Strain-dependent interactions of Streptococcus suis and Glaesserella parasuis in co-culture.

Streptococcus suis (S. suis) and Glaesserella parasuis (G. parasuis) are ubiquitous colonizers of swine tonsils that can cause systemic disease and death, under undefined conditions. It is not known, however, whether these 2 species interact during initial infection. To investigate whether such interactions occur, the objective of this study was to assess phenotypic differences between mono-and co-cultures of S. suis and G. parasuis when representative strains with different virulence potential were co-cultured in vitro. In cross-streak screening experiments, some G. parasuis (GP) serovar strains (GP3, GP4, GP5) exhibited altered morphology with some S. suis (SS) serovar strains, such as SS2, but not with SS1. Co-culture with GP5 reduced hemolytic activity of SS1, but not of SS2. Although both SS strains outgrew GP isolates in biofilm co-cultures, strain type affected the number of planktonic or sessile cells in co-culture biofilms. Numbers of sessile SS1 increased in co-cultures, but not of GP3. Both planktonic and sessile SS2 increased in co-culture, whereas GP5 decreased. Sessile SS1 increased, but planktonic GP5 decreased in co-culture and planktonic SS2 increased, but sessile GP3 decreased when grown together. The SS2 strain had a competitive advantage over GP3 during mid-exponential co-culture in broth. Streptococcus suis is predicted to use more unique carbon sources, suggesting that S. suis outcompetes G. parasuis in growth and nutrient consumption. This work provides direction for future studies of phenotypic and genotypic interactions between these and other swine tonsil co-colonizers.

Streptococcus suis (S. suis) et Glaesserella parasuis (G. parasuis) sont des colonisateurs omniprésents des amygdales porcines qui peuvent provoquer des maladies systémiques et la mort, dans des conditions non définies. On ne sait pas cependant si ces 2 espèces interagissent lors de l'infection initiale. Pour déterminer si de telles interactions se produisent, l'objectif de cette étude était d'évaluer les différences phénotypiques entre les mono- et cocultures de S. suis et G. parasuis lorsque des souches représentatives ayant un potentiel de virulence différent étaient cocultivées in vitro. Dans les expériences de dépistage par stries croisées, certaines souches des sérotypes de G. parasuis (GP) (GP3, GP4, GP5) présentaient une morphologie altérée avec certaines souches de sérovars de S. suis (SS), telles que SS2, mais pas avec SS1. La coculture avec GP5 a réduit l'activité hémolytique de SS1, mais pas de SS2. Bien que la croissance des deux souches SS ait surpassé celle des isolats de GP dans les cocultures de biofilms, le type de souche a affecté le nombre de cellules planctoniques ou sessiles dans les biofilms de coculture. Le nombre de SS1 sessiles a augmenté dans les cocultures, mais pas de GP3. Les SS2 planctoniques et sessiles ont augmenté en coculture, tandis que GP5 a diminué. La SS1 sessile a augmenté, mais la GP5 planctonique a diminué en coculture et la SS2 planctonique a augmenté, mais la GP3 sessile a diminué lorsqu'elles sont cultivées ensemble. La souche SS2 avait un avantage compétitif sur GP3 lors de la coculture mi-exponentielle en bouillon. On prévoit que S. suis utilise plus des sources de carbone uniques, ce qui suggère que S. suis surpasse G. parasuis en termes de croissance et de consommation de nutriments. Ce travail fournit une orientation pour les études futures des interactions phénotypiques et génotypiques entre ces derniers et d'autres co-colonisateurs des amygdales porcines.(Traduit par Docteur Serge Messier).

Comparative genomics study of Staphylococcus aureus isolated from cattle and humans reveals virulence patterns exclusively associated with bovine clinical mastitis strains.

