Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC.

Escherichia coli O78 strains are frequently associated with extraintestinal diseases, such as airsacculitis and septicemia, in poultry, livestock, and humans. To understand the influence of the pst operon in the virulence of E. coli, we introduced mutations into the pst genes of the avian pathogenic E. coli (APEC) O78:K80 strain 7122 by allelic exchange. The mutation of pst genes led to the constitutive expression of the Pho regulon. Furthermore, the virulence of APEC strain 7122 in a chicken infection model was attenuated by inactivation of the Pst system. The pst mutant caused significantly fewer extraintestinal lesions in infected chickens, and bacterial numbers isolated from different tissues after infection were significantly lower for the mutant than for the wild-type strain. Moreover, resistance to the bactericidal effects of rabbit serum and acid shock was impaired in the pst mutant, in contrast to the wild-type strain. In addition, the MIC of polymyxin was twofold lower for the mutant than for the wild-type strain. Although the pst mutant demonstrated an increased susceptibility to rabbit serum, this strain was not killed by chicken serum, suggesting the presence of differences in host innate immune defenses and complement-mediated killing. In APEC O78 strain 7122, a functional Pst system is required for full virulence and resistance to acid shock and polymyxin. Our results suggest that the mutation of pst genes induces a deregulation of phosphate sensing and changes in the cell surface composition that lead to decreased virulence, indicating the importance of the Pst system for the virulence of pathogenic E. coli strains from different hosts.

The production of a haemolysin by Streptococcus suis capsular type 2 was investigated. Human group 0 erythrocytes were the most susceptible, followed by horse, sheep, cow and pig red blood cells, which exhibited similar susceptibilities; rabbit erythrocytes were the least susceptible. The haemolysin was produced at the end of the exponential growth phase. The toxin described in this paper was purified by affinity chromatography using a thiopropylSepharose 6B column. It is an extracellular protein with a molecular mass of 65 kDa. The haemolysin belongs to the family of toxins known as antigenically related cholesterol-binding cytolytic toxins, since it shares common characteristics with other members of this family, such as sensitivity to oxygen and oxidizing agents, activation by reducing agents, inhibition by low concentrations of cholesterol, formation of transmembrane pores and a 'multihit' mechanism of action. In addition, anti-streptolysin antibodies inhibited the haemolytic activity caused by the S. suis haemolysin. Antibodies against the haemolysin could not be detected in pigs experimentally infected with a haemolytic positive strain of 5. suis capsular type 2. To our knowledge, this is the only Lancefield group D Streptococcus producing a haemolysin with these characteristics. The role of this haemolysin in the pathogenesis of 5. suis infections remains to be investigated.

Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectinbinding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis. INTRODUCTIONStreptococcus suis is a major swine pathogen that causes septicaemia, meningitis, endocarditis and arthritis (Higgins & Gottschalk, 2005). Of the 35 known serotypes, serotype 2 is the most frequently isolated and associated with disease (Higgins & Gottschalk, 2005). It has been proposed that two serotypes (serotypes 32 and 34) be excluded from S. suis and redesignated Streptococcus orisratti (Hill et al., 2005). S. suis, especially serotype 2, has also been described as an important zoonotic agent that affects people in close contact with infected pigs or pork-derived products (Lun et al., 2007). Indeed, an important number of cases of human disease with a high rate of mortality in China were linked directly to a concurrent outbreak of S. suis infection in pigs (Ye et al., 2006).Little is known about S. suis virulence factors. The capsule polysaccharide (CPS) is a critical virulence factor, given that unencapsulated isogenic mutants are completely avirulent and rapidly cleared from the circulation in pig and mouse infection models (Charland et al., 2000;Smith et al., 1999). However, non-virulent strains are also encapsulated, indicating that the virulence of this pathogen is a multifactorial process . Other potential virulence factors have also been described in S. suis, including a haemolysin (suilysin), a 136 kDa muramidase-released protein (MRP), a 110 kDa extracellular factor (EF) protein, a hyaluronidase, a superoxide dismutase, various proteases, a serum opacity factor and different adhesins (Baums et al., 2006;.The pathogenesis of S. suis infection is not fully understood and likely involves many steps . Binding between b...

