Andrew A. Potter

Analysis of the Contribution of Salmonella Pathogenicity Islands 1 and 2 to Enteric Disease Progression Using a Novel Bovine Ileal Loop Model and a Murine Model of Infectious Enterocolitis

ABSTRACT We have developed a novel ileal loop model for use in calves to analyze the contribution of Salmonella enterica serovar Typhimurium type III secretion systems to disease processes in vivo. Our model involves constructing ileal loops with end-to-end anastamoses to restore the patency of the small intestine, thereby allowing experimental animals to convalesce following surgery for the desired number of days. This model overcomes the time constraint imposed by ligated ileal loop models that have precluded investigation of Salmonella virulence factors during later stages of the infection process. Here, we have used this model to examine the enteric disease process at 24 h and 5 days following infection with wild-type Salmonella and mutants lacking the virulence-associated Salmonella pathogenicity island 1 (SPI-1) or SPI-2 type III secretion systems. We show that SPI-2 mutants are dramatically attenuated at 5 days following infection and report a new phenotype for SPI-1 mutants, which induce intestinal pathology in calves similar to wild-type Salmonella in the 5-day ileal loop model. Both of these temporal phenotypes for SPI-1 and SPI-2 mutants were corroborated in a second animal model of enteric disease using streptomycin-pretreated mice. These data delineate novel phenotypes for SPI-1 and SPI-2 mutants in the intestinal phase of bovine and murine salmonellosis and provide working models to further investigate the effector contribution to these pathologies.

Protection of chickens against Escherichia coli infections by DNA containing CpG motifs.

ABSTRACT Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate of Escherichia coli was applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after the E. coli challenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN(2007) had a survival rate of 15%. In contrast, groups that received CpG-ODN(2007), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P < 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN(2007) by the subcutaneous route (P < 0.01). A dose of as little as 3.16 μg of CpG-ODN(2007), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds against E. coli infection (P < 0.01). This study demonstrates that CpG-ODN(2007) has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN(2007) has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.

Salmonella vaccines in poultry: past, present and future

Salmonella species are important zoonotic pathogens that cause gastrointestinal disease in humans and animals. Poultry products contaminated with these pathogens are one of the major sources of human Salmonella infections. Vaccination of chickens, along with other intervention measures, is an important strategy that is currently being used to reduce the levels of Salmonella in poultry flocks, which will ultimately lead to lower rates of human Salmonella infections. However, despite numerous studies that have been performed, there is still a need for safer, well-defined Salmonella vaccines. This review examines the different classes of Salmonella vaccines that have been tested, highlighting the merits and problems of each, and provides an insight into the future of Salmonella vaccines and the platforms that can be used for delivery.

Genome to Kinome: Species-Specific Peptide Arrays for Kinome Analysis

Marrying bioinformatics and phosphorylation assays enables the empirical analysis of kinomes of multiple species. Tools for conducting high-throughput kinome analysis do not exist for many species. For example, two commonly used techniques for monitoring phosphorylation events are phosphorylation-specific antibodies and peptide arrays. The majority of phosphorylation-specific antibodies are for human or mouse targets, and the construction of peptide arrays relies on information from phosphorylation databases, which are similarly biased toward human and mouse data. This is a substantial obstacle because many species other than mouse represent important biological models. On the basis of the observation that phosphorylation events are often conserved across species with respect to their relative positioning within proteins and their biological function, we demonstrate that it is possible to predict the sequence contexts of phosphorylation events in other species for the production of peptide arrays for kinome analysis. Through this approach, genomic information can be rapidly used to create inexpensive, customizable, species-specific peptide arrays for high-throughput kinome analysis. We anticipate that these arrays will be valuable for investigating the conservation of biological responses across species, validating animal models of disease, and translating research to clinical applications.

Iron-repressible Outer-membrane Proteins of Pasteurella haemolytica

The outer-membrane protein (OMP) profile of Pasteurella haemolytica grown under iron-replete and iron-restricted conditions was studied by polyacrylamide gel electrophoresis and immunoblotting. A serotype 1 isolate induced the synthesis of a new 77,000 Mr OMP in iron-restricted media while two other proteins of 100,000 Mr and 71,000 Mr were synthesized in increased amounts. None of these proteins were peptidoglycan-associated or heat-modifiable, and only the 100,000 Mr protein showed some degree of disulphide cross-linking. Kinetic analysis revealed that the iron-repressible proteins appeared in the outer membrane within 15 min of establishment of iron-restricted conditions. Analysis of P. haemolytica isolates representing serotypes 1 to 12 showed that iron-repressible OMPs of 77,000 Mr and 71,000 Mr could be induced in all 12 serotypes but that there was some variability in the expression of the 100,000 Mr protein. Immunoblotting of OMPs with convalescent sera from P. haemolytica-infected calves indicated that antibodies directed against all three iron-repressible OMPs were present, suggesting that these proteins were expressed in vivo.

Protection of Neonatal Chicks Against a Lethal Challenge of Escherichia coli Using DNA Containing Cytosine-Phosphodiester-Guanine Motifs

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. We recently have shown that CpG ODN protects against extracellular bacterