Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

This thesis represents the first anthropological perspective to be offered on the nature of the Klondike Gold Rush population. In order to better understand the experience of the average gold rusher, morbidity and mortality patterns are examined for the residents of the Yukon Territory following the discovery of gold in the region (1898-1904). Infectious diseases such as measles, pneumonia, smallpox and typhoid fever are the primary focus of this study, however local factors such as the severe climate and the seclusion of the gold fields from the outside world also offers an interesting opportunity to examine the consequences of leading a particularly harsh and physically demanding lifestyle in an inhospitable environment. / October 2008

This thesis represents the first anthropological perspective to be offered on the nature of the Klondike Gold Rush population. In order to better understand the experience of the average gold rusher, morbidity and mortality patterns are examined for the residents of the Yukon Territory following the discovery of gold in the region (1898-1904). Infectious diseases such as measles, pneumonia, smallpox and typhoid fever are the primary focus of this study, however local factors such as the severe climate and the seclusion of the gold fields from the outside world also offers an interesting opportunity to examine the consequences of leading a particularly harsh and physically demanding lifestyle in an inhospitable environment.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

This thesis represents the first anthropological perspective to be offered on the nature of the Klondike Gold Rush population. In order to better understand the experience of the average gold rusher, morbidity and mortality patterns are examined for the residents of the Yukon Territory following the discovery of gold in the region (1898-1904). Infectious diseases such as measles, pneumonia, smallpox and typhoid fever are the primary focus of this study, however local factors such as the severe climate and the seclusion of the gold fields from the outside world also offers an interesting opportunity to examine the consequences of leading a particularly harsh and physically demanding lifestyle in an inhospitable environment.

Submitted by Lucas Bocchini Rodrigues Alves null (lucasbocchini@gmail.com) on 2017-12-05T19:45:59Z No. of bitstreams: 1 Dissertação_Lucas_Bocchini_Rodrigues_Alves.pdf: 48834271 bytes, checksum: 5031c0180284664e9a7c99d9701586b4 (MD5) / Submitted by Lucas Bocchini Rodrigues Alves null (lucasbocchini@gmail.com) on 2017-12-11T18:47:10Z No. of bitstreams: 1 Dissertação_Lucas_Bocchini_Rodrigues_Alves.pdf: 48834271 bytes, checksum: 5031c0180284664e9a7c99d9701586b4 (MD5) / Approved for entry into archive by Karina Gimenes Fernandes null (karinagi@fcav.unesp.br) on 2017-12-13T11:36:05Z (GMT) No. of bitstreams: 1 rodriguesalves_lb_jabo.pdf: 48291587 bytes, checksum: dba569cb90d71bd85abca6cc394cb228 (MD5) / Made available in DSpace on 2017-12-13T11:36:05Z (GMT). No. of bitstreams: 1 rodriguesalves_lb_jabo.pdf: 48291587 bytes, checksum: dba569cb90d71bd85abca6cc394cb228 (MD5) Previous issue date: 2017-07-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / RESUMO – Salmonella Gallinarum (SG) é um patógeno hospedeiro-específico que causa o tifo aviário, doença sistêmica severa que é considerada uma das principais preocupações da indústria avícola mundial. Quando infecta a ave, SG utiliza mecanismos de evasão para sobreviver e replicar no interior de macrófagos. Nesse contexto, os genes phoPQ codificam o sistema regulatório de dois componentes (PhoPQ) que regula genes de virulência responsáveis pela adaptação de Salmonella spp. a fatores antimicrobianos como baixo pH, peptídeos antimicrobianos e baixas concentrações de cátions bivalentes. No presente estudo, objetivou-se investigar a função desses genes para SG. Assim, uma estirpe de SG com genes phoPQ defectivos (SG ∆phoPQ) foi construída e sua patogenicidade avaliada em aves poedeiras de 20 dias de vida susceptíveis ao tifo aviário. SG ∆phoPQ não causou sinais clínicos nem mortalidade em aves desafiadas oralmente, sendo não-patogênica. Ademais, essa estirpe não foi recuperada de fígados e baços. Por outro lado, aves desafiadas subcutaneamente com a estirpe mutante tiveram alterações patológicas discretas a moderadas e baixas contagens bacterianas em tecidos de fígado e baço. A partir dos dados, observa-se que SG ∆phoPQ é atenuado para aves o que sugere que ambos os genes são importantes durante a infecção sistêmica em aves por SG. / ABSTRACT – Salmonella Gallinarum (SG) 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. Herein, we aimed to investigate the role of the mentioned genes to SG. Thus, a phoPQ-depleted SG strain (SG ∆phoPQ) was constructed and its virulence assessed in twenty-day-old laying hens susceptible to fowl typhoid. SG ∆phoPQ 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 ∆phoPQ is attenuated to susceptible chickens and suggest that both genes are important during chicken systemic infection by SG.