Staphylococcus aureus causes nosocomial and intramammary infections in humans and cattle, respectively. A large number of virulence factors are thought to play important roles in the pathogenesis of this bacterium. Currently, genome-wide and data-analysis studies are being used to better understand its epidemiology. In this study, we conducted a genome wide comparison and phylogenomic analyses of S. aureus to find specific virulence patterns associated with clinical and subclinical mastitis strains in cattle and compare them with those of human origin. The presence/absence of key virulence factors such as adhesin, biofilm, antimicrobial resistance, and toxin genes, as well as the phylogeny and sequence type of the isolates were evaluated. A total of 248 genomes (27 clinical mastitis, 43 subclinical mastitis, 21 milk, 53 skin-related abscesses, 49 skin infections, and 55 pus from cellulitis) isolated from 32 countries were evaluated. We found that the cflA, fnbA, ebpS, spa, sdrC, coa, emp, vWF, atl, sasH, sasA, and sasF adhesion genes, as well as the aur, hglA, hglB, and hglC toxin genes were highly associated in clinical mastitis strains. The strains had diverse genetic origins (72 protein A and 48 sequence types with ST97, ST8 and ST152 being frequent in isolates from clinical mastitis, abscess, and skin infection, respectively). Further, our phylogenomic analyses suggested that zoonotic and/or zooanthroponotic transmission may have occurred. These findings contribute to a better understanding of S. aureus epidemiology and the relationships between adhesion mechanisms, biofilm formation, antimicrobial resistance, and toxins and could aid in the development of improved vaccines and strain genotyping methods.

Innate response of rainbow trout gill epithelial (RTgill-W1) cell line to ultraviolet-inactivated VHSV and FliC and rhabdovirus infection.

Gills reportedly play a crucial role in induction of an antiviral immune response in fish. We investigated the expression of innate response genes in the rainbow trout gill epithelial cell line RTgill-W1 36 h after pretreatment with ultraviolet-inactivated viral hemorrhagic septicemia virus (UV-VHSV), flagellin C protein from Edwardsiella tarda (FliC), VHSV and SVCV using an Agilent 4 × 44k cGRASP salmonid microarray. RTgill-W1 cells pretreated with UV-VHSV, triggered an independent gene expression profile from those treated with a recombinant flagellin C protein from Edwardsiella tarda. In addition, exposure of RTgill-W1 cells to live viruses spring viremia of carp virus and viral hemorrhagic septicemia virus induced a less robust transcriptional change of 24 and 22 gene probes, respectively, when compared to 123 genes for UV-VHSV. Further the pretreatment of RTgill-W1 cells with (UV-VHSV) significantly reduced VHSV genome copy number at 6 d post infection (dpi) relative to the FliC-treated and untreated control. A quantitative PCR was used to study the transcriptional modulation of a set of 25 innate immune-related genes highlighted by the microarray data and a panel of 7 established antiviral genes in the protected cells. Notably, the expression of ifn1, ifn2, mx1 and mx3 were expressed more in untreated cells than in UV-VHSV-treated cells where virus replication was inhibited. The results from this study shed light on the mechanisms and pathways used by teleost gill epithelium innate immunity in combating viral and bacterial infection.

Genomic comparisons and phylogenetic analysis of mastitis-related staphylococci with a focus on adhesion, biofilm, and related regulatory genes.

Mastitis is a common and costly disease on dairy farms, commonly caused by Staphylococcus spp. though the various species are associated with different clinical outcomes. In the current study, we performed genomic analyses to determine the prevalence of adhesion, biofilm, and related regulatory genes in 478 staphylococcal species isolated from clinical and subclinical mastitis cases deposited in public databases. The most prevalent adhesin genes (ebpS, atl, pls, sasH and sasF) were found in both clinical and subclinical isolates. However, the ebpS gene was absent in subclinical isolates of Staphylococcus arlettae, S. succinus, S. sciuri, S. equorun, S. galinarum, and S. saprophyticus. In contrast, the coa, eap, emp, efb, and vWbp genes were present more frequently in clinical (vs. subclincal) mastitis isolates and were highly correlated with the presence of the biofim operon (icaABCD) and its transcriptional regulator, icaR. Co-phylogenetic analyses suggested that many of these adhesins, biofilm, and associated regulatory genes could have been horizontally disseminated between clinical and subclinical isolates. Our results further suggest that several adhesins, biofilm, and related regulatory genes, which have been overlooked in previous studies, may be of use for virulence profiling of mastitis-related Staphylococcus strains or as potential targets for vaccine development.

'Big Data' in animal health research - opportunities and challenges.

