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Made available in DSpace on 2021-06-25T11:11:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries. School of Agricultural and Veterinary Sciences Post Graduate Program in Veterinary Medicine and Agricultural and Livestock Microbiology Sao Paulo State University (FCAV/UNESP) School of Veterinary Medicine and Science University of Nottingham School of Agricultural and Veterinary Sciences Post Graduate Program in Veterinary Medicine and Agricultural and Livestock Microbiology Sao Paulo State University (FCAV/UNESP)

Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases.

Among the important recent observations involving anaerobic respiration was that an electron acceptor produced as a result of an inflammatory response to Salmonella Typhimurium generates a growth advantage over the competing microbiota in the lumen. In this regard, anaerobically, salmonellae can oxidize thiosulphate (S

We analysed intestinal tissues from groups of fast growing (Ross 308) broilers with natural or experimental coccidiosis, by genomic microarray. We identified genes that were differentially expressed (DE) in all groups and analysed expression of a panel of these, by qPCR, in Ross 308 and slow growing (Ranger classic) broilers, infected with 2500 or 7000 oocysts of Eimeria maxima for 6 or 13 days post-infection (dpi). Four genes (ADD3, MLLT10, NAV2 and PLXNA2) were upregulated (P

© 2020 Elsevier Inc. One promising strategy to combat antimicrobial resistance is to use bacteriophages that attach to the sex pili produced by transmissible antimicrobial resistance (AMR) plasmids, infect AMR bacteria and select for loss of the AMR plasmids, prolonging the life of existing antimicrobials. The maturation protein of the bacteriophage MS2 attaches to the pili produced by Incompatibility group F plasmid-containing bacteria. This interaction initiates delivery of the viral genetic material into the bacteria. Using protein-protein docking we constructed a model of the F pilus comprising a trimer of subunits binding to the maturation protein. Interactions between the maturation protein and the F pilus were investigated using molecular dynamics simulations. In silico alanine scanning and in silico single-point mutations were explored, with the longer term aim of increasing the affinity of the maturation protein to other Incompatibility group pili, without reducing the strength of binding to F pilin. We report our computational findings on which residues are required for the maturation protein and F pilin to interact, those which had no effect on the interaction and the mutations which led to a stronger interaction.

RESUMO Embora Salmonella Enteritidis (SE) seja capaz de metabolizar 1,2-propanodiol (1,2-Pd), utilizado como fonte de carbono e de energia ao longo de uma rota dependente de vitamina B12, a importância deste composto na infeção de Gallus gallus domesticus por SE permanece desconhecida. No presente estudo, foram construídos um mutante de SE sem os genes pduCDE, que codifica a propanodiol desidratase (Pdu), e outro contendo as deleções no pduCDE e também nos genes cobS e cbiA, responsáveis pela síntese de vitamina B12. Em seguida, avaliou-se a importância do metabolismo do 1,2-Pd em SE para colonização intestinal de infecção sistêmica de poedeiras comerciais. As estirpes mutantes de SE foram capazes de colonizar o intestino, de serem excretadas nas fezes e de invadir o baço e o fígado na mesma intensidade que a estirpe selvagem, o que sugere que os produtos dos genes pduC, pduD, pduE, cobS e cbiA não são essenciais durante infecção por Salmonella Enteritidis nessa espécie.

Salmonella enterica serovar Gallinarum (S. Gallinarum) is the cause of typhoid in chickens but the immune factors that may facilitate the development of typhoid have not been fully elucidated. We show that, in contrast to non-typhoid S. Enteritidis infection, S. Gallinarum significantly reduced nitrite ion production and expression of mRNA for heterophil granulocyte chemoattractants CXCLi2 and IL-6 in chicken monocyte-derived macrophages (chMDMs) (p &lt, 0.05) at 6 h post-infection (pi). S. Gallinarum also reduced IFN-&gamma, and IL-17 expression by CD4+ lymphocytes cultured with infected chMDMs for 5 days but did not induce a Th2 phenotype or anergy. In vivo, S. Gallinarum also induced significantly lower expression of CXCLi1, CXCLi2, IL-1&beta, IL-6 and iNOS mRNA in the caecal tonsil by day 2 pi (p &lt, 0.05&ndash, 0.01) and consistently lower levels of IFN-&gamma, IL-18, IL-12, and IL-17. In the spleen, S. Gallinarum induced significantly lower levels of iNOS and IFN-&gamma, (p &lt, 0.01 and 0.05 respectively) and consistently lower levels of IL-18 and IL-12 but significantly greater (p &lt, 0.01) expression of anti-inflammatory IL-10 at day 4 and 5 pi when compared to S. Enteritidis. This immune phenotype was associated with transit from the intestinal tissues to the liver by S. Gallinarum, not observed following S. Enteritidis infection. In conclusion, we report an immune mechanism that may facilitate typhoid disease in S. Gallinarum-infected chickens. However, down-regulation of inflammatory mediators, upregulation of IL-10, and associated liver colonisation are also characteristic of human typhoid, suggesting that this may also be a useful model of typhoid in humans.

