Genome-resolved metagenomics suggests a mutualistic relationship between Mycoplasma and salmonid hosts.

Salmonids are important sources of protein for a large proportion of the human population. Mycoplasma species are a major constituent of the gut microbiota of salmonids, often representing the majority of microbiota. Despite the frequent reported dominance of salmonid-related Mycoplasma species, little is known about the phylogenomic placement, functions and potential evolutionary relationships with their salmonid hosts. In this study, we utilise 2.9 billion metagenomic reads generated from 12 samples from three different salmonid host species to I) characterise and curate the first metagenome-assembled genomes (MAGs) of Mycoplasma dominating the intestines of three different salmonid species, II) establish the phylogeny of these salmonid candidate Mycoplasma species, III) perform a comprehensive pangenomic analysis of Mycoplasma, IV) decipher the putative functionalities of the salmonid MAGs and reveal specific functions expected to benefit the host. Our data provide a basis for future studies examining the composition and function of the salmonid microbiota.

MOLECULAR IDENTIFICATION OF MEMBERS OF THE FAMILY PASTEURELLACEAE FROM THE ORAL CAVITY OF KOALAS (PHASCOLARCTOS CINEREUS) AND THEIR RELATIONSHIP WITH ISOLATES FROM KOALA BITE WOUNDS IN HUMANS.

A total of 22 Pasteurellaceae isolates obtained from the oral cavity of koalas (Phascolarctos cinereus) at different wildlife centers in Australia were investigated using amplification and sequencing of two housekeeping genes, rpoA and recN. The available sequences from the Lonepinella koalarum type strain (ACM3666T) and the recent isolates of Lonepinella-like bacteria obtained from human infected wounds associated with koala bites were also included. Phylogenetic analysis was performed on the concatenated rpoA-recN genes and genome relatedness was calculated based on the recN sequences. The oral cavity isolates, the koala bite wound isolates, and L. koalarum ACM3666T resulted in four clusters (Clusters 1-4). Clusters 1-3 were clearly not members of the genus Lonepinella. Cluster 1 was closely related to the genus Fredericksenia, and Clusters 2 and 3 appeared to be novel genera. Cluster 4 consisted of three subclusters: Cluster 4a with one koala bite wound isolate and L. koalarum ACM3666T, Cluster 4b with three oral cavity isolates and two Lonepinella-like wound isolates, and Cluster 4c with three nearly identical oral cavity isolates that may represent a different species within the genus Lonepinella. The rich Pasteurellaceae population, including potential novel taxa in the oral cavity of koalas supports an important role of these highly adapted microorganisms in the physiology of koalas. Moreover, the pathogenic potential of Lonepinella-like species is an important consideration when investigating infected koala bites in humans.

Immune Suppression Induced by Gallibacterium anatis GtxA During Interaction with Chicken Macrophage-Like HD11 Cells.

The RTX toxin GtxA expressed by Gallibacterium anatis biovar haemolytica has been proposed a major virulence factor during disease manifestations in the natural host, the chicken. To better understand the role of GtxA in the pathogenesis of G. anatis, we compared the GtxA expressing wildtype strain with its isogenic ∆gtxA mutant that was unable to express GtxA during exposure to chicken macrophage-like HD11 cells. From adhesion and invasion assays, we showed that GtxA appears to promote adhesion and invasion of HD11 cells. By using quantitative RT-PCR, we also demonstrated that the G. anatis expressing GtxA induced a mainly anti-inflammatory (IL-10) host cell response as opposed to the pro-inflammatory (IL-1ß, IL-6 and TNF-α) response induced by the GtxA deletion mutant. Interestingly, these results, at least partly, resemble recent responses observed from spleen tissue of chickens infected with the same two bacterial strains. The effect of the GtxA toxin on the type of cell death was less clear. While GtxA clearly induced cell death, our efforts to characterize whether this was due to primarily necrosis or apoptosis through expression analysis of a broad range of apoptosis genes did not reveal clear answers.