Automated systems for high-input data collection and data storage have led to exponential growth in the availability of information. Such datasets and the tools applied to them have been referred to as 'big data'. Starting with a systematic review of the terms 'informatics, bioinformatics and big data' in animal health this special issue of AHRR illustrates some big-data applications with papers on how the use of various omics methods may be used to facilitate the development of improved diagnostics, therapeutics, and vaccines for foodborne pathogens in poultry and on how a better understanding of rumen microbiota could lead to improved feed absorption while minimizing methane production. Other papers in this issue cover the use of big data modeling in dairy cattle for more effective disease interventions and machine learning tools for livestock breeding. The final two reviews describe the use of big data in better vector-borne pathogen forecasts with canine seroprevalence maps and modeling approaches to understand the transmission of avian influenza virus. Although a lot of technical and ethical issues remain with the use of big data, these reviews illustrate the tremendous potential that big-data systems have to revolutionize animal health research.

Complete Genome Sequences of 11 Staphylococcus sp. Strains Isolated from Buffalo Milk and Milkers' Hands.

Here, we present data on the complete genome sequences of 11 Staphylococcus sp. isolates (three S. chromogenes isolates and one isolate each of S. saprophyticus, S. xylosus, S. hominis, S. agnetis, S. caprae, S. aureus, and S. warneri), obtained as part of a mastitis study of buffalo milk (from healthy animals and from those with subclinical mastitis) and milkers' hands.

Effect of tracheal antimicrobial peptide on the development of Mannheimia haemolytica pneumonia in cattle.

Bacterial pneumonia causes significant economic loss to the beef industry and occurs at times of stress and viral infection. Administering antibiotics to at-risk calves is often used to prevent the disease, but alternatives to mass treatment with antibiotics are needed. Tracheal antimicrobial peptide (TAP), a ß-defensin naturally produced by bovine airways, has bactericidal activity against the pathogens that cause pneumonia in cattle. However, TAP expression is suppressed by glucocorticoid (stress) and viral infection. We hypothesized that delivering TAP to the respiratory tract would prevent development of pneumonia in calves infected with Mannheimia haemolytica. Clean-catch calves (i.e. obtained prior to contact with the dam) were challenged by aerosol with M. haemolytica, and TAP or water was delivered to the respiratory tract at 0.3, 2 and 6 hours post-infection. TAP treatment did not protect against development of disease. Calves treated with TAP had similar bacterial loads in the nasal cavity and lung compared to calves treated with water. Similarly, TAP treatment did not affect the development of clinical signs, elevated rectal temperatures, or increased levels of blood neutrophils, haptoglobin and fibrinogen that occurred after bacterial challenge. Postmortem gross and histologic lung lesions were also similar in the two groups. To determine why there was a lack of protective effect, we tested the effect of substances in respiratory lining fluid on the bactericidal activity of TAP. Physiologic concentrations of sodium chloride inhibited TAP bactericidal activity in vitro, as did serum at concentrations of 0.62 to 2.5%, but concentrated bronchoalveolar lavage fluid had no consistent effect. These findings suggest that TAP does not have in vivo bactericidal activity against M. haemolytica because of interference by physiological sodium chloride levels and by serum. Thus, administration of TAP may not be effective for prevention of M. haemolytica pneumonia.

Short communication: Detection of antibiotic resistance, mecA, and virulence genes in coagulase-negative Staphylococcus spp. from buffalo milk and the milking environment.

The aim of this study was to determinate whether coagulase-negative staphylococci (CNS) from buffalo milk or the milking environment possess virulence factors that are associated with intramammary infections or antimicrobial resistance. Milk samples (n = 320) from 80 lactating buffalo were evaluated for clinical and subclinical mastitis by physical examination, the strip cup test, California Mastitis Test (CMT), and somatic cell count (SCC) over a 4-mo period. In addition, swabs were obtained from the hands of consenting milkers (16), liners (64), and from the mouths (15) and nostrils (15) of buffalo calves. No clinical cases of mastitis were observed; however, CMT together with SCC results indicated that 8 animals had subclinical mastitis. Eighty-four CNS isolates were identified by MALDI-TOF MS and cydB real-time PCR (qPCR) and then evaluated by qPCR for presence of the eta, etb, sea, sec, cna, seb, sei, seq, sem, seg, see, and tst toxin genes, adhesion- and biofilm-associated genes (eno, ebps, fib, fnbA, coa), and the methicillin resistance gene (mecA). Resistance to antibiotics commonly used for mastitis treatment in Brazil was determined using the Kirby-Bauer test. Two strains were positive for the see and eta toxin genes; and mecA (1), eno (27), ebps (10), fnbA (10), and coa (5) genes were also detected. A notable number of isolates were resistant to erythromycin (30), penicillin (26), and cotrimoxazole (18); importantly, 10 vancomycin-resistant isolates were also detected. A smaller number of isolates were resistant to rifampicin (8), oxacillin (7), clindamycin (5), cefepime (4), tetracycline (3), ciprofloxacin (2), and chloramphenicol (1), and none were resistant to gentamicin or ciprofloxacin. Isolates with resistance to 2 (13 isolates), 3 (3), 4 (3), 5 (1), and 6 (1) antibiotics were detected. Taken together, our findings suggest that CNS isolates may not be a significant cause of clinical or even subclinical mastitis in buffaloes, but they may be a reservoir of virulence and antibiotic resistance genes.

