A commercial autogenous injection vaccine protects ballan wrasse (Labrus bergylta, Ascanius) against Aeromonas salmonicida vapA type V.

Atypical Aeromonas salmonicida (aAs) and Vibrionaceae related species are bacteria routinely recovered from diseased ballan wrasse used as cleaner fish in the Atlantic salmon farming industry. Autogenous (i.e. farm specific inactivated) multivalent vaccines formulated from these microorganisms are widely used to protect farmed wrasse despite limited experimental proof that they are primary pathogens. In this study, the components of a commercial multivalent injection vaccine containing four strains of Aeromonas salmonicida and one strain of Vibrio splendidus previously isolated from ballan wrasse in Scotland, were tested for infectivity, pathogenicity and virulence via intra peritoneal injection at pre-deployment size (25-50 g) and the efficacy of the vaccine for protection against aAs assessed. Injection with 3.5 × 109, 8 × 109 1.8 × 109 and 5 × 109 cfu/fish of Vibrio splendidus, V. ichthyoenteri, Aliivibrio logeii and A. salmonicida, respectively, did not cause significant mortalities, lesions or clinical signs after a period of 14 days. IP injection with both aAs and Photobacterium indicum successfully reproduced the clinical signs and internal lesions observed during natural outbreaks of the disease. Differences in virulence (LD50 at day 8-post infection of 3.6 × 106 cfu/fish and 1.6 × 107 cfu/fish) were observed for two aAs vapA type V isolates. In addition, the LD50 for Photobacterium indicum was 2.2 × 107 cfu/fish. The autogenous vaccine was highly protective against the two aAs vapA type V isolates after 700-degree days of immunisation. The RPSFINAL values for the first isolate were 95 and 91% at 1 × 106 cfu/fish and 1 × 107 cfu/fish, respectively, and 79% at 1 × 107 cfu/fish for the second isolate tested. In addition, significantly higher anti aAs seral antibodies (IgM), were detected by ELISA in vaccinated fish in contrast with control (mock vaccinated) fish. These results suggest wrasse can be effectively immunised and protected against aAs infection by injection with oil adjuvanted vaccines prepared with inactivated homologous isolates.

Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes beta or gamma via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int(280)-beta) or EHEC O157:H7 (Int(280)-gamma) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1') from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1') or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int(280)-gamma and Efa-1' or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.

The fibronectin binding protein ShdA is not a prerequisite for long term faecal shedding of Salmonella typhimurium in pigs.

Porcine carcasses contaminated with Salmonella typhimurium pose significant public health problems. Prolonged faecal shedding of Salmonella in pigs contributes to the contamination level of carcasses. Although the mechanism of prolonged faecal shedding is not yet clarified, the CS54 Island, and more specifically the shdA gene encoding a fibronectin binding autotransporter protein, was identified as an important locus for intestinal colonization and persistence of Salmonella typhimurium in mice. The aim of this study was to assess the contribution of ShdA in faecal shedding of Salmonella typhimurium in pigs. Pigs were orally inoculated with a Salmonella typhimurium wild type field strain or its isogenic shdA mutant strain. For the first few days after inoculation, the shdA mutant strain was excreted more, the diarrhoea was more pronounced and higher numbers of internal organs were infected. No effect on long-term shedding was found. In a porcine ileal loop model, the wild type strain and shdA mutant strain did not show any differences in the induction of neutrophil influx into the intestinal wall and lumen. In conclusion, we have shown that a Salmonella typhimurium deletion mutant in shdA is more virulent during the first days after inoculation and is not significantly impaired in persistence or prolonged shedding in pigs.

Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants.

BACKGROUND: Salmonella enterica serovar typhimurium (S typhimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. AIMS: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). METHODS: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. RESULTS: S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.S typhimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading S typhimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. CONCLUSIONS: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis.

Vaccination against salmonella, enterohaemorrhagic E. coli and Campylobacter in food-producing animals.

The threat of zoonotic infection with food-poisoning enteric pathogens remains a major threat to public health throughout the world. Control of enteric pathogens within food-producing animals remains an obvious strategy to prevent the introduction of these pathogens into the human food chain. Vaccines are currently available for the control of Salmonella; however such vaccines vary in their efficacy and acceptability to the food production industries and consumers. Vaccines for the control of enterohaemorrhagic E. coli and Campylobacter are currently unavailable. Understanding the molecular basis of how these organisms colonise the intestines and cause disease is essential for the development of effective multivalent vaccines. The mechanisms by which these organisms colonise the gut will be reviewed. Bacterial Type Three Secretion Systems (TTSS) have been shown to be major virulence factors influencing the colonisation and pathogenicity of Salmonella and enterohaemorrhagic E. coli in some but not all animal species. Thus, understanding the specific nature of host/pathogen interactions is crucial in the development of cross-species vaccines. TTSSs act by delivering effector proteins into intestinal epithelial cells, which act to modify signalling events in host cells causing cytoskeletal and pathophysiological changes to the benefit of the pathogen. Disruption of TTSSs and/or related secreted effector proteins can be adopted as a strategy for the attenuation of live vaccine strains. Furthermore secreted effector proteins have the potential to be incorporated into sub-unit vaccines. As many of such effector proteins are conserved between different serotypes, such vaccines offer the hope of cross-serotype protective immune responses. Novel experimental vaccines are currently being developed to exploit these and other recent discoveries in bacterial virulence mechanisms. The potential of such vaccines to control zoonotic pathogens will be discussed.

Characterisation of a resource population of pigs screened for resistance to salmonellosis.

The degree of resistance to Salmonella choleraesuis infection in a reference family purposely bred to map resistance genes was assessed. Aspects of the innate and specific immune system were studied to find a parameter that might predict the resistance of pigs to salmonellosis. The family was bred from commercial full-sister pairs of F1-gilts and four boars. One boar (G398) was identified as breeding susceptible offspring, and one boar (G402) as breeding resistant offspring on the basis of pyrexial responses and numbers of Salmonella in liver and spleen post mortem. The other two boars were classified as 'possible resistant' (Y2008) and 'unknown' (Y6101) respectively. Functional differences in immune cells (neutrophils and lymphocytes) between the offspring of G398 and G402 were detected. The most resistant piglets had a higher number of circulating neutrophils and better polymorphonuclear neutrophils (PMNs) function, but a lower mitogenic response of lymphocytes both pre- and post-infection and a lower antibody response. Between the offspring groups of Y2008 and Y6101 no differences were found in the number of viable Salmonella in liver and spleen at post mortem or in immune cell function, however, the survival rate of these offspring groups was clearly different. Twenty three percent of the Y2008-offspring and 33% of the Y6101-offspring reached the predetermined humane clinical endpoint before the end of the experiment. Our findings suggest a role for several inherited immunological traits, including PMN function and lecithin-induced mitogenic proliferation, which appear to influence resistance to salmonellosis.

An improved method for preparing thick sections for immuno/histochemistry and confocal microscopy and its use to identify rare events.

Detection of rare events within solid tissues by immunocytochemistry is aided by imaging thick sections. Sections of 40--100 microm thickness of paraformaldehyde-fixed solid tissue can be prepared by use of a vibrating microtome and when immunolabelled these sections can be imaged in a confocal microscope. This approach provides excellent preservation of the structure of the sample and imposes minimal antigenic damage. In studies of the invasion of the bovine intestinal epithelium by Salmonella, this method has allowed detection of individual invading bacteria within large samples. The thick vibrating microtome sections were also used for the detection of rare apoptotic cell nuclei identified by TUNEL staining.

Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis.

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

Salmonella enterica serovar-host specificity does not correlate with the magnitude of intestinal invasion in sheep.

The colonization of intestinal and systemic tissues by Salmonella enterica serovars with different host specificities was determined 7 days after inoculation of 1 to 2-month-old lambs. Following oral inoculation, S. enterica serovars Abortusovis, Dublin, and Gallinarum were recovered in comparable numbers from the intestinal mucosa, but serovar Gallinarum was recovered in lower numbers than the other serovars from systemic sites. The pattern of bacterial recovery from systemic sites following intravenous inoculation was similar. The magnitude of intestinal invasion was evaluated in ovine ligated ileal loops in vivo. Serovars Dublin and Gallinarum and the broad-host-range Salmonella serovar Typhimurium were recovered in comparable numbers from ileal mucosa 3 h after loop inoculation, whereas the recovery of serovar Abortusovis was approximately 10-fold lower. Microscopic analysis of intestinal mucosae infected with serovars Typhimurium and Dublin showed dramatic morphological changes and infiltration of inflammatory cells, whereas mucosae infected with serovars Abortusovis and Gallinarum were indistinguishable from uninfected mucosae. Together these data suggest that Salmonella serovar specificity in sheep correlates with bacterial persistence at systemic sites. Intestinal invasion and avoidance of the host's intestinal inflammatory response may contribute to but do not determine the specificity of serovar Abortosovis for sheep. Intestinal invasion by serovar Abortusovis was significantly reduced after mutation of invH but was not reduced following curing of the virulence plasmid, suggesting that the Salmonella pathogenicity island 1 influences but the virulence plasmid genes do not influence the ability of serovar Abortusovis to invade the intestinal mucosa in sheep.

Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves.

We have used signature-tagged mutagenesis to identify mutants of the host-specific Salmonella enterica serotype Dublin which were avirulent in calves and/or BALB/c mice. A mutant with a transposon insertion in the sseD gene of Salmonella pathogenicity island 2 (SPI-2), which encodes a putative secreted effector protein, was identified. This mutant was recovered from the bovine host but not from the murine host following infection with a pool of serotype Dublin mutants. However, a pure inoculum of the sseD mutant was subsequently shown to be attenuated in calves following infection either by the intravenous route or by the oral route. The sseD mutant was fully invasive for bovine intestinal mucosa but was subsequently unable to proliferate to the same numbers as the parental strain in vivo. Both the sseD mutant and a second SPI-2 mutant, with a transposon insertion in the ssaT gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that SPI-2 is required by serotype Dublin for the induction of both systemic and enteric salmonellosis in calves.

