Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

Rational Design of Live-Attenuated Vaccines against Genome-Reduced Pathogens.

To develop safe and highly effective live vaccines, rational vaccine design is necessary. Here, we sought a simple approach to rationally develop a safe attenuated vaccine against the genome-reduced pathogen Erysipelothrix rhusiopathiae. We examined the mRNA expression of all conserved amino acid biosynthetic genes remaining in the genome after the reductive evolution of E. rhusiopathiae. Reverse transcription-quantitative PCR (qRT-PCR) analysis revealed that half of the 14 genes examined were upregulated during the infection of murine J774A.1 macrophages. Gene deletion was possible only for three proline biosynthesis genes, proB, proA, and proC, the last of which was upregulated 29-fold during infection. Five mutants bearing an in-frame deletion of one (ΔproB, ΔproA, or ΔproC mutant), two (ΔproBA mutant), or three (ΔproBAC mutant) genes exhibited attenuated growth during J774A.1 infection, and the attenuation and vaccine efficacy of these mutants were confirmed in mice and pigs. Thus, for the rational design of live vaccines against genome-reduced bacteria, the selective targeting of genes that escaped chromosomal deletions during evolution may be a simple approach for identifying genes which are specifically upregulated during infection. IMPORTANCE Identification of bacterial genes that are specifically upregulated during infection can lead to the rational construction of live vaccines. For this purpose, genome-based approaches, including DNA microarray analysis and IVET (in vivo expression technology), have been used so far; however, these methods can become laborious and time-consuming. In this study, we used a simple in silico approach and showed that in genome-reduced bacteria, the genes which evolutionarily remained conserved for metabolic adaptations during infection may be the best targets for the deletion and construction of live vaccines.

Human Erysipelothrix rhusiopathiae infection via bath water - case report and genome announcement.

Erysipelothrix rhusiopathiae is a facultative anaerobic, environmentally stable, Gram-positive rod that causes swine and avian erysipelas as a zoonotic pathogen. In humans, the main manifestations described are circumscribed erysipeloid, generalized erysipeloid, and endocarditis. Here, we report a 46-year-old female patient who presented to the physician because of redness and marked functio laesa of the hand, in terms of a pain-related restricted range of motion, and was treated surgically. E. rhusopathiae was detected in tissue biopsy. The source of infection was considered to be a pond in which both swine and, later, her dog bathed. The genome of the isolate was completely sequenced and especially the presumptive virulence associated factors as well as the presumptive antimicrobial resistance genes, in particular a predicted homologue to the multiple sugar metabolism regulator (MsmR), several predicted two-component signal transduction systems, three predicted hemolysins, two predicted neuraminidases, three predicted hyaluronate lyases, the surface protective antigen SpaA, a subset of predicted enzymes that potentially confer resistance to reactive oxygen species (ROS), several predicted phospholipases that could play a role in the escape from phagolysosomes into host cell cytoplasm as well as a predicted vancomycin resistance locus (vex23-vncRS) and three predicted MATE efflux transporters were investigated in more detail.

Erysipelas in a stranded common bottlenose dolphin: first report in a South American odontocete.

Erysipelas is a zoonotic disease caused by Erysipelothrix rhusiopathiae. In cetaceans, this disease has two main clinical forms: a cutaneous one, grossly characterized by rhomboid lesions, and a septicemic and often fatal form. Erysipelas is considered an important cause of morbidity and mortality in captive cetaceans; however, information in free-ranging cetaceans is limited. An adult common bottlenose dolphin (Tursiops truncatus) was found dead and in advanced autolysis in Paraíba state, northeastern Brazil, on July 19th, 2020. Upon gross examination, 80% of the body surface presented disseminated rhomboid cutaneous lesions ranging from 4 to 6 cm-width, characterized by well-defined edges and occasional ulceration, consistent with erysipelas. Additionally, anthropic-made postmortem linear cuts and partial mechanical removal of the flank musculature were noted. Skin samples were collected for histopathologic and molecular analyses. Microscopically, it was possible to observe multifocal dermatitis with vasculitis. Erysipelothrix sp. was detected by PCR. Despite previous reports of human consumption of cetacean meat in northeastern Brazil, the observed marks and advanced carcass autolysis suggested that the animal was most likely used as bait for fishing instead of human intake. This case highlights the value of postmortem examination and PCR even in poorly preserved cadavers and contributes to the understanding of the epidemiology of cutaneous erysipelas in free-ranging cetaceans (first report in an odontocete from the Southern Hemisphere). Due to the zoonotic potential of certain Erysipelothrix species (i.e., E. rhusiopathiae), active public health policies are required to inform field professionals and the general public about the health threats associated with marine mammal manipulation and consumption.

Erysipelothrix anatis sp. nov., Erysipelothrix aquatica sp. nov. and Erysipelothrix urinaevulpis sp. nov., three novel species of the genus, and emended description of Erysipelothrix.

Seven genotypically distinct strains assigned to the genus Erysipelothrix were isolated in different laboratories from several animal sources. Strain D17_0559-3-2-1T and three further strains were isolated from samples of duck, pig and goose. The strains had >99 % 16S rRNA gene sequence similarity to each other and to strain VA92-K48T and two further strains isolated from samples of medical leech and a turtle. The closest related type strains to the seven strains were those of Erysipelothrix inopinata (96.74 %) and Erysipelothrix rhusiopathiae (95.93 %). Average nucleotide identity, amino acid identity and in silico DNA-DNA hybridization results showed that the strains represented two separate novel species. One further phylogenetically distinct strain (165301687T) was isolated from fox urine. The strain had highest 16S rRNA gene sequence similarity to the type strains of Erysipelothrix tonsillarum (95.67 %), followed by Erysipelothrix piscisicarius (95.58 %) and Erysipelothrix larvae (94.22 %) and represented a further novel species. Chemotaxonomic and physiological data of the novel strains were assessed, but failed to unequivocally differentiate the novel species from existing members of the genus. MALDI-TOF MS data proved the discrimination of at least strain 165301687T from all currently described species. Based on the presented phylogenomic and physiological data, we propose three novel species, Erysipelothrix anatis sp. nov. with strain D17_0559-3-2-1T (=DSM 111258T= CIP 111884T=CCM 9044T) as type strain, Erysipelothrix aquatica sp. nov. with strain VA92-K48T (=DSM 106012T=LMG 30351T=CIP 111492T) as type strain and Erysipelothrix urinaevulpis sp. nov. with strain 165301687T (=DSM 106013T= LMG 30352T= CIP 111494T) as type strain.

Diversity and dynamics of bacteria at the Chrysomya megacephala pupal stage revealed by third-generation sequencing.

Characterization of the microbial community is essential for understanding the symbiotic relationships between microbes and host insects. Chrysomya megacephala is a vital resource, a forensic insect, a pollinator, and a vector for enteric bacteria, protozoa, helminths, and viruses. However, research on its microbial community is incomprehensive, particularly at the pupal stage, which comprises approximately half of the entire larval development stage and is important entomological evidence in forensic medicine. For the first time, this study investigated the bacterial communities of C. megacephala pupae at different ages using third-generation sequencing technology. The results showed that C. megacephala has a diverse and dynamic bacterial community. Cluster analysis at ≥ 97% similarity produced 154 operational taxonomic units (OTUs) that belonged to 10 different phyla and were distributed into 15 classes, 28 orders, 50 families, 88 genera, and 130 species. Overall, the number of bacterial OTUs increased with the development of pupae, and the relative abundance of Wolbachia in the Day5 group was significantly lower than that in the other groups. Within the pupal stage, Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla of bacteria. At the genus level, Wolbachia and Ignatzschineria coexisted, a rarely known feature. In addition, we found Erysipelothrix rhusiopathiae, the etiological agent of swine erysipelas, which is rarely identified in insects. This study enriches the understanding of the microbial community of C. megacephala and provides a reference for better utilization and control of C. megacephala.

A PCR assay to specifically detect serovar 1a strains of Erysipelothrix rhusiopathiae and differentiate them from serovar 2 strains possessing an intact ERH_1440 gene.

The Erysipelothrix rhusiopathiae ERH_1440 gene, which encodes CDP-glycerol:poly (glycerophosphate) glycerophosphotransferase, is conserved in serovar 1a strains. The gene is usually missing or truncated in other serovar strains and therefore has been used for PCR detection of serovar 1a strains. We have previously reported a rare case of an E. rhusiopathiae serovar 2 strain possessing an intact ERH_1440. In this study, we analyzed three additional serovar 2 strains with an intact ERH_1440 and developed a new PCR assay for the specific detection and differentiation of serovar 1a strains from these serovar 2 strains. PCR with primers designed based on serovar 1a-specific gene sequences upstream of ERH_1440 showed 100% specificity for four hundred thirty Erysipelothrix strains isolated from extensive origins.

