A 14th century CE Brucella melitensis genome and the recent expansion of the Western Mediterranean clade.

Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species-Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14th-century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14th CE and suggest that its divergence was due to human and ruminant co-migration.

Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice.

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B. melitensis. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B. melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B. melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and ß oxidation of fatty acids are no longer required in Interleukin (IL)-17RA-/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B. melitensis in vivo.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains.

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.

Molecular examination for Coxiella burnetii and Brucella spp. infections in Iranian women experiencing spontaneous miscarriage.

BACKGROUND: Spontaneous miscarriage, a leading health concern globally, often occurs due to various factors, including infections. Among these, Coxiella burnetii and Brucella spp. may have adverse effects on pregnancy outcomes. While previous research has established a link between infections and spontaneous miscarriage, our study aimed specifically to investigate the presence of these two pathogens in abortion samples from women who experienced spontaneous miscarriages in Iran. Our study can add to the existing knowledge by focusing on Iran, a region with a high prevalence of C. burnetii and Brucella spp. As a result, it could provide a better understanding and unique insights into the relationship of these pathogens with spontaneous miscarriages in endemic regions. METHODS: From March 2021 to March 2022, a total of 728 abortion samples (including placenta and cotyledon) were collected from 409 women who had experienced spontaneous miscarriages in the provinces of Tehran, Fars, and West Azerbaijan in Iran. The specimens included 467 Formalin-Fixed Paraffin-Embedded (FFPE) and 261 fresh frozen samples. After DNA extraction from abortion samples, the quantitative real-time PCR (qPCR) assay targeted a specific fragment of the IS1111 and IS711 elements for molecular identification of C. burnetii and Brucella spp., respectively. Furthermore, the qPCR assay employing specific primers for different species was used to determine the species of Brucella. RESULTS: Among the studied women, 1 out of 409 (0.24%) samples tested positive for Brucella spp., specifically Brucella melitensis. There were no positive specimens for C. burnetii. CONCLUSIONS: Our study contributes to understanding the potential involvement of Brucella species in spontaneous infectious abortion within endemic regions. The identification of B. melitensis in this study highlights the need for further research in this area. However, while our results suggest a relatively low or zero identification of these pathogens in our sample population, this does not rule out the possibility of undetected infections. Therefore, it is critical to acknowledge the limitations of the molecular techniques used (qPCR), which may have potential limitations such as sensitivity and specificity. Moreover, because 64.15% of our samples were FFPE, the sensitivity of the qPCR test may be reduced. These raise concerns about the accuracy of the reported prevalence rates and the potential for false positives or negatives.

Molecular epidemiological characteristics of osteoarthritis-associated Brucella melitensis in China: evidence from whole-genome sequencing-based analysis.

BACKGROUND: Brucellosis, developing complications including arthritis, spondylitis, sacroiliitis, and osteomyelitis, is one of the most common zoonotic diseases in the current world which causes economic losses to the livestock industry and is a great public health concern. Brucella melitensis are the main pathogen of brucellosis epidemics in China, most of which are located in northern China. However, there is limited knowledge about the epidemiology of osteoarthritis-associated brucellosis. This study was aimed to reveal the prevalence of osteoarthritis-associated brucellosis in Inner Mongolia and also to investigate the molecular characteristics of B. melitensis isolates. METHODS AND RESULTS: In 2018, the osteoarthritis symptoms of brucellosis in the Brucellosis department of a hospital in Inner Mongolia were investigated. Twenty osteoarthritis-associated B. melitensis strains, isolated from the inpatients in Inner Mongolia during 2013-2017, were subjected to whole genome sequencing. The multilocus sequence type (MLST) and core genome SNP (cgSNP) analysis were conducted to detect molecular epidemiological characteristics. The incidence of brucellosis osteoarthritis symptoms in males (85/120, 70.8%) was significantly higher than that in females (35/120, 29.2%), and the age of patients was concentrated between 41 and 60 years old. In silico analyses indicated ST8 was the prevalent sequence type and the transmission of osteoarthritis-associated B. melitensis among different geographical areas. All strains carry virulence genes, including cgs, lpsA, manCoAg, pgm, pmm, virB4, wbdA and wboA. CONCLUSION: Our study showed the close epidemiologically connection of osteoarthritis-associated B. melitensis strains in northern China. And ST8 was the prevalent sequence type which need our attention.