GtxA is a virulence factor that promotes a Th2-like response during Gallibacterium anatis infection in laying hens.

GtxA, a leukotoxic RTX-toxin, has been proposed a main virulence factor of Gallibacterium anatis. To evaluate the impact of GtxA during infection, we experimentally infected laying hens with a G. anatis wild-type (WT) strain and its isogenic gtxA deletion mutant (ΔgtxA), respectively, and monitored the birds during a 6 day period. Birds inoculated with ΔgtxA had significantly reduced gross lesions and microscopic changes compared to the birds inoculated with the WT strain. To assess the host response further, we quantified the expression of pro-inflammatory cytokines and apoptosis genes by RT-qPCR. In the ovarian tissue, the expression levels of IL-4 and TNF-α were significantly lower in the ΔgtxA group compared to the WT group, while IL-6 and IL-10 levels appeared similar in the two groups. In the spleen tissue of ΔgtxA infected chickens, IL-4 expression was also lower compared to the WT infected chickens. The results indicated that GtxA plays a key role in an acute cytokine-mediated Th2-like response against G. anatis infection in the ovary tissue. The pro-inflammatory response in the ovary tissue of birds inoculated with ΔgtxA mutant was thus significantly lower than the wild-type response. This was, at least partly, supported by the apoptosis gene expression levels, which were significantly higher in the ΔgtxA mutant compared to the wild-type infected chickens. In conclusion, GtxA clearly plays an important role in the pathogenesis of G. anatis infections in laying hens. Further investigations into the specific factors regulating the host response is however needed to provide a more complete understanding of the bacteria-host interaction.

Citrus flavonoids, ß-Glucan and organic acid feed additives decrease relative risk during Yersinia ruckeri O1 biotype 2 infection of rainbow trout (Oncorhynchus mykiss).

Whether through direct supplementation of bacteria or by prebiotic supplementation thought to favour subsets of bacteria, modulation of gut microbiota constitutes an important and promising alternative to the use of prophylactic and growth promoting antibiotics in worldwide aquaculture. We fed a commercial base feed, alone or supplemented with either proprietary ß-glucan, ß-glucan and organic acids, citrus flavonoid or yeast cell wall supplements, to rainbow trout over a period of four weeks. Fish from each feed group were then subjected to experimental, waterborne infection with Yersinia ruckeri O1 biotype 2. Following experimental feeding, the ß-glucan and organic acids supplemented group showed significantly improved feed conversion and lipid efficiency ratios. Furthermore, the ß-glucan, ß-glucan and organic acids and citrus flavonoid supplements proved to significantly reduce the risk of mortality in rainbow trout during experimental infection as shown by hazard ratio analysis. Resulting in 33.2%, 30.6% and 30.5% reduction in risk relative to the non-supplemented base feed, respectively, these three supplements show a promising potential either as stand-alone feed supplements, or as components in complex feed formulations.

Human Milk Fortification with Bovine Colostrum Is Superior to Formula-Based Fortifiers to Prevent Gut Dysfunction, Necrotizing Enterocolitis, and Systemic Infection in Preterm Pigs.

