis demonstrated the presence of the novel blaOXA-919 in the chromosome of several other Ochrobactrum species. Our study indicated that Ochrobactrum chromosomes may be a reservoir of blaOXA-919 ß-lactamases.

Emerging diversity and ongoing expansion of the genus Brucella.

Remarkable genetic diversity and breadth of host species has been uncovered in the Brucella genus over the past decade, fundamentally changing our concept of what it means to be a Brucella. From ocean fishes and marine mammals, to pond dwelling amphibians, forest foxes, desert rodents, and cave-dwelling bats, Brucella have revealed a variety of previously unknown niches. Classical microbiological techniques have been able to help us classify many of these new strains but at times have limited our ability to see the true relationships among or within species. The closest relatives of Brucella are soil bacteria and the adaptations of Brucella spp. to live intracellularly suggest that the genus has evolved to live in vertebrate hosts. Several recently discovered species appear to have phenotypes that are intermediate between soil bacteria and core Brucella, suggesting that they may represent ancestral traits that were subsequently lost in the traditional species. Remarkably, the broad relationships among Brucella species using a variety of sequence and fragment-based approaches have been upheld when using comparative genomics with whole genomes. Nonetheless, genomes are required for fine-scale resolution of many of the relationships and for understanding the evolutionary history of the genus. We expect that the coming decades will reveal many more hosts and previously unknown diversity in a wide range of environments.

Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

New Brucella variant isolated from Croatian cattle.

BACKGROUND: A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme. CASE PRESENTATION: Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3. CONCLUSIONS: Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.

Comparative study of IS711 and bcsp31-based polymerase chain reaction (PCR) for the diagnosis of human brucellosis in whole blood and serum samples.

BACKGROUND: Clinical diagnosis of human brucellosis (HB) is often difficult due to non-specific symptoms. Immunological tests have been the most common method used in HB diagnosis, but molecular methods based on quantitative polymerase chain reaction (qPCR) have largely replaced these diagnostic methods. The aim of this study was to validate a HB diagnostic qPCR method; assessing different target Brucella genes, and the influence of biological matrices (serum vs. whole blood) on analytical parameters. MATERIAL AND METHODS: Two target genes, IS711 and bcsp31, for HB molecular diagnosis were evaluated, together with biological matrix type (whole blood and serum) using samples spiked with Brucella abortus. In addition, diagnostic parameters of this qPCR method were evaluated in paired whole blood and serum samples from patients with suspected HB. RESULTS: Both genes could be potential diagnostic targets, but IS711 showed a lower limit of detection. In spiked matrix experiments, whole blood showed a lower limit of detection than serum after probit regression (224 vs. 3681 CFU/mL) and ANOVA analysis showed a significant (p < 0.001) difference between the Cq of whole blood at all dilutions and that of serum. In 12 paired clinical samples, no serum samples and only one whole blood sample tested positive for Brucella using this qPCR detection method. CONCLUSIONS: This standardized qPCR-based Brucella detection method could improve diagnosis of HB, serving as a rapid, highly sensitive, and specific test. Whole blood is better suited to qPCR-based HB diagnosis due to the presence of higher target DNA loads in this matrix, compared to serum.

The one hundred year journey of the genus Brucella (Meyer and Shaw 1920).

The genus Brucella, described by Meyer and Shaw in 1920, comprises bacterial pathogens of veterinary and public health relevance. For 36 years, the genus came to include three species that caused brucellosis in livestock and humans. In the second half of the 20th century, bacteriologists discovered five new species and several 'atypical' strains in domestic animals and wildlife. In 1990, the Brucella species were recognized as part of the Class Alphaproteobacteria, clustering with pathogens and endosymbionts of animals and plants such as Bartonella, Agrobacterium and Ochrobactrum; all bacteria that live in close association with eukaryotic cells. Comparisons with Alphaproteobacteria contributed to identify virulence factors and to establish evolutionary relationships. Brucella members have two circular chromosomes, are devoid of plasmids, and display close genetic relatedness. A proposal, asserting that all brucellae belong to a single species with several subspecies debated for over 70 years, was ultimately rejected in 2006 by the subcommittee of taxonomy, based on scientific, practical, and biosafety considerations. Following this, the nomenclature of having multiples Brucella species prevailed and defined according to their molecular characteristics, host preference, and virulence. The 100-year history of the genus corresponds to the chronicle of scientific efforts and the struggle for understanding brucellosis.

