S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes. Thus, we expressed and produced recombinant S1 protein to protect newborn piglets by immunization of sows. METHODS: Affinity tagged PEDV S1 protein was expressed in a secretory form in yeast, insect and mammalian cells to identify the most suitable production system. Purified recombinant protein was analysed by SDS-PAGE, Western blot and deglycosylation assay. A pregnant sow was intramuscularly immunized three times with adjuvanted recombinant protein prior to farrowing. PEDV-specific immune responses in sera and colostrum of the sow and piglets were assayed by ELISA and virus neutralization assays. Piglets were challenged orally with PEDV, and clinical parameters were monitored for 6 days post-challenge. RESULTS AND CONCLUSION: Of three eukaryotic expression systems tested (yeast, insect-cell, and mammalian), expression by HEK-293 T cells gave the highest yield of protein that was N-glycosylated and was the most appropriate candidate for vaccination. Administration of the subunit vaccine in a sow resulted in induction of S1-specific IgG and IgA that were passively transferred to the suckling piglets. Also, high virus neutralization titres were observed in the serum of the vaccinated sow and its piglets. After PEDV challenge, piglets born to the vaccinated sow exhibited less severe signs of disease and significantly lower mortality compared to the piglets of a control sow. However, there were no significant differences in diarrhea, body weight and virus shedding. Thus, vaccination with S1 subunit vaccine failed to provide complete protection to suckling piglets after challenge exposure, and further improvements are needed for the development of a subunit vaccine that fully protects against PEDV infection.

Role of motAB in adherence and internalization in polarized Caco-2 cells and in cecal colonization of Campylobacter jejuni.

Campylobacter jejuni, a gram-negative motile bacterium commonly found in the chicken gastrointestinal tract, is one of the leading causes of bacterial gastroenteritis in humans worldwide. An intact and functional flagellum is important for C. jejuni virulence and colonization. To understand the role of C. jejuni motility in adherence and internalization in polarized Caco-2 cells and in cecal colonization of chickens we constructed a C. jejuni NCTC11168 V1 deltamotAB mutant. The motAB genes code for the flagellar motor, which enables the rotation of the flagellum. The nonmotile deltamotAB mutant expressed a full-length flagellum, which allowed us to differentiate between the roles of full-length flagella and motility in the ability of C. jejuni to colonize. To study the adherence and invasion abilities of the C. jejuni deltamotAB mutant we chose to use polarized Caco-2 cells, which are thought to be more representative of in vivo intestinal cell architecture and function. Although the C. jejuni deltamotAB mutant adhered significantly better than the wild type to the Caco-2 cells, we observed a significant reduction in the ability to invade the cells. In this study we obtained evidence that the flagellar rotation triggers C. jejuni invasion into polarized Caco-2 cells and we believe that C. jejuni is propelled into the cell with a drill-like rotation. The deltamotAB mutant was also tested for its colonization potential in a 1-day-old chicken model. The nonmotile C. jejuni deltamotAB mutant was not able to colonize any birds at days 3 and 7, suggesting that motility is essential for C. jejuni colonization.

Innate immune cocktail partially protects broilers against cellulitis and septicemia.

Antimicrobial/host defense peptides (A/HDP) are natural compounds that are found in leucocyte cells and on the skin and bodily fluids of birds, reptiles, and mammals. Not only do they possess antibacterial, antiviral, and antiparasitic characteristics but they also stimulate the host immune system to combat infectious diseases and may play a role in the promotion of wound repair. Gamma-amino butyric acid (GABA) is an amino acid-based inhibitory neurotransmitter in the brain that has also been shown to promote wound healing on skin. The objective of this study was to establish a therapeutic cocktail that protects birds against Escherichia coli-related disease and lesions in broilers. We injected a cocktail of six A/HDPs with or without GABA into 3-wk-old broilers by a subcutaneous or intramuscular route followed 24 hr later by challenge with a field isolate of serogroup O2 E. coli. Birds were examined for 5-6 days post-E. coli challenge and clinical, pathologic, and bacteriologic assessments were conducted. Birds that were subcutaneously injected with an A/HDP plus GABA cocktail had significantly higher survival rates and lower levels of bacteremia (P < 0.05), but a similar percentage of the surviving birds had large cellulitis lesions compared to the surviving phosphate-buffered saline-injected control birds. When this cocktail was administered intramuscularly, there was a trend towards protection against E. coli-related death, although the results were not statistically significant and there was no reduction in bacteremia. A significant number of birds had a reduced bacterial load on cellulitis lesions but no reduction in lesion size, which suggests that when the cocktail was administered intramuscularly it failed to protect against cellulitis. These results suggest that the route of administration of the cocktail influences disease outcome. Gene expression analysis was performed to investigate whether the cocktail induced immunomodulatory functions in avian cells that complemented their antimicrobial and anti-endotoxic effects. A/HDP plus GABA mediated temporal induction of pro-inflammatory cytokines but no induction of any of the chemokines under investigation. This cocktail shows potential to protect against E. coli-related death, which is a major economic burden to the poultry industry.