Prevalence of Brucella melitensis and Brucella abortus tetracyclines resistance: A systematic review and meta-analysis.

INTRODUCTION: Brucellosis is a zoonotic disease that can be transmitted from animals to humans. Brucellosis is caused by bacteria of the genus Brucella, which are typically transmitted through contact with infected animals, unpasteurized dairy products, or airborne pathogens. Tetracyclines (tetracycline and doxycycline) are antibiotics commonly used to treat brucellosis; however, antibiotic resistance has become a major concern. This study assessed the worldwide prevalence of tetracycline-resistant Brucella isolates. METHODS: A systematic search was conducted in Scopus, PubMed, Web of Science, and EMBASE using relevant keywords and Medical Subject Headings (MeSH) terms until August 13, 2022, to identify relevant studies for meta-analysis. A random effects model was used to estimate the proportion of resistance. Meta-regression analysis, subgroup analysis, and examination of outliers and influential studies were also performed. RESULTS: The prevalence rates of resistance to tetracycline and doxycycline were estimated to be 0.017 (95% confidence interval [CI], 0.009-0.035) and 0.017 (95%CI, 0.011-0.026), respectively, based on 51 studies conducted from 1983 to 2020. Both drugs showed increasing resistance over time (tetracycline: r = 0.077, P = 0.012; doxycycline: r = 0.059, P = 0.026). CONCLUSION: The prevalence of tetracycline and doxycycline resistance in Brucella was low (1.7%) but increased over time. This increase in tetracycline and doxycycline resistance highlights the need for further research to understand resistance mechanisms and develop more effective treatments.

The canonical Brucella species-host dependency is changing, however, the antibiotic susceptibility profiles remain unchanged.

Brucellosis is a chronic disease caused by Brucella species with a wide range of hosts, from marine mammals to terrestrial species, but with strict host preferences. With the zoonotic character, the prevalence of human brucellosis cases is a reflection of animal infections. This study aimed to identify 192 Brucella isolates obtained from various sources by Bruce-ladder PCR and to determine their antibiotic susceptibilities by gradient diffusion method (E-test). As a result of the PCR, all human isolates (n = 57) were identified as B. melitensis. While 58 (82.9%) of the cattle isolates were identified as B. abortus, 59 (90.8%) of the sheep isolates were identified as B. melitensis. In addition, 12 (17.1%) of the cattle isolates and 6 (9.2%) of the sheep isolates were determined as B. melitensis and B. abortus, respectively. The primary host change behavior of B. melitensis was 1.9 times higher than that of B. abortus. While gentamicin and ciprofloxacin susceptibilities of Brucella isolates were 100%, tetracycline, doxycycline, streptomycin, trimethoprim/sulfamethoxazole and rifampicin susceptibilities were 99%, 99%, 97.4%, 91.7% and 83.9%, respectively. The lowest sensitivity of the isolates was determined against to cefoperazone as 26%. A triple-drug resistance was detected in 1 B. abortus isolate that included simultaneous resistance to cefoperazone, rifampicin, and trimethoprim/sulfamethoxazole. The high susceptibility profiles we found against to antibiotics such as tetracycline, doxycycline gentamicin and ciprofloxacin, used widely in treatment, are encouraging. However, the change in the canonical Brucella species-primary host preference suggests the need to reconsider eradication program, including updating vaccine formulations.

Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

MLVA typing of Brucella melitensis and B. abortus isolates of animal and human origin from India.