Brucella Genomics: Macro and Micro Evolution.

Brucella organisms are responsible for one of the most widespread bacterial zoonoses, named brucellosis. The disease affects several species of animals, including humans. One of the most intriguing aspects of the brucellae is that the various species show a ~97% similarity at the genome level. Still, the distinct Brucella species display different host preferences, zoonotic risk, and virulence. After 133 years of research, there are many aspects of the Brucella biology that remain poorly understood, such as host adaptation and virulence mechanisms. A strategy to understand these characteristics focuses on the relationship between the genomic diversity and host preference of the various Brucella species. Pseudogenization, genome reduction, single nucleotide polymorphism variation, number of tandem repeats, and mobile genetic elements are unveiled markers for host adaptation and virulence. Understanding the mechanisms of genome variability in the Brucella genus is relevant to comprehend the emergence of pathogens.

Detection of Brucella spp. in raw milk from various livestock species raised under pastoral production systems in Isiolo and Marsabit Counties, northern Kenya.

INTRODUCTION: Brucellosis is an important zoonotic disease in Kenya, and identifying the bacteria in milk is important in assessing the risk of exposure in people. METHODS: A cross-sectional study that involved 175 households was implemented in the pastoral counties of Marsabit and Isiolo in Kenya. Pooled milk samples (n = 164) were collected at the household level, and another 372 were collected from domesticated lactating animals (312 goats, 7 sheep, 50 cattle and 3 camels). Real-time polymerase chain reaction (qPCR) testing of the milk samples was performed to identify Brucella species. Brucella anti-LPS IgG antibodies were also detected in bovine milk samples using an indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Based on the qPCR, the prevalence of the pathogen at the animal level (considering samples from individual animals) was 2.4% (95% confidence interval (CI) 1.1-4.5) and 3.0% (CI: 1.0-7.0) in pooled samples. All 14 samples found positive by qPCR were from goats, with 10 contaminated with B. abortus and 4 with B. melitensis. The Brucella spp. antibody prevalence in bovine milk using the milk ELISA was 26.0% (95% CI: 14.6-40.3) in individual animal samples and 46.3% (95% CI: 30.7-62.6) in pooled samples. CONCLUSION: The study is the first in Kenya to test for Brucella spp. directly from milk using qPCR without culturing for the bacteria. It also detected B. abortus in goats, suggesting transmission of brucellosis between cattle and goats. The high prevalence of Brucella spp. is a significant public health risk, and there is a need for intervention strategies necessary in the study area.

Expanding the host range: infection of a reptilian host (Furcifer pardalis) by an atypical Brucella strain.

Atypical brucellae show deviant phenotypes and/or genotypes. Besides Brucella inopinata, B. microti and B. vulpis, atypical strains have been described infecting humans, rodents, amphibians and fish. They represent potential zoonotic agents. Here, we provide evidence that reptiles as the remaining poikilothermic vertebrate class also represent susceptible hosts for atypical Brucella.

Molecular characterization of Brucella species detected in humans and domestic ruminants of pastoral areas in Kagera ecosystem, Tanzania.