Background Enteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively. Methods Salmonella-free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 (S. Enteritidis), Tϕ7 phage suspension (S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonellas was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S. Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h. Results The median levels of Salmonella reduction following phage treatment were 1.38 log10 MPN (Enteritidis) and 1.83 log10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer (p Salmonella recombinant strain, compared with buffer-treated controls (p Salmonella was occurring on the skin surface. Conclusions The results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observed was due to lysis occurred on the skin surface.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs., The O-antigen of LPS is known to limit the binding of antibody to bacterial surface antigens. Here the AUs show that the chemical and physical structure of the O-antigen are central factors in limiting the exposure of surface antigens to antibodies during Salmonella infection, thus defining their protective qualities.

New findings What is the central question of this study? How do gastric stretch and gastric cooling stimuli affect cardiac autonomic control? What is the main finding and its importance? Gastric stretch causes an increase in cardiac sympathetic activity. Stretch combined with cold stimulation result in an elimination of the sympathetic response to stretch and an increase in cardiac parasympathetic activity, in turn resulting in a reduction in heart rate. Gastric cold stimulation causes a shift in sympathovagal balance towards parasympathetic dominance. The cold-induced bradycardia has the potential to decrease cardiac workload, which might be significant in individuals with cardiovascular pathologies. Abstract Gastric distension increases blood pressure and heart rate in young, healthy humans, but little is known about the effect of gastric stretch combined with cooling. We used a randomized crossover study to assess the cardiovascular responses to drinking 300 ml of ispaghula husk solution at either 6 or 37°C in nine healthy humans (age 24.08 ± 9.36 years) to establish the effect of gastric stretch with and without cooling. The effect of consuming peppermint oil capsules to activate cold thermoreceptors was also investigated. The ECG, respiratory movements and continuous blood pressure were recorded during a 5 min baseline period, followed by a 115 min post-drink period, during which 5 min epochs of data were recorded. Cardiac autonomic activity was assessed using time and frequency domain analyses of respiratory sinus arrhythmia to quantify parasympathetic autonomic activity, and corrected QT (QTc) interval analysis to quantify sympathetic autonomic activity. Gastric stretch only caused a significant reduction in QTc interval lasting up to 15 min, with a concomitant but non-significant increase in heart rate, indicating an increased sympathetic cardiac tone. The additional effect of gastric cold stimulation was significantly to reduce heart rate for up to 15 min, elevate indicators of cardiac parasympathetic tone and eliminate the reduction in QTc interval seen with gastric stretch only. Stimulation of gastric cold thermoreceptors with menthol also caused a significant reduction in heart rate and concomitant increase in the root mean square of successive differences. These findings indicate that gastric cold stimulation causes a shift in the sympathovagal balance of cardiac control towards a more parasympathetic dominant pattern.

Production disease in pigs is caused by a variety of different pathogens, mainly enteric and respiratory and can result in significant economic loss. Other factors such as stress, poor husbandry and nutrition can also contribute to an animal's susceptibility to disease. Molecular biomarkers of production disease could be of immense value by improving diagnosis and risk analysis to determine best practice with an impact on increased economic output and animal welfare. In addition to the use of multiplex PCR or microarrays to detect individual or mixed pathogens during infection, these technologies can also be used to monitor the host response to infection via gene expression. The patterns of gene expression associated with cellular damage or initiation of the early immune response may indicate the type of pathology and, by extension the types of pathogen involved. Molecular methods can therefore be used to monitor both the presence of a pathogen and the host response to it during production disease. The field of biomarker discovery and implementation is expanding as technologies such as microarrays and next generation sequencing become more common. Whilst a large number of studies have been carried out in human medicine, further work is needed to identify molecular biomarkers in veterinary medicine and in particular those associated with production disease in the pig industry. The pig transcriptome is highly complex and still not fully understood. Further gene expression studies are needed to identify molecular biomarkers which may have predictive value in identifying the environmental, nutritional and other risk factors which are associated with production diseases in pigs.

