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.

Genome-wide Core Proteome Analysis of Brucella melitensis Strains for Potential Drug Target Prediction.

INTRODUCTION: Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. In humans, chronic infection occurs through contact with infected animals or their waste products. METHODS: The subtractive genomic approach is considered as a powerful and useful method for the identification of potential drug and vaccine targets. In this study, an attempt has been made through a subtractive proteomic strategy to identify novel drug targets in Brucella melitensis strains. Total 2604 core proteins of 56 strains of B. melitensis were taken, of which 545 non-human homologs were found to be essential for pathogen growth. Metabolic pathway analysis of these essential proteins revealed that 129 proteins are exclusively involved in 21 unique metabolic pathways in B. melitensis reference strain. RESULTS: Of these, 31 proteins were found to be involved in 10 metabolic pathways that are unique to the pathogen. We selected Nitrate reductase subunit-ß, Urease subunit α-2, Pantoate-ß-alanine ligase, Isochorismatase, 2-dehydro-3-deoxyphosphooctonate aldolase and Serine O-acetyltransferase as drug targets in Brucella melitensis strains. Among these druggable targets, we selected only Pantoate-ß- alanine ligase as high confidence target based on intensive literature curation, which is nonhomologous to the human gut metagenome involved in biosynthesis of secondary metabolites pathway. Pantothenate synthetase is the best chemotherapeutic target to combat Brucellulosis. CONCLUSION: Furthermore, in vitro and in vivo validation is needed for the evaluation of lead compounds against Brucella melitensis strains.

Evolutionary history and current distribution of the West Mediterranean lineage of Brucella melitensis in Italy.

Ovine and caprine brucellosis, caused by Brucella melitensis, is one of the world's most widespread zoonoses and is a major cause of economic losses in domestic ruminant production. In Italy, the disease remains endemic in several southern provinces, despite an ongoing brucellosis eradication programme. In this study, we used whole-genome sequencing to detail the genetic diversity of circulating strains, and to examine the origins of the predominant sub-lineages of B. melitensis in Italy. We reconstructed a global phylogeny of B. melitensis, strengthened by 339 new whole-genome sequences, from Italian isolates collected from 2011 to 2018 as part of a national livestock surveillance programme. All Italian strains belonged to the West Mediterranean lineage, which further divided into two major clades that diverged roughly between the 5th and 7th centuries. We observed that Sicily serves as a brucellosis burden hotspot, giving rise to several distinct sub-lineages. More than 20 putative outbreak clusters of ovine and caprine brucellosis were identified, several of which persisted over the 8 year survey period despite an aggressive brucellosis eradication campaign. While the outbreaks in Central and Northern Italy were generally associated with introductions of single clones of B. melitensis and their subsequent dissemination within neighbouring territories, we observed weak geographical segregation of genotypes in the southern regions. Biovar determination, recommended in routine analysis of all Brucella strains by the World Organisation for Animal Health (OIE), could not discriminate among the four main global clades. This demonstrates a need for updating the guidelines used for monitoring B. melitensis transmission and spread, both at the national and international level, and to include whole-genome-based typing as the principal method for identification and tracing of brucellosis outbreaks.

Brucella melitensis, a latent "travel bacterium," continual spread and expansion from Northern to Southern China and its relationship to worldwide lineages.

Brucellosis caused by Brucella melitensis is considered to be one of the most important zoonotic diseases in China. In this study, Conventional bio-typing, MLVA (multiple locus variable-number tandem repeat analysis), and WGS (whole-genome sequencing)-SNP (single nucleotide polymorphism) were used to study the genetic similarity of B. melitensis in northern and southern China and analyze its relationship with worldwide lineages. Currently, the distribution of species/biovars of B. melitensis has obviously changed, and B. melitensis has become the dominant species in southern regions of China. Strains from the southern had a common geographic origin with strains from the northern. Many MLVA-16 events were shared in the genotypes of the southern and northern strains, suggest that genotypic movement occurred from north to south. Based on WGS-SNP analysis, strains from different provinces were closely related and may have descended from one common ancestor, suggests that the southern strains originated from northern China. These data indicate that B. melitensis is a latent "travel bacterium" that spread and expanded from North China to South China. Moreover, B. melitensis strains from China are also genetically related to strains from other Asian regions (Kazakhstan, Russia, Mongolia, and India). The movement of infected sheep and their products requires control.

Molecular characterization of Brucella species from Zimbabwe.

Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.

Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections.