Resumo: Este trabalho objetivou avaliar a anatomopatologia, o hemograma e o perfil bioquímico sérico de aves de postura inoculadas por Salmonella Gallinarum (SG) contendo os genes cobS e cbiA inoperantes (SGcobScbiA) que mostrou ser avirulenta em trabalhos anteriores, comparando-a com cepas virulentas SGNalr e SGcobS, para mostrar se SGcobScbiA pode ser componente de vacina contra cepas selvagens de SG e S.Enteritidis. 280 pintainhas foram distribuídas em 4 grupos (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) e G4 (controle). Com exceção do G4, os grupos receberam 0,2 mL de suas respectivas cepas contendo aproximadamente 108 UFC/mL de inóculo, aos 5 dias de idade. A eutanásia foi realizada 24h antes (1DAI) e após a inoculação (1DPI), e 3 (3DPI), 5 (5DPI), 7 (7DPI), 10 (10DPI) e 15 (15DPI) dias após a administração do inóculo, sacrificando-se, em cada momento, dez aves de cada grupo. As aves foram sacrificadas, obtendo-se amostras de sangue utilizadas para os exames hematológicos e bioquímicos. Fragmentos de fígado, baço, timo, bursa de Fabricius, rins e coração foram destinados aos exames histológicos. As aves inoculadas com a cepa SGcobS tiveram comportamento semelhante às aves inoculadas por SGNalr, porém com algumas respostas diferentes nos exames hematológicos e bioquímicos. As aves inoculadas com a cepa SGcobScbiA tiveram comportamento semelhante ao grupo controle, entretanto foi verificado alterações brandas em alguns parâmetros, mostrando que estudos futuros devem ser feitos, verificando se as alterações constatadas não irão interferir no desempenho de aves vacinadas com a cepa SGcobScbiA. / Abstract: The aim of the present study was to evaluate anatomopathology, hemogram and blood serum components of commercial layers experimentally inoculated with SGcobScbiA, which is a Salmonella Gallinarum (SG) strain it shows attenuation of the virulence in previous research and it was compared with high virulence SGNalr and SGcobS strains in order to show if SGcobScbiA has potential to be use as a vaccine against SG and S. Enteritidis wild strains. 280 commercial layers were divided into 4 groups (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) and G4 (control group). With exception of G4, all the other groups received 0,2 mL of their respective strain containing about 108 CFU/mL of the inoculum with five days of age. Birds were sacrificed 24 hours before (1DBI) and 24 hours after the inoculation (1DAI), and three (3DAI), five (5DAI), seven (7DAI) ten (10DAI), and fifteen (15DAI) days after the administration of the inoculum, slaughtering ten birds at a time in each group. Birds were submitted to euthanasia and blood samples were collected in order to make the hematological and blood serum components test. Samples of liver, spleen, thymus, bursa of Fabricius, kidneys and heart were collected for the histological test. The birds inoculated with SGcobS strain had similar behavior when compared with that ones who received SGNalr strain, however some different responses in the hematological and blood serum components were found. On the other hand, the birds inoculated with SGcobScbiA strain had similar behavior when compared with the control group, however, lower alterations in some parameters were found. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SGcobScbiA strain. / Orientador: Ângelo Berchieri Júnior / Coorientador: José Jurandir Fagliari / Banca: Antonio Carlos Alessi / Banca: Raimundo Souza Lopes / Mestre

The problem of this thesis consists primarily of a bacteriological survey of the eating utensils of Hardin College Cafeteria and twenty-five other eating establishments in the city of Wichita Falls, Texas. This investigation was made primarily with reference to a determination of the possible presence of typhoid and related organisms, and secondarily to an investigation of the actual presence of those bacterial organisms associated with the more common outbreaks of food poisoning.

