In-Vitro Adhesion and Invasion Properties of Salmonella Typhimurium Competing with Bacteriophage in Epithelial Cells and Chicken Macrophages

HY Lee D Biswas J Ahn About the authors

ABSTRACT

This study was designed to assess the role of bacteriophage P22 in the adhesion, invasion, intracellular survival of, and cellular immune response to Salmonella Typhimurium in intestinal epithelial INT-407 and chicken macrophage-like HD11 cells. The ability of S. Typhimurium to adhere, invade, and survive to INT-407 and HD11cells was evaluated under Salmonella infection alone (control), phage treatment followed by Salmonella infection (PS), Salmonella infection followed by phage treatment (SP), and a combination treatment with Salmonella and phage (S+P). The number of S. Typhimurium associated on INT-407 cells was reduced from 4.2 to 2.7 log cfu/cm2 by phage treatment (SP). The number of intracellular S. Typhimurium within INT-407 cells was significantly reduced to below the detection limit (0.7 log cfu/cm2) compared with the control (3.4 log cfu/cm2). S. Typhimurium remained inside HD11 cells at 49% and 17% levels in the absence and presence of phages, respectively, at 24 h post-infection (hpi). The expression levels of IFN-g, IL-10, IL-1b, IL-6, IL-8, iNOS, and IL-12 increased in HD11 cells regardless the absence and presence of phages, while those of IL-16, TLR2-1, TLR3, and TLR7 were decreased at 0 and 24 hpi. This study sheds new light on our understanding of the role of phages in Salmonella adhesion, invasion, survival, and cellular immune responses.

Keywords:
Adhesion; Bacteriophage; Invasion; Salmonella; Toll-like receptor

INTRODUCTION

Salmonella enterica serovars are major causes of the human infectious disease known as salmonellosis, resulting from the ingestion of contaminated food (Vikram et al., 2012Vikram A, Jayaprakasha GK, Jesudhasan PR, Pillai SD, Patil BS. Obacunone represses Salmonella pathogenicity islands 1 and 2 in an envZ-dependent fashion. Applied and Environmental Microbiology2012;78:7012-7022.).The ingested Salmonella undergoes the infection process, including bacterial adherence, colonization, invasion, and propagation in the intestinal epithelium and macrophage cells (Antunes et al., 2010Antunes LC, Buckner MM, Auweter SD, Ferreira RB, Lolic P, Finlay BB. Inhibition of Salmonella host cell invasion by dimethyl sulfide. Applied and Environmental Microbiology 2010;76:5300-5304.). Although innate immune responses are the first line of host defense against bacterial infection, Salmonella can effectively induce cytoskeletal rearrangement and membrane ruffling of host cells by several effector proteins that are responsible for Salmonella internalization into the epithelial cells (Kubori & Galan, 2002Kubori T, Galan JE. Salmonella type III secretion-associated protein InvE controls translation of effector proteins into host cells. Journal of Bacteriology 2002;184:4699-4708., Yano & Kurata, 2011Yano T, Kurata S. Intracellular recognition of pathogens and autophagy as an innate immune host defence. Journal of Biochemistry 2011;150:143-149.).The intestinal adhesion and invasion of the epithelium is an important stage to initiate the Salmonella pathogenesis, depending on the type III secretion system (TTSS) encoded by Salmonella pathogenicity island (SPI) (Baxter et al., 2003Baxter MA, Fahlen TF, Wilson RL, Jones BD. HilE interacts with HilD and negatively regulates hilA transcription and expression of the Salmonella enterica serovar Typhimurium invasion phenotype. Infection and Immunity 2003;71:1295-1305.). Therefore, the effective control of Salmonella infection is a high priority worldwide.

