Novel atypical Aeromonas salmonicida bath challenge model for juvenile ballan wrasse (Labrus bergylta, Ascanius).

Atypical Aeromonas salmonicida (aAs) is currently one of the most routinely recovered bacterial pathogens isolated during disease outbreaks in farmed cleaner fish, ballan wrasse (Labrus bergylta, Ascanius). Vibrionaceae family bacteria have also been isolated from ballan wrasse in Scotland. This study determined the infectivity, pathogenicity and virulence of aAs and Vibrionaceae isolates in juvenile farmed ballan wrasse (n = 50; approx. 2 g) using a bath challenge, and fish were monitored for a period of 16 days. Atypical As caused significant mortalities in contrast to Vibrionaceae isolates. Notably, differential virulence was observed between two aAs vapA type V strains at similar challenge doses. Diseased fish exhibited a systemic infection where aAs was detected in all analysed tissues (liver, spleen and kidney) by PCR and qPCR. Macroscopically, moribund and survivor fish exhibited hepatomegaly and splenomegaly. In moribund and surviving fish, histopathology showed granulomatous hepatitis with eosinophilic granular cells surrounding bacterial colonies and endocarditis along with splenic histiocytosis. This is the first report of a successful aAs bath challenge model for juvenile ballan wrasse which provides an important tool for future studies on vaccine efficacy and immunocompetence.

Genomic tailoring of autogenous poultry vaccines to reduce Campylobacter from farm to fork.

Campylobacter is a leading cause of food-borne gastroenteritis worldwide, linked to the consumption of contaminated poultry meat. Targeting this pathogen at source, vaccines for poultry can provide short-term caecal reductions in Campylobacter numbers in the chicken intestine. However, this approach is unlikely to reduce Campylobacter in the food chain or human incidence. This is likely as vaccines typically target only a subset of the high genomic strain diversity circulating among chicken flocks, and rapid evolution diminishes vaccine efficacy over time. To address this, we used a genomic approach to develop a whole-cell autogenous vaccine targeting isolates harbouring genes linked to survival outside of the host. We hyper-immunised a whole major UK breeder farm to passively target offspring colonisation using maternally-derived antibody. Monitoring progeny, broiler flocks revealed a near-complete shift in the post-vaccination Campylobacter population with an ~50% reduction in isolates harbouring extra-intestinal survival genes and a significant reduction of Campylobacter cells surviving on the surface of meat. Based on these findings, we developed a logistic regression model that predicted that vaccine efficacy could be extended to target 65% of a population of clinically relevant strains. Immuno-manipulation of poultry microbiomes towards less harmful commensal isolates by competitive exclusion, has major potential for reducing pathogens in the food production chain.

Delineating associations of progressive pleuroparenchymal fibroelastosis in patients with pulmonary fibrosis.

Background: Computer quantification of baseline computed tomography (CT) radiological pleuroparenchymal fibroelastosis (PPFE) associates with mortality in idiopathic pulmonary fibrosis (IPF). We examined mortality associations of longitudinal change in computer-quantified PPFE-like lesions in IPF and fibrotic hypersensitivity pneumonitis (FHP). Methods: Two CT scans 6-36 months apart were retrospectively examined in one IPF (n=414) and one FHP population (n=98). Annualised change in computerised upper-zone pleural surface area comprising radiological PPFE-like lesions (Δ-PPFE) was calculated. Δ-PPFE >1.25% defined progressive PPFE above scan noise. Mixed-effects models evaluated Δ-PPFE against change in visual CT interstitial lung disease (ILD) extent and annualised forced vital capacity (FVC) decline. Multivariable models were adjusted for age, sex, smoking history, baseline emphysema presence, antifibrotic use and diffusion capacity of the lung for carbon monoxide. Mortality analyses further adjusted for baseline presence of clinically important PPFE-like lesions and ILD change. Results: Δ-PPFE associated weakly with ILD and FVC change. 22-26% of IPF and FHP cohorts demonstrated progressive PPFE-like lesions which independently associated with mortality in the IPF cohort (hazard ratio 1.25, 95% CI 1.16-1.34, p<0.0001) and the FHP cohort (hazard ratio 1.16, 95% CI 1.00-1.35, p=0.045). Interpretation: Progression of PPFE-like lesions independently associates with mortality in IPF and FHP but does not associate strongly with measures of fibrosis progression.

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

First detection of infectious spleen and kidney necrosis virus (ISKNV) associated with massive mortalities in farmed tilapia in Africa.

In late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalization of chromatin and fine granulation. Transmission electron microscopy showed cells containing conspicuous virions with typical iridovirus morphology, that is enveloped, with icosahedral and/or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as infectious spleen and kidney necrosis virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow-up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR-positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.

Exercise Training Induces a Shift in Extracellular Redox Status with Alterations in the Pulmonary and Systemic Redox Landscape in Asthma.

Redox dysregulation and oxidative stress have been implicated in asthma pathogenesis. Exercise interventions improve symptoms and reduce inflammation in asthma patients, but the underlying mechanisms remain unclear. We hypothesized that a personalised exercise intervention would improve asthma control by reducing lung inflammation through modulation of local and systemic reactive species interactions, thereby increasing antioxidant capacity. We combined deep redox metabolomic profiling with clinical assessment in an exploratory cohort of six female patients with symptomatic asthma and studied their responses to a metabolically targeted exercise intervention over 12 weeks. Plasma antioxidant capacity and circulating nitrite levels increased following the intervention (p = 0.028) and lowered the ratio of reduced to oxidised glutathione (p = 0.029); this was accompanied by improvements in physical fitness (p = 0.046), symptoms scores (p = 0.020), quality of life (p = 0.046), lung function (p = 0.028), airway hyperreactivity (p = 0.043), and eosinophilic inflammation (p = 0.007). Increased physical fitness correlated with improved plasma antioxidant capacity (p = 0.019), peak oxygen uptake and nitrite changes (p = 0.005), the latter also associated with reductions in peripheral blood eosinophil counts (p = 0.038). Thus, increases in "redox resilience" may underpin the clinical benefits of exercise in asthma. An improved understanding of exercise-induced alterations in redox regulation offers opportunities for greater treatment personalisation and identification of new treatment targets.

Time taken from primary care referral to a specialist centre diagnosis of idiopathic pulmonary fibrosis: an opportunity to improve patient outcomes?

For patients with IPF, length of time in healthcare systems prior to review in an ILD clinic reflects disease severity and may impact upon patient outcome https://bit.ly/2TkO26r.

Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle.

Salmonella enterica serovar Dublin is a host-restricted serovar associated with typhoidal disease in cattle. In contrast, the fowl-associated serovar S. enterica serovar Gallinarum is avirulent in calves, yet it invades ileal mucosa and induces enteritis at levels comparable to those induced by S. enterica serovar Dublin. Suppression subtractive hybridization was employed to identify S. enterica serovar Dublin strain SD3246 genes absent from S. enterica serovar Gallinarum strain SG9. Forty-one S. enterica serovar Dublin fragments were cloned and sequenced. Among these, 24 mobile-element-associated genes were identified, and 12 clones exhibited similarity with sequences of known or predicted function in other serovars. Three S. enterica serovar Dublin-specific regions were homologous to regions from the genome of Enterobacter sp. strain 638. Sequencing of fragments adjacent to these three sequences revealed the presence of a 21-kb genomic island, designated S. enterica serovar Dublin island 1 (SDI-1). PCR analysis and Southern blotting showed that SDI-1 is highly conserved within S. enterica serovar Dublin isolates but rarely found in other serovars. To probe the role of genes identified by subtractive hybridization in vivo, 24 signature-tagged S. enterica serovar Dublin SD3246 mutants lacking loci not present in Salmonella serovar Gallinarum SG9 were created and screened by oral challenge of cattle. Though attenuation of tagged SG9 and SD3246 Salmonella pathogenicity island-1 (SPI-1) and SPI-2 mutant strains was detected, no obvious defects of these 24 mutants were detected. Subsequently, a DeltaSDI-1 mutant was found to exhibit weak but significant attenuation compared with the parent strain in coinfection of calves. SDI-1 mutation did not impair invasion, intramacrophage survival, or virulence in mice, implying that SDI-1 does not influence fitness per se and may act in a host-specific manner.

EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells.

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC.

Vaccination of calves with EspA, a key colonisation factor of Escherichia coli O157:H7, induces antigen-specific humoral responses but does not confer protection against intestinal colonisation.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are frequently associated with direct or indirect contact with ruminant faeces and may result in haemorrhagic colitis and severe renal and neurological sequelae. Broadly cross-protective vaccines for control of EHEC do not yet exist and the molecular mechanisms that influence bacterial persistence in the intestines of ruminants are incompletely understood. We sought to determine the role in colonisation and protective efficacy of EspA, which forms a filamentous extension of the locus of enterocyte effacement-encoded type III secretion system that injects EHEC proteins into enterocytes. A non-polar deletion of espA severely impaired the ability of E. coli O157:H7 to colonise the intestines of calves. Vaccination of calves with highly purified recombinant EspA induced high-titre antigen-specific IgG1 (also reactive to native EspA) and salivary IgA responses, however these responses did not protect calves against intestinal colonisation by E. coli O157:H7 upon experimental infection.

Functional analysis of lymphostatin homologues in enterohaemorrhagic Escherichia coli.

Enteropathogenic Escherichia coli contain a large chromosomal gene (lifA) that encodes lymphostatin, a predicted 365 kDa protein that inhibits the mitogen-activated proliferation of peripheral blood lymphocytes and lamina propria mononuclear cells and the synthesis of proinflammatory cytokines. Non-O157 serotypes of enterohaemorrhagic E. coli (EHEC) contain a highly homologous gene, designated efa1 (EHEC factor for adherence), which influences adherence to epithelial cells in vitro and intestinal colonization in calves. Serotype O157:H7 EHEC strains contain a truncated version of this gene (efa1') and a pO157-encoded homologue of lifA/efa1 (toxB). Here we report for the first time that efa1 inhibits mitogen-activated proliferation of bovine peripheral blood lymphocytes by EHEC O103:H2, but that E. coli K-12 strains expressing the N-terminal and central portions of the protein lack activity. While a Shiga toxin-negative E. coli O157:H7 strain was shown to possess lymphostatin-like activity, deletion of efa1' or toxB, singly or in combination, failed to significantly relieve the inhibitory effect.

Reduced amounts of LPS affect both stress tolerance and virulence of Salmonella enterica serovar Dublin.

Signature-tagged mutagenesis (STM) is a widely used technique for identification of virulence genes in bacterial pathogens. While this approach often generates a large number of mutants with a potential reduction in virulence a major task is subsequently to determine the mechanism by which the mutations influence virulence. Presently, we have characterised a Salmonella enterica serovar Dublin STM mutant that, in addition to having reduced virulence, was also impaired when growing under various stress conditions. The mutation mapped to the manC (rfbM) gene of the O-antigen gene cluster involved in O-antigen synthesis. The O-antigen is a component of the lipopolysaccharide (LPS) forming a unique constituent of the outer membrane of Gram-negative bacteria. While mutations in the O-antigen genes usually eliminate the entire O-antigen side chain we found that the transposon mutant produced intact O-antigen, however, the mutation reduced the amount of LPS.

Human intestinal tissue tropism of intimin epsilon O103 Escherichia coli.

Human intestinal in vitro organ culture was used to assess the tissue tropism of human isolates of Escherichia coli O103:H2 and O103:H- that express intimin epsilon. Both strains showed tropism for follicle associated epithelium and limited adhesion to other regions of the small and large intestine. This is similar to the tissue tropism shown by intimin gamma enterohaemorrhagic (EHEC) O157:H7, but distinct from that of intimin alpha enteropathogenic (EPEC) O127:H6.

Phenotypic and functional characterization of intraepithelial lymphocytes in a bovine ligated intestinal loop model of enterohaemorrhagic Escherichia coli infection.

