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.

Origins and global context of Brucella abortus in Italy.

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.

Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy.

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

Survey of Mycobacterium spp. in Eurasian Badgers (Meles meles) in Central Italy.

A survey to determine the presence of Mycobacterium spp. in the Abruzzo and Molise regions was conducted by testing samples from 124 badgers found dead or road-killed during the 2013-2021 period. Head lymph nodes were collected from all carcasses, as well as mediastinal lymph nodes from 20 of them, for bacteriological and molecular tests; tissues were inoculated onto a set of solid egg-based Lowenstein-Jensen media and in a liquid culture system (BACTEC) and were analyzed by polymerase chain reactions (PCRs). Organs and lymph nodes from 31 carcasses were collected for histological tests. During post-mortem examinations, macroscopic lesions consistent with a Mycobacterium tuberculosis complex (MTBC) and with nontuberculous mycobacteria (NTM) infections were not detected. Mycobacteria were isolated from four animals (3.22%). M. avium subsp. avium was isolated by head lymph nodes from two badgers (1.61%), M. avium subsp. paratuberculosis (0.80%) from one, and Mycobacterium spp. from another (0.80%). The significance of nontuberculous mycobacteria (NTM) in wildlife hosts in the absence of clinical signs and gross pathology has yet to be assessed. The most critical aspect came from isolates belonging to the Mycobacterium avium complex infection in wildlife due to the possible interference with tuberculin skin tests in cattle.

Genetic Diversity of Brucella melitensis Isolated from Domestic Ruminants in Iraq.

The control and eradication of brucellosis represents a critical objective for Veterinary and Health Authorities across several countries globally. Efficient surveillance programs play a pivotal role in detecting and managing outbreaks. Epidemiological investigations significantly benefit from standardized and efficient molecular typing techniques and analytical tools, enabling public health laboratories to identify the origin of outbreaks. This study aimed to sequence Brucella spp. strains isolated in Iraq from different ruminant species to verify their molecular epidemiological correlations and, above all, to shed a light on how these Iraqi isolates are positioned in the phylogenetic context of Brucella spp. The 35 isolates under study were from abortion, milk, placenta, and the fetal membranes of sheep, cattle, and buffalo. Genotyping involved various techniques: MLVA-16, Whole Genome Sequencing, MLST, and cgMLST. All the Iraqi isolates from our study clustered in MLVA-16 within the East Mediterranean clade, and all but one grouped together in the same branch of the MST tree. MST analysis showed the minimum distance of one allele between the studied isolates, except for one strain from buffalo, which was positioned farther away from the rest of the isolates. In cgMLST, the majority of strains grouped within a large cluster predominantly comprising genotypes from the Middle East. The application of different control measures in different territories based on molecular epidemiological studies would increase the chances of maximizing public health benefits and minimizing the spread of infection to disease-free or lower prevalence areas.

Genomic and Antimicrobial Surveillance of Campylobacter Population in Italian Poultry.

Campylobacter is one of the most common foodborne diseases worldwide with increasing rates of antibiotic resistance. Most cases of campylobacteriosis can be traced back to the consumption of poultry meat. Despite many efforts to reduce contamination in farms and in slaughterhouses, the persistence of this pathogen in poultry products remains a problem. This study aimed to evaluate the genetic diversity and antibiotic resistance of 542 C. jejuni and C. coli in Italian poultry, in the framework of two National Monitoring Programs. Genomes were screened for antibiotic resistance, virulence determinants and contextualized within a global collection of C. jejuni. ST2116, ST2863 and ST 832 were the most prevalent and significantly associated with Italian poultry. A worrying increase in resistance to quinolones, fluoroquinolones and tetracycline was observed in C. jejuni, while an increased occurrence of multidrug resistant (MDR) strains and strains resistant to macrolides was detected in C. coli. Low resistance rates were found for aminoglycosides. Molecular resistance determinants were consistent with the phenotypic resistance for tetracycline and quinolones. In silico analysis revealed 119 genes associated with virulence factors, with a notably higher prevalence of some genes in ST2863 genomes. This study highlights the increased resistance to macrolides and the emergence of MDR strains for C. coli, the genetic basis of AMR and the predominance of two genotypes among Campylobacter strains isolated from the Italian poultry farms.

The Current Landscape of Antibiotic Resistance of Salmonella Infantis in Italy: The Expansion of Extended-Spectrum Beta-Lactamase Producers on a Local Scale.

Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene bla CTX-M-1 as well as aadA1, dfrA1, dfrA14, sul1, and tet(A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20-80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone.

Identification of Mycoplasma mycoides subspecies mycoides from slaughtered cattle in two transboundary states of North­eastern Nigeria.