Bacteriophages have recently been received great attention as an alternative biocontrol to antibiotics due to the increasing emergence of antibiotic-resistant bacteria (Lu and Koeris, 2011Lu TK, Koeris MS. The next generation of bacteriophage therapy. Current Opinion in Microbiology 2011;14:524-531., Golkar et al., 2014Golkar Z, Bagasra O, Pace DG. Bacteriophage therapy: a potential solution for the antibiotic resistance crisis. Journal of Infection in Developing Countries 2014;8:129-236.). From the viewpoint of phage therapy against extracellular and intracellular pathogens, however, there is a challenging question as whether phages can affect extracellular S. Typhimurium adhesion and invasion of epithelium cells and evade the cellular immune responses in the host cells. Therefore, a systematic approach for studying the adherence and invasion properties of extracellular S. Typhimurium competing with phages, the intracellular survival of S. Typhimurium,and macrophage cellular immune responses to phages is essential to elucidate the pathogen-phage-macrophage interactions at cellular immune response levels and to design an effective phage therapeutic strategy.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs), including lipoteichoic acid (TLR2), double-stranded RNA (TLR3), lipopolysaccharide (LPS; TLR4), flagell in (TLR5), single-stranded RNA (TLR7), and CpG oligodeoxynucleotides (ODN; TLR9) (Ishii et al., 2005Ishii K, Coban C, Akira S. Manifold mechanisms of Toll-like receptor-ligand recognition. Journal of Clinical Immunology 2005;25:511-521.). TLRs, as pattern recognition receptors (PRRs), can trigger inflammatory immune responses in macrophages, inducing the production of cytokines and chemokines (Iqbal et al., 2005Iqbal M, Philbin VJ, Withanage GSK, Wigley P, Beal RK, Goodchild MJ, Barrow P, McConnell I, Maskell DJ, Young J, Bumstead N, Boyd Y, Smith AL. Identification and functional characterization of chicken Toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar Typhimurium. Infection and Immunity2005;73:2344-2350.; Kawai & Akira, 2006Kawai T, Akira S. Innate immune recognition of viral infection. Nature Immunology 2006;7:131-137.). However, the potential role of phages in macrophage-mediated immune response to intracellular pathogens still remains unclear. Therefore, the aims of this study were to examine the effect of phage P22 on the adhesion and invasion of S. Typhimurium into the intestinal epithelium cell line INT-407, and assess the cellular immune response of Salmonella-infected macrophage cell line HD11 against phage P22.

MATERIALS AND METHODS

Bacterial strain and bacteriophage

The strain of Salmonella enteric subsp. enteric serovar Typhimurium LT2 (ATCC 19585) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in Trypticase Soy broth (TSB) (Difco, Becton, Dickinson and Co., Sparks, MD, USA) at 37oC for 20 h. After cultivation, cultures were centrifuged at 3,000 × g for 20 min at 4oC and resuspended in 0.1% sterile buffered peptone water (BPW) at approximately 108 cfu/mL. Salmonella bacteriophage P22 (ATCC 97540) was purchased from ATCC and propagated with S. Typhimurium at 37oC for 24 h. The culture was centrifuged at 13,000 × g for 2 min, and the supernatant was filtered by using a 0.2-mm filter to eliminate bacterial lysates. The phages was purified according to the polyethylene glycol (PEG) precipitation method (Yamamoto et al., 1970Yamamoto KR, Alberts BM, Benzinger R, Lawhorne L, Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 1970;40:734-744.).

Phage plaque assay

Phagetiter was determined by using a soft-agar overlay method (Bielke et al., 2007Bielke L, Higgins S, Donoghue A, Donoghue D, Hargis BM. Salmonella host range of bacteriophages that infect multiple genera. Poultry Science 2007;86:2536-2540.). The phages were serially (1:10) diluted with phosphate buffered saline (PBS, pH 7.2) buffer. Each dilution was mixed with S. Typhimurium cells in TSB (0.5% agar), pour-plated onto the surface of pre-warmed TSB (1.5% agar), and incubated 37oC for 24 h to enumerate lytic phages expressed as plaque-forming unit (pfu).

Cell lines and culture conditions

Human intestinal epithelium cell line, INT-407 CCL-6, and chicken macrophage-like cell line, HD11, were purchased from American Type Culture Collection (ATCC). INT-407 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; HyClone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and 100 µg/mL gentamicin. HD11 cells were cultured in DMEM supplemented with 2 mM L-glutamine, 10% heat-inactivated chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were incubated at 37oC with 5% CO2.

