Search

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.

Swine brucellosis due to Brucella suis biovar 2 (bv2) is enzootic in wild boar and hare in continental Europe and may cause major economic losses to the pig industry, mainly in free-ranged pig farms. The high nucleotide identity found among the B. suis biovar 2 isolates has long hindered the full understanding of the epidemiology and the phylogeography of the disease. Here, we used multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) and whole-genome analysis to identify single-nucleotide polymorphisms (SNPs) in order to gain insights from the largest B. suis bv2 dataset analyzed so far composed of domestic pigs and wildlife isolates collected throughout Europe since the 1970s. We found four major clades with a specific phylogeographic pattern. The Iberian clade contains isolates exclusively from the Iberian Peninsula. The Central European clade includes most isolates from France, Northern Italy, Switzerland and an important proportion of those of Northern Spain. The Eastern European clade clustered isolates from Croatia and Hungary mainly but also from areas of France, Germany, Italy and Poland. Finally, a separated Sardinian clade grouped three isolates from this island. At fine scale, MLVA demonstrated an endemic status of the infection in Europe and it allowed tracking a large outbreak formed by different farms from Spain linked to the same infection source. The whole genome SNP analysis showed that the strains form genetically distinct clades, shared between wild boar and pigs, in agreement with the MLVA clades. Interestingly, all hare isolates clustered together within two groups composed exclusively of wildlife isolates. Our results support the hypothesis that maintenance and spread of B. suis bv2 in Europe is a dynamic process linked to the natural expansion of wild boar as the main wild reservoir of the infection, while spread over long distances is found largely dependent on anthropogenic activities., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Brucellosis is a major public health problem still prevalent as a neglected endemic zoonosis requiring proactive attention in many communities worldwide. The present study involved analysis of Brucella field strains submitted for typing to the Italian National Reference Laboratory for Brucellosis from 2007 to 2015. Strains were identified at the species and biovar levels by classic and molecular techniques according to the World Organisation for Animal Health Manual. In total, 5,784 strains were typed: 3,089 Brucella abortus (53.4%), 2,497 B. melitensis (43.2%), 10 B. ovis (0.2%), 181 B. suis (3.1%), and 7 B. ceti (0.1%). The 2,981 strains from cattle were typed as B. abortus biovars 1, 3, and 6 (90.1%) and B. melitensis biovar 3 (9.9%). The 318 strains from water buffalo were typed as B. abortus biovars 1, 3 (95.9%) and B. melitensis biovar 3 (4.1%). The 2,279 strains from sheep and goats were typed as B. abortus biovars 1 and 3 (4.3%); B. melitensis biovars 1, 3, (95.3%); and B. ovis (0.4%). The 173 strains from wild boar were typed as B. suis biovar 2 (98.3%) and B. melitensis biovar 3 (1.7%). The 11 strains from pigs were typed as B. suis biovar 2. The 13 strains from humans were typed as B. melitensis biovar 3. The two strains from horses were typed as B. abortus biovar 1, while the seven strains from dolphins were typed as B. ceti. This additional knowledge on the epidemiology of brucellosis in Italy may be useful to formulate policies and strategies for the control and eradication of the disease in animal populations. The animal species affected, biovars typed, geographical origins, and spatial distributions of isolates are herein analyzed and discussed., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Janowicz et al.)