Coccidiosis in broiler chickens, caused by infection with Eimeria spp. remains one of the most economically important production diseases. Development of a genetic biomarker panel of sub-clinical infection would be an important biological tool for the management of broiler flocks. We analysed expression of MicroRNAs (miRNAs) to determine the potential for these in diagnosing coccidiosis in broiler flocks. miRNA expression, in the ilea of Ross 308 broilers, was compared between chickens naturally clinically or sub-clinically infected with Eimeria maxima and Eimeria acervulina using NextSeq 500 sequencing. 50 miRNAs with greatest coefficient of variance were determined and principal component analysis showed that these miRNAs clustered within the clinical and sub-clinical groups much more closely than uninfected controls. Following false detection rate analysis and quantitative PCR we validated 3 miRNAs; Gallus gallus (gga)-miR-122-5p, gga-miR-205b and gga-miR-144-3p, which may be used to diagnose sub-clinical coccidiosis.

Aims:Beta2-Microglobulin (B2M) forms part of the HLA class I complex and plays a role in metastatic biology. B2M mutations occur frequently in mismatch repair-deficient colorectal cancer (dMMR CRC) with limited data suggesting they may protect against recurrence. Our experimental study tested this hypothesis by investigating B2M mutation status and B2M protein expression and recurrence in patients in the stage II QUASAR clinical trial. Methods:Sanger sequencing was performed for the three coding exons of B2M on 121 dMMR and a subsample of 108 pMMR tumours; 52 with recurrence and 56 without. B2M protein expression was assessed by immunohistochemistry. Mutation status and protein expression were correlated with recurrence and compared to proficient mismatch repair (pMMR) CRCs. Results:Of 121 dMMR CRCs, deleterious B2M mutations were detected in 39 (32%). Five tumours contained missense B2M-variants of unknown significance, and were thus excluded from further analyses, leaving 116 dMMR tumours. With median follow-up 7.4 years, none of the 39 B2M-mutant tumours recurred, compared with 14/77 (18%) B2M-wildtype tumours (p=0.005); six at local and eight at distant sites. Sensitivity and specificity of IHC in detecting B2M mutations was 87% and 71% respectively. Significantly (p

1. There is huge emphasis in veterinary and agricultural science in understanding the basics of processes and exploiting them for benefits to the economy and human and animal welfare. It is always valuable to be able to step back from existing or favourite hypotheses and paradigms to look at an area of work or problem and see whether a different approach might be productive particularly by drawing parallels with other sometimes unrelated problems. 2. This approach has been used to explore (i) the use of live, attenuated Salmonella vaccines to generate a new form of competitive exclusion, (ii) gene expression technology for the design of improved inactivated vaccines (iii) use of cytokine therapy to reduce persistent carriage by Salmonella, (iv) using bacteriophages to reduce carcass contamination by food-borne pathogens and reduce carriage of antibiotic resistance plasmids. 3. The potential for extending virus therapy to parasite infections is also discussed.

Immune evasion is a critical survival mechanism for bacterial colonization of deeper tissues and may lead to life-threatening conditions such as endotoxaemia and sepsis. Understanding these immune evasion pathways would be an important step for the development of novel anti-microbial therapeutics. Here, we report a hitherto unknown mechanism by which Salmonella exploits an anti-inflammatory pathway in human immune cells to obtain survival advantage. We show that Salmonella enterica serovar Typhimurium strain 4/74 significantly (P < 0·05) increased expression of mRNA and surface protein of the type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in human monocytes. However, we also show that S. Typhimurium induced retrograde recycling of VPAC1 from early endosomes to Rab11a-containing sorting endosomes, associated with the Golgi apparatus, and anterograde trafficking via Rab3a and calmodulin 1. Expression of Rab3a and calmodulin 1 were significantly increased by S. Typhimurium infection and W-7 (calmodulin antagonist) decreased VPAC1 expression on the cell membrane while CALP-1 (calmodulin agonist) increased VPAC1 expression (P < 0·05). When infected monocytes were co-cultured with VIP, a significantly higher number of S. Typhimurium were recovered from these monocytes, compared with S. Typhimurium recovered from monocytes cultured only in cell media. We conclude that S. Typhimurium infection exploits host VPAC1/VIP to gain survival advantage in human monocytes.