Cell invasion assay

For the invasion assay, INT-407 and HD11 cells were seeded at 2 × 106 cells/mL into 24-well plates and 25 cm2 T-flask, respectively, and incubated to approximately 90% confluence for 24-48 h at 37oC under 5% CO2. The post confluent INT-407 or HD11 cultures were rinsed twice with PBS buffer and then stabilized in serum- and antibiotic-free DMEM for 1 h prior to bacterial infection. On the INT-407 cell monolayers, S. Typhimurium and phage P22 were infected simultaneously or sequentially at approximately 106 cfu/cm2 and 107 pfu/cm2, respectively: i) 1 h of pre-treatment of INT-407 cell monolayers with phage P22 followed by 1 h of Salmonella infection (PS), ii) 1 h of Salmonella infection of INT-407 cell monolayers followed by 1 h of phage treatment (SP), and iii) 1 h of combined treatment of phage and S. Typhimurium (S+P). INT-407 cells infected with S. Typhimurium or phage P22 alone were used as control. On the HD11 cell monolayers, S. Typhimurium was infected for 1 h at 37oC. The infected HD11 cells were treated with 100 µg/mL of gentamicin at 37oC for 1 h to eliminate adherent S. Typhimurium cells and then incubated with or without phage P22 at 37oC for 24 h.

Quantification of bacterial adhesion and invasion

The infected INT-407 and HD11 cell monolayers were rinsed 3 times with PBS to eliminate non-adherent S. Typhimurium cells. For associated bacterial count, the INT-407 and HD11 cell monolayers were lysed with 1% Triton X-100 for 15 min at 37oC. For intracellular bacteria count, the INT-407 and HD11 cell monolayers were treated with 100 µg/mL of gentamicin at 37oC for 1 h and then lysed with 1% Triton X-100 for 15 min at 37oC to release intracellular bacteria. The collected lysates were serially diluted with PBS to plate on TSA, incubated for 24 to 48 h at 37oC, and enumerate the adherent and intracellular.

RNA extraction and cDNA synthesis

HD11 cell monolayers at 0 and 24 h post-infection (hpi) were rinse with PBS, lysed with 1 mLof TRIzol reagent (Life Technologies Co. Carlsbad, CA, USA), and then collected using a cell scrapper. The cell lysates were mixed with 200 µL of chloroform and incubated for 3 min at 25oC. The mixtures were centrifuged at 12,000 × g for 15 min at 4oC to collect the upper aqueous phase (500 µL), which were mixed with 500 µL of isopropanol for 10 min at 25oC and centrifuged at 12,000 × g for 10 min at 4oC. The pellet was rinsed with 1 mL of 75% ethanol, air-dried to remove ethanol, and dissolved in 20 µL of RNase-free water at 40oC. According to the protocol of qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA), the RNA extract (1 µg) was mixed with 4 µL of 5X qScript cDNA SuperMix (MgCl2, dNTPs, RNase inhibitor protein, qScript reverse transcriptase, and oligo(dT) primer). The mixture was incubated subsequently at 42oC for 30 min and 95oC for 3 min.

Quantitative RT-PCR assay

The reaction mixture containing 10 µL of 2× QuantiTect SYBR Green PCR Master, 2 µL of each primer, and 2 µL of cDNA, and 4 µL of RNase-free water was amplified using an iCycler iQ(tm) system (Bio-Rad Laboratories, Hemel Hempstead, UK). The PCR mixture was denatured at95 °C for 30 sec, followed by 40 cycles of 95 °C for 5 sec, 55 °C for 15 sec, and 72 °C for 10 sec. The relative expression levels of genes were estimated by the comparative method (Livak & Schmittgen, 2001Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods 2001;25:402-408.). The CT values of target genes in Salmonella-infected HD11 cells with and without phage P22 were compared to those in non-infected HD11 cells. The reference gene (GAPDH) was used for normalization of inflammatory mediator gene expression.

Statistical analysis

All experiments were conducted with three replicates. Data were analyzed using the Statistical Analysis System software. The general linear model and least significant difference (LSD) procedures were used to evaluate the treatment as a fixed effect. Significant mean differences were calculated by Fisher's LSD at p< 0.05.

RESULTS

Adhesion and invasion abilities of S. Typhimurium to INT-407 and HD11 cells

The adhesion ability of S. Typhimurium to INT-407 cells were evaluated under different treatment conditions: i) INT-407 infected with Salmonella or phage alone (control), ii) phage treatment followed by Salmonella infection (PS), iii) Salmonella infection followed by phage treatment (SP), and iv) combination treatment with Salmonella and phage (S+P) (Fig. 1A).Compared with the control, the ability of S. Typhimurium to adhere to INT-407 cells significantly decreased in SP treatment, followed by S+P treatment. The associated numbers of S. Typhimurium were 4.24, 3.99, 2.65, and 3.63 cfu/cm2, respectively, for the control, PS, SP, and S+P.