SEABIRDS AS POSSIBLE RESERVOIRS OF ERYSIPELOTHRIX RHUSIOPATHIAE ON ISLANDS USED FOR CONSERVATION TRANSLOCATIONS IN NEW ZEALAND.

Erysipelothrix rhusiopathiae, the causative agent of the disease erysipelas, is a gram-positive bacillus, and an opportunistic pathogen in diverse species of animals. In New Zealand, E. rhusiopathiae has killed endangered birds on offshore islands, including Kakapo (Strigops habroptilus), Takahe (Porphyrio hochstetteri), and Kiwi (Apteryx spp.). The source of infection is uncertain, and the prevalence of E. rhusiopathiae among wild birds is currently unknown. During October 2018 to December 2018, we surveyed dead and live seabirds that visit two of New Zealand's offshore islands used for Kakapo conservation with the goal of determining the prevalence of E. rhusiopathiae. Bone marrow from dead birds was cultivated on selective agar, and organisms cultured were identified using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. The prevalence of E. rhusiopathiae was calculated in different species for each island. We surveyed live birds using PCR with Erysipelothrix spp.-specific primers on blood samples. The prevalence of E. rhusiopathiae in dead seabirds on Whenua Hou and Te Hauturu-o-Toi was 3.4% (3/86) and 11.4% (5/44), respectively. On Whenua Hou, E. rhusiopathiae was detected in Sooty Shearwaters (Puffinus griseus; 5.9%, 2/34) and Mottled Petrels (Pterodroma inexpectata; 2.7%, 1/36) while it was detected only in Cook's Petrels (Pterodroma cookie; 13.5%, 5/37) on Te Hauturu-o-Toi. Blood samples were collected from two seabird species; only one of 50 Mottled Petrels (2.0%) was positive for the presence of Erysipelothrix spp. Our findings confirm that burrowing seabirds are possible reservoirs of E. rhusiopathiae on both islands studied and may be the source of spillover to other species on the island. The differences in observed prevalence suggest the species composition of the reservoir of E. rhusiopathiae may vary geographically.

Comparative genomics of a novel clade shed light on the evolution of the genus Erysipelothrix and characterise an emerging species.

Erysipelothrix sp. isolates obtained from a deadly outbreak in farmed turkeys were sequenced and compared to representatives of the genus. Phylogenetic trees-supported by digital DNA:DNA hybridization and Average Nucleotide Identity-revealed a novel monophyletic clade comprising isolates from pigs, turkeys, and fish, including isolates previously described as E. sp. Strain 2. Genes coding for the SpaC protein, typically found in E. sp. Strain 2, were detected in all isolates of the clade. Therefore, we confirm E. sp. Strain 2 represents a unique species that may be isolated from a broad host range, and the name "Erysipelothrix takahashiae" is suggested. Core genome analysis showed that the pathogenic species of this genus, E. rhusiopathiae and the clade E. sp. Strain 2, are enriched in core functionalities related to nutrient uptake and transport, but not necessarily homologous pathways. For instance, whereas the aerobic DctA transporter may uptake C4-dicarboxylates in both species, the anaerobic DcuC transporter is exclusive of the E. sp. Strain 2. Remarkably, the pan-genome analysis uncovered that genes related to transport and metabolism, recombination and repair, translation and transcription in the fish isolate, within the novel clade, have undergone a genomic reduction through pseudogenization. This reflects distinct selective pressures shaping the genome of species and strains within the genus Erysipelothrix while adapting to their respective niches.

Serovars and SpaA Types of Erysipelothrix rhusiopathiae Isolated from Pigs in Japan from 2012 to 2019.