Very little has been reported comparing resistance to coccidiosis in fast or slow growing broilers, the latter of which are becoming more prevalent in the broiler industry. We examined mRNA expression in the intestines of fast and slow growing broilers following Eimeria infection. We show that by day 13 post-infection (d pi) with 2500 or 7000 oocysts of Eimeria maxima, slower-growing (Ranger Classic) broilers significantly (P < 0.01) upregulated expression of proinflammatory cyclooxygenase genes (LTB4DH, PTSG1 and PTSG2) above that detected in fast growing (Ross 308) broilers. Expression of CD8α mRNA was downregulated in Ross 308 at day 6d pi with either 2500 or 7000 oocysts of E maxima (P < 0.05), compared to uninfected controls, but was not differentially expressed in Ranger Classic. CD4 genes were not differentially expressed in either chicken line infected with either infectious oocyst dose at d6 pi, compared to uninfected controls. However, at d13 pi, CD4 expression was significantly upregulated in both chicken lines infected with either infectious oocyst dose, compared to uninfected controls (P < 0.05) but this was significantly greater in Ranger Classic broilers compared to Ross 308 (P < 0.05). At d13 pi, expression of CD3 chains (required for T lymphocyte activation) was significantly increased in Ranger Classic compared to Ross 308, infected with either oocyst dose (P < 0.05-0.01). Expression of IL-2 and IL-15 mRNA, required for T lymphocyte proliferation was also significantly upregulated, or maintained longer, in Ranger Classic broilers compared to Ross 308. These differences in immune response to E maxima corresponded with a reduction in E maxima genome detected in the intestines of Ranger Classic compared to Ross 308.

In addition to evaluating the efficacy potential of a combined use of vaccination and competitive exclusion (CE) against Salmonella exposure in chicks at 3-days of age, a live Salmonella Enteritidis vaccine (SE-LV) and a CE culture were tested for their ability to induce parameters of the innate immunity. Whereas the invasive SE-LV induced an influx of granulocytes and macrophages as well as an increased transcription of several cytokines in the caecal mucosa, the CE culture did not demonstrate any differences in these parameters compared to controls. It is therefore highly probable that the effects observed with CE cultures are not due to the rapid stimulation of the immune system. The combined use of both preparations did not result in an additive intestinal exclusion effect of the challenge strain more pronounced than that after single administration of the CE culture. The combined use of the Salmonella live vaccine and the CE culture resulted in an additive protective effect and prevented completely the systemic dissemination of the Salmonella challenge strain. To exploit the potential of combined use of CE and vaccination further and most effectively, live Salmonella vaccines are needed that are despite their attenuation in virulence still capable to induce both intestinal colonisation- and invasion-inhibition effects against Salmonella exposure.

Tuberculosis (TB) remains a major cause of mortality and morbidity in the worldwide. The endoplasmic-reticulum stress (ERS) response constitutes a cellular process that is triggered by mycobacterial infection that disturbs the folding of proteins in the endoplasmic reticulum (ER). The unfolded protein response (UPR) is induced to suspend the synthesis of early proteins and reduce the accumulation of unfolded- or misfolded proteins in the ER restoring normal physiological cell function. Prolonged or uncontrolled ERS leads to the activation of three signaling pathways (IRE1, PERK and ATF6) which directs the cell towards apoptosis. The absence of this process facilitates spread of the mycobacteria within the body. We summarize here recent advances in understanding the signaling pathway diversity governing ERS in relation to TB.

© 2019 Elsevier Ltd Studies have shown that administration of vasoactive intestinal peptide (VIP) in mice rescues them from lethal endotoxaemia and that this is correlated with decreased concentration of inflammatory cytokines. VIP has, therefore, been proposed as a novel anti-inflammatory which could be used in the treatment of Gram negative sepsis. However, the effect of VIP has not been reported in mice infected with viable Gram negative bacteria. Here, we show that Salmonella enterica serovar Typhimurium 4/74 significantly increased expression of mRNA of a type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in murine ileum and mesenteric lymph nodes at day 6 post-infection (d6 pi) and in the spleen at d3 pi. When VIP (5 nmol/ml) was administered to S. Typhimurium-infected mice, there was a significant increase in the number of S. Typhimurium cultured from murine faeces and ileum at d3 and 6 pi and in MLN and spleen at d3 dpi, compared to faeces and tissues examined from mice infected with S. Typhimurium (without VIP administration). Administration of VIP to S. Typhimurium-infected mice also altered the splenic architecture, resulting in a lack of discernable periarterial lymphoid sheaths or marginal zones at d6 pi but liver histology appeared similar on both d3 and d6 pi. The effects of VIP administration were correlated with a significant decrease in expression of inflammatory cytokine mRNA, associated with systemic inflammatory response syndrome (SIRS) of bacteraemia and acute sepsis. We conclude that VIP inhibits expression of diagnostic/prognostic cytokine biomarkers of sepsis in S. Typhimurium-infected mice. However, this occurred with a concomitant increase in Salmonella growth in tissues and increased bacterial shedding in faeces. Thus, VIP may have potential as an adjunctive therapy to antibiotics in sepsis.