<p>The  Pakistan  poultry  industry  has  developed  into  the  11thlargest  poultry  industry  in  the world  and  poultry  products  provide  high-quality  and  affordable  protein  sources  to  communities throughout the country. However, <em>Salmonella </em>Gallinarum, the etiological agent for fowl typhoid, is  endemic  in  Pakistan  with  infections  leading  to  high  mortality  and  substantial  economic  loss. Currently, <em>Salmonella </em>Gallinarum  infectionsin  Pakistan  poultry  are  controlled  with  antibiotics. The continued emergence of antibiotic resistance, however, has led to global initiatives to reduce the  use  of  antibiotics  in  both  human  and  veterinary  medicine.  Concurrently,  the  Pakistan government  recently  introduced  new  national  policies  that  limit  the  use  of  antibiotics  for performance  in  livestock  and  poultry  production.  As  such,  controlling  bacterial  infections  in poultry  without  increasing  the  likelihood  of  antibiotic use could  ensure  the  sustainability  of Pakistan  poultry  production  without  posing  risks  to  public  health.  Toward  this  end,  we hypothesized that <em>Salmonella</em> Gallinarum infections inchickens could be prevented or otherwise controlled through the use of phages. To test this hypothesis, wastewater samples were collected from Lahore, Pakistan and different cities of Indiana, US and processed to isolate bacteriophages. The  phages  were  characterized  in  terms  of  morphology,  host  spectra,  lytic  capacity,  genomic sequencing,  and  survivability  in  different  environments. Transmission  electron  microscopy showed these phages belonged to myoviridae (n = 5) and podoviridae (n = 1) families. Spectrum analysis  revealed  that  each  phage  lysed  at  least  8  out  of  10  different  strains  of <em>Salmonella </em>Gallinarum and significantly reduced (P < 0.05) <em>Salmonella </em>Gallinarum when co-cultured in liquid medium with the bacterium. Stability of the phages was tested insimulated gastric fluid (SGF; pH= 2.5) andsimulated intestinal fluid (SIF; pH~6.8). Results showed that phage concentrationswere reduced to undetectable levels when exposed to SGF for more than 5 minutes. However, exposure to SIF did not result in appreciable reductions in phage concentrations. To mitigate potential effects of  gastric  environments,  phages  were  encapsulated  using  a  sodium  alginate-based  method.  In contrast  to  unprotected  phages,  encapsulated  phages  remained  viable  (~100%)  after  30  minutes exposure to SGF. Additionally, encapsulation efficiencies ranged between 90-99%. Encapsulated phages were sequentially incubated in SGF (30 minutes) and SIF(120 minutes) to determine the rate  of  release  of  the  phages  from  capsules. All  phages  were  released from  capsules after  60 minutes  of  exposureto  SIF. To  determine  if  the  phages  effectively  controlled <em>Salmonella </em>Gallinarum infections in chickens, 100, day-old Jumbo Cornish Rock Cross birds were randomly assigned  to  one  of  four  treatments:  1)  Control 1  (bacterial  challenge,  no  phage  treatment);  2) Control 2 (no phage or bacterial challenge); 3) challenged with SalmonellaGallinarum and treated with  unprotected  phages;  and  4)  challenged  with <em>Salmonella</em> Gallinarum  and  treated  with encapsulated phages. At7 d of age, chicks receiving the bacterial challenge were administered 5 X106CFU (500 μL) of <em>Salmonella</em> Gallinarum. For birds in phage treatment groups, the phages were administered (500 uL; 5 X108 PFU/mL or g) at 0, 12, and 24 hours post-challenge. Six birds from each group were euthanized at 1, 2, and 4 days post-challenge (dpc) and cecal SalmonellaGallinarum  concentrations  were  quantified.  