Antimicrobial resistance of Streptococcus suis isolates recovered from clinically ill nursery pigs and from healthy pigs at different stages of production.

Streptococcus suis isolates (N = 379) from clinically ill pigs and from healthy pigs were tested for antimicrobial susceptibility using a disk diffusion method. Isolates from clinical cases had a higher prevalence of resistance compared with isolates from healthy pigs. There was a high to moderate prevalence of resistance to antibiotics commonly used in nursery pig diets such as tetracycline (84.2%), tiamulin (65.2%), and spectinomycin (40.4%). There was a low prevalence of resistance, however, to antimicrobials that are only used as parenteral treatments and not added to feed (e.g., ceftiofur, florfenicol). These findings should help practitioners in choosing appropriate drugs for use on Ontario swine farms.

Résistance antimicrobienne des isolats de Streptococcus suis récupérés de porcs en santé à différents stades de production et de porcelets de pouponnière cliniquement malades. Des isolats de Streptococcus suis (N = 379) provenant de porcs cliniquement malades et de porcs en santé ont été testés pour la susceptibilité antimicrobienne en utilisant une méthode de diffusion par disque. Les isolats des cas cliniques avaient une prévalence accrue de résistance comparativement aux isolats de porcs en santé. Il y avait une prévalence élevée à modérée de résistance aux antibiotiques communément utilisés dans les diètes des porcelets de pouponnière, dont la tétracycline (84,2 %), la tiamuline (65,2 %) et la spectinomycine (40,4 %). Par contre, il y avait une faible prévalence de la résistance aux antimicrobiens qui étaient seulement utilisés comme traitements parentéraux et non ajoutés aux aliments (p. ex., ceftiofur, florfénicol). Ces constatations devraient aider les praticiens à choisir les médicaments appropriés pour utilisation dans les fermes porcines de l'Ontario.(Traduit par Isabelle Vallières).

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum.

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 108 cfu/fish and after mortality had begun, fish were given erythromycin- and florfenicol-medicated feed at a rate of 75 mg kg- 1 day- 1 and 10 mg kg- 1  day- 1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30-day trial. Relative to antibiotic-free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.

Investigation of putative invasion determinants of Actinobacillus species using comparative genomics.

Actinobacillus spp. are Gram-negative bacteria associated with mucosal membranes. While some are commensals, others can cause important human and animal diseases. A. pleuropneumoniae causes severe fibrinous hemorrhagic pneumonia in swine but not systemic disease whereas other species invade resulting in septicemia and death. To understand the invasive phenotype of Actinobacillus spp., complete genomes of eight isolates were obtained and pseudogenomes of five isolates were assembled and annotated. Phylogenetically, A. suis isolates clustered by surface antigen type and were more closely related to the invasive A. ureae, A. equuli equuli, and A. capsulatus than to the other swine pathogen, A. pleuropneumoniae. Using the LS-BSR pipeline, 251 putative virulence genes associated with serum resistance and invasion were detected. To our knowledge, this is the first genome-wide study of the genus Actinobacillus and should contribute to a better understanding of host tropism and mechanisms of invasion of pathogenic Actinobacillus and related genera.

An epidemiological study of Streptococcus suis serotypes of pigs in Ontario determined by a multiplex polymerase chain reaction.

The purpose of the study was to identify the serotypes of Streptococcus suis from tonsil swabs in clinically ill and healthy pigs in Ontario using a multiplex polymerase chain reaction (PCR) method. Although 22 different serotypes were identified, most isolates were S. suis-like bacteria or untypable.