Brucellosis is a widely prevalent zoonotic disease of major public health significance. A collection of Brucella melitensis and Brucella abortus field isolates of animal and human origin were subjected to MLVA-15 typing followed by phylogeography studies. The MLVA-15 analysis of B. melitensis (n = 65) field isolates resulted in 48 different profiles. The panel I marker bruce45 was found to be most conserved, while the rest of the panel I markers showed low to moderate length polymorphism. Among the panel II markers, bruce04, bruce16 and bruce30 showed a high discriminatory index. The MLVA-15 typing of 13 B. abortus field isolates revealed 13 different genotypes with panel II markers showing higher discriminatory ability vis-à-vis panel I. The minimum spanning tree analysis (MST) in comparison with isolates from the international database revealed that all B. melitensis and B. abortus isolates from this study belonged to the 'Eastern Mediterranean' and the 'abortus C' lineage, respectively. The MLVA-15 typing could differentiate field isolates of B. abortus and B. melitensis originating from different regions, reaffirming the technique's potential of high resolution and suitability for local epidemiological studies. The MLVA scheme also has the advantage of comparison of local isolates with a worldwide database, allowing for phylogeographical studies.

Core Genome Multilocus Sequence Typing Scheme for Improved Characterization and Epidemiological Surveillance of Pathogenic Brucella.

Brucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.

A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams.

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.

Trans-species transmission of Brucellae among ruminants hampering brucellosis control efforts in Egypt.

AIMS: This study aimed to identify the genotypic fingerprinting of Brucella melitensis biovar 3 isolates from ruminants in Kafr El-Sheikh, Egypt, to compare with other peers globally and to highlight the epidemiology and potential causes of brucellosis control failure. METHODS AND RESULTS: A multilocus variable-number tandem-repeat analysis (MLVA 16) was carried out on 41 B. melitensis bv3 isolates, 31 from the preferential hosts (28 sheep and three goats) and 10 from atypical hosts (nine cattle and one buffalo), identified by bacteriological and molecular techniques. MLVA-16 analysis revealed 19 genotypes with nine as singletons. The most prevalent genotypes were M3_K.E (3,5,3,13,1,1,3,3,7,43,8,7,6,7,5,3), M13_K.E (3,5,3,13,1,1,3,3,7,43,8,5,8,7,7,3) and M5_K.E (3,5,3,13,1,1,3,3,7,43,8,4,8,7,11,3) circulating between different animal species. The B. melitensis isolation from aborted cows in farms that had never reared small ruminants indicates the likelihood of cow to cow B. melitensis transmission. Different genotypes of B. melitensis could be isolated from the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with genotypes reported outside the study area. Worldwide, our genotypes were mostly related to the Western Mediterranean lineage and less likely to the America's clonal lineage. CONCLUSION: There is a high genetic similarity of B. melitensis bv3 genotypes among different ruminant species, and the same animal could be infected with different genotypes. There is a high probability of spreading of B. melitensis among atypical hosts in the absence of the original hosts. The genetic relatedness of B. melitensis bv3 genotypes in the study area with other different geographic areas highlighted the national and international ruminants movement role as a potential factor for maintaining B. melitensis infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations are required to understand the impact of the presence of more than one genotype of B. melitensis in the same animal on the efficacy of brucellosis control strategies.

Identification and molecular characterization of Brucella abortus and Brucella melitensis isolated from milk in cattle in Azerbaijan.

BACKGROUND: Azerbaijan currently ranks thirteenth in global incidence of human brucellosis, with an estimated annual incidence through 2000 at over 50 cases per million. Brucella melitensis has been isolated from patients and is thought to have been acquired through contact with small ruminants or as a foodborne infection. To reduce the burden of human brucellosis, the Azerbaijani government began in 2002, a nationwide vaccination control campaign in small ruminants. There is serological evidence of bovine brucellosis (presumably due to Brucella abortus) in Azerbaijan, but no prevalence estimates were available when this study started in March 2017. The aim of this study was to isolate and identify Brucella spp. from cow milk in the Ganja region, where brucellosis takes a heavy toll on humans and livestock. RESULTS: Blood and milk samples were collected from cows (n = 1075) in early lactation (up to 90-days) in farms that had a history of previous positive serological results and abortions. Twenty-two out of 57 milk samples collected from seropositive cows, showed growth on Farrell's media, when incubated with 5% CO2. Eight additional milk samples showed growth in the absence of CO2. The classical biotyping classified them as Brucella abortus (22) and Brucella melitensis (8). RT-PCR confirmed that strains belonged to the genus Brucella. MLVA profiles were obtained for DNA extracted from two B. abortus and six B. melitensis strains. While the B. abortus genetic profile was described in the MLVA database, matching the profile of B. abortus strains isolated in East Europe, Central Asia and China, we found a new genotype for the B. melitensis strains isolated in Azerbaijan, clustering with strains belonging to the American clade, rarely identified in the region. CONCLUSION: Despite the implementation of the vaccination program in small ruminants, our results suggest that spill-over events of B. melitensis from small ruminants to cattle have occurred. However, cattle are likely to be primarily infected with B. abortus, which warranted the implementation of a bovine brucellosis program. Such a program started in fall 2017. In the Ganja region, cattle should be considered as a potential source of B. abortus and B. melitensis for humans.