High Affinity Iron Acquisition Systems Facilitate but Are Not Essential for Colonization of Chickens by Salmonella Enteritidis.

The roles of TonB mediated Fe3+ (ferric iron) uptake via enterobactin (involving biosynthesis genes entABCDEF) and Fe2+ (ferrous iron) uptake through the FeoABC transporter are poorly defined in the context of chicken-Salmonella interactions. Both uptake systems are believed to be the major contributors of iron supply in the Salmonella life cycle. Current evidence suggests that these iron uptake systems play a major role in pathogenesis in mammals and as such, they represent promising antibacterial targets with therapeutic potential. We investigated the role of these iron uptake mechanisms regarding the ability of Salmonella Enteritidis (SEn) strains to colonize in a chicken infection model. Further we constructed a bioluminescent reporter to sense iron limitation during gastrointestinal colonization of Salmonella in chicken via ex vivo imaging. Our data indicated that there is some redundancy between the ferric and ferrous iron uptake mechanisms regarding iron acquisition during SEn pathogenesis in chicken. We believe that this redundancy of iron acquisition in the host reservoir may be the consequence of adaptation to unique avian environments, and thus warrants further investigation. To our knowledge, this the first report providing direct evidence that both enterobactin synthesis and FeoABC mediated iron uptake contribute to the virulence of SEn in chickens.

Characterization of colonization kinetics and virulence potential of Salmonella Enteritidis in chickens by photonic detection.

The light emitting module lux operon (luxCDABE) of Photorhabdus luminescens can be integrated into a "dark" bacterium for expression under a suitable promoter. The technique has been used to monitor kinetics of infection, e.g., by studying gene expression in Salmonella using mouse models in vivo and ex vivo. Here, we applied the bioluminescence imaging (BLI) technique to track Salmonella Enteritidis (SEn) strains carrying the lux operon expressed under a constitutive promoter sequence (sigma 70) in chicken after oral challenge. Detectable photon signals were localized in the crop, small intestine, cecum, and yolk sac in orally gavaged birds. The level of colonization was determined by quantification of signal intensity and SEn prevalence in the cecum and yolk sac. Furthermore, an isogenic SEn mutant strain tagged with the lux operon allowed for us to assess virulence determinants regarding their role in colonization of the cecum and yolk sac. Interestingly, mutations of SPI-1(Salmonella Pathogenicity Island 1) and fur (ferric uptake regulator) showed significantly decreased colonization in yolk sac that was correlated with the BLI data. A similar trend was detected in a ΔtonB strain by analyzing enrichment culture data. The inherently low quantum yield, light scattering, and absorption by tissues did not facilitate detection of signals from live birds. However, the detection limit of lux operon has the potential to be improved by resonance energy transfer to a secondary molecule. As a proof-of-concept, we were able to show that sensitization of a fluorescent-bound molecule known as the lumazine protein (LumP) improved the limit of detection to a certain extent.

Enhancement of protective efficacy of innate immunostimulant based formulations against yolk sac infection in young chicks.

This study was conducted to characterize and compare the protective effects of various innate immune stimulants against yolk sac infection (YSI) caused by an avian pathogenic Escherichia coli in young chicks. The immune stimulants were administered alone or in various combinations of unmethylated CpG oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (Poly I:C), and avian antimicrobial peptides (AMPs). Routes included in ovo or in ovo followed by a subcutaneous (S/C) injection. CpG alone and in combination with Poly I:C, truncated avian cathelicidin (CATH)-1(6-26), avian beta defensin (AvBD)1, and CATH-1(6-26) + AvBD1, were administered in ovo to 18-day-old embryonated eggs for gene expression and challenge studies. Next, CpG alone and the potentially effective formulation of CpG + Poly I:C, were administrated via the in ovo route using 40 embryonated eggs. At 1 day post-hatch, half of each group also received their respective treatments via the S/C route. Four hours later, all chicks were challenged using E. coli strain EC317 and mortalities were recorded for 14 d. The first challenge study revealed that amongst the single use and combinations of CpG with different innate immune stimulants, a higher protection and a lower clinical score were offered by the combination of CpG + Poly I:C. The second challenge study showed that this combination (CpG + Poly I:C) provides an even higher level of protection when a second dose is administered via the S/C route at 1 day post-hatch. The current research highlights the efficacy of a combination of CpG + Poly I:C administered either in ovo or in ovo along with a S/C injection and its potential use as an alternative to antibiotics against yolk sac infection in young chicks.