Molecular basis of Salmonella-induced enteritis.

Salmonella pathogenesis is a complex and multifactorial phenomenon. Many genes required for full virulence in mice have been identified, but only a few of these have been shown to be necessary for the induction of enteritis. Likewise, at least some of the Salmonella virulence factors affecting enteritis do not appear to be required for infection of systemic sites in mice. This suggests that subsets of virulence genes influence distinct aspects of Salmonella pathogenesis. Recently, considerable progress has been made in characterizing the virulence mechanisms influencing enteritis caused by non-typhoid Salmonella spp. The Salmonella pathogenicity island-1-encoded type III secretion system mediates the translocation of secreted effector proteins into target epithelial cells. These effector proteins are key virulence factors required for Salmonella intestinal invasion and the induction of fluid secretion and inflammatory responses.

Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis.

Salmonella enterica serovars Typhimurium and Dublin lysed primary bovine alveolar macrophages and immortalized J774.2 macrophage-like cells in the absence of either the morphological changes or DNA fragmentation characteristic of apoptosis. Macrophage lysis was dependent on a subset of caspases and an intact sipB gene.

Mutation of waaN reduces Salmonella enterica serovar Typhimurium-induced enteritis and net secretion of type III secretion system 1-dependent proteins.

Mutation of waaN, a gene involved in lipid A biosynthesis, reduced enteropathogenic responses induced by Salmonella enterica serovar Typhimurium in bovine ligated ileal loops. However, the secretion of key virulence determinants was also reduced, and therefore the reduction in enteropathogenicity cannot be solely attributed to a reduction in biological activity of lipid A.

Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells.

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.

The Salmonella YopJ-homologue AvrA does not possess YopJ-like activity.

The YopJ protein of Yersinia pseudotuberculosis inhibits several eukaryotic signalling pathways that are normally activated in cells following their contact with bacteria. Salmonella encodes a protein, AvrA, that is secreted by the typeIII inv/spa secretion system which is clearly homologous to YopJ (56% identical, 87% similarity). Since AvrA and YopJs similarity also encompassed a region of YopJ that had previously been shown to be critical for its biological activity, we were interested whether AvrA and YopJ provoked similar responses in eukaryotic cells. Two different approaches were used to determine whether AvrA possesses YopJ-like activity in modulating cytokine expression or killing macrophages. An avrA strain of Salmonella dublin was constructed and its activity was compared to an isogenic wildtype counterpart in cellular response assays. In a complementary approach, AvrA was expressed in and delivered into eukaryotic cells by a yopJ strain of Yersinia pseudotuberculosis. We show here that AvrA affects neither cytokine expression or plays a role in macrophage killing when expressed by either Salmonella or Yersinia. Additionally, AvrA does not possess SopB/D-like activity in promoting fluid secretion into infected calf ileal loops. These data indicate that Salmonella and Yersinia trigger and/or modulate eukaryotic cell responses by different typeIII-secreted proteins and suggests that despite their close evolutionary relatedness, AvrA and YopJ perform different functions for Salmonella and Yersinia, respectively.

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis.

Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.

Differential regulation of enteric and systemic salmonellosis by slyA.

Mutation of slyA, which reduces Salmonella typhimurium virulence in mice, caused only minor attenuation of S. typhimurium virulence in orally inoculated calves. This correlated with modest reductions in intestinal invasion and enteropathogenic responses in bovine ligated ileal loops. slyA appears to regulate virulence genes involved in systemic, but not enteric, salmonellosis.

Salmonella SirA is a global regulator of genes mediating enteropathogenesis.

SirA of Salmonella typhimurium is known to regulate the hilA and prgH genes within Salmonella pathogenicity island 1 (SPI1). To identify more members of the SirA regulon, we screened 10,000 random lacZY fusions (chromosomal MudJ insertions) for regulation by SirA and identified 10 positively regulated fusions. Three fusions were within the SPI1 genes hilA (an SPI1 transcriptional regulator), spaS (a component of the SPI1 type III export apparatus) and sipB (a substrate of the SPI1 export apparatus). Two fusions were within the sopB gene (also known as sigD). sopB is located within SPI5, but encodes a protein that is exported via the SPI1 export apparatus. In addition, five fusions were within genes of unknown function that are located in SPI4. As spaS and sipB were likely to be hilA dependent, we tested all of the fusions (except hilA) for hilA dependence. Surprisingly, we found that all of the fusions require hilA for expression and that plasmid-encoded SirA cannot bypass this requirement. Therefore, SirA regulates hilA, the product of which regulates genes within SPI1, SPI4 and SPI5. Both sirA and hilA mutants are dramatically attenuated in a bovine model of gastroenteritis, but have little or no effect in the mouse model of typhoid fever. This study establishes the SirA/HilA regulatory cascade as the primary regulon controlling enteropathogenic virulence functions in S. typhimurium. Because S. typhimurium causes gastroenteritis in both cattle and humans, we believe that this information may be directly applicable to the human disease.