The use of whole-genome sequencing (WGS) using next-generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through single-nucleotide polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome multilocus sequence typing (cgMLST) has not been explored so far. The current study developed an allele-based cgMLST method and compared its performance to that of the genome-wide SNP approach and the traditional multilocus variable-number tandem repeat analysis (MLVA) on a defined sample collection. The data set was comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 isolates with unknown epidemiological status, composed of human and animal samples collected in Italy. The cgMLST scheme generated in this study contained 2,704 targets of the B. melitensis 16M reference genome. We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤6 loci in the cgMLST and ≤7 in WGS SNP analysis. Higher phylogenetic distance resolution was achieved with cgMLST and SNP analysis than with MLVA, particularly for strains belonging to the same lineage, thereby allowing diverse and unrelated genotypes to be identified with greater confidence. The application of a cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen, and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

Global evolution and phylogeography of Brucella melitensis strains.

BACKGROUND: Brucellosis is a bacterial zoonotic disease. Annually in the world more than 500,000 new cases of brucellosis in humans are registered. In this study, we propose an evolutionary model of the historical distribution of B. melitensis based on the full-genomic SNP analysis of 98 strains. RESULTS: We performed an analysis of the SNP of the complete genomes of 98 B. melitensis strains isolated in different geographical regions of the world to obtain relevant information on the population structure, genetic diversity and the evolution history of the species. Using genomic sequences of 21 strains of B. melitensis isolated in Russia and WGS data from the NCBI database, it was possible to identify five main genotypes and 13 species genotypes for analysis. Data analysis based on the Bayesian Phylogenetics and Phylogeography method allowed to determine the regions of geographical origin and the expected pathways of distribution of the main lines (genotypes and subgenotypes) of the pathogen. CONCLUSIONS: Within the framework of our study, the model of global evolution and phylogeography of B. melitensis strains isolated in various regions of the planet was proposed for the first time. The sets of unique specific SNPs described in our study, for all identified genotypes and subgenotypes, can be used to develop new bacterial typing and identification systems for B. melitensis.

A case report of spontaneous abortion caused by Brucella melitensis biovar 3.

BACKGROUND: Brucellosis is a worldwide zoonotic disease caused by Brucella spp. Brucella invades the body through the skin mucosa, digestive tract, and respiratory tract. However, only a few studies on human spontaneous abortion attributable to Brucella have been reported. In this work, the patient living in Shanxi Province in China who had suffered a spontaneous abortion was underwent pathogen detection and Brucella melitensis biovar 3 was identified. CASE PRESENTATION: The patient in this study was 22 years old. On July 16, 2015, she was admitted to Shanxi Grand Hospital, Shanxi Province, China because of one day of vaginal bleeding and three days of abdominal distension accompanied by fever after five months of amenorrhea. A serum tube agglutination test for brucellosis and blood culture were positive. At the time of discharge, she was prescribed oral doxycycline (100 mg/dose, twice a day) and rifampicin (600 mg/dose, once daily) for 6 weeks as recommended by the World Health Organization (WHO). No recurrence was observed during the six months of follow-up after the cessation of antibiotic treatment. CONCLUSIONS: This is the first reported case of miscarriage resulting from Brucella melitensis biovar 3 isolated from a pregnant woman who was infected through unpasteurized milk in China. Brucellosis infection was overlooked in the Maternity Hospital because of physician unawareness. Early recognition and prompt treatment of brucellosis infection are crucial for a successful outcome in pregnancy.

Genotyping of Brucella melitensis and Brucella abortus strains in Kazakhstan using MLVA-15.

Currently, although the prevalence of brucellosis in Kazakhstan remains high, there are limited data available on the genetic diversity of circulating Brucella strains. Here, MLVA was employed to genotype a panel of 102 Brucella isolates collected from eight Kazakh regions and neighboring countries (Russia, Kyrgyzstan) during the period 1935-2017. MLVA-11 analysis classified 64 B. abortus strains into genotypes 72, 82, 331, 71, 341 and 69, while one genotype was novel, having no correspondence within the MLVA international database. MLVA-11 analysis of 37 B. melitensis strains showed 100% identity with genotypes 116, 114 and 11. One B. suis strain was classified into genotype 33. Phylogeography based on MLVA-15 demonstrated that all B. abortus and B. melitensis strains belonged to "Abortus C" and "East Mediterranean" lineages, respectively. B. abortus strains from Kazakhstan and Russia resulted genetically related to Portuguese, Brazilian and US isolates, suggesting ancient spread of these lineages from Europe westwards to South America and eastwards to Turkey, Russia and Asia. Most of Kazakh B. melitensis isolates were related to strains circulating in China, likely due to long-term trading partnerships between the two countries. In fine-scale MLVA-15 analysis, 17 B. abortus and 12 B. melitensis genotypes were identified; among them 12 are novel. Interestingly, epidemiological information supporting molecular data were retrieved for two clusters within the B. abortus group, thus proving that MLVA is an appropriate tool for effective traceback analyses. Our findings suggest that molecular genotyping should be applied systematically to support control plans for eradication of brucellosis in Kazakhstan.