This study aimed to perform molecular typing of Mycoplasma mycoides subsp. mycoides from slaughtered cattle in Adamawa and Taraba States, north­eastern Nigeria. A total of four hundred and eighty (480) samples of lung tissues, nasal swabs, ear swabs and pleural fluids were collected from cattle at slaughter and processed according to standard laboratory protocols. Identification and confirmation were achieved with specific PCR and PCR­RFLP. An overall M. mycoides subsp. mycoides isolation rate of 6.87% (33/480) was obtained. In Adamawa State, 12 (10.91%) isolates of M. mycoides subsp. mycoides came from both, lung tissues and pleural fluids. While in Taraba State, 5 (7.14%) and 4 (5.71%) isolates of M. mycoides subsp. mycoides came from lung tissues and pleural fluids, respectively. The samples from nasal and ear swabs from the study states were negative for M. mycoides subsp. mycoides. Thirty­three out of the 37 culture positive isolates were confirmed to be Mycoplasma mycoides subspecies mycoides with the production of a band equivalent to 574­bp. Molecular typing with restriction endonuclease Vsp1 results in the two bands of 180­bp and 380­bp. In conclusion, the study has established an isolation rate of 6.87% for M. mycoides subsp. mycoides. Measures to strengthen movement control in order to minimise the spread of this dreaded disease of cattle were recommended.

Characterization of Salmonella Typhimurium and monophasic Salmonella Typhimurium isolated in Abruzzo and Molise regions, Italy, from 2012 to 2017.

Salmonellosis is currently the second most common zoonosis in European Union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease trend in the yearly number of infections caused by Salmonella. In Italy, S. Typhimurium and monophasic S. Typhimurium represent the most prevalent serotypes. In this paper, we investigated these two serovars isolated from 2012 to 2017 in Abruzzo and Molise regions. A set of 345 strains isolated from human sporadic cases, surface water, food and animals were collected and analyzed. Monophasic S. Typhimurium strains were found to be resistant to streptomycin, sulfisoxazole, ampicillin, tetracycline and nalidixic acid, while S. Typhimurium isolates showed high levels of resistance to tetracycline, sulfisoxazole and ampicillin. The 5-loci Multilocus Variable-Number Tandem Repeat Analysis (MLVA) identified 88 genotypes (GT), six of which were common for the two serovars. Several MLVA profiles were shared by human and non-human isolates. MLVA had sufficient typing resolution for epidemiological studies of S. Typhimurium but demonstrated poor discriminatory in trace-back study of monophasic S. Typhimurium.

The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death.

This work highlights how our silver ultra nanoclusters (ARGIRIUM-SUNc) hand-made synthesized, are very useful as a bactericide and anti-biofilm agent. The Argirium-SUNc effective antibacterial concentrations are very low (< 1 ppm) as compared to the corresponding values reported in the literature. Different bacterial defense mechanisms are observed dependent on ARGIRIUM-SUNc concentrations. Biochemical investigations (volatilome) have been performed to understand the pathways involved in cell death. By using fluorescence techniques and cell viability measurements we show, for the first time, that membrane depolarization and calcium intracellular level are both primary events in bacteria death. The ARGIRIUM-SUNc determined eradication of different biofilm at a concentration as low as 0.6 ppm. This suggests that the effect of the nanoparticles follows a common mechanism in different bacteria. It is highly probable that the chemical constitution of the crosslinks could be a key target in the disrupting mechanism of our nanoparticles. Since the biofilms and their constituents are essential for bacterial survival in contact with humans, the silver nanoparticles represent a logical target for new antibacterial treatments.

Whole Genome Sequence Analysis of Brucella abortus Isolates from Various Regions of South Africa.

The availability of whole genome sequences in public databases permits genome-wide comparative studies of various bacterial species. Whole genome sequence-single nucleotide polymorphisms (WGS-SNP) analysis has been used in recent studies and allows the discrimination of various Brucella species and strains. In the present study, 13 Brucella spp. strains from cattle of various locations in provinces of South Africa were typed and discriminated. WGS-SNP analysis indicated a maximum pairwise distance ranging from 4 to 77 single nucleotide polymorphisms (SNPs) between the South African Brucella abortus virulent field strains. Moreover, it was shown that the South African B. abortus strains grouped closely to B. abortus strains from Mozambique and Zimbabwe, as well as other Eurasian countries, such as Portugal and India. WGS-SNP analysis of South African B. abortus strains demonstrated that the same genotype circulated in one farm (Farm 1), whereas another farm (Farm 2) in the same province had two different genotypes. This indicated that brucellosis in South Africa spreads within the herd on some farms, whereas the introduction of infected animals is the mode of transmission on other farms. Three B. abortus vaccine S19 strains isolated from tissue and aborted material were identical, even though they originated from different herds and regions of South Africa. This might be due to the incorrect vaccination of animals older than the recommended age of 4-8 months or might be a problem associated with vaccine production.

Whole-Genome Sequences of SARS-CoV-2 Lineage B.1.525 Strains (Variant η) Detected from Patients in the Abruzzo Region (Central Italy) during Spring 2021.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are emerging worldwide. Here, we report the complete genome sequences of 13 severe acute SARS-CoV-2 strains belonging to lineage B.1.525 (variant η).

Genomic Comparison of Salmonella Enteritidis Strains Isolated from Laying Hens and Humans in the Abruzzi Region during 2018.