Figure 1
Adhesion ability of S. Typhimurium (A) intracellular survival at 0 hpi () and 24 (■) hpi (B) to INT-407 cells infected with Salmonella or phage alone (control), phage treatment followed by Salmonella infection (PS), Salmonella infection followed by phage treatment (SP), and combination treatment with Salmonella and phage (S+P). Means with different letters (A-C and a-b) are significantly different at p < 0.05. The asterisk (*) indicates the bacterial count below the detection limit (0.73 log cfu/cm2).

The invasion and survival properties of S. Typhimurium were evaluated in INT-407 cells treated with PS, SP, and S+P at 0 and 24 hpi (Fig. 1B). S. Typhimurium cells were more invasive in the control than in the phage treatments, including PS, SP, and S+P. The invasive ability was significantly decreased in SP, corresponding to the adhesion ability. The numbers of intracellular S. Typhimurium were 2.66, 2.05, 1.34, and 2.05 log cfu/cm2, respectively, at 0 hpi. The phage treatment after Salmonella-infection effectively blocked the internalization of S. Typhimurium in theINT-407 cells. Compared with the control, the intracellular survival of S. Typhimurium in INT-407 cells was slightly increased in the control and PS at 24 hpi, while that was marginally decreased in SP and S+P. The intracellular survival of S. Typhimurium was evaluated in HD11 treated with and without phage (Fig. 2). The number of intracellular S. Typhimurium in HD11 was 3.76 log cfu/cm2 at 0 hpi, which was reduced to 3.44 log cfu/cm2 (51% reduction) in the absence of phage and 2.93 log cfu/cm2 (83% reduction) in the presence of phage at 24 hpi.

Figure 2
Survival of intracellular S. Typhimurium infected in HD11 cells treated without (-) and with (+) phage P22 for 24 h at 37oC. Means with different letters (a-b) are significantly different at p < 0.05.

Inflammatory-mediated gene expression in Salmonella -infected HD11 cells

The relative expression profiles of pro-inflammatory and TLR genes were determined in HD11 cells cultured with and without phage at 24 hpi (Fig. 3). The relative expression levels IFN-g, IL-10, IL-1b, IL-6, IL-8, iNOS, and IL-12 mRNAs increased, while those of IL-16, TLR2-1, TLR3, and TLR7decreased in HD11 cells at 24 hpi. The levels of IL-13, IL-15, IL-17, IL-18, IL-3, TLR2-2, TLR4, and TLR5 mRNAs remained constant in HD11 cells at 24 hpi. The mRNA levels increased between 2 to 7-fold for IFN-g, IL-10, IL-1b, IL-6, IL-8, iNOS, and IL-12. Although no significant differences were observed in the expression levels of pro-inflammatory and TLR mRNAs between absence and presence of phages, the expression levels of pro-inflammatory cytokines were slightly higher in the presence of phages than the absence of phages.

Figure 3
Relative expression of inflammatory-mediated genes in Salmonella-infected HD11 cells treated with (+) and without (-) phage P22 at 24 hpi.

DISCUSSION

Enteric bacteria can pass through the epithelial barrier, infect macrophage, and then develop survival strategies against the phagocytosis-associated cellular defense system. Phage can be a potential approach for controlling intracellular pathogens. This study describes the potential role of phage P22 in the adhesion and invasion of S. Typhimurium in the intestinal epithelial cells and the macrophage-mediated immune response to intracellular S. Typhimurium.

The phage treatment after Salmonella infection effectively excluded the attached S. Typhimurium from the INT-407 cells (Fig. 1A). Phage can be used as a possible approach to treat rather than prevent bacterial infections. This observation indicates that there were no significant changes in the lytic activity or binding affinity of phages against the adherent S. Typhimurium, resulting in the bacterial invasion of host cells.