Erysipelothrix rhusiopathiae causes swine erysipelas (SE), which results in considerable economic loss on pig farms. During SE outbreaks that occurred sporadically from 2008 to 2011 in Japan, new E. rhusiopathiae strains were isolated with a specific surface protective antigen (Spa)A protein characterized by methionine at position 203 and isoleucine at position 257 (M203/I257 SpaA type). To determine whether strains with the M203/I257 SpaA type are still prevalent in Japan, we collected 79 strains of E. rhusiopathiae from pigs showing various SE symptoms from 2012 to 2019 and classified them based on serovar typing, spaA gene sequence analysis, and lineage typing. We found that the majority of recent E. rhusiopathiae strains (59/79) belonged to the serovar 1a strain, and that the M203/I257 SpaA type (56/59) was predominant continuing from 2008 to 2011. Furthermore, serovar 1a strains with IVb-1 and IVb-2 lineages that had been isolated in specific regions of Japan were no longer local but were found across Japan. The pathogenicity of recent isolates tested in mice was not significantly changed when compared to that of previously isolated strains. Our results suggest that recent SE outbreaks were not due to changes in the SpaA protein or to altered virulence of E. rhusiopathiae but were rather caused by the persistent presence of E. rhusiopathiae with the M203/I257 SpaA type.

An invasive infection with an unusual spaB-possessing Erysipelothrix rhusiopathiae in a human.

Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a variety of animals. In humans, in contrast to the cutaneous form called erysipeloid, which is an occupational disease and common in individuals who handle raw meat and fish, invasive systemic infections are unusual. E. rhusiopathiae expresses an immunogenic surface protein, Spa (surface protective antigen), which is involved in virulence. Among the antigenically different Spa proteins (SpaA, B and C), which are mostly associated with serovars, SpaA is by far the most prevalent in E. rhusiopathiae isolates from diseased animals. However, the Spa type has not been examined for human isolates, and it is unknown whether SpaB- or SpaC-possessing isolates can cause disease in humans. A Gram-positive, rod-shaped bacterium isolated from a case of human pyogenic spondylitis was analysed. The bacterium was identified as E. rhusiopathiae by a routine biochemical test and MS, and ultimately confirmed by an E. rhusiopathiae-specific PCR assay. Spa typing by sequencing revealed the SpaB type, and the serovar of the strain was identified as untypeable by a conventional agar gel precipitation test, but determined to be serovar 6 by a serotyping PCR assay. Sequence analysis of the serovar-defining chromosomal region revealed that the isolate displayed the same gene organization as the serovar 6 reference strain, but the region was disrupted by an insertion sequence element, suggesting that the isolate originated from a serovar 6 strain. These results highlight that unusual, spaB-possessing E. rhusiopathiae strains can potentially pose serious risks to humans.

Comparative genome analysis of Erysipelothrix rhusiopathiae isolated from domestic pigs and wild boars suggests host adaptation and selective pressure from the use of antibiotics.

The disease erysipelas caused by Erysipelothrix rhusiopathiae (ER) is a major concern in pig production. In the present study the genomes of ER from pigs (n=87), wild boars (n=71) and other sources (n=85) were compared in terms of whole-genome SNP variation, accessory genome content and the presence of genetic antibiotic resistance determinants. The aim was to investigate if genetic features among ER were associated with isolate origin in order to better estimate the risk of transmission of porcine-adapted strains from wild boars to free-range pigs and to increase our understanding of the evolution of ER. Pigs and wild boars carried isolates representing all ER clades, but clade one only occurred in healthy wild boars and healthy pigs. Several accessory genes or gene variants were found to be significantly associated with the pig and wild boar hosts, with genes predicted to encode cell wall-associated or extracellular proteins overrepresented. Gene variants associated with serovar determination and capsule production in serovars known to be pathogenic for pigs were found to be significantly associated with pigs as hosts. In total, 30 % of investigated pig isolates but only 6 % of wild boar isolates carried resistance genes, most commonly tetM (tetracycline) and lsa(E) together with lnu(B) (lincosamides, pleuromutilin and streptogramin A). The incidence of variably present genes including resistance determinants was weakly linked to phylogeny, indicating that host adaptation in ER has evolved multiple times in diverse lineages mediated by recombination and the acquisition of mobile genetic elements. The presented results support the occurrence of host-adapted ER strains, but they do not indicate frequent transmission between wild boars and domestic pigs. This article contains data hosted by Microreact.

Genetic analysis of an Erysipelothrix rhusiopathiae swine isolate determined to be serovar 2 by a gel double diffusion test but serovar 1a/2 by a serotyping PCR assay.