Porcine brucellosis occurs in many countries where pigs are farmed, often representing an underrated problem. B. suis biovar 2 is the most common isolate in Europe, with high prevalence reported in wild boars in which it is generally isolated in the absence of gross lesions. In the last five years, we tested for Brucella spp. 389 lymph nodes of wild boars collected during hunting seasons or during necropsy procedures. In this paper, we describe the first case of isolation of B. suis biovar 2 from a wild boar aborted foetus, and we analyse the genomic relationships with B.suis biovar 2 strains isolated in the past five years in Abruzzi Region, Central Italy. The genetic fingerprint revealed that the isolates under study belong to the MLST ST16 and to the MLVA11 Gt 57, similar to the Central-Eastern European strains. Massive restocking (for hunting purpose) of wild boars from Eastern Europe have been done since 1950 in Italy contributing to the increasing of population size and distribution, as well as to the interbreeding between these foreign breeds and the local population. The contamination of pastures with infected material such as aborted wild boars foetuses can increase the risk of transmission of Brucella among wild and domestic animals. The contact of B. suis with domestic ruminants may also cause serological reactions to brucellosis serological testing, and even unapparent infection, thus hampering the efforts made in the brucellosis eradication campaign., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Brucellosis is a common and chronic disease of cattle and other bovids that often causes reproductive disorders. Natural infection in cattle is caused by Brucella abortus and transmission typically occurs during abortions, calving, or nursing. Brucellosis is also a major zoonotic disease due to contamination of dairy products or contact with the tissues of infected animals. Brucellosis has been eradicated from most of the developed world in the last 40 years but persists in many regions-the disease remains prevalent in portions of Africa, the Middle East, Asia, and Central and South America, as well as in the Mediterranean basin. In Italy, B. abortus has persisted in southern regions in both cattle and water buffalo. Previous attempts at analyzing the phylogenetics of B. abortus in Italy have been challenging due to limited genetic variability and unresolved global population genetic structure of this pathogen., Results: We conducted genome-wide phylogenetic analyses on 11 representative strains of B. abortus from Italy, and compared these sequences to a worldwide collection of publically available genomes. Italian isolates belong to three clades that are basal to the main and global B. abortus lineage. Using six SNP-based assays designed to identify substructure within the Italian clades, we surveyed a collection of 261 isolates and found that one clade predominates throughout endemic districts in the country, while the other two clades are more geographically restricted to portions of southern Italy., Conclusions: Although related strains exist worldwide, B. abortus isolates from Italy are substantially different than those found in much of the rest of Europe and North America, and are more closely related to strains from the Middle East and Asia. Our assays targeting genetic substructure within Italy allowed us to identify the major lineages quickly and inexpensively, without having to generate whole genome sequences for a large isolate collection. These findings highlight the importance of genetic studies to assess the status and the history of pathogens.

Background: Human brucellosis cases are still reported each year in Sweden despite eradication of the disease in animals. Epidemiological investigation has never been conducted to trace back the source of human infection in the country. The purpose of the study was to identify the source of infection for 16 human brucellosis cases that occurred in Sweden, during the period 2008-2012., Results: The isolates were identified as Brucella melitensis and MLVA-16 genotyping revealed 14 different genotypes of East Mediterranean and Africa lineages. We also reported one case of laboratory-acquired brucellosis (LAB) that was shown to be epidemiological linked to one of the cases in the current study., Conclusions: Brucella melitensis was the only species diagnosed, confirming its highest zoonotic potential in the genus Brucella, and MLVA-16 results demonstrated that the cases of brucellosis in Sweden herein investigated, are imported and linked to travel in the Middle East and Africa. Due to its zoonotic concerns, any acute febrile illness linked to recent travel within those regions should be investigated for brucellosis and samples should be processed according to biosafety level 3 regulations.

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., (© 2015 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

This communication describes the isolation of Brucella suis (B. suis) biovar 2 in semi‑free‑range pigs located in the province of Rome, Italy. Sera of 28 pigs from a herd with reproductive problems were tested for brucellosis. Twenty-five sera (89%) were found positive to Rose Bengal Test (RBT), while 22 (79%) were positive to Complement Fixation Test (CFT). Two positive pigs were slaughtered, organs were collected and tested for the presence of bacteria. Brucella spp. was isolated from the spleens and the abdominal lymph nodes of the 2 subjects. The isolates were identified as B. suis biovar 2 by biochemical and Polymerase Chain Reaction (PCR) tests. The frequent infringement in the fences of the premises and the birth of striped piglets provided evidence that sows mated with wild boar, the major reservoir of B. suis biovar 2. Conversely, the isolation of B. suis biovar 2 from spleens and lymphnodes of seropositive slaughtered animals only, as well as the constant negative results from all vaginal swabs and the abortion materials tested, raise doubts on the implication of B. suis biovar 2 in the infertility of the holding.

Since 1994, when Brucella ceti was first isolated from an aborted dolphin fetus, several cases have been reported worldwide. The first case of B. ceti in the Mediterranean (and in Italy), however, was recorded only in 2012, off the coast of Tuscany. Extensive studies, using serological and microbiological methods, have documented this bacterium in dolphins and demonstrated its zoonotic potential. We describe the typing of two B. ceti strains isolated from striped dolphins (Stenella coeruleoalba) stranded on the southern Apulia coastline. B. ceti isolates were conventionally typed, and then genotyped by both the multilocus sequence typing (MLST) and the multilocus variable number of tandem repeats typing (MLVA) methodologies to infer phylogeny and potential epidemiological links between the two cases. The two isolates were identified through MLST analysis as belonging to the common sequence type 26 (ST26), while MLVA analysis, having established that the two isolates have identical profiles, assigned them to a novel genotype within cluster A - a unique representative of a new Mediterranean subcluster. The results thus revealed a link between the two cases studied, demonstrating the usefulness of MLST and MLVA for the epidemiological investigation of brucellae among marine mammals.