There are two bacterial invasion mechanisms, including zippering (affinity binding of bacteria adhesins and host receptors) and triggering (injection of effector proteins into the host cells via TTSS) events (Ó Cróinín & Backert, 2012ÓCróinín T, Backert S. Host epithelial cell invasion by Campylobacter jejuni: trigger or zipper mechanism? Frontiers in Cellular and Infection Microbiology 2012;2:1-13.). Bacterial cells can internalize and even reproduce within the nonphagocytic epithelial cells (Kim et al., 2011Kim JS, Eom JS, Jang JI, Kim HG, Seo DW, Bang I-S, Bang SH, Lee IS, Park YK. Role of Salmonella pathogenicity island 1 protein iacP in Salmonella enterica serovar Typhimurium pathogenesis. Infection and Immunity2011;79:1440-1450.).

The numbers of intracellular S. Typhimurium were reduced to below detection limit in the SP treatment (Fig. 1B). The phage treatment after Salmonella-infection most successfully inhibited the growth of intracellular S. Typhimurium in INT-407 cells. This implies that the phage-infected S. Typhimurium entered and was then lysed within the INT-407 cells. The number of intracellular S. Typhimurium in HD11 was significantly reduced to 2.93 log cfu/cm2 (83% reduction) in the presence of phage at 24 hpi (Fig. 2). The internalized S. Typhimurium cells undergo a series of survival processes, SPI-1 and SPI-2 (Flannagan et al., 2009Flannagan RS, Cosio G, Grinstein S. Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nature Reviews Microbiology 2009;7:355-366.). The significant reduction in the number of internalized S. Typhimurium in HD11 cells might be due to the inflammatory immune responses to the phage P22.

The phage P22 marginally induced immune responses in HD11 cells at 24 hpi (Fig. 3). The expression of IL-1b, IL-6, and IL-8 was up regulated in Salmonella-infected macrophages (Wigley, 2004Wigley P. Genetic resistance to Salmonella infection in domestic animals. Research in Veterinary Science 2004;76:165-169., Withanage et al., 2004Withanage GSK, Kaiser P, Wigley P, Powers C, Mastroeni P, Brooks H, Barrow P, Smith A, Maskell D, McConnell I. Rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with Salmonella enterica Serovar Typhimurium. Infection and Immunity2004;72:2152-2159.). IL-1β is a pro-inflammatory cytokine and IL-6 is a pro- and anti-inflammatory cytokine (Lee et al., 2010 Lee SH, Lillehoj HS, Hong YH, Jang SI, Lillehoj EP, Ionescu C, Mazuranok L, Bravo D. In vitro effects of plant and mushroom extracts on immunological function of chicken lymphocytes and macrophages. British Poultry Science2010;51:213-221.). The inflammatory cytokines were inhibited by IL-10 and IL-13, which were increased during the initial stage of bacterial infection (Rothwell et al., 2004Rothwell L, Young JR, Zoorob R, Whittaker CA, Hesketh P, Archer A, Smith AL, Kaiser P. Cloning and characterization of chicken IL-10 and its role in the immune response to Eimeria maxima. Journal of Immunology 2004;173:2675-2682., Setta et al., 2012Setta AM, Barrow PA, Kaiser P, Jones MA. Early immune dynamics following infection with Salmonella enterica serovars Enteritidis, Infantis, Pullorum and Gallinarum: Cytokine and chemokine gene expression profile and cellular changes of chicken cecal tonsils. Comparative Immunology, Microbiology and Infectious Diseases 2012;35:397-410.). The expression level of iNOS mRNA in bacteria-infected HD11 cells was enhanced by IFN-g but not IL-18 (He et al., 2011He H, Genovese KJ, Kogut MH. Modulation of chicken macrophage effector function by TH1/TH2 cytokines. Cytokine 2011;53:363-369.). IL-18 acts as an inducer of cellular immune responses against intracellular pathogens when combined with IL-12 (Biet et al., 2002Biet F, Locht C, Kremer L. Immunoregulatory functions of interleukin 18 and its role in defense against bacterial pathogens. Journal of Molecular Medicine 2002;80:147-162.). Lipopolysaccharide-induced TNF-afactor (LITAF) is involved in the initiation of cytokine cascade in response to Salmonella infection (Ma et al., 2010Ma J, Zhang Y-g, Xia Y, Sun J. The inflammatory cytokine tumor necrosis factor modulates the expression of Salmonella typhimurium effector proteins. Journal of Inflammation 2010;7:42.). TLR2, TLR4, and TLR5 are activated on the host cell surface, whereas TLR3 and TLR7 are activated within the host cells in response to bacterial infections (Ishii et al., 2005Ishii K, Coban C, Akira S. Manifold mechanisms of Toll-like receptor-ligand recognition. Journal of Clinical Immunology 2005;25:511-521.). TLR2 was primarily involved in the induction of anti-inflammatory cytokine, IL-10 (Netea et al., 2004Netea MG, van der Graaf C, Van der Meer JWM, Kullberg BJ. Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system. Journal of Leukocyte Biology 2004;75:749-755.). TLR2 was stimulated by TLR-4 which was not up-regulated after Salmonella infection (Higgs et al., 2006Higgs R, Cormican P, Cahalane S, Allan B, Lloyd AT, Meade K, James T, Lynn DJ, Babiuk LA, O'Farrelly C. Induction of a novel chicken Toll-like receptor following Salmonella enterica serovar Typhimurium infection. Infection and Immunity2006;74:1692-1698.). The TLR activation is mainly responsible for the cytokine induction in macrophages. However, the levels of TLR transcripts were decreased in this study. This result implies that the pro-inflammatory gene expression may be induced by a non-TLR-mediated recognition pathway (Bliss et al., 2005Bliss TW, Dohms JE, Emara MG, Keeler CL. Gene expression profiling of avian macrophage activation. Veterinary Immunology and Immunopathology 2005;105:289-299.).