Avian toll-like receptors.

Analysis of the genomes of two distantly related bird species, chicken and zebra finch (divergence of about 100 million years), indicate that there are ten avian toll-like receptors and that five of these, TLR2a, 2b, 3, 4, 5 and 7, are clear orthologs to TLRs found in mammals. Duplication of genes has led to TLR1La and 1Lb, TLR2a and 2b, and two TLR7s in the zebra finch. Avian TLR21 may be orthologous to TLR21 found in fish and amphibians, and avian TLR15, which is phylogenetically related to the TLR2 family, appears to be unique to avian species. While TLR2 is conserved between mammalian and avian species, the other TLR2 family members evolved independently. Dimerization between either of the two avian TLR2 species and TLR1La or 1Lb permits recognition of the same broad range of molecules as recognized by mammalian TLR2 dimerized with either TLR1, 6 and 10. Similarly, while TLR9 has been lost from the avian genome, DNA high in unmethylated CpG motifs is immunostimulatory through avian TLR21 which is absent in mammals. Thus, while some TLR members were commonly retained in both mammals and birds, others were separately lost or gained, or diverged independently; but broadly speaking the TLRs of the two classes of vertebrates evolved to recognize very similar spectra of microbial products. Components of downstream TLR signaling are also mostly conserved but with some losses in avian species; notably, TRAM is absent in avian genomes and, hence, the TRIF/TRAM-dependent signaling pathway utilized by mammals in LPS activation appears to be absent in birds.

Genes coding for virulence determinants of Campylobacter jejuni in human clinical and cattle isolates from Alberta, Canada, and their potential role in colonization of poultry.

Forty nine Campylobacter jejuni isolates from cattle feces collected from Alberta feedlots and 50 clinical C. jejuni isolates from people in Alberta were tested for the presence of 14 genes encoding putative virulence factors by PCR. These included genes implicated in adherence and colonization (flaC, cadF, docC, racR, jlpA, peb1, and dnaJ), invasion (virB11, ciaB, pldA, and iamA) and protection against harsh conditions (htrA, cbrA, and sodB). The genes examined were widely distributed in both the cattle fecal isolates and the human isolates. Of the isolates tested, 67% contained all of the genes except virB11. The cadF gene was found in 100% of the isolates tested. The presence or absence of virulence-associated genes was not associated with the ability of the organism to colonize birds. All of the C. jejuni isolates used to challenge birds were able to colonize the animals regardless of virulence gene profile. While some diversity in the profile of the occurrence of virulence-associated genes in C. jejuni exists, the distribution of these putative virulence-associated genes isolates from feedlot cattle feces and humans in Alberta was similar. In addition it was not possible to predict the ability of the selected isolates to colonize young chicks based on the presence of these genes coding for virulence determinants.

Avian antimicrobial peptides: in vitro and in ovo characterization and protection from early chick mortality caused by yolk sac infection.

Increasing antibiotic resistance is a matter of grave concern for consumers, public health authorities, farmers, and researchers. Antimicrobial peptides (AMPs) are emerging as novel and effective non-antibiotic tools to combat infectious diseases in poultry. In this study, we evaluated six avian AMPs including 2 truncated cathelicidins, [CATH-1(6-26) and CATH-2(1-15)], and 4 avian ß-defensins (ABD1, 2, 6 and 9) for their bactericidal and immunomodulatory activities. Our findings have shown CATH-1(6-26) and ABD1 being the two most potent avian AMPs effective against Gram-positive and Gram-negative bacteria investigated in these studies. Moreover, CATH-1(6-26) inhibited LPS-induced NO production and exhibited dose-dependent cytotoxicity to HD11 cells. While, ABD1 blocked LPS-induced IL-1ß gene induction and was non-toxic to HD11 cells. Importantly, in ovo administration of these AMPs demonstrated that ABD1 can offer significant protection from early chick mortality (44% less mortality in ABD1 treated group versus the control group) due to the experimental yolk sac infection caused by avian pathogenic Escherichia coli. Our data suggest that in ovo administration of ABD1 has immunomodulatory and anti-infection activity comparable with CpG ODN. Thus, ABD1 can be a significant addition to potential alternatives to antibiotics for the control of bacterial infections in young chicks.

Salmonella enterica serovar Enteritidis tatB and tatC mutants are impaired in Caco-2 cell invasion in vitro and show reduced systemic spread in chickens.

Salmonella enterica subsp. enterica serovar Enteritidis is a leading causative agent of gastroenteritis in humans. This pathogen also colonizes the intestinal tracts of poultry and can spread systemically in chickens. Transfer to humans usually occurs through undercooked or improperly handled poultry meat or eggs. The bacterial twin-arginine transport (Tat) pathway is responsible for the translocation of folded proteins across the cytoplasmic membrane. In order to study the role of the Tat system in the infection and colonization of chickens by Salmonella Enteritidis, we constructed chromosomal deletion mutants of the tatB and tatC genes, which are essential components of the Tat translocon. We observed that the tat mutations affected bacterial cell morphology, motility, and sensitivity to albomycin, sodium dodecyl sulfate (SDS), and EDTA. In addition, the mutant strains showed reduced invasion of polarized Caco-2 cells. The wild-type phenotype was restored in all our Salmonella Enteritidis tat mutants by introducing episomal copies of the tatABC genes. When tested in chickens by use of a Salmonella Enteritidis Delta tatB strain, the Tat system inactivation did not substantially affect cecal colonization, but it delayed systemic infection. Taken together, our data demonstrated that the Tat system plays a role in Salmonella Enteritidis pathogenesis.

The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization.

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacter planktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Delta cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Delta cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Delta cprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.

Iron-Uptake Systems of Chicken-Associated Salmonella Serovars and Their Role in Colonizing the Avian Host.

Iron is an essential micronutrient for most bacteria. Salmonella enterica strains, representing human and animal pathogens, have adopted several mechanisms to sequester iron from the environment depending on availability and source. Chickens act as a major reservoir for Salmonella enterica strains which can lead to outbreaks of human salmonellosis. In this review article we summarize the current understanding of the contribution of iron-uptake systems to the virulence of non-typhoidal S. enterica strains in colonizing chickens. We aim to address the gap in knowledge in this field, to help understand and define the interactions between S. enterica and these important hosts, in comparison to mammalian models.

Comparative in vivo infection models yield insights on early host immune response to Campylobacter in chickens.

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian beta-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter.

Genomics-based molecular epidemiology of Campylobacter jejuni isolates from feedlot cattle and from people in Alberta, Canada.

Feedlot cattle in Alberta, Canada, have been identified as reservoirs for Campylobacter jejuni, an important human pathogen. Oligonucleotide DNA microarrays were used as a platform to compare C. jejuni isolates from feedlot cattle and human clinical cases from Alberta. Comparative genomic hybridization (CGH) analysis was performed on 87 isolates (46 bovine, 41 human) obtained within the same geographical regions and time frame. Thirteen CGH clusters were obtained based on overall comparative genomic profile similarity. Nine CGH clusters contained human and cattle isolates, three contained only human isolates, and one contained only cattle isolates. The study isolates clustered regardless of temporal or geographical frameworks. In addition, array genes (n = 1,399) were investigated on a gene-by-gene basis to see if any were unequally distributed between human and cattle sources or between clusters dominated by either human or cattle isolates ("human enriched" versus "cattle enriched"). Using Fisher's exact test with the Westfall and Young correction for these comparisons, a small number of differentially distributed genes were identified. Our findings suggest that feedlot cattle and human C. jejuni strains are very similar and may be endemic within Alberta. Further, the common distribution of human clinical and bovine C. jejuni isolates within the same genetically based clusters suggests that dynamic and important transmission routes between cattle and human populations may exist.