Étude épidémiologique des sérotypes Streptococcus suisdes porcs en Ontario déterminés par amplification en chaîne par polymérase multiplexe. Le but de cette étude consistait à identifier les sérotypes de Streptococcus suis provenant d'écouvillons des amygdales chez des porcs cliniquement malades et en santé en Ontario en utilisant une méthode multiplexe d'amplification en chaîne par polymérase (PCR). Même si 22 sérotypes différents ont été identifiés, la plupart des isolats étaient des bactéries de type S. suis ou non typables.(Traduit par Isabelle Vallières).

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum).

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) in freshwater-reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 108  cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 106 , 107 or 108  cfu/fish of F. psychrophilum FPG 101, the 108  cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R2  = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety-seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log10 gene/copies and >3.0 log10 gene copies/reaction, respectively, following infection with 108  cfu/fish.

Some coagulase negative Staphylococcus spp. isolated from buffalo can be misidentified as Staphylococcus aureus by phenotypic and Sa442 PCR methods.

OBJECTIVE: Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes. RESULTS: From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.

Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization-time of flight mass spectrometry and cydB real-time quantitative PCR.

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species.

Differential expression of putative adhesin genes of Actinobacillus suis grown in in vivo-like conditions.

Actinobacillus suis is an opportunistic pathogen that resides in the tonsils of the soft palate of swine. Unknown stimuli can cause this organism to invade the host, resulting in septicaemia and sequelae including death. To better understand its pathogenesis, the expression of several adhesin genes was evaluated by semi-quantitative real-time PCR in A. suis grown in conditions that mimic the host environment, including different nutrient and oxygen levels, exponential and stationary phases of growth, and in the presence of the stress hormone epinephrine. Fifty micromolar epinephrine did not affect the growth rate or expression of A. suis adhesin genes, but there was a significant growth phase effect for many genes. Most adhesin genes were also differentially expressed during anoxic static growth or aerobic growth, and in this study, all genes were differentially expressed in either exponential or stationary phase. Based on the time*treatment interactions observed in the anoxic study, a model of persistence of A. suis in the host environment in biofilm and planktonic states is proposed. Biofilm dynamics were further studied using wild type and isogenic mutants of the type IVb pilin (Δ flp1), the OmpA outer membrane protein (ΔompA), and the fibronectin-binding (ΔcomE1) genes. Disruption of these adhesin genes affected the early stages of biofilm formation, but in most cases, biofilm formation of the mutant strains was similar to that of the wild type by 24h of incubation. We postulate that other adhesins may have overlapping functions that can compensate for those of the missing adhesins.

Attachment of Actinobacillus suis H91-0380 and Its Isogenic Adhesin Mutants to Extracellular Matrix Components of the Tonsils of the Soft Palate of Swine.

Tonsils conduct immune surveillance of antigens entering the upper respiratory tract. Despite their immunological function, they are also sites of persistence and invasion of bacterial pathogens. Actinobacillus suis is a common resident of the tonsils of the soft palate in pigs, but under certain circumstances it can invade, causing septicemia and related sequelae. Twenty-four putative adhesins are predicted in the A. suis genome, but to date, little is known about how they might participate in colonization or invasion. To better understand these processes, swine tonsil lysates were characterized by mass spectrometry. Fifty-nine extracellular matrix (ECM) proteins were identified, including small leucine-rich proteoglycans, integrins, and other cell surface receptors. Additionally, attachment of the wild type and 3 adhesin mutants to 5 ECM components was evaluated. Exponential cultures of wild-type A. suis adhered significantly more than stationary cultures to all ECM components studied except collagen I. During exponential growth, the A. suis Δflp1 mutant attached less to collagen IV while the ΔompA mutant attached less to all ECMs. The ΔcomE1 strain attached less to collagen IV, fibronectin, and vitronectin during exponential growth and exhibited differential attachment to collagen I over short adherence time points. These results suggest that Flp1, OmpA, and ComE1 are important during early stages of attachment to ECM components found in tonsils, which supports the notion that other adhesins have compensatory effects during later stages of attachment.

Identification of putative adhesins of Actinobacillus suis and their homologues in other members of the family Pasteurellaceae.

BACKGROUND: Actinobacillus suis disease has been reported in a wide range of vertebrate species, but is most commonly found in swine. A. suis is a commensal of the tonsils of the soft palate of swine, but in the presence of unknown stimuli it can invade the bloodstream, causing septicaemia and sequelae such as meningitis, arthritis, and death. It is genotypically and phenotypically similar to A. pleuropneumoniae, the causative agent of pleuropneumonia, and to other members of the family Pasteurellaceae that colonise tonsils. At present, very little is known about the genes involved in attachment, colonisation, and invasion by A. suis (or related members of the tonsil microbiota). RESULTS: Bioinformatic analyses of the A. suis H91-0380 genome were done using BASys and blastx in GenBank. Forty-seven putative adhesin-associated genes predicted to encode 24 putative adhesins were discovered. Among these are 6 autotransporters, 25 fimbriae-associated genes (encoding 3 adhesins), 12 outer membrane proteins, and 4 additional genes (encoding 3 adhesins). With the exception of 2 autotransporter-encoding genes (aidA and ycgV), both with described roles in virulence in other species, all of the putative adhesin-associated genes had homologues in A. pleuropneumoniae. However, the majority of the closest homologues of the A. suis adhesins are found in A. ureae and A. capsulatus--species not known to infect swine, but both of which can cause systemic infections. CONCLUSIONS: A. suis and A. pleuropneumoniae share many of the same putative adhesins, suggesting that the different diseases, tissue tropism, and host range of these pathogens are due to subtle genetic differences, or perhaps differential expression of virulence factors during infection. However, many of the putative adhesins of A. suis share even greater homology with those of other pathogens within the family Pasteurellaceae. Similar to A. suis, these pathogens (A. capsulatus and A. ureae) cause systemic infections and it is tempting to speculate that they employ similar strategies to invade the host, but more work is needed before that assertion can be made. This work begins to examine adhesin-associated factors that allow some members of the family Pasteurellaceae to invade the bloodstream while others cause a more localised infection.

Complete genome sequence of Actinobacillus equuli subspecies equuli ATCC 19392(T).

Actinobacillus equuli subsp. equuli is a member of the family Pasteurellaceae that is a common resident of the oral cavity and alimentary tract of healthy horses. At the same time, it can also cause a fatal septicemia in foals, commonly known as sleepy foal disease or joint ill disease. In addition, A. equuli subsp. equuli has recently been reported to act as a primary pathogen in breeding sows and piglets. To better understand how A. equuli subsp. equuli can cause disease, the genome of the type strain of A. equuli subsp. equuli, ATCC 19392(T), was sequenced using the PacBio RSII sequencing system. Its genome is comprised of 2,431,533 bp and is predicted to encode 2,264 proteins and 82 RNAs.

Validation of reference genes for quantitative real-time PCR (qPCR) analysis of Actinobacillus suis.

BACKGROUND: Quantitative real-time PCR is a valuable tool for evaluating bacterial gene expression. However, in order to make best use of this method, endogenous reference genes for expression data normalisation must first be identified by carefully validating the stability of expression under experimental conditions. Therefore, the objective of this study was to validate eight reference genes of the opportunistic swine pathogen, Actinobacillus suis, grown in aerobic cultures with (Epinephrine) or without (Aerobic) epinephrine in the growth medium and in anoxic static cultures (Anoxic), and sampled during exponential and stationary phases. RESULTS: Using the RefFinder tool, expression data were analysed to determine whether comprehensive stability rankings of selected reference genes varied with experimental design. When comparing Aerobic and Epinephrine cultures by growth phase, pyk and rpoB were both among the most stably expressed genes, but when analysing both growth phases together, only pyk remained in the top three rankings. When comparing Aerobic and Anoxic samples, proS ranked among the most stable genes in exponential and stationary phase data sets as well as in combined rankings. When analysing the Aerobic, Epinephrine, and Anoxic samples together, only gyrA ranked consistently among the top three most stably expressed genes during exponential and stationary growth as well as in combined rankings; the rho gene ranked as least stably expressed gene in this data set. CONCLUSIONS: Reference gene stability should be carefully assessed with the design of the experiment in mind. In this study, even the commonly used reference gene 16S rRNA demonstrated large variability in stability depending on the conditions studied and how the data were analysed. As previously suggested, the best approach may be to use a geometric mean of multiple genes to normalise qPCR results. As researchers continue to validate reference genes for various organisms in multiple growth conditions and sampling time points, it may be possible to make informed predictions as to which genes may be most suitable to validate for a given experimental design, but in the meantime, the reference genes used to normalise qPCR data should be selected with caution.