Ruminants are a major reservoir of enterohaemorrhagic Escherichia coli (EHEC), which cause acute gastroenteritis in humans with potentially life-threatening sequelae. The mechanisms underlying EHEC persistence in ruminant hosts are poorly understood. EHEC produce several cytotoxins that inhibit the proliferation of bovine lymphocytes in vitro and influence EHEC persistence in calves, suggesting that bacterial suppression of mucosal inflammation may be important in vivo. In order to address this hypothesis, intraepithelial lymphocytes (IEL) obtained from ligated intestinal loops of five 9-14 day old calves were characterized 12 h after inoculation with E. coli strains. Loops were inoculated with an EHEC O103 : H2 strain, an isogenic Deltastx1 mutant incapable of producing Shiga toxin 1 (Stx1) and a porcine non-pathogenic E. coli strain. The IEL mainly comprised activated CD2(+) CD3(+) CD6(+) CD8alpha(+) T cells and resembled IEL obtained from the intestinal mucosa of orally challenged calves. Forty per cent of all IEL were potentially sensitive to Stx1 in that they expressed the receptor for Stx1. Nevertheless, analysis of IEL from inoculated loops failed to detect a significant effect of the different E. coli strains on proliferative capacity, natural killer cell activity or the cytokine mRNA profile. However, the EHEC wild-type strain reduced the percentage of CD8alpha(+) T cells in the ileal mucosa compared with loops inoculated with the Deltastx1 mutant. This shift in IEL composition was not associated with inhibition of IEL proliferation in situ, since the majority of the IEL from all loops were in the G(0)/G(1) phase of the cell cycle. These studies indicate that the ligated ileal loop model will be a useful tool to dissect the mechanisms underlying suppression of mucosal inflammation by EHEC in the reservoir host.

Net replication of Salmonella enterica serovars Typhimurium and Choleraesuis in porcine intestinal mucosa and nodes is associated with their differential virulence.

Salmonella enterica is a facultative intracellular pathogen of worldwide importance and causes a spectrum of diseases depending on serovar- and host-specific factors. Oral infection of pigs with S. enterica serovar Typhimurium strain 4/74 produces acute enteritis but is rarely fatal, whereas serovar Choleraesuis strain A50 causes systemic disease with a high mortality rate. With a porcine ligated ileal loop model, we observed that systemic virulence of serovar Choleraesuis A50 is not associated with enhanced intestinal invasion, secretory responses, or neutrophil recruitment compared to serovar Typhimurium 4/74. The net growth in vivo of serovar Choleraesuis A50 and serovar Typhimurium 4/74 was monitored following oral inoculation of pigs with strains harboring pHSG422, which exhibits temperature-sensitive replication. Analysis of plasmid partitioning revealed that the enteric virulence of serovar Typhimurium 4/74 relative to that of serovar Choleraesuis A50 is associated with rapid replication in the intestinal wall, whereas systemic virulence of serovar Choleraesuis A50 is associated with enhanced persistence in intestinal mesenteric lymph nodes. Faster replication of serovar Typhimurium, compared to that of serovar Choleraesuis, in the intestinal mucosa was associated with greater induction of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-8 (IL-8), and IL-18 as detected by reverse transcriptase PCR analysis of transcripts from infected mucosa. During replication in batch culture and porcine alveolar macrophages, transcription of genes encoding components of type III secretion systems 1 (sipC) and 2 (sseC) was observed to be significantly higher in serovar Typhimurium 4/74 than in serovar Choleraesuis A50, and this may contribute to the differences in epithelial invasion and intracellular proliferation. The rapid induction of proinflammatory responses by strain 4/74 may explain why pigs confine serovar Typhimurium infection to the intestines, whereas slow replication of serovar Choleraesuis may enable it to evade host innate immunity and thus disseminate by stealth.