We studied the effects of two Salmonella enterica serovar Typhimurium (host-adapted) strains (14028 and 4/74) and three S. Choleraesuis (non-host-adapted) strains (A50, A45, and B195) in human monocytes between 2 and 24 h postinfection (p.i.) to investigate whether differences in immune response may explain the much higher prevalence of sepsis in individuals infected with S. Choleraesuis. Both serovars significantly increased the production of cytokines associated with acute sepsis (tumor necrosis factor alpha [TNF-α], interleukin β [IL-β], and IL-6), but temporal differences occurred between these serovars and between different S. Choleraesuis strains. Generally, all S. Choleraesuis strains induced significantly higher production of inflammatory cytokines than S. Typhimurium strains (P < 0.01 to 0.05). All S. Choleraesuis strains very significantly increased IL-10 production by monocytes at 6 and 24 h p.i. in comparison to S. Typhimurium strains (P < 0.01). In addition, ∼80% of monocytes were viable at 24 h p.i. with S. Choleraesuis A50, compared to only ∼40% following S. Typhimurium infection. Using S. Typhimurium 14028 and S. Choleraesuis A50 as examples of these two serovars, we also showed differential expression of genes within the Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) (JAK/STAT) pathway via quantitative PCR (qPCR) microarray analysis. High serum IL-10 concentration and monocyte survival have been reported as markers of the development of human sepsis. We therefore conclude that high production of IL-10 by monocytes may, in part, explain the greater propensity for S. Choleraesuis to induce human sepsis and that this may be greater in strains such as A50, which induces both high IL-10 production and monocyte survival.

To persist in the host, Salmonella is known to facultatively parasitize cells to escape the immune response. Intracellular Salmonella enterica can replicate using effector proteins translocated across the Salmonella-containing vacuolar membrane via a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). One of these factors, Salmonella secreted factor L (SseL), is a deubiquitinase that contributes to the virulence of Salmonella Typhimurium in mice by inhibiting the cellular NF-κB inflammatory pathway. However, the nature of its effect on the NF-κB pathway is controversial, and little research has been performed in other animal models. In this study, the SseL of Salmonella Pullorum was studied, and chickens were used as an infection model. An sseL gene deletion strain, a complementation strain and a eukaryotic expression plasmid were used to clarify the means by which SseL regulates Salmonella virulence and the cellular inflammatory response. SseL significantly enhanced the virulence of Salmonella Pullorum in chickens and suppressed activation of the cellular NF-κB pathway, thus inhibiting cellular inflammatory cytokine expression.

Made available in DSpace on 2018-12-11T16:52:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-01. Added 1 bitstream(s) on 2021-07-15T15:21:28Z : No. of bitstreams: 1 S1517-83822018000300601.pdf: 692251 bytes, checksum: b1b3eaf5bece488e7ed9ca1ca902e1c6 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Salmonella Gallinarum is a host-restrict pathogen that causes fowl typhoid, a severe systemic disease that is one of the major concerns to the poultry industry worldwide. When infecting the bird, SG makes use of evasion mechanisms to survive and to replicate within macrophages. In this context, phoPQ genes encode a two-component regulatory system (PhoPQ) that regulates virulence genes responsible for adaptation of Salmonella spp. to antimicrobial factors such as low pH, antimicrobial peptides and deprivation of bivalent cations. The role of the mentioned genes to SG remains to be investigated. In the present study a phoPQ-depleted SG strain (SG ΔphoPQ) was constructed and its virulence assessed in twenty-day-old laying hens susceptible to fowl typhoid. SG ΔphoPQ did cause neither clinical signs nor mortality in birds orally challenged, being non-pathogenic. Furthermore, this strain was not recovered from livers or spleens. On the other hand, chickens challenged subcutaneously with the mutant strain had discreet to moderate pathological changes and also low bacterial counts in liver and spleen tissues. These findings show that SG ΔphoPQ is attenuated to susceptible chickens and suggest that these genes are important during chicken infection by SG. São Paulo State University (Unesp) School of Agricultural and Veterinary Sciences Post Graduate Program in Agricultural and Livestock Microbiology Federal University of Paraiba Department of Veterinary Sciences University of Nottingham School of Veterinary Medicine and Science, Sutton Bonington São Paulo State University (Unesp) School of Agricultural and Veterinary Sciences Post Graduate Program in Agricultural and Livestock Microbiology FAPESP: 2016/10369-0