Brucellosis is a zoonotic disease of importance to both public health and the livestock industry. The disease is likely to be endemic in Tanzania and little is reported on molecular characterization of Brucella species in pastoral settings. This study aimed at characterizing Brucella species (targeting genus Brucella) infecting humans, cattle and goat in Kagera region (Ngara and Karagwe districts) using real-time PCR, PCR amplification of 16S rRNA genes and Sanger sequencing. Brucella spp. were detected in 47 samples (19 sera and 28 milk) out of 125 samples (77 sera, 35 milk and 13 aborted materials) using real-time PCR. All aborted materials (13 samples) were negative to real-time PCR. Out of the 47 real-time PCR positive samples (28 milk and 19 sera), 20 samples (10 milk and 10 sera) showed an expected 16S rRNA gene PCR product. Sequence analysis and blasting confirmed the presence of Brucella spp. in pastoral areas of Kagera region. The Brucella spp. from Kagera were phylogenetically grouped in two clades and three branches all closer to B. melitensis, B. abortus and B. suis from USA, Sudan and Iran. However, they were distinct from other species isolated also in USA, New Zealand, Germany and Egypt. This was expected based on the distance between the geographical regions from which the data (nucleotides sequences from 16S gene sequencing) for the phylogeny reconstruction were obtained. This is the first study to report Brucella species identified using 16S rRNA gene sequencing in East and Central Africa. A livestock vaccination program re-inforced with a high index of Brucella diagnosis is needed to eradicate brucellosis in animals and minimize suffering from Brucella infections in humans in Tanzania.

Brucellosis in the Middle East: Current situation and a pathway forward.

Brucellosis is a bacterial endemic zoonotic disease of global significance with detrimental impacts on public health and food animal production. It is caused by Brucella spp., an expanding group of pathogens able to infect various host species. Bovines and small ruminants, which excrete the bacteria in milk and in reproductive discharges, are major sources of infection for humans and other animals. Contact with contaminated animals and consumption of unpasteurized dairy products are the main routes for human infection. In spite of the considerable progress of knowledge gained and success achieved in brucellosis control in the developed world, this disease continues to be an important burden in the Middle East (ME). Common risk factors implicated in the difficulty and complexity of brucellosis control within the region include (1) social and political instabilities; (2) insufficient resources and infrastructure for appropriate diagnosis, reporting, and implementation of control measures; (3) variation of livestock husbandry systems and their commingling with other livestock and wildlife; and (4) traditional cultural practices, including consumption of unpasteurized dairy products. Development of core interdisciplinary competencies is required for a true One Health-based endeavor against the disease. National awareness and educational programs addressing all population sectors from consumers to decision-makers seem to be the next logical, sustainable, and economically viable approach toward improving disease status in this region. In the present review, we describe the current situation of brucellosis in the ME, focusing on the major limitations and shortcomings regarding disease control. We propose a regional approach toward public awareness of brucellosis as the first step in mitigating the disease and discuss the potential benefits, and components of such a strategy, which can further be used as a model for other endemic zoonotic diseases.

Survival of Brucella abortus S19 and other Brucella spp. in the presence of oxidative stress and within macrophages.

The evolutionary "success" of the genus Brucella depends on the ability to persist both in the environment as well as inside of even activated macrophages of the animal host. For that, the Brucellae produce catalase and superoxide dismutase to defend against oxidative stress. Since the deletion of the mglA gene in the B. abortus S19 vaccine strain resulted not only in an increased tolerance to H2O2 but also in the induction of cytokines in macrophages, we here investigated the effect of oxidative stress (Fe2+ and H2O2) on the survival of B. abortus S19 and the isogenic B. abortus S 19 ∆mglA 3.14 deletion mutant in comparison with B. neotomae 5K33, Brucella strain 83/13, and B. microti CCM4915. These Brucellae belong to different phylogenetic clades and show characteristic differences in the mgl-operon. From the various Brucellae tested, B. abortus S19 showed the highest susceptibility to oxidative stress and the lowest ability to survive inside of murine macrophages. B. abortus S19 ∆mglA 3.14 as well as B. neotomae, which also belongs to the classical core clade of Brucella and lacks the regulators of the mgl-operon, presented the highest degree of tolerance to H2O2 but not in the survival in macrophages. The latter was most pronounced in case of an infection with B. 83/13 and B. microti CCM4915. The various Brucellae investigated here demonstrate significant differences in tolerance against oxidative stress and different survival in murine macrophages, which, however, do not correlate directly.

Molecular investigation of infection sources and transmission chains of brucellosis in Zhejiang, China.