At 1  dpc, birds  treated  with  unprotected  and encapsulated  phages  had significantly lower (P  <  0.05) SalmonellaGallinarum concentrations(4.36 ± 0.20and 5.05 ± 0.22 logCFU/g, respectively) than those found in untreated birds (5.71 ± 0.13). Likewise,  at4  dpc, <em>Salmonella </em>Gallinarum concentrationsin  ceca  of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 0.05; 3.26 ± 0.62 and 4.02 ± 0.15 log  CFU/g,  respectively)  than  those  found  in untreated  birds(4.65  ±  0.08log  CFU/g). A second trial was conducted with higher challenge doses (1 mL at 1× 109CFU) and an additional treatment including a mixture (1:1) of unprotected and encapsulated phages. At1dpc, <em>Salmonella</em> Gallinarum concentrations  in the ceca  of  birds  treated  with unprotected  phages,  encapsulated phages, and a mixture of unprotected  and encapsulated phages  were significantly lower(4.28 ± 0.11, 3.72 ± 0.40, and 3.81 ± 0.36log CFU/g, respectively) than found in those of untreated birds (5.26 ± 0.19log CFU/g). At 2 dpc, concentrations of<em> Salmonella </em>Gallinarumin the ceca of birds treated  with  unprotected,  encapsulated,  and  a mixture  of  unprotected  and  encapsulated  phages were significantly  lower  (P  <  0.05; 4.31  ±0.53, 3.96  ±0.61,  and 4.38  ±  0.44logCFU/g, respectively) than those found in the ceca of untreated birds (5.72 ± 0.27logCFU/g).However, no significant differences were found in concentrations of <em>Salmonella</em> Gallinarum in the ceca of birds treated with encapsulated phages versus those treated with unprotected phagesor a mixture of   encapsulated   and   unprotected   phages.   Similarly,   at   4   dpc, <em>Salmonella </em>Gallinarum concentrations in the ceca  of  birds  treated  with unprotected  phages, encapsulated  phages,  and  a mixture of unprotected and encapsulated phages were significantly lower (3.17 ± 0.45, 3.56 ± 0.51, and 3.81 ± 0.54log CFU/g, respectively) than found in those of untreated birds (5.79 ± 0.08log CFU/g). At  7  d  post-challenge,  concentrations of <em>Salmonella</em> Gallinarum in  the  ceca  of  birds treated  with mixture  of  unprotected  and  encapsulated phages(2.40  ±  0.55log  CFU/g)  were significantly lower (P  <  0.05) than  those  found  in the ceca  of  untreated  birds(7.08  ±  0.19log CFU/g). Similarly,  concentrations of<em> Salmonella</em> Gallinarum  in the  ceca of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 0.05; 4.29 ± 0.39and 4.60 ± 0.37 log  CFU/g,  respectively)  than  those  found  in  untreated  birds.  Taken  together,  these  data indicate that <em>Salmonella </em>Gallinarum infections could be controlled with phage-based treatments. Additionally, the use of a mixture of unprotected and encapsulated phages may be more effective, presumably  by  allowing  unprotected  phages  to  act  immediately  in  the  proximal  gastrointestinal tract  (GIT;  e.g.,  crop)  with  encapsulated  phages  having  greater  activity  once  released  from capsules in the distal small intestine. While no deleterious effects of the phages were observed on the chickens themselves, continuing studies should more comprehensively assess host-response to phage treatment including potential impact on microbial communities throughout the chicken GIT.</p>

Salmonella enterica sérovar Typhi (Typhi) est une bactérie pathogène spécifique à l’homme. Typhi est l’agent étiologique de la fièvre typhoïde chez l’humain, causant plus de 16 millions de nouveaux cas par année et plus de 600 000 morts. Il a été démontré que pour causer une infection systémique, Salmonella doit nécessairement survivre dans les macrophages de l'hôte. Paradoxalement, S. enterica sérovar Typhimurium, très apparenté à Typhi (près de 90 % d’homologie), n’a pas la capacité de se disséminer dans l’organisme humain et peut infecter plusieurs espèces animales. Nous avons antérieurement identifié 36 gènes uniques à Typhi (absents chez Typhimurium) situés sur 15 régions différentes et exprimés sélectivement lors de l’infection de macrophages humains. Ainsi, l’une de ces régions a suscité notre attention, soit la région sty4217-4222 et plus particulièrement le produit du gène sty4221, une aminotransférase hypothétique. Ce dernier gène est d’intérêt dû à l’homologie qu’il détient avec une