Typing and comparative genome analysis of Brucella melitensis isolated from Lebanon.

Brucella melitensis is the main causative agent of the zoonotic disease brucellosis. This study aimed at typing and characterizing genetic variation in 33 Brucella isolates recovered from patients in Lebanon. Bruce-ladder multiplex PCR and PCR-RFLP of omp31, omp2a and omp2b were performed. Sixteen representative isolates were chosen for draft-genome sequencing and analyzed to determine variations in virulence, resistance, genomic islands, prophages and insertion sequences. Comparative whole-genome single nucleotide polymorphism analysis was also performed. The isolates were confirmed to be B. melitensis. Genome analysis revealed multiple virulence determinants and efflux pumps. Genome comparisons and single nucleotide polymorphisms divided the isolates based on geographical distribution but revealed high levels of similarity between the strains. Sequence divergence in B. melitensis was mainly due to lateral gene transfer of mobile elements. This is the first report of an in-depth genomic characterization of B. melitensis in Lebanon.

[HOOF genotyping characteristics of Brucella melitensis strains isolated in Ulanqab of Inner Mongolia Autonomous Region].

Objective: To investigate the HOOF genotyping characteristics of 83 Brucella (B.) melitensis strains isolated in Ulanqab of Inner Mongolia Autonomous Region from 2012 to 2015. Methods: A total of 83 B. melitensis strains were detected by convention identification and AMOS-PCR, then HOOF protocol with eight VNTR locus were used for the genotyping of the strains, and the allelic diversity of each VNTR locus and the discriminatory power of VNTR typing of HOOF were assessed by Hunter-Gaston Discriminatory index. BioNumerics 5.0 was used for phylogenetic analysis and constructing dendrogram. Results: All of the isolates were identified as B. melitensis strains by two identification methods. The complete eight VNTR locus had higher polymophism and diversity index was 0.998; and diversity index of six locus (1, 2 and 4-7) were ≥0.678, discriminatory power of HOOF was mainly from this six higher diversity index locus. The 83 B. melitensis strains were classified into eight clusters and 76 genotypes, 6 shared genotypes included 13 isolates, indicating that these brucellosis cases had epidemiological link, the other 70 strains had distinct genotypes, indicating that these cases had no epidemiological link. Conclusions: The epidemic of human brucellosis in Ulanqab was characterized by local and sporadic outbreaks. Cross infection was related with the transfer of the sources of infection.

Evaluation of a hypervariable octameric oligonucleotide fingerprints assay for identification of and discrimination between wild-type and vaccine strains of Brucella melitensis.

OBJECTIVE To evaluate a hypervariable octameric oligonucleotide fingerprints (HOOF-Prints) assay for identification of and discrimination between wild-type and vaccine strains of Brucella melitensis. SAMPLE Brucella melitensis vaccine strain M5 and wild-type strain M43. PROCEDURES 8 pairs of primers (alterable, octameric nucleotides) were designed on the basis of a biological analysis of 8 flanking sequences in the DNA of B melitensis. The HOOF-Prints technique was used to identify wild-type and vaccine strains of B melitensis. Phylogenetic analysis of short, polymorphic fragments of DNA from B melitensis strains M5 and M43 was performed. RESULTS Variable-number tandem repeat DNA segments of B melitensis vaccine strain M5 and wild-type strain M43 were successfully amplified by means of PCR assay. All target gene fragments ranged in size from 100 to 300 bp. Separate phylogenetic analysis of each Brucella strain revealed considerable differences between the vaccine and wild-type strains. CONCLUSIONS AND CLINICAL RELEVANCE The results of this study suggested the HOOF-Prints assay may be useful for discriminating vaccine strains of B melitensis from wild-type strains. This ability could allow discrimination between animals that are seropositive because of vaccination against B melitensis and those that are seropositive because of B melitensis infection and could decrease the likelihood of importing Brucella-infected animals.

Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16.

Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries.

Genotyping of Brucella melitensis and Brucella abortus strains currently circulating in Xinjiang, China.