We previously developed a multiplex PCR assay for the differentiation of serovar 1a, 1b, 2 and 5 strains of Erysipelothrix rhusiopathiae. In this study, we analyzed the serovar-defining chromosomal region of a serovar 2 swine isolate, which was PCR-positive for both serovars 1a and 2 by the multiplex PCR assay. Genetic analysis of the chromosomal region revealed that, as in serovar 1a strains, the ERH_1440 gene, which is usually truncated or missing in serovar 2 strains, was intact in this strain. This paper first shows an E. rhusiopathiae serovar 2 strain possessing an intact ERH_1440 gene and suggests that care may be needed when determining the serovar of such rare strains by PCR assay.

Development of a Multiplex PCR-Based Assay for Rapid Serotyping of Erysipelothrix Species.

The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.

Genome sequence of multidrug-resistant Erysipelothrix rhusiopathiae ZJ carrying several acquired antimicrobial resistance genes.

OBJECTIVES: This study aimed to determine the genetic environment of antimicrobial resistance genes (ARGs) in Erysipelothrix rhusiopathiae strain ZJ isolated from a pig with symptoms of swine erysipelas in China. METHODS: Illumina MiSeq (200× coverage) and PacBio RS II (100× coverage) platforms were used for genome sequencing. ARGs and prophages were identified using ResFinder 3.0 and PHASTER, respectively. A conjugation experiment, induced prophage infection and long-term passage assay were performed to determine the transferability and stability of ARGs in this strain. RESULTS: The assembled circular genome of E. rhusiopathiae ZJ was 1 945 689 bp with a GC content of 36.48%; no plasmid sequence was detected. Eleven acquired ARGs were identified in the genome. A novel integrative and conjugative element (ICE) encoding a multidrug resistance (MDR) gene cluster [aadE-apt-spw-lsa(E)-lnu(B)-aadE-sat4-aphA3] was identified in strain ZJ. A prophage Φ1605 harbouring mef(A)-msr(D) and tet(M) was also found in this strain, which can take a circular form and can be induced by mitomycin C to infect E. rhusiopathiae G4T10 for ARG transfer. CONCLUSION: To our knowledge, this is the first report of a complete genome sequence of E. rhusiopathiae carrying multiple ARGs obtained from a pig farm. This is the first identification of a novel chimeric ICE carrying a MDR gene cluster and a prophage carrying ARGs in E. rhusiopathiae, which will provide a valuable reference to understand the potential transfer mechanism of MDR gene clusters carried by ICEs and prophages in Gram-positive bacteria.

A putative transcription regulator involved in the virulence attenuation of an acriflavine-resistant vaccine strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas.

Acriflavine, an acridine dye that causes frameshift mutations, has been used to attenuate various veterinary pathogens for the development of live vaccines. Erysipelothrix rhusiopathiae Koganei 65-0.15 strain (Koganei) (serovar 1a) is the acriflavine-resistant live vaccine currently used in Japan for the control of swine erysipelas. To investigate the attenuation mechanisms of the Koganei strain, we analyzed the draft genome sequence of the Koganei strain against the reference genome sequence of the E. rhusiopathiae Fujisawa strain (serovar 1a). The sequence analysis revealed a high degree of sequence similarity between the two strains and identified a total of 98 sequence differences within 80 protein-coding sequences. Among them, insertions/deletions (indels) were identified in 9 genes, of which 7 resulted in frameshift and premature termination. To investigate whether these mutations resulted in the attenuation of the Koganei strain, we focused on the indel mutation identified in ERH_0661, an XRE family transcriptional regulator. We introduced the mutation into ERH_0661 of the Fujisawa strain and restored the mutation of the Koganei strain. Animal experiments using the recombinant strains showed that mice survived inoculation with 103 colony forming units (CFUs) (equivalent to approximately 100 50% lethal doses [LD50] of the wild-type Fujisawa) of the recombinant Fujisawa strain, and the mice became ill after inoculation with 108 CFUs of the recombinant Koganei strain. These results suggest that the transcriptional regulator ERH_0661 is involved in the virulence of E. rhusiopathiae and that the ERH_0661 mutation is partially responsible for the attenuation of the Koganei strain.

Genome-Wide Identification of Virulence Genes in Erysipelothrix rhusiopathiae: Use of a Mutant Deficient in a tagF Homolog as a Safe Oral Vaccine against Swine Erysipelas.