Enhancement of immunoprotective effect of CpG-ODN by formulation with polyphosphazenes against E. coli septicemia in neonatal chickens.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents and vaccine adjuvants in a variety of bacterial and protozoan diseases in different animal species. The objective of this study was to investigate the immunoprotective effect of formulated CpG-ODN with polyphosphazene, liposome or oil-in-water emulsion against E. coli infections in neonatal chickens. Eighteen-day-old embryonating eggs were inoculated with 50 microg CpG-ODN or formulated CpG-ODN with polyphophazene, liposome or oil-in-water emulsion. Four days after exposure to formulated CpG-ODN or day-1 post-hatch, 1 x 10(4) or 1 x l0(5) cfu of a virulent isolate of E. coli was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for eight days following challenge with E. coli. The survival rate of birds following E. coli infection was 0% in groups receiving either non-CpG-ODN or saline. In contrast, birds receiving either CpG-ODN or CpG-ODN formulated with polyphosphazene had significantly higher survival of 55% (P<0.0001). The relative risk of mortality was significantly reduced for birds treated with CpG-ODN formulated in PCPP (0.25), in PCEP (0.33), or unformulated CpG-ODN (0.39) in comparison to the group treated with saline (p<0.01). Although formulation of CpG-ODN with liposomes or oil-in-water emulsion did not increase the immunoprotective effect against E. coli infection, no adverse reactions or poor hatchability were observed in embryos. This is the first time that CpG-ODN formulated with polyphosphazene has been demonstrated to have an immunoprotective effect against an extra cellular bacterial infection in neonatal broiler chickens following in ovo delivery.

Carbon nanotubes significantly enhance the biological activity of CpG ODN in chickens.

Synthetic unmethylated cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) is an effective immune stimulant in chicken. To be effective CpG dosage requirement is high. High dosage increases cost of treatment and introduces toxicity. A delivery system using multi-walled carbon nanotubes (MWCNT) is utilized in this study to aid in lowering the effective dose of the immune stimulant. CpG ODNs were attached non-covalently in different ways to multi-walled carbon nanotubes (MWCNT). We assessed and selected an appropriate linking method of CpG ODN with MWCNT followed by cellular uptake studies to establish that MWCNT-conjugated CpG ODNs were delivered better than free CpG ODNs into the cell. It was observed that MWCNT-conjugated CpG ODNs were equally effective in priming the cells in vitro at 1000-fold less concentration than free CpG ODN. In vivo studies revealed that a significantly lower dose of CpG ODN, when given subcutaneously, was enough to protect chickens from a lethal challenge of bacteria. The mechanism of immune stimulation was examined by in vivo cell recruitment and in vitro cytokine production studies. MWCNT-conjugated CpG ODNs are significantly more efficacious and less toxic than free CpG ODN to qualify as a potential immune stimulant.

Immunoproteomic analysis of Lawsonia intracellularis identifies candidate neutralizing antibody targets for use in subunit vaccine development.

Lawsonia intracellularis is an obligate intracellular microorganism and the causative agent of porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to achieve using conventional microbiological techniques. In this work, we used 2-dimensional gel electrophoresis coupled with Western-immunoblotting, mass spectrometry and bioinformatics to identify bacterial proteins that interact in vitro with pig intestinal cells (IPEC-1), have immunogenic properties and the potential to be used as subunit vaccine antigens. We detected eleven immunogenic bacterial proteins from which fliC (LI0710), LI1153 (annotated by NCBI as Putative protein N), and LI0649 (annotated as autotransporter) were predicted to be expressed on the outer membrane while LI0169 (oppA; annotated as ABC dipeptide transport system) was predicted to be periplasmic with a transmembrane domain forming a central pore through the plasma membrane. Genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole Lawsonia lysate established that all four recombinant proteins were immunogenic. Further, rabbit hyperimmune sera generated against the vaccine strain of L. intracellularis and rabbit serum specific for each recombinant protein showed an inhibitory effect on the attachment and penetration of live, avirulent L. intracellularis, thus indicating that each protein is a potential neutralizing antibody target and a candidate for subunit vaccine formulation.

Protection of neonatal broiler chicks against Salmonella Typhimurium septicemia by DNA containing CpG motifs.