Comprehensive Proteomic Analysis of Brucella melitensis ATCC23457 Strain Reveals Metabolic Adaptations in Response to Nutrient Stress.

In the present study, a comprehensive proteomic analysis of Brucella melitensis (B. melitensis) strain ATCC23457 was carried out to investigate proteome alterations in response to in vitro-induced nutrient stress. Our analysis resulted in the identification of 2440 proteins, including 365 hypothetical proteins and 850 potentially secretory proteins representing ~77.8% of the B. melitensis proteome. Utilizing a proteogenomics approach, we provide translational evidence for eight novel putative protein-coding genes and confirmed the coding potential of 31 putatively annotated pseudogenes, thus refining the existing genome annotation. Further, using a label-free quantitative proteomic approach, new insights into the cellular processes governed by nutrient stress, including enrichment of amino acid metabolism (E), transcription (K), energy production and conversion (C), and biogenesis (J) processes were obtained. Pathway analysis revealed the enrichment of survival and homeostasis maintenance pathways, including type IV secretion system, nitrogen metabolism, and urease pathways in response to nutrient limitation. To conclude, our analysis demonstrates the utility of in-depth proteomic analysis in enabling improved annotation of the B. melitensis genome. Further, our results indicate that B. melitensis undergoes metabolic adaptations during nutrient stress similar to other Brucella. sp, and adapts itself for long-term persistence and survival.

The Pseudogene BMEA_B0173 Deficiency in Brucella melitensis Contributes to M-epitope Formation and Potentiates Virulence in a Mice Infection Model.

It is widely acknowledged that pseudogenes play important roles in bacterial diversification and evolution and participate in gene regulation and RNA interference (RNAi). However, the function of most pseudogenes in Brucella spp remains poorly understood, warranting further studies.To comprehensively analyze the function of the pseudogenes BMEA_B0173 in Brucella melitensis strain 63/9, a BMEA_B0173 in-frame deleted mutant strain was constructed. Then, the phenotypes of the mutant strain, such as growth characteristics and bacterial virulence, were assessed in mice infection models. Finally, iTRAQ analysis was performed to investigate the gene expression profile affected by the pseudogenes BMEA_B0173. In this study, we found that BMEA_B0173 deletion exhibited increased agglutination with M monospecific sera. In a mouse model of chronic infection, the BMEA_B0173 deletion strain displayed increased colonization in the spleen compared to the wild-type pathogen. The iTRAQ assay revealed that 252 proteins were differentially expressed between the BMEA_B0173 deletion and the wild-type strains. In addition, deletion of BMEA_B0173 significantly increased the expression of proteins involved in the denitrification pathway, iron metabolism, and several transcriptional regulators, which might cause increased virulence of the mutant strain. In conclusion, this study preliminary uncovered the function of the pseudogene BMEA_B0173 in Brucella melitensis 63/9 and provided novel insights for studying the pathogenesis of Brucella strains.

Use of touch-down polymerase chain reaction to enhance the sensitivity of Brucella melitensis detection in raw milk.

Brucellosis is a highly contagious bacterial zoonotic infectious disease severely affecting the public health and economic features of endemic and non-endemic countries. The present study assessed the potentials of using the touch-down polymerase chain reaction (TD-PCR) compared to the conventional PCR and culture methods in order to detect Brucella melitensis in raw milk samples of 55 sheep and 45 goats through deriving the primers from the omp31 element of the Brucella genome. In addition, nine isolates of B. melitensis were identified using the culture method. No positive cases were found in sediment samples, while the fatty tap layer test by conventional PCR and TD-PCR revealed 6 and 16 positive samples, respectively. Based on the survey of the limits of detection by TD-PCR and conventional PCR, TD protocol had a detection threshold of three logs higher than the conventional protocol under the experimental condition. The developed protocol of this study was highly sensitive and extremely fast. Therefore, this TD-PCR protocol could detect even a very low number of bacteria in milk samples. To our best knowledge, this is the first report on the use of the TD-PCR method to identify B. melitensis in milk.

Occurrence and risk factors of brucellosis among domestic animals: an artificial neural network approach.

Brucellosis is a zoonotic infectious disease with a worldwide distribution. This cross-sectional study aimed to determine the occurrence of Brucella in milk and blood samples of ruminants and the importance factors associated with animal brucellosis in Eastern Iran. A total of 200 paired samples, including blood (100) and milk (100), were obtained from the goats, sheep, and cows in Eastern Iran. Serum agglutination (SAT) and 2-mercapto ethanol (2-ME) tests were performed on the sera. A multiplex-polymerase chain reaction (m-PCR) assay was performed to identify the following species of Brucella, including B. abortus biovar 1, 2, 4, 3b, 5, 6, and 9, B. abortus S19, B. melitensis, and B. melitensis Rev.1 vaccine strain. B. abortus RB51 vaccine strain was also investigated in a second PCR assay. Risk factors for infection with Brucella spp. including the effect of abortion, contact with the wild animals, herd type, age, and previous vaccination in predicting blood contamination with B. abortus biovar 1, 2, and 4 were modeled by use of the artificial neural network. A total of 23 samples were seropositive regarding SAT and 2-ME tests. In total, B. abortus was detected in 35% and 15% of blood and milk samples, respectively, by the m-PCR assay. One sample of each of milk and blood was detected to have B. melitensis. Some samples were simultaneously contaminated with two Brucella species or two biovars of B. abortus. B. abortus S19 and B. melitensis Rev.1 vaccine strains were also detected in milk and blood samples. The sensitivity and specificity of the ANN model were 81% and 62%, respectively. In conclusion, B. abortus had higher frequency than B. melitensis in blood and milk samples. ANN determined herd type, previous vaccination, and age of the animal as the largest predictors of blood contamination with B. abortus.

Isolation of Brucella melitensis biovar 1 from human milk confirms breastfeeding as a possible route for infant infection.

Brucellosis during pregnancy is associated with major concerns for mothers but, it is still not clear whether the infection could be transmitted through breastfeeding to newborns. This study aimed at evaluating the shedding of Brucella melitensis through human milk. We describe phenotypic and genotypic characterization of Brucella isolate from human milk. The characterized isolate by Bruce-ladder PCR, AMOS PCR and biotyping confirmed the presence of Brucella melitensis biovar 1 from human milk. However, the breastfeeding of newborn baby induced no serious abnormality, although occasional weakness, loss of appetite and vomiting were reported by the parents. All Brucella serological tests including RBT, SAT and 2 ME test were also positive for the baby and her mother, although the blood culture was negative for the baby. Evaluation of the blood DNA from mother and her baby also confirm the presence of Brucella melitensis in the blood samples. Therefore, the isolation of B. melitensis biovar 1 from human milk as well as presence of Brucella melitensis in the blood samples confirms breastfeeding as a possible route for infant infection.

Brucella melitensis invA gene (BME_RS01060) transcription is promoted under acidic stress conditions.

The invA gene of Brucella melitensis codes for a NUDIX (nucleoside diphosphate linked to moiety X) hydrolase related to invasiveness. The objective of this work was to evaluate invA transcription under acidic conditions. The invA gene transcription was up regulated at pH 3 and pH 5 observed with semiquantitative real-time PCR in B. melitensis 133 strain. Results indicated that invA gene transcription at pH 3 showed a basal and decreased transcription compared to that of pH 5 incubation. Transcription levels of the dnaK gene were similar to those obtained with invA gene. The survival rates of wild type and invA mutant strains at pH 5 were above 90% in all post-incubation times. In contrast, at pH 3 there was a time-dependent reduction on both strains at 15 min (P < 0.05). These results suggest that invA gene transcription is promoted under acidic conditions in Brucella melitensis.