Swine erysipelas is caused by the Gram-positive pathogen Erysipelothrix rhusiopathiae The swine erysipelas live vaccine in Japan, the E. rhusiopathiae Koganei 65-0.15 strain (Koganei), has been reported to cause arthritis and endocarditis. To develop a vaccine with increased safety, we used a virulent Fujisawa strain to construct transposon mutants for a total of 651 genes, which covered 38% of the coding sequence of the genome. We screened the mutants for attenuation by inoculating mice with 108 CFU of each mutant and subsequently assessed protective capability by challenging the surviving mice with 103 CFU (102 times the 50% lethal dose) of the Fujisawa strain. Of the 23 attenuated mutants obtained, 6 mutants were selected and evaluated for protective capability in pigs by comparison to that of the Koganei strain. A mutant in the ERH_0432 (tagF) gene encoding a putative CDP-glycerol glycerophosphotransferase was found to be highly attenuated and to induce humoral and cell-mediated immune responses in conventional pigs. An in-frame deletion mutant of the gene, the Δ432 mutant, was constructed, and attenuation was further confirmed in germfree piglets; three of four piglets subcutaneously inoculated with 109 CFU of the Δ432 mutant showed no apparent clinical symptoms, whereas all four of the Koganei-inoculated piglets died 3 days after inoculation. It was confirmed that conventional pigs inoculated orally or subcutaneously with the Δ432 strain were almost completely protected against lethal challenge infection. Thus, the tagF homolog mutant of E. rhusiopathiae represents a safe vaccine candidate that can be administered via the oral and subcutaneous routes.

Detection and quantification of Erysipelothrix rhusiopathiae in blood from infected chickens - addressing challenges with detection of DNA from infectious agents in host species with nucleated red blood cells.

PURPOSE: The present study aimed to establish pretreatment protocols as well as real-time and droplet digital polymerase chain reaction (PCR) methodologies to detect and quantify Erysipelothrix rhusiopathiae (ER) DNA in blood samples from infected chickens, as tools for routine diagnostics and monitoring of experimental infections. Chicken blood is a problematic matrix for PCR analysis because nucleated erythrocytes contribute large amounts of host DNA that inhibit amplification. METHODOLOGY: Using artificially spiked samples of fresh chicken blood, as well as blood samples from three experimental infection studies, the performance of pretreatment protocols, including choice of blood stabilization agent, centrifugation speeds and Ficoll gradient separation, was evaluated. The results were compared with those from traditional culture-based protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results/Key findings. Simple preparations producing cell-free samples performed well on artificial spike-in samples, providing high sensitivity. However, performance was poor in clinical samples or artificial samples where the bacteria were incubated for 4 h or more in fresh blood prior to DNA extraction. In these samples, a Ficoll separation protocol that creates samples rich in lymphocytes, monocytes and thrombocytes prior to DNA extraction was far more effective. CONCLUSIONS: Our results indicate that ER bacteria undergo rapid phagocytosis in chicken blood and that analysis of a blood fraction enriched for phagocytic cells is necessary for reliable detection and quantification. The presented results explain the poor performance of PCR detection reported in previously published experimental ER infection studies, and the proposed solutions are likely to have broader implications for PCR-based veterinary diagnostics in non-mammalian host species such as poultry and fish.

Disassociation of Spa type and serovar of an Erysipelothrix rhusiopathiae serovar 6 strain isolated from a diseased pig.

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae is an important component in protecting pigs against swine erysipelas. The Spa protein has been antigenically divided into 3 types: SpaA, SpaB, and SpaC. Swine erysipelas vaccines are formulated with strains of serovar 1 and/or 2, both of which are SpaA-possessing serovars. The association of Spa type with E. rhusiopathiae serovar has been reported, and therefore, the determination of the Spa type and the serovar of clinical isolates are important to assess vaccine efficacy. An E. rhusiopathiae strain, designated Ireland, was isolated from a diseased pig and identified as serovar 6 by a conventional agar gel precipitation test. Sequence analysis of the chromosomal locus presumably defining the serovar antigenicity of E. rhusiopathiae revealed that the gene content and organization of the chromosomal regions of the Ireland strain were identical to those of the serovar 6 reference strain (Tuzok). Sequence analysis of the spa gene and dot blots using a SpaA-specific monoclonal antibody confirmed that, unlike the Tuzok strain possessing SpaB, the Ireland strain expressed SpaA, indicating that the Spa type is not associated with the serovar in this strain. Thus, further investigation into the association between Spa type and serovar of clinical swine isolates is warranted.