hémolysine connue (Hly) produite par Treponema denticola, possédant elle-même une activité d’aminotransférase. Chez T. denticola, Hly dégrade la cystéine et produit du H2S qui est toxique pour l’hôte. Notre hypothèse est que la spécificité d’hôte et la capacité de produire une infection systémique de Typhi sont dues à l’expression de gènes qui ne se retrouvent pas chez d’autres salmonelles. Le but de cette étude était donc de caractériser le gène sty4221 quant à son activité hémolytique, cytotoxique et tenter de déterminer son rôle dans la virulence de cette bactérie. Le gène sty4221 a été cloné sous le contrôle d’un promoteur inductible à l’arabinose et exprimé par E. coli. L’activité hémolytique du clone a été déterminée par simple observation sur gélose sang. Ce clone a également permis d’observer l’effet cytotoxique du surnageant de culture sur différentes lignées cellulaires, par quantification de la relâche de LDH. Le gène sty4221 a été muté chez la souche sauvage de Typhi, ISP1820, l’implication pathogénique du gène a ainsi pu être étudiée. Des tests de phagocytose, d’invasion et de survie dans des macrophages humains ont été effectués, ainsi que des tests d’adhésion et d’invasion sur des cellules HeLa. Par ailleurs, une première tentative de purification de la protéine a été entreprise. En somme, nous savons maintenant que STY4221 a des propriétés hémolytiques, augmentées par la présence de cystéine. De plus, STY4221 a un effet cytotoxique sur les macrophages THP-I, mais aucun effet sur les HeLa. Or, sty4221 ne semble pas impliqué dans les étapes d’adhésion, d’invasion, de phagocytose ou de survie. La caractérisation de sty4221 permettra sans doute d’approfondir nos connaissances sur les toxines trouvées uniquement chez Typhi. / Salmonella enterica serovar Typhi (Typhi) is a human restricted pathogen causing typhoid fever, a systemic infection. Annually, at least 16 million new cases with 600, 000 associated deaths are reported. It has been demonstrated that Salmonella has to survive in the macrophages of its host, in order to produce a systemic disease. This ability to cause a disseminated infection in human is unique to Typhi. Our laboratory had isolated 36 genes that were unique to Typhi (absent from Typhimurium’s genome), and that were expressed during human macrophages infection. One of these genes, sty4221, a putative aminotransferase, was of high interest since it shares sequence similarities with a known hemolysin (Hly), which also possesses an aminotransferase activity. That hemolysin is produced by Treponema denticola, it catabolizes cysteine and produces H2S, a toxic metabolite for the host. Our hypothesis is that host specificity and the ability to cause a systemic infection might be explained by the expression of genes that are not found in other salmonellas. The goal of this study was to characterize the gene sty4221, in terms of hemolytic and cytotoxic activity and to determine its role in virulence. The sty4221gene has been cloned in a vector under an arabinose inducible promoter and transformed in a strain of E. coli. The hemolytic activity has been investigated on blood-agar medium. To evaluate the cytotoxicity of the STY4221 protein on human cultured cells, direct observation by photonic microscopy was done. The cytotoxicity activity on human cultured cells has been quantitatively measured with a lactate dehydrogenase release assay. Moreover, the sty4221 gene has been deleted in order to study its implication in the infection and the survival within human macrophages and for adhesion/invasion on epithelial. Protein purification was also attempted. We now know that protein STY4221 has a hemolytic activity that is enhanced by cysteine. Also, we proved that the expression of sty4221 has a cytotoxic effect on THP-I macrophages, but not on epithelial HeLa cells. Meanwhile, sty4221 does not seem to be important during adhesion, invasion, infection nor survival. The characterization of protein STY4221 might extend the list of known exotoxin of Typhi.