In conclusion, the most significant findings of this in-vitrostudy were:1) the phage treatment after Salmonella infection effectively inhibited the invasion ability and intracellular survival of S. Typhimurium in epithelial INT-407 cells, and 2) although no significant macrophage-mediated inflammatory immune responses were observed in HD11 cells exposed to phage, the number of intracellular S. Typhimurium was significantly reduced in HD11 cells at 24 hpi. This in-vitro study provides useful information for understanding the role of phage in inhibiting extracellular and intracellular pathogens and also improving phage therapy at the cellular level. However, further in-vivo studies are needed to elucidate the complex gene regulatory networks of the inflammatory immune system, which is the ongoing investigation of our laboratory.

ACKNOWLEDGEMENT

This study was supported by Research Grant from Kangwon National University

REFERENCES

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  • Kim JS, Eom JS, Jang JI, Kim HG, Seo DW, Bang I-S, Bang SH, Lee IS, Park YK. Role of Salmonella pathogenicity island 1 protein iacP in Salmonella enterica serovar Typhimurium pathogenesis. Infection and Immunity2011;79:1440-1450.
  • Kubori T, Galan JE. Salmonella type III secretion-associated protein InvE controls translation of effector proteins into host cells. Journal of Bacteriology 2002;184:4699-4708.
  • Lee SH, Lillehoj HS, Hong YH, Jang SI, Lillehoj EP, Ionescu C, Mazuranok L, Bravo D. In vitro effects of plant and mushroom extracts on immunological function of chicken lymphocytes and macrophages. British Poultry Science2010;51:213-221.
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  • Setta AM, Barrow PA, Kaiser P, Jones MA. Early immune dynamics following infection with Salmonella enterica serovars Enteritidis, Infantis, Pullorum and Gallinarum: Cytokine and chemokine gene expression profile and cellular changes of chicken cecal tonsils. Comparative Immunology, Microbiology and Infectious Diseases 2012;35:397-410.
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  • Wigley P. Genetic resistance to Salmonella infection in domestic animals. Research in Veterinary Science 2004;76:165-169.
  • Withanage GSK, Kaiser P, Wigley P, Powers C, Mastroeni P, Brooks H, Barrow P, Smith A, Maskell D, McConnell I. Rapid expression of chemokines and proinflammatory cytokines in newly hatched chickens infected with Salmonella enterica Serovar Typhimurium. Infection and Immunity2004;72:2152-2159.
  • Yamamoto KR, Alberts BM, Benzinger R, Lawhorne L, Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 1970;40:734-744.
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Publication Dates

  • Publication in this collection
    Oct-Dec 2015

History

  • Received
    Oct 2014
  • Accepted
    Apr 2015
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