Brucellosis is a well-known zoonotic disease that can cause severe economic and healthcare losses. Xinjiang, one of the biggest livestock husbandry sectors in China, has gone through increasing incidence of brucellosis in cattle and small ruminants recently. In this paper, 50 B. melitensis strains and 9 B. abortus strains collected from across Xinjiang area (from 2010 to 2015) were genotyped using multiple locus variable-number tandem-repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST). Based on 8 loci (MLVA-8), 50 B. melitensis strains were classified into three genotypes. Genotypes 42 (n=38, 76%) and 63 (n=11, 22%) were part of the East Mediterranean group, and one genotype with pattern of 1-5-3-13-2-4-3-2 represents a single-locus variant from genotype 63. MLVA-16 resolved 50 B. melitensis strains into 28 genotypes, of which 15 are unique to Xinjiang and 10 are in common with those in adjacent country Kazakhstan and neighboring provinces of China. Minimum Spanning Tree (MST) analysis implies that B. melitensis strains collected from across Kazakhstan, Xinjiang and China areas may share a common origin. Nine B. abortus strains were sorted into three genotypes by MLVA-8, genotypes 36 (n=7, 77.8%), 86 (n=1, 11.1%) and a new genotype with pattern of 4-5-3-13-2-2-3-1. Each B. abortus strain showed distinct MLVA-16 genotypes, suggesting that B. abortus species may possess more genetic diversity than B. melitensis. Using MLST, most B. melitensis strains (n=49) were identified as sequence type ST8, and most B. abortus strains (n=8) were recognized as ST2. Two new sequence types, ST37 and ST38, represented by single strain from B. melitensis and B. abortus species respectively, were also detected in this study. These results could facilitate the pathogen surveillance in the forthcoming eradication programs and serve as a guide in source tracking in case of new outbreaks occur.

Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis.

Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs.

MLVA as an Epidemiological Tool To Trace Back Brucella melitensis Biovar 1 Re-Emergence in Italy.

Brucellosis is an important zoonosis caused by Brucella spp., still prevalent in most areas of the world. Brucellosis control in animals is the key to protect humans. The knowledge of Brucella spp. prevailing genotypes in a territory represents an important epidemiological tool to formulate policies and strategies for disease control and to trace back the introduction of new strains previously considered as exotic. In the last years, multiple-locus variable number tandem repeat analysis (MLVA) has been proposed as complementary to classical biotyping methods. MLVA may add important information to the classical epidemiological investigation techniques, to help in tracing back sources of infection in brucellosis outbreaks. Sardinia is an Italian region officially free from sheep and goats brucellosis since 1998. In 2011, Brucella melitensis biovar 1, a biotype not reported in Italy since 1995, was isolated in one flock in the region. The genotyping MLVA-16 showed that isolates belonged to a rare American lineage, confirming it was introduced from other countries. The strain was considered as probably originating from Spain, where this lineage is endemic. BrucellaMLVA-16 has been proved to be useful to analyse the epidemiological correlation of strains enabling to trace its geographic origin by comparing their previously reported genetic patterns.

Full genome SNP-based phylogenetic analysis reveals the origin and global spread of Brucella melitensis.

BACKGROUND: Brucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis. RESULTS: Single sequencing runs of each genome resulted in draft genome sequences of MY1483/09 and Phil1136/12, which covered 99.85% and 99.92% of the complete genome sequences, respectively. The B. melitensis genome sequences, and two B. abortus strains used as the outgroup strains, yielded a total of 13,728 SNP sites. Phylogenetic analysis using whole-genome SNPs and geographical distribution of the isolates revealed spatial clustering of the B. melitensis isolates into five genotypes, I, II, III, IV and V. The Mediterranean strains, identified as genotype I, occupied the basal node of the phylogenetic tree, suggesting that B. melitensis may have originated from the Mediterranean regions. All of the Asian B. melitensis strains clustered into genotype II with the SEA strains, including the two isolates sequenced in this study, forming a distinct clade denoted here as genotype IId. Genotypes III, IV and V of B. melitensis demonstrated a restricted geographical distribution, with genotype III representing the African lineage, genotype IV representing the European lineage and genotype V representing the American lineage. CONCLUSION: We showed that SNPs retrieved from the B. melitensis draft full genomes were sufficient to resolve the interspecies relationships between B. melitensis strains and to discriminate between the vaccine and endemic strains. Phylogeographic reconstruction of the history of B. melitensis global spread at a finer scale by using whole-genome SNP analyses supported the origin of all B. melitensis strains from the Mediterranean region. The possible global distribution of B. melitensis following the ancient trade routes was also consistent with whole-genome SNP phylogeny. The whole genome SNP phylogenetics analysis, hence is a powerful tool for intraspecies discrimination of closely related species.