Salmonella enterica infection affects a wide range of animals and humans, and a small number of serovars cause typhoid-like infections, one characteristic of which is persistent infection in convalescents. Avian-specific S. enterica serovar Pullorum produces systemic disease in young chickens, which is followed by a carrier state in convalescent birds, leading to infection of the ovary at sexual maturity and vertical transmission. However, the immunological basis of persistent infection remains unclear. S. enterica serovar Enteritidis is taxonomically closely related but does not show this characteristic. Differences in the immune responses between S Pullorum and S Enteritidis were compared by using Salmonella-infected chicken monocyte-derived macrophages (chMDMs) and CD4+ T lymphocytes that had been cocultured with infected chMDMs or chicken splenocytes in vitro and also in 2-day-old chickens in vivo In comparison with S Enteritidis, S Pullorum-infected chMDMs showed reduced mRNA expression levels of interleukin-12α (IL-12α) and IL-18 and stimulated the proliferation of Th2 lymphocytes, with reduced expression of gamma interferon (IFN-γ) and IL-17 and increased expression levels of IL-4 and IL-13 There was little evidence of clonal anergy or immune suppression induced by S Pullorum in vitro. S Pullorum also increased the levels of expression of IL-4 and decreased the levels of IFN-γ in the spleen and cecal tonsil of infected birds. This suggests that S Pullorum is able to modulate host immunity from a dominant IFN-γ-producing Th17 response toward a Th2 response, which may promote persistent infection in chickens. S Pullorum in chickens is presented as a good model of the typhoid group to study persistent infection.

Made available in DSpace on 2019-10-06T15:55:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Biotechnology and Biological Sciences Research Council Antimicrobial resistance (AMR) is now a major global problem largely resulting from the overuse of antibiotics in humans and livestock. In some AMR bacteria, resistance is encoded by conjugative plasmids expressing sex-pili that can readily spread resistance through bacterial populations. The aim of this study was to use sex pilus-specific (SPS) phage to reduce the carriage of AMR plasmids. Here, we demonstrate that SPS phage can kill AMR Escherichia coli and select for AMR plasmid loss in vitro. For the first time, we also demonstrate that SPS phage can both prevent the spread of AMR Salmonella Enteritidis infection in chickens and shift the bacterial population towards antibiotic sensitivity. School of Veterinary Medicine and Science University of Nottingham School of Agricultural and Veterinary Sciences São Paulo State University (FCAV/UNESP) School of Agricultural and Veterinary Sciences São Paulo State University (FCAV/UNESP) Biotechnology and Biological Sciences Research Council: BB/M028399/1

Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log(10) colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.

Murine/LPS models of Gram negative sepsis indicate that vasoactive intestinal peptide (VIP) has therapeutic potential. We investigated the unknown effect of VIP on JAK/STAT proteins and genes in human monocytes infected with Salmonella Typhimurium 14028. S. Typhimurium 14028 increased expression of both IL-6 receptor (IL-6R) and interferon gamma receptor 1 (IFNγR1) on monocytes but co-culture of infected monocytes with VIP (10-7 M) only decreased expression of IFNγR1 (P < 0.05). In contrast, S. Typhimurium 14028 infection or co-culture with VIP had no effect on IL-10 receptor expression on the monocyte surface. However, S. Typhimurium 14028 down regulated IFNGR1 gene expression and this was not altered by co-culture with VIP, suggesting that changes in IFNγR1 protein may be due to an effect on cytoplasmic transport. 15 JAK/STAT genes, out of 84 studied, were up-regulated by S. Typhimurium 14028 infection and five were down-regulated. Co-culture with VIP significantly decreased expression of two genes (IFNG and IL-20) and increased expression of three genes (SOCS1, SOCS3 and STAT4) (P < 0.05). S. Typhimurium 14028 also increased expression of PTPN1, which dephosphorylates JAK2 and TYK2. This was unaltered by co-culture with VIP but S. Typhimurium 14028-induced expression of ISG15, associated with susceptibility to Gram negative infection, was further increased by VIP. We conclude that the effect of VIP on JAK/STAT genes may preclude its therapeutic use in human Gram negative sepsis.