Salmonellosis is a major cause of bacterial foodborne infection. Since 2016, an increased number of cases of gastroenteritis caused by Salmonella enterica serovar Enteritidis linked to eggs produced in Poland has been reported in Europe. In Italy, S. Enteritidis is one of the three most commonly reported serotypes, associated mainly with the consumption of contaminated eggs and derived products. In our work, we analysed 61 strains of S. Enteritidis obtained from humans and farms in the Abruzzi region, Italy, in 2018. We used Multiple-Loci Variable-Number Tandem Repeat (VNTR) analysis (MLVA)-based typing and Whole-Genome Sequencing (WGS) tools to identify closely related strains and perform cluster analysis. We found two clusters of genetically similar strains. The first one was present in the local farms and isolated from human cases and had single-linkage distance of no more than two core genes and less than five Single-Nucleotide Polymorphisms (SNPs). The second cluster contained strains isolated from humans and from a dessert (tiramisù) sample that shared identical core genome and were assigned the same SNP address. Cluster 2 isolates were found to be genetically similar to an S. Enteritidis strain from a multi-country outbreak linked to Polish eggs.

Prevalence, Population Diversity and Antimicrobial Resistance of Campylobacter coli Isolated in Italian Swine at Slaughterhouse.

Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Swine are known to be the main reservoir of Campylobacter coli and a possible source infection of humans as a result of carcass contamination at slaughter. The aim of this study was to evaluate the prevalence of C. coli contamination in swine carcasses, the antimicrobial resistance (AMR) patterns of isolates and the genetic diversity between strains obtained from swine and those isolated from humans. The prevalence of contamination was higher on carcasses (50.4%) than in faeces (32.9%). The 162 C. coli isolated from swine were examined by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The results of PFGE indicated a high genetic diversity among the isolates, with 25 different PFGE types. MLST assigned 51 sequence types (STs) to isolates. The most common genotype was ST-854 (16.04%), ST-9264 (10.49 %) and ST-1016 (6.08 %). Results of AMR showed a high resistance to quinolones and fluoroquinolones together with aminoglycosides and tetracycline. Many strains were multi-resistant with predominant R-type TeSCipNa (57%). Five resistance genes were detected along with mutation in the gyrA gene. A strong correlation between phenotypic and genotypic resistance was found for fluoroquinolone and tetracycline. Genetic profiles obtained in swine isolates were compared to those of 11 human strains. All human strains and 64.19% of animal strains (104/162) were assigned to the ST-828 clonal complex.

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.

Occurrence of Brucella ceti in striped dolphins from Italian Seas.

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian waters reported to date and show the usefulness of WGS for understanding of the evolution of this emerging pathogen.

Whole Genome Sequencing for Tracing Geographical Origin of Imported Cases of Human Brucellosis in Sweden.

Human infections with Brucella melitensis are occasionally reported in Sweden, despite the fact that the national flocks of sheep and goats are officially free from brucellosis. The aim of our study was to analyze 103 isolates of B. melitensis collected from patients in Sweden between 1994 and 2016 and determine their putative geographic origin using whole genome sequencing (WGS)-based tools. The majority of the strains were assigned to East Mediterranean and African lineages. Both in silico Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) and core genome Multilocus Sequence Typing (cgMLST) analyses identified countries of the Middle East as the most probable source of origin of the majority of the strains. Isolates collected from patients with travel history to Iraq or Syria were often associated with genotypes from Turkey, as the cgMLST profiles from these countries clustered together. Sixty strains were located within a distance of 20 core genes to related genotypes from the publicly available database, and for eighteen isolates, the closest genotype was different by more than 50 loci. Our study showed that WGS based tools are effective in tracing back the geographic origin of infection of patients with unknown travel status, provided that public sequences from the location of the source are available.

Distribution of Brucella field strains isolated from livestock, wildlife populations, and humans in Italy from 2007 to 2015.

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.

Phylogeography and epidemiology of Brucella suis biovar 2 in wildlife and domestic swine.

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.

Cross-reactivity in serological tests for brucellosis: a comparison of immune response of Escherichia coli O157:H7 and Yersinia enterocolitica O:9 vs Brucella spp.

According to European Union (EU) regulations, the serological tests for the eradication of bovine and ovine brucellosis are the Rose Bengal Test, Complement Fixation Test, and i-ELISA. These methods, also recommended by the World Organization for Animal Health (OIE) for international trades, have limitations related to the use of suspensions of smooth Brucellae or LPS extracts. Limitations include false-positive serological reactions to brucellosis, which in turn impedes accurate diagnosis in some herds. False positive reactions should be considered carefully during the final stages of an eradication programme and for surveillance purposes in brucellosis-free areas. In this study, we produced specific sera through the experimental infection of sheep with Y. enterocolitica O:9 and E. coli O157:H7. These are the most important cross-reactive bacteria with Brucella. We then evaluated the antibody response of groups of sheep that had been immunised towards homologous antigens and official antigens for brucellosis, in order to identify a differential diagnostic protocol to distinguish cross-reaction in Brucella-infected animals.