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG-ODN) motifs have been shown to stimulate the innate immune system against a variety of bacterial and protozoan infections in a variety of vertebrate species. The objective of this study was to investigate the immunostimulatory effect of CpG-ODN in neonatal broilers against Salmonella Typhimurium septicemia. Day-old broiler chicks, or embryonated eggs that had been incubated for 18 days, received 50 microg of CpG-ODN, 50 microg of non-CpG-ODN, or saline. Four days after exposure to CpG-ODN or day 2 posthatch, 1 x 10(6) or 1 x 10(7) colony-forming units (cfu) of a virulent isolate of Salmonella Typhimurium was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 10 days following challenge with Salmonella Typhimurium. The survival rate of birds in groups receiving either non-CpG-ODN or saline following Salmonella Typhimurium infection was 40%-45%. In contrast, birds receiving CpG-ODN had significantly higher survival rate of 80%-85% (P < 0.0001). Bacterial loads and pathology were low in groups treated with CpG-ODN compared to the groups receiving saline or non-CpG-ODN. Colony-forming units of Salmonella Typhimurium in the peripheral blood were significantly lower in birds treated with CpG-ODN compared to the group that received saline. This is the first time that CpG-ODN has been demonstrated to have an immunoprotective effect against an intracellular bacterial infection in neonatal broiler chickens following in ovo delivery.

In Ovo Administration of Innate Immune Stimulants and Protection from Early Chick Mortalities due to Yolk Sac Infection.

Omphalitis or yolk sac infection (YSI) and colibacillosis are the most common infectious diseases that lead to high rates of early chick mortalities (ECMs) in young chicks. Out of numerous microbial causes, avian pathogenic Escherichia coli (APEC) or extraintestinal pathogenic E. coli infections are considered the most common cause of these conditions. YSI causes deterioration and decomposition of yolk, leading to deficiency of necessary nutrients and maternal antibodies, retarded growth, poor carcass quality, and increased susceptibility to other infections, including omphalitis, colibacillosis, and respiratory tract infection. Presently, in ovo injection of antibiotics, heavy culling, or after hatch use of antibiotics is practiced to manage ECM. However, increased antibiotic resistance and emergence of "super bugs" associated with use or misuse of antibiotics in the animal industry have raised serious concerns. These concerns urgently require a focus on host-driven nonantibiotic approaches for stimulation of protective antimicrobial immunity. Using an experimental YSI model in newborn chicks, we evaluated the prophylactic potential of three in ovo-administered innate immune stimulants and immune adjuvants for protection from ECM due to YSI. Our data have shown >80%, 65%, and 60% survival with in ovo use of cytosine-phosphodiester-guanine (CpG) oligodeoxynucleotides (ODN), polyinosinic:polycytidylic acid, and polyphosphazene, respectively. In conclusion, data from these studies suggest that in ovo administration of CpG ODN may serve as a potential candidate for replacement of antibiotics for the prevention and control of ECM due to YSI in young chicks.

Tetrasodium EDTA Is Effective at Eradicating Biofilms Formed by Clinically Relevant Microorganisms from Patients' Central Venous Catheters.

Central venous access devices (CVADs) are an essential component of modern health care. However, their prolonged use commonly results in microbial colonization, which carries the potential risk of hospital-acquired bloodstream infections. These infections complicate the treatment of already sick individuals and cost the existing health care systems around the world millions of dollars. The microbes that colonize CVADs typically form multicellular biofilms that are difficult to dislodge and are resistant to antimicrobial treatments. Clinicians are searching for better ways to extend the working life span of implanted CVADs, by preventing colonization and reducing the risk of bloodstream infections. In this study, we analyzed 210 bacterial and fungal isolates from colonized CVADs or human bloodstream infections from two hospitals geographically separated in the east and west of Canada and screened the isolates for biofilm formation in vitro Twenty isolates, representing 12 common, biofilm-forming species, were exposed to 4% tetrasodium EDTA, an antimicrobial lock solution that was recently approved in Canada for use as a medical device. The EDTA solution was effective at eradicating surface-attached biofilms from each microbial species, indicating that it could likely be used to prevent biofilm growth within CVADs and to eliminate established biofilms. This new lock solution fits with antibiotic stewardship programs worldwide by sparing the use of important antibiotic agents, targeting prevention rather than the expensive treatment of hospital-acquired infections.IMPORTANCE The colonization of catheters by microorganisms often precludes their long-term use, which can be a problem for human patients that have few body sites available for new catheters. The colonizing organisms often form biofilms, and increasingly these organisms are resistant to multiple antibiotics, making them difficult to treat. In this article, we have taken microorganisms that are associated with biofilm formation in catheters from two Canadian hospitals and tested them with tetrasodium EDTA, a new antimicrobial catheter lock solution. Tetrasodium EDTA was effective at eliminating Gram-positive, Gram-negative, and fungal species and represents a promising alternative to antibiotic treatment with less chance of the organisms developing resistance. We expect that our results will be of interest to researchers and clinicians and will lead to improved patient care.