Made available in DSpace on 2018-11-26T17:42:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01. Added 1 bitstream(s) on 2019-10-09T18:27:05Z : No. of bitstreams: 1 S1517-83822017000400754.pdf: 1519580 bytes, checksum: 67cd3c5a0368af87b475ca6002ce88ae (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE Delta motB) or non-flagellated (SE Delta fliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE Delta motB and SE Delta fliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE Delta motB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE Delta fliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken. (C) 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Patol Vet, Jaboticabal, SP, Brazil Univ Fed Paraiba, Dept Ciencias Vet, Areia, Paraiba, Brazil Univ Nottingham, Sch Vet Med & Sci, Sutton Bonington Campus, Nottingham, Leics, England Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Patol Vet, Jaboticabal, SP, Brazil FAPESP: 2014/02014-1 FAPESP: 2013/26127-7

Domestic ducks remain a major source of zoonotic Salmonella enterica infections for man worldwide and approaches to protection should include vaccine-mediated immunity. With this in mind we developed several genetically defined mutants in a virulent duck Salmonella typhimurium isolate TT-1. From initial tests for virulence in day-old ducks, ΔrpoS, ΔhilA, and ΔslyA mutants retained some virulence so were not studied further. Amongst the mutants showing greater attenuation, ΔssrB, ΔphoPQ, ΔompR, and ΔclpP also showed high levels of protection when 1-day-old ducks, which were vaccinated orally, were challenged 1 week later demonstrating the capacity to protect ducks in the first few weeks of life when they are most susceptible and when the risk of infection is greatest. Immunized ducks triggered Omp-specific IgG, IgM, and IgA responses and raised IL-2 and IFN-γ levels in the serum coupled with IL-4 suppression.

We compared the effect of VIP on human blood monocytes infected with Salmonella typhimurium 4/74 or stimulated with LPS. VIP (10−7 M) increased monocyte viability by 24% and 9% when cultured for 24 h with 4/74 or Salmonella LPS (100 ng/ml), respectively. Significantly increased (P < 0.05) numbers of 4/74 were also recovered from monocytes co-cultured with VIP after 6 h post-infection (pi) and this remained high after 24 h pi. Both 4/74 and LPS increased (P < 0.05) the concentration of TNF-α, IL-1β and IL-6 measured in monocyte supernatants. However, LPS induced this effect more rapidly while, with the exception of IL-6, 4/74 induced higher concentrations (P < 0.05). VIP significantly decreased (P < 0.05) TNF-α and IL-1β production by 4/74-infected monocytes after 6 pi, but only after 24 h in LPS-cultured monocytes. This trend was reversed for IL-6 production. However, TNF-α and IL-1β production by 4/74-infected monocytes, cultured with VIP, still remained higher (P < 0.05) than concentrations measured in supernatants cultured only with LPS. VIP also increased (P < 0.05) production of anti-inflammatory IL-10 in both 4/74 and LPS cultures after 24 h. We also show a differential effect of VIP on the expression of TNFα and IL-6 receptors, since VIP was only able to decreased expression in LPS-stimulated monocytes but not in 4/74-infected monocytes. In conclusion, we show a differential effect of VIP on human monocytes infected with virulent Salmonella or stimulated with LPS. Our study suggests that the use of VIP in bacteraemia and/or sepsis may be limited to an adjunctive therapy to antibiotic treatment.

We show that IL-36α induced maturation of human MDDCs and stimulated differentiation of IFN-γ producing (Type 1) CD3+ lymphocytes but was not as effective as IL-36β in doing so. For the first time, we also show that IL-36α induced expression of CD14 by MDDCs and this was highly potentiated by co-cultured with IFN-γ. In contrast, lipopolysaccharide (LPS) did not increase CD14 expression by MDDCs, suggesting that if MDDCs represent a physiologically relevant population in vivo, they need to be stimulated by relevant inflammatory cytokines prior to CD14 expression and detection of LPS, expressed by Gram negative bacteria. IFN-γ synergised with IL-36α to restore the high levels of CD11c expression by MDDCs, which was reduced by culture with these cytokines in isolation. IL-36α/IFN-γ synergy also correlated with increased binding of the opsonic complement protein (iC3b) to MDDCs. However although IL-36α increased the phagocytic capacity of MDDCs for Salmonella Typhimurium 4/74 this was not synergistically increased by IFN-γ (P > 0.05). In conclusion we report the hitherto unknown effects of IL-36α on the innate cell function of human MDDCs.

Lynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome with a 60-80% lifetime risk of colorectal cancer. We assessed the uptake of predictive testing and colorectal screening among first-degree relatives (FDRs) in LS families and explored novel methods for informing and engaging at-risk relatives. Uptake of predictive testing was explored using Kaplan-Meier analysis and engagement with colorectal screening was ascertained. A questionnaire was distributed to 216 general practitioners (GPs) of registered LS family members to determine their prior experience and opinion of an enhanced role. Of 591, 329 (55.7%) FDRs had undergone predictive testing. Uptake was significantly lower in males (p = 0.012) and individuals

The complete genome sequences of seven closely related Vibrio cholerae phages isolated from environmental sites in southeastern China are reported here. Phages QH, CJY, H1, H2, H3, J2, and J3 are members of the Podoviridae family and are highly similar to the previously sequenced Vibrio phages VP2, VP5, and phiVC8.