BACKGROUND: Fortification of donor human milk (DHM) is required for optimal growth of very preterm infants, but there are concerns of more gut dysfunction and necrotizing enterocolitis (NEC) when using formula-based fortifiers (FFs), especially soon after birth. Intact bovine colostrum (BC) is rich in nutrients and bioactive factors, and protects against NEC in preterm pigs. We hypothesized that fortification of DHM with BC is superior to FFs to prevent gut dysfunction and infections when provided shortly after preterm birth. METHODS: Two FF products, Enfamil (ENF; intact protein, vegetable oil) and PreNAN+Nutrilon (NAN; extensively hydrolyzed protein, maltodextrin), were compared with BC as fortifier to DHM fed to preterm pigs for 5 days. RESULTS: Relative to the DHM+BC group, DHM+FF groups had higher diarrhea score and lower hexose uptake and lactase activity, and specifically the DHM+NAN group showed higher gut permeability, NEC score, more mucosa-adherent bacteria with altered gut microbiota structure (ie, lower diversity, increased Enterococcus, decreased Staphylococcus abundance). Both DHM+FF groups showed higher expression of intestinal cytokine and inflammation-related genes, more gut-derived bacteria in the bone marrow, lower density of mucin-containing goblet cells, and slightly higher colon lactate, stomach pH and acetate, and blood neutrophil-to-lymphocyte levels than the DHM+BC group. CONCLUSIONS: Used as a fortifier to DHM, BC is superior to FFs to support gut function, nutrient absorption, and bacterial defense mechanisms in preterm pigs. It is important to optimize the composition of nutrient fortifiers for preterm infants fed human milk.

Vaccination with outer membrane vesicles and the fimbrial protein FlfA offers improved protection against lesions following challenge with Gallibacterium anatis.

Gallibacterium anatis is an opportunistic poultry pathogen belonging to the Pasteurellaceae family. It has been shown to cause oophoritis, salpingitis and peritonitis in hens, as well as being associated with reduced semen quality in cockerels. Widespread multidrug resistance and substantial antigenic variation among strains of Gallibacterium anatis is a major constraint to treatment with antimicrobials and prevention of infection by vaccination. Novel vaccine strategies targeting G. anatis are therefore necessary. Outer membrane vesicles (OMVs) are nanosized vesicles formed from the outer membrane of Gram-negative bacteria. These vesicles have shown promising potential as both adjuvants and as vaccine candidates against numerous bacterial species. A high vesiculating mutant of G. anatis (G. anatis ΔtolR) has previously been made, enabling production of OMVs in large scale. In this study, we elucidated the potential of G. anatis ΔtolR OMVs as adjuvant for the conserved antigens GtxA-N (the N-terminal part of the RTX like toxin Gallibacterium toxin A) and FlfA (F17-like fimbria), as well as evaluated if combinations of OMVs together with antigens could facilitate cross-protective immunity against three different strains of G. anatis. We showed that ΔtolR OMVs function as an adjuvant for GtxA-N by inducing antigen specific antibody production. However, OMVs in combination with GtxA-N failed to induce protection against lesions after challenge infection. In contrast, vaccination with OMVs in combination with FlfA protected against lesions, especially in the salpinx, caused by two diverse strains of G. anatis, thereby indicating a cross-protective potential. No protection against the third G. anatis strain 7990 could be obtained in any of the experimental settings. In conclusion, ΔtolR OMVs and FlfA could serve as potential future vaccine components againt G. anatis.

Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus, and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with lysosomes to form a phagolysosome. The results indicate that an intracellular phase may be one way S. zooepidemicus survives in the host, and could in part explain how S. zooepidemicus can cause recurrent/persistent infections. Future studies should reveal the ability of S. zooepidemicus to internalize and survive in primary equine endometrial cells and during in vivo conditions.

Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens.

Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens.

Immunogenic and protective efficacy of recombinant protein GtxA-N against Gallibacterium anatis challenge in chickens.

Gallibacterium anatis is a major cause of reproductive tract infections in chickens. Here, we aimed to evaluate the efficacy of the recombinant protein GtxA-N at protecting hens, by addressing three objectives; (i) evaluating the antibody response following immunization (ii) scoring and comparing lesions, following challenge with G. anatis, in immunized and non-immunized hens and (iii) investigating if the anti-GtxA-N antibody titre in individual hens correlated with the observed lesions. Two consecutive experiments were performed in hens. In the first experiment hens were immunized with GtxA-N on day 0 and day 14, infected with G. anatis on day 28 and euthanized on day 56. The GtxA-N antibody response was assessed in pooled serum samples throughout the experiment, using an indirect enzyme-linked immunosorbent assay (ELISA). In the second experiment the GtxA-N antibody titres were assessed in individual hens before and after immunization. Subsequently, the hens were inoculated with G. anatis and finally all hens where euthanized and submitted for post mortem examination 48 h after inoculation. Immunization elicited strong antibody responses that lasted at least 8 weeks (P < .0001). The individual antibody titres observed in response to immunization varied considerably among hens (range: 174,100-281,500). Lesion scores following G. anatis infection were significantly lower in immunized hens compared to non-immunized hens (P = .004). Within the immunized group, no correlation was found between the individual antibody titres and the lesion scores. This study clearly demonstrated GtxA-N as a vaccine antigen able of inducing protective immunity against G. anatis.

Bacterial determinants of importance in the virulence of Gallibacterium anatis in poultry.

Gallibacterium anatis, a member of the Pasteurellaceae family, constitute a part of the normal micro-flora of the upper respiratory tract and the lower genital tract in chickens. However, increasing evidence indicate that G. anatis is also associated with a wide range of pathological changes, particularly in the reproductive organs, which leads to decreased egg production, lowered animal welfare and increased mortality. As a recently defined opportunistic pathogen limited focus has been placed on the pathogenesis and putative virulence factors permitting G. anatis to cause disease. One of the most studied virulence determinants is a large RTX-like toxin (GtxA), which has been demonstrated to induce a strong leukotoxic effect on avian macrophages. A number of fimbria of different sizes and shapes has been described. Particularly fimbriae belonging to the F17-like family appears to be common in a diverse selection of G. anatis strains. Mutants lacking the FlfA fimbria were severely attenuated in experimentally infected chickens. Additional characteristics including the ability to express capsular material possibly involved in serum resistance; secretion of metalloproteases capable of degrading immunoglobulins, and hemagglutinins, which may promote biofilm formation are all factors likely linked to the virulence of G. anatis. A major advantage for the study of how G. anatis interact with its host is the ability to perform biologically relevant experimental infections where natural routes of exposure allows reproduction of lesions observed during spontaneous infections. This review summarizes the current understanding of the G. anatis pathogenesis and discusses the contribution of the established and putative virulence factors described for this bacterium to date.

Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS.

Streptococcus equi includes very important animal and human pathogens. S. equi subsp. equi (SEE) is a highly pathogenic equine specific subspecies, while S. equi subsp. zooepidemicus (SEZ) and S. equi subsp. ruminatorum are opportunistic pathogens of various animal species and humans. Due to great phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies in horses. Superspectra created in this study enabled correct identification of 86 strains belonging to different subspecies of S. equi, isolated from various hosts, infection sites and years. In general, higher average identification accuracy was achieved for SEE (99.0±3.0%) than for SEZ (93.3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method with spectra analyses using the SARAMIS database. Additionally, first results on subtyping of SEZ indicated that a more refined discrimination, for example for epidemiological surveys, may be possible.

Chaperone-usher fimbriae in a diverse selection of Gallibacterium genomes.

BACKGROUND: Fimbriae are bacterial cell surface organelles involved in the pathogenesis of many bacterial species, including Gallibacterium anatis, in which a F17-like fimbriae of the chaperone-usher (CU) family was recently shown to be an important virulence factor and vaccine candidate. To reveal the distribution and variability of CU fimbriae 22 genomes of the avian host-restricted bacteria Gallibacterium spp. were investigated. Fimbrial clusters were classified using phylogeny-based and conserved domain (CD) distribution-based approaches. To characterize the fimbriae in depth evolutionary analysis and in vitro expression of the most prevalent fimbrial clusters was performed. RESULTS: Overall 48 CU fimbriae were identified in the genomes of the examined Gallibacterium isolates. All fimbriae were assigned to γ4 clade of the CU fimbriae of Gram-negative bacteria and were organized in four-gene clusters encoding a putative major fimbrial subunit, a chaperone, an usher and a fimbrial adhesin. Five fimbrial clusters (Flf-Flf4) and eight conserved domain groups were defined to accommodate the identified fimbriae. Although, the number of different fimbrial clusters in individual Gallibacterium genomes was low, there was substantial amino acid sequence variability in the major fimbrial subunit and the adhesin proteins. The distribution of CDs among fimbrial clusters, analysis of their flanking regions, and evolutionary comparison of the strains revealed that Gallibacterium fimbrial clusters likely underwent evolutionary divergence resulting in highly host adapted and antigenically variable fimbriae. In vitro, only the fimbrial subunit FlfA was expressed in most Gallibacterium strains encoding this protein. The absence or scarce expression of the two other common fimbrial subunits (Flf1A and Flf3A) indicates that their expression may require other in vitro or in vivo conditions. CONCLUSIONS: This is the first approach establishing a systematic fimbria classification system within Gallibacterium spp., which indicates a species-wide distribution of γ4 CU fimbriae among a diverse collection of Gallibacterium isolates. The expression of only one out of up to three fimbriae present in the individual genomes in vitro suggests that fimbriae expression in Gallibacterium is highly regulated. This information is important for future attempts to understand the role of Gallibacterium fimbriae in pathogenesis, and may prove useful for improved control of Gallibacterium infections in chickens.

In silico prediction of Gallibacterium anatis pan-immunogens.

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

Outer membrane vesicles reflect environmental cues in Gallibacterium anatis.

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg-production worldwide. Increased knowledge of the pathogenesis and virulence factors is important to better understand and prevent the negative effects of G. anatis. To this end outer membrane vesicles (OMVs) are natural secretion products of Gram-negative bacteria, displaying an enormous functional diversity and promising results as vaccine candidates. This is the first study to report that G. anatis secretes OMVs during in vitro growth. By use of transmission electron microscopy (TEM) and SDS-PAGE, we showed that changes in in vitro growth conditions, including incubation time, media composition and temperature, affected the OMV production and protein composition. A large protein band was increased in its concentration after prolonged growth. Analysis by LC-MS/MS indicated that the band contained two proteins; the 320.1 kDa FHA precursor, FhaB, and a 407.8 kDa protein containing a von Willebrand factor type A (vWA) domain. Additional two major outer-membrane (OM) proteins could be identified in all samples; the OmpH-homolog, OmpC, and OmpA. To understand the OMV formation better, a tolR deletion mutation (ΔtolR) was generated in G. anatis. This resulted in a constantly high and growth-phase independent production of OMVs, suggesting that depletion of peptidoglycan linkages plays a role in the OMV formation in G. anatis. In conclusion, our results show that G. anatis produce OMVs in vitro and the OMV protein profile suggests that the production is an important and well-regulated ability employed by the bacteria, which may be used for vaccine production purposes.

Insight from molecular, pathological, and immunohistochemical studies on cellular and humoral mechanisms responsible for vaccine-induced protection of rainbow trout against Yersinia ruckeri.

The immunological mechanisms associated with protection of vaccinated rainbow trout, Oncorhynchus mykiss, against enteric redmouth disease (ERM), caused by Yersinia ruckeri, were previously elucidated by the use of gene expression methodology and immunochemical methods. That approach pointed indirectly to both humoral and cellular elements being involved in protection. The present study correlates the level of protection in rainbow trout to cellular reactions in spleen and head kidney and visualizes the processes by applying histopathological, immunohistochemical, and in situ hybridization techniques. It was shown that these cellular reactions, which were more prominent in spleen than in head kidney, were associated with the expression of immune-related genes, suggesting a Th2-like response. Y. ruckeri, as shown by in situ hybridization (ISH), was eliminated within a few days in vaccinated fish, whereas nonprotected fish still harbored bacteria for a week after infection. Vaccinated fish reestablished normal organ structure within a few days, whereas nonprotected fish showed abnormalities up to 1 month postinfection. Protection in the early phase of infection was mainly associated with the expression of genes encoding innate factors (complement factors, lysozyme, and acute phase proteins), but in the later phase of infection, increased expression of adaptive immune genes dominated. The histological approach used has shown that the cellular changes correlated with protection of vaccinated fish. They comprised transformation of resident cells into macrophage-like cells and increased occurrence of CD8α and IgM cells, suggesting these cells as main players in protection. Future studies should investigate the causality between these factors and protection.

A simple and novel method for retrieval of Pasteurellaceae from swab samples collected in the field.

Traditionally it has been difficult or impossible to collect and preserve bacterial samples of especially fastidious bacteria in mixed primary cultures, unless the samples could be transported to a laboratory within approximately 24 h. Therefore, a simple novel method for preserving swab samples until bacterial isolation can be completed in the laboratory was developed and evaluated. Pasteurellaceae bacteria were used as a representative for fastidious bacteria. A 7.5% glucose serum medium was used as freeze medium. Swab samples were soaked in the medium a maximum of 2 h after collection and stored at -20°C. As a control study, 15 samples were collected from the oral cavity of a captive brown bear. One was immediately plated, while the remaining 12 swabs were stored at -20°C for 7 days and multiples of 30 days up to 330 days prior to plating. Two samples were stored without the medium for 7 and 30 days prior to plating. From a field setting in Greenland, eight polar bear samples were collected and subsequently stored for 240 to 259 days at -20°C before incubation. Pasteurellaceae bacteria were isolated and genotyped from all samples stored in the freeze medium, indicating that the medium enabled the bacteria to survive for at least 330 days at -20°C. The 100% recovery of target organisms in the polar bear samples even following lengthy storage and transport demonstrates that the method is very useful under remote field conditions.

The fimbrial protein FlfA from Gallibacterium anatis is a virulence factor and vaccine candidate.

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production worldwide. Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents, while antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was demonstrated by flow cytometry and immunofluorescence microscopy. The predicted function of FlfA as a fimbrial subunit protein was confirmed by immunogold electron microscopy. An flfA deletion mutant (ΔflfA) was generated in G. anatis 12656-12, and importantly, this mutant was significantly attenuated in the natural chicken host. Furthermore, protection against G. anatis 12656-12 could be induced by immunizing chickens with recombinant FlfA. Finally, in vitro expression of FlfA homologs was observed in a genetically diverse set of G. anatis strains, suggesting the potential of FlfA as a serotype-independent vaccine candidate This is the first study describing a fimbrial subunit protein of G. anatis with a clear potential as a vaccine antigen.

Genome analysis and phylogenetic relatedness of Gallibacterium anatis strains from poultry.

Peritonitis is the major disease problem of laying hens in commercial table egg and parent stock operations. Despite its importance, the etiology and pathogenesis of this disease have not been completely clarified. Although avian pathogenic Escherichia coli (APEC) isolates have been incriminated as the causative agent of laying hen peritonitis, Gallibacterium anatis are frequently isolated from peritonitis lesions. Despite recent studies suggesting a role for G. anatis in the pathogenesis of peritonitis, little is known about the organism's virulence mechanisms, genomic composition and population dynamics. Here, we compared the genome sequences of three G. anatis isolates in an effort to understand its virulence mechanisms and identify novel antigenic traits. A multilocus sequence typing method was also established for G. anatis and used to characterize the genotypic relatedness of 71 isolates from commercial laying hens in Iowa and 18 international reference isolates. Genomic comparisons suggest that G. anatis is a highly diverse bacterial species, with some strains possessing previously described and potential virulence factors, but with a core genome containing several antigenic candidates. Multilocus sequence typing effectively distinguished 82 sequence types and several clonal complexes of G. anatis, and some clones seemed to predominate among G. anatis populations from commercial layers in Iowa. Biofilm formation and resistance to antimicrobial agents was also observed in several clades. Overall, the genomic diversity of G. anatis suggests that multiple lineages exist with differing pathogenic potential towards birds.

Natural transformation of Gallibacterium anatis.

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.