Actinobacillus pleuropneumoniae is an important pig pathogen that is responsible for swine pleuropneumonia, a highly contagious respiratory infection. Knowledge of the importance, composition and structural determination of the major antigens involved in virulence provides crucial information that could lead to the development of a rationale for the production of specific serodiagnostic tools as well as vaccine development. Thus, efforts have been devoted to study mainly A. pleuropneumoniae virulence determinants with special emphasis on the Apx toxins (for A. pleuropneumoniae RTX toxins). In comparison, little attention has been given to the surface polysaccharides, which include capsular polysaccharides (CPS) and cell-wall lipopolysaccharides (LPS). Here, we review current knowledge on CPS and LPS of A. pleuropneumoniae used as diagnostic tools to monitor the infection and as immunogens for inclusion in vaccine preparations for animal protection.

A Streptococcus suis surface protein reacting with convalescent-phase sera from pigs clinically infected by S. suis type 2 was identified. The apparent 110-kDa protein, designated Sao, exhibits typical features of membraneanchored surface proteins of gram-positive bacteria, such as a signal sequence and an LPVTG membrane anchor motif. In spite of high identity with the partially sequenced genomes of S. suis Canadian strain 89/1591 and European strain P1/7, Sao does not share significant homology with other known sequences. However, a conserved avirulence domain that is often found in plant pathogens has been detected. Electron microscopy using an Sao-specific antiserum has confirmed the surface location of the Sao protein on S. suis. The Sao-specific antibody reacts with cell lysates of 28 of 33 S. suis serotypes and 25 of 26 serotype 2 isolates in immunoblots, suggesting its high conservation in S. suis species. The immunization of piglets with recombinant Sao elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.Streptococcus suis is an important swine pathogen that causes many pathological conditions, such as arthritis, endocarditis, meningitis, pneumonia, and septicemia (19,21). It is also an important zoonotic agent for humans in contact with colonized, otherwise healthy pigs or their by-products, causing meningitis and endocarditis (1, 53). Thirty-three serotypes (types 1 to 31, 33, and 1/2) based on capsular antigens are currently known (15-17, 22, 24, 43). Type 2 is considered the most virulent and prevalent type in diseased pigs. The mechanisms involved in the pathogenesis and virulence of S. suis are not completely understood (19), and attempts to control the infection are hampered by the lack of an effective vaccine.Several approaches have been used to develop vaccines for S. suis. However, little success was achieved because the protection was either serotype or strain dependent, and results in most instances were equivocal (23, 42). For example, some protection with killed whole cells or live avirulent vaccines was reported, but this required repeated immunization, and the protection against heterologous challenges was not evaluated (25, 56). Exposure of young pigs to live virulent strains showed a positive effect in reducing clinical signs characteristics of S. suis infection (52). Since the S. suis capsule plays an important role in virulence, attempts have been made to develop a vaccine based on capsular material. However, this vaccination approach was unsatisfactory because the capsular polysaccharide is poorly immunogenic (9). More recently, interest has shifted toward protein antigens of S. suis as vaccine candidates. Subunit vaccines using suilysin (27) or muramidase-released protein and extracellular protein factor (57) have been shown to protect pigs from homologous and heterologous serotype 2 strains, but their use is hindered by ...

Sao is a Streptococcus suis surface protein recently identified as a potential vaccine candidate. In this study, recombinant Sao in combination with Quil A provided cross-protection against S. suis serotype 2 disease in mouse and pig vaccination protocols. Subcutaneous immunization of mice elicited strong immunoglobulin G (IgG) antibody responses. All four IgG subclasses were induced, with the IgG2a titer being the highest, followed by those of IgG1, IgG2b, and IgG3. Challenge of the mice with S. suis strain 31533 resulted in a mortality rate of 80% for the control group, which received Quil A only. In contrast, all of the mice immunized with Sao survived. In a pig vaccination protocol, intramuscular immunization with Sao also elicited significant humoral antibody responses, and both the IgG1 and IgG2 subclasses were induced, with a predominance of IgG2 production. In vitro assay showed that Sao-induced antibodies significantly promoted the ability of porcine neutrophils in opsonophagocytic killing of S. suis. An aerosol challenge of the pigs with S. suis strain 166 resulted in clinical signs characteristic of S. suis infection in diseased pigs. The vaccine group showed significantly better survival, lower clinical scores, and less S. suis recovery from postmortem tissue samples than did the control group. Furthermore, this study also revealed that although challenge S. suis strains express Sao size variants, recombinant Sao conferred cross-protection. These data demonstrate that recombinant Sao formulated with Quil A triggers strong opsonizing antibody responses which confer efficient immunity against challenge infection with heterologous S. suis type 2.

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