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

Despite the eradication of brucellosis from most of Europe, the disease remains relatively common in a variety of livestock in southern European countries. It is therefore surprising that with such high prevalence rates, there have been few genetic characterizations of brucellosis outbreaks in this region. We conducted a genetic assessment of 206 isolates of Brucella abortus and B. melitensis from Italy using Variable Number Tandem Repeats (VNTRs). We determined genetic diversity and geographic distribution of these Brucella VNTR genotypes from 160 farms in eight regions of Southern Italy in a fine-scale analysis using 16 VNTR loci in a MLVA-16 methodology. In a broad scale analysis, we then used a reduced dataset of 11 VNTR loci (MLVA-11) to compare genotypes from Italy to a global database. In the 84 isolates of B. melitensis, there were 56 genotypes using MLVA-16; 43 of these genotypes were found only once. At a broad scale, 81 of these isolates were part of an Italian sub-group within the West Mediterranean group. One of the two B. melitensis isolates from a human patient shared the same genotype as a livestock isolate, suggesting a possible epidemiological connection. In 122 B. abortus isolates, there were 34 genotypes by MLVA-16; 16 of these genotypes were found only once. At a broad scale with MLVA-11, one genotype was predominant, comprising 77.8% of the isolates and was distributed throughout Southern Italy. These data on the current lineages of Brucella present in Italy should form the basis for epidemiological studies of Brucella throughout the country, while placing these strains in a global context., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology., Results: The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created., Conclusion: In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer.Finally, this platform can be considered a valid alternative to standard genotyping techniques, particularly useful dealing with a large number of samples in short time. These data confirmed that this technology represents a significative advancement in high-throughput accurate Brucella genotyping.

Approximately 250,000 water buffalo (Bubalus bubalis) live in the Campania region of southern Italy where the breeding of this species is very popular. Of these animals, almost 150,000 are concentrated in the Caserta province where the prevalence of Brucella abortus in this species represents approximately 20% at herd level. The Italian brucellosis eradication programme provides a slaughter and vaccination strategy for this province. B. abortus strain RB51 (RB51) has become the official vaccine for the prevention of brucellosis in cattle in several countries. The aim of this study was to evaluate the efficacy of RB51 in water buffalo compared to the B. abortus S19 vaccine (S19). The study was performed in accordance with a protocol described in mice. Female buffalo aged five months were inoculated. Five received a RB51 dosage on two occasions that was three times greater than that approved for use in cattle and a booster after one month, five received B. abortus S19 vaccine at the standard dosage and three controls received a phosphate buffer solution. Buffalo were then challenged with a virulent B. abortus strain 544 thirty days post vaccination. Antibodies that developed in the five animals vaccinated with RB51 were not detected by the Rose Bengal test or complement fixation test (CFT) and were also tested by CFT prepared with RB51 antigen. After culling, B. abortus was cultured from the spleen, retropharyngeal and supra-mammary lymph nodes. A statistical evaluation was performed to assess the immunogenicity values obtained in buffalo vaccinated with S19, compared to those obtained in buffalo vaccinated with the RB51 vaccine and in the unvaccinated control group.

In 2001, antibodies to Brucella spp. were detected in the wild boar (Sus scrofa) population of Regional Park in the Piedmont, northwest Italy. This was the first report of swine brucellosis in Italian wildlife, and in response, we conducted a survey on wild boars culled in the park to evaluate the presence and epidemiologic pattern of Brucella. In total, 2,267 serum samples and 1,841 tissue samples were collected and tested from 2001 to 2007. Differences in antibody and infection prevalence among sampling years were statistically significant (chi(2)=28.2, P<0.0001 and chi(2)=27.8, P<0.0001, respectively). Serologic and culture results also differed between gender and age groups, and for serology, a positive age trend was observed in both male and female age classes (chi(2(trend))=90.9, P<0.00005 OR: 1, 2.5, 4.9, 7.5; chi(2(trend))=43.1, P<0.00002, OR: 1, 2.5, 2.9, 4.8, respectively).