The complete genomes of two Vibrio cholerae bacteriophages of potential interest for cholera bacteriophage (phage) therapy were sequenced and annotated. The genome size of phage 24 is 44,395 bp encoding 71 putative proteins, and that of phage X29 is 41,569 bp encoding 68 putative proteins.

A pediatric assessment is now a required component of every drug marketing application in North America, Europe, and Japan, unless a waiver has been granted previously. Nonclinical juvenile toxicity studies are often required as part of this assessment. The protocols for juvenile toxicity studies are best devised in consultation with the regulatory authorities. It is important to submit the pediatric investigation plan (PIP) or pediatric study plan (PSP) early, in order not to delay the marketing authorization of the drug in adults. The choice of species and the design of juvenile toxicity studies are based on a series of complex considerations, including the therapeutic use of the drug, age at which children will be treated, duration of treatment, and potential age- or species-specific differences in efficacy, pharmacokinetics, or toxicity.

Made available in DSpace on 2018-12-11T17:11:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-04 Salmonella Gallinarum (SG) causes fowl typhoid (FT), a disease responsible for economic losses to the poultry industry worldwide. FT has been considered to be under control in Brazil; nevertheless, since 2012 it has frequently been identified in poultry farming of several Brazilian states. The present study was aimed at assessing (i) the pathogenicity of a SG strain recently isolated from an FT outbreak affecting chickens of both white and brown layers; (ii) the transmission of SG through eggs and hatching; (iii) the effects of antibiotic therapy on SG persistence in poultry tissues and on its vertical transmission and (iv) the genetic profiles of strains isolated over 27 years by Pulsed Field Gel Electrophoresis. Clinical signs, mortality and gross pathologies were very marked amongst brown-egg layers. In contrast, clinical manifestation of FT and mortality were barely present amongst the white-egg layers, although bacteria could be re-isolated from their tissues up to 35 days after infection. No bacteria were re-isolated from the laid eggs, so vertical transmission was not achieved, although newly hatched uninfected chicks became infected spontaneously after hatching. Antibiotic therapy was shown to be effective at reducing mortality, but was not able to clear infection or to favour SG transmission via eggs. Our pulsed field gel electrophoresis results revealed an endemic SG clone that may have been circulating in the Brazilian poultry flocks in the south and southeast regions for more than 20 years. The results suggest that the industrial incubation of SG-contaminated eggs could be one of the factors responsible for the spread of FT in Brazil. Department of Veterinary Pathology from the School of Agricultural and Veterinarian Sciences Universidade Estadual Paulista (FCAV/Unesp) School of Veterinary Medicine and Science The University of Nottingham Department of Veterinary Sciences Federal University of Paraiba Department of Veterinary Pathology from the School of Agricultural and Veterinarian Sciences Universidade Estadual Paulista (FCAV/Unesp)

The prevalence of

Salmonella enterica can be considered as one of the most important causes of food- poisoning with poultry thought to be the main source. Although S. Typhimurium, S. Enteritidis and the vast majority of other Salmonella serovars generally produce little systemic disease in adult chickens, they are able to colonize the alimentary tract of poultry. The two caeca are the main sites of the colonization of Salmonellae in chickens, and the bacteria can be easily harvested from the caeca for analysis. Bacterial proteins analyses utilizing SDS-PAGE showed differences between in vitro and in vivo that out of about 40 protein bands of in vitro preparation only a few (3-5) bands can be visualized from in vivo preparations. We suggested that some avian proteases might be responsible. Accordingly, and to investigate the hypothesis that an inactivated vaccine harvested from chickens is thought to be more protective than bacteria grown in broth culture, the immunogenicity of inactivated vaccines, either proteins or formalin killed bacteria (S. Enteritidis) harvested from chicken intestine and those from broth culture (in vitro), were compared using bacterial proteins or formalin killed bacteria as an orally inoculated vaccine candidate in chicken. The results demonstrated that sonicated proteins extracts in general have a stronger effect as vaccines against S. Enteritidis colonization in chickens than formalin killed bacteria. In contrast, the in vitro sonicated proteins obtained from a nutrient broth culture had a much better protective vaccine effect than the in vivo sonicated proteins preparations harvested from bacteria grown in chickens.

Salmonella enterica subsp. enterica comprises more than 2500 separate serovars that differ genetically and in their pathogenicity. From the point of view of infection biology they may be divided into two groups or pathovars.

Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease.