Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

[Case report of fatal sepsis caused by Actinobacillus suis/equuli in an adult male].

This is a case report of sepsis caused by the species Actinobacillus suis/equuli in a male agriculture worker that ended fatally. The article also contains information on identification and results of antibiotic susceptibility testing. This is a rare case of human infection and probably the first case of a human being infected by this species in the Czech Republic.

Phenotypic and genotypic characterization of Actinobacillus suis sensu stricto isolated from a dairy calf.

The species of the genus Actinobacillus have so far been associated with specific animal hosts, and A. suis sensu stricto, an opportunistic pathogen of swine, is rarely isolated from ruminants. We describe here the isolation of A. suis sensu stricto from a newborn calf that died on a dairy farm in Japan. Identification of the isolate was performed by phenotypic and genotypic characterization, with the latter consisting of nucleotide sequence analyses of the 16S rRNA gene plus three housekeeping genes, rpoB, infB and recN.

Septicemic Actinobacillus suis infection in a neonatal piglet with multifocal necrotic glossitis.

Five-day-old neonatal piglets presented with debilitation and ananastasia. At the necropsy of one piglet, the apex of the tongue was found to be discolored dark red, and disseminated white foci were found on the cut surface. Many white foci were also found in the lungs and on the serosa of the liver and spleen. Histopathological findings revealed multifocal necrotic glossitis and pneumonia with Gram-negative bacilli. The bacilli were identified as Actinobacillus suis through immunohistochemical, biochemical, and genetic tests, including 16S rRNA gene sequencing. Although A. suis usually causes inflammation in thoracic and abdominal organs, lesions were also found in the tongue in the present case. This study is the first report of glossitis caused by A. suis.

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.

Sequence and structural diversity of transferrin receptors in Gram-negative porcine pathogens.

Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis are bacterial pathogens from the upper respiratory tract that are responsible for a substantial burden of porcine disease. Although reduction of disease has been accomplished by intensive management practices, immunization remains an important strategy for disease prevention, particularly when intensive management practices are not feasible or suitable. An attractive target for vaccine development is the surface receptor involved in acquiring iron from host transferrin, since it is common to all three pathogenic species and has been shown to be essential for survival and disease causation. It has also recently been demonstrated that an engineered antigen derived from the lipoprotein component of the receptor, transferrin-binding protein B (TbpB), was more effective at preventing infection by H. parasuis than a commercial vaccine product. This study was initiated to explore the genetic and immunogenic diversity of the transferrin receptor system from these species. Nucleic acid sequences were obtained from a geographically and temporally diverse collection of isolates, consisting of 41 A. pleuropneumoniae strains, 30 H. parasuis strains, and 2 A. suis strains. Phylogenetic analyses demonstrated that the receptor protein sequences cluster independently of species, suggesting that there is genetic exchange between these species such that receptor-based vaccines should logically target all three species. To evaluate the cross-reactive response of TbpB-derived antigens, pigs were immunized with the intact TbpB, the TbpB N-lobe and the TbpB C-lobe from A. pleuropneumoniae strain H49 and the resulting sera were tested against a representative panel of TbpBs; demonstrating that the C-lobe induces a broadly cross-reactive response. Overall our results indicate that there is a common reservoir for transferrin receptor antigenic variation amongst these pathogens. While this could present a challenge to future vaccine development, our results suggest a rationally designed TbpB-based vaccine may provide protection against all three pathogens.

First Human Case of Meningitis and Sepsis in a Child Caused by Actinobacillus suis or Actinobacillus equuli.

We report the first human case of meningitis and sepsis caused in a child by Actinobacillus suis or A. equuli, a common opportunistic pathogen of swine or horses, respectively. Identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and real-time PCR assay. A previous visit to a farm was suspected as the source of infection.

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.

Complete genome sequence of Actinobacillus suis H91-0380, a virulent serotype O2 strain.

Here, we report the first complete genome sequence of Actinobacillus suis, an important opportunistic pathogen of swine. By comparing the genome sequence of A. suis with those of other members of the family Pasteurellaceae, we hope to better understand the role of these organisms in health and disease in swine.

Development of a real-time polymerase chain reaction assay for detection of Actinobacillus suis in porcine lung.

In the current study, the development and validation of a real-time polymerase chain reaction (PCR) assay using a TaqMan-labeled probe for the detection of Actinobacillus suis from porcine lung samples is described. This real-time PCR amplified a 110-bp region of the 23S ribosomal RNA gene from A. suis but not from other bacteria. First, the assay was validated with 183 bacterial strains representing different species of bacteria. Subsequently, 85 porcine lung specimens that were declared A. suis-positive and -negative by bacterial culture and identification were tested to assess whether it can be performed directly on tissue specimens. The bacterial culture results and real-time PCR results agreed across all the samples tested assigning 100% positive and negative predictive values to the PCR. Further, the detection limit of the assay was 380 colony-forming units (CFU) per ml or approximately 1 CFU per reaction. In conclusion, the TaqMan real-time PCR assay described herein is a highly specific, sensitive, and reproducible test, which can be used to detect A. suis DNA in porcine lung specimens, thus providing a timely diagnosis.

The lipopolysaccharide core of Actinobacillus suis and its relationship to those of Actinobacillus pleuropneumoniae.

The Gram-negative bacteria Actinobacillus suis colonizes the upper respiratory and genital tracts of swine. Along with capsular polysaccharides, lipopolysaccharides (O-chain→core→lipid A~cell) are a main cell-surface component of A. suis. In this study, we determined that A. suis lipopolysaccharide incorporates a conserved core that shares some structural features with several core types of A. pleuropneumoniae . These common core structural features likely account for the observed serological cross-reactivity between A. suis and A. pleuropneumoniae, and the data suggest that the structural epitopes responsible for immunogenicity are those in the outer core domain.