In the present study, a total of 7793 samples from 5 different types of hosts were collected and tested, with a seroprevalence of 2.4% (184/7793). Although the seroprevalence of human and animal brucellosis is relatively low, numbers of human brucellosis cases reported have increased continuously from 2004 to 2018. A total of 118 Brucella strains containing 4 biotypes were obtained, including Brucella melitensis bv.1 (n = 8) and bv.3 (n = 106), Brucella abortus bv.3 (n = 3) and bv.7 (n = 1). Twenty-one shared MLVA-16 genotypes, each composed of 2 to 19 strains obtained from different hosts, suggest the occurrence of a brucellosis outbreak epidemic with multiple source points and laboratory infection events. Moreover, 30 shared MLVA-16 genotypes were observed among 59.6% (68/114) B. melitensis isolates from Zhejiang and strains from other 21 different provinces, especially northern provinces, China. The analysis highlighted the imported nature of the strains from all over the northern provinces with a dominant part from the developed areas of animal husbandry. These data revealed a potential transmission pattern of brucellosis in this region, due to introduced infected sheep leading to a brucellosis outbreak epidemic, and eventually causing multiple laboratory infection events. It is urgent to strengthen the inspection and quarantine of the introduced animals.

Genome-wide unique insertion sequences among five Brucella species and demonstration of differential identification of Brucella by multiplex PCR assay.

Brucellosis is a neglected zoonotic disease caused by alpha proteobacterial genus Brucella comprising of facultative intracellular pathogenic species that can infect both animals and humans. In this study, we aimed to identify genome-wide unique insertion sequence (IS) elements among Brucella abortus, B. melitensis, B. ovis, B. suis and B. canis for use in species differentiation by conducting an intensive in silico-based comparative genomic analysis. As a result, 25, 27, 37, 86 and 3 unique ISs were identified respectively and they had a striking pattern of distribution among them. To explain, a particular IS would be present in four species with 100% identity whereas completely absent in the fifth species. However, flanking regions of that IS element would be highly identical and conserved in all five species. Species-specific primers designed on these flanking conserved regions resulted in two different amplicons grouping the species into two: one that possesses IS and the other that lacks it. Seeking for species-specific amplicon size for particular species was sufficient to identify it irrespective of biovar. A multiplex PCR developed using these primers resulted in successful differentiation of the five species irrespective of biovars with significant specificity and sensitivity when examined on clinical samples.

Evaluation of serological diagnostic tests of human brucellosis for prevention and control in Mexico.

Brucellosis is a zoonosis mainly present in developing countries. The WHO reports 500,000 new cases every year. From 2012 to 2016, 13,677 cases were reported in Mexico, with 2.00 to 2.64 rate per 100,000 inhabitants. To analyze the diagnostic algorithm of brucellosis in Mexico, we compared the commercial laboratory tests ELISA, Brucellacapt®, and lateral flow test (LFT) in a study of 473 individuals from two endemic Mexican populations. All patients were treated in first-level medical units for presenting brucellosis compatible symptoms and without a history of the disease. Clinical-epidemiological information was gathered and initial serum samples were obtained to react with anti-Brucella antibodies; subsequent samples were collected at follow-up treatment visits. Using the Rose Bengal screening, we found 165 negative samples and 308 positive reactive samples, of which 222 cases were confirmed and 234 were positive on at least one marker (IgG or IgM) or LFT. When Brucellacapt® was used, similar results to those observed with the conventional algorithm were found as judged by the Cohen's kappa coefficient (κ) (0.813, 95% CI 0.7788-0.8472). Similar κ indices between conventional algorithm and ELISA pair were found, 0.7038 (95% CI 0.6555-0.7521), representing high similarity between both groups of diagnosis. We conclude that conventional serodiagnoses, Brucellacapt® and LFT, presented inconclusive results and poor correlation between them. By contrast, ELISA test pair (IgG + IgM) presented high correlation with the conventional algorithm and greater capacity for correct positive and negative classification.

Investigation of the molecular characteristics of Brucella isolates from Guangxi Province, China.

BACKGROUND: Human brucellosis has become a severe public health problem in China's Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates. RESULTS: A total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012-2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group. CONCLUSIONS: Our investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region.