Role in virulence and protective efficacy in pigs of Salmonella enterica serovar Typhimurium secreted components identified by signature-tagged mutagenesis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic enteric pathogen of worldwide importance and pigs are a significant reservoir of human infection. Signature-tagged transposon mutagenesis (STM) was used to identify genes required by S. Typhimurium to colonize porcine intestines. A library of 1045 signature-tagged mutants of S. Typhimurium ST4/74 Nal(R) was screened following oral inoculation of pigs in duplicate. A total of 119 attenuating mutations were identified in 95 different genes, many of which encode known or putative secreted or surface-anchored molecules. A large number of attenuating mutations were located within Salmonella pathogenicity islands (SPI)-1 and -2, confirming important roles for type III secretion systems (T3SS)-1 and -2 in intestinal colonization of pigs. Roles for genes encoded in other pathogenicity islands and islets, including the SPI-6-encoded Saf atypical fimbriae, were also identified. Given the role of secreted factors and the protection conferred against other pathogens by vaccination with extracellular and type III secreted proteins, the efficacy of a secreted protein vaccine from wild-type S. Typhimurium following intramuscular vaccination of pigs was evaluated. Serum IgG responses against type III secreted proteins were induced following vaccination and a significant reduction in faecal excretion of S. Typhimurium was observed in the acute phase of infection compared to mock-vaccinated animals. Vaccination with secreted proteins from an isogenic S. Typhimurium prgH mutant produced comparable levels of protection to vaccination with the preparation from the parent strain, indicating that protection was not reliant on T3SS-1 secreted proteins. The data provide valuable information for the control of Salmonella in pigs.

SiiE is secreted by the Salmonella enterica serovar Typhimurium pathogenicity island 4-encoded secretion system and contributes to intestinal colonization in cattle.

Here we report that Salmonella enterica serovar Typhimurium pathogenicity island 4 carries a type I secretion system (siiCDF) which secretes an approximately 600-kDa protein (encoded by siiE). SiiE is surface expressed, and its production is regulated by HilA. SiiE and SiiF influence colonization in cattle and the invasion of bovine enterocytes.

Systemic translocation of Salmonella enterica serovar Dublin in cattle occurs predominantly via efferent lymphatics in a cell-free niche and requires type III secretion system 1 (T3SS-1) but not T3SS-2.

Salmonella enterica is an important diarrheal pathogen, and infections may involve severe systemic sequelae depending on serovar- and host-specific factors. The molecular mechanisms underlying translocation of host-restricted and -specific serovars of S. enterica from the intestines to distal organs are ill defined. By surgical cannulation of lymph and blood vessels draining the distal ileum in cattle, S. enterica serovar Dublin was observed to translocate predominantly via mesenteric lymph nodes to efferent lymphatics in a manner that correlates with systemic virulence, since the fowl typhoid-associated serovar Gallinarum translocated at a significantly lower level. While both S. enterica serovars Dublin and Gallinarum were intracellular while in the intestinal mucosa and associated with major histocompatibility complex class II-positive cells, the bacteria were predominantly extracellular within efferent lymph. Screening of a library of signature-tagged serovar Dublin mutants following oral inoculation of calves defined the role of 36 virulence-associated loci in enteric and systemic phases of infection. The number and proportion of tagged clones reaching the liver and spleen early after oral infection were identical to the values in efferent lymph, implying that this may be a relevant mode of dissemination. Coinfection studies confirmed that lymphatic translocation requires the function of type III secretion system 1 (T3SS-1) but, remarkably, not T3SS-2. This is the first description of the mode and genetics of systemic translocation of serovar Dublin in its natural host.

Adhesins of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) was first recognized as a cause of human disease in 1983 and is associated with diarrhea and hemorrhagic colitis, which may be complicated by life-threatening renal and neurological sequelae. EHEC are defined by their ability to produce one or more Shiga-like toxins (Stx), which mediate the systemic complications of EHEC infections, and to induce characteristic attaching and effacing lesions on intestinal epithelia, a phenotype that depends on the locus of enterocyte effacement. Acquisition of Stx-encoding bacteriophages by enteropathogenic E. coli is believed to have contributed to the evolution of EHEC, and consequently some virulence factors are conserved in both pathotypes. A key requirement for E. coli to colonize the intestines and produce disease is the ability to adhere to epithelial cells lining the gastrointestinal tract. Here, we review knowledge of the adhesins produced by EHEC and other Stx-producing E. coli, with emphasis on genetic, structural, and mechanistic aspects and their contribution to pathogenesis.