Discovery and characterization of a fructosylated capsule polysaccharide and sialylated lipopolysaccharide in a virulent strain of Actinobacillus suis.

We are developing a serotyping system for Actinobacillus suis based on its capsule (K) and lipopolysaccharide O-chain (O) structures. Previously, we have shown that less virulent strains of this swine pathogen express a (1→6)-ß-D-glucan as both K- and O-chain polysaccharides and were serologically classified as K:1/O:1. Here, we show that representative A. suis strains with a high (H91-0380; serotype K:2/O:2) and intermediate (C84; serotype K:2/O:1) degree of virulence possess a capsule polysaccharide (K:2) composed of an O-acetylated diglycosyl phosphate repeat decorated with fructose: [→4)-3-O-Ac-ß-D-GlcpNAc-(1→3)-[ß-D-Fruf-(2→2)]-α-D-Galp-(1→PO(4)(-)→]. In addition, the serotype O:2 lipopolysaccharide was shown to express a sialylated O-chain [→3)-ß-D-Galp-(1→4)-[Neu5Ac-(2→3)-α-D-Galp-(1→6)]-ß-D-Glcp-(1→6)-ß-D-GlcpNAc-(1→]. As (1→6)-ß-D-glucan is ubiquitous in the environment, low levels of antibodies in the animals are predicted to prevent disease by K:1/O:1 strains. The greater potential associated with K:2/O:2 and K:2/O:1 strains is most likely due to the absence of (1→6)-ß-D-glucan as the K antigen and, in the case of K:2/O:2, the presence of sialic acid in the lipopolysaccharide, a nonulosonic acid known to promote evasion of host recognition.

Conserved interaction between transferrin and transferrin-binding proteins from porcine pathogens.

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.

Structural variations within the transferrin binding site on transferrin-binding protein B, TbpB.

Pathogenic bacteria acquire the essential element iron through specialized uptake pathways that are necessary in the iron-limiting environments of the host. Members of the Gram-negative Neisseriaceae and Pasteurellaceae families have adapted to acquire iron from the host iron binding glycoprotein, transferrin (Tf), through a receptor complex comprised of transferring-binding protein (Tbp) A and B. Because of the critical role they play in the host, these surface-exposed proteins are invariably present in clinical isolates and thus are considered prime vaccine targets. The specific interactions between TbpB and Tf are essential and ultimately might be exploited to create a broad-spectrum vaccine. In this study, we report the structure of TbpBs from two porcine pathogens, Actinobacillus pleuropneumoniae and suis. Paradoxically, despite a common Tf target, these swine related TbpBs show substantial sequence variation in their Tf-binding site. The TbpB structures, supported by docking simulations, surface plasmon resonance and hydrogen/deuterium exchange experiments with wild-type and mutant TbpBs, explain why there are structurally conserved elements within TbpB homologs despite major sequence variation that are required for binding Tf.

Common sialylated glycan in Actinobacillus suis.

A sialylated oligosaccharide was identified in four representative strains of the Gram-negative swine pathogen, Actinobacillus suis. As characterized, the glycan consists of a free oligosaccharide with a N-acetyl-lactosamine-like backbone decorated with sialic acid, phosphoethanolamine (PEA) and O-acetyl units: 9-O-Ac-Neu5Ac-(2-->6)-beta-d-Galp-(1-->4)-beta-d-6-O-Ac-GlcpNAc-(1-->3)-[PEA-->6]-beta-d-Galp-(1-->3)-beta-d-GlcpNAc-(1-->2)-[9-O-Ac-Neu5Ac-(2-->6)]-beta-d-Galp-(1-->4)-beta-d-6-O-Ac-GlcpNAc-(1-->3)-[PEA-->6]-beta-d-Galp-(1-->3)-d-GlcpNAc. The ubiquitous expression of this sialylated glycan suggests that this carbohydrate may play an important role in the survival of A. suis in the host.

Characterization of colonization-deficient mutants of Actinobacillus suis.

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P=0.02) and to PBMEC (P=0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.

Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds.

Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.

Enhanced resistance to bacterial infection in protegrin-1 transgenic mice.

Antibiotic-resistant bacteria have become a public health concern. It was suggested that one source of resistant pathogens may be food-producing animals. Alternative approaches are therefore needed to enhance the resistance of farm animals to bacterial infection. Protegrin-1 (PG-1) is a neutrophil-derived antimicrobial peptide that possesses activity against a wide range of bacteria and enveloped viruses. Here we report on the production of transgenic mice that ectopically expressed PG-1 and compare their susceptibilities to Actinobacillus suis infection with those of their wild-type (WT) littermates. Of the 126 mice that were challenged with A. suis, 87% of the transgenic mice survived, whereas 31% of their WT littermates survived. The PG-1 transgenic mice had significantly lower bacterial loads in their lungs and reduced numbers of pulmonary pathological lesions. The antimicrobial function of PG-1 was confirmed in vitro by using fibroblast cells isolated from the transgenic mice but not the WT mice. Moreover, differential blood cell counts in bronchoalveolar lavage fluid indicated greater number of neutrophils in PG-1 transgenic mice than in their WT littermates after bacterial challenge. Our data suggest that the ectopic expression of PG-1 in mice confers enhanced resistance to bacterial infection, laying the foundation for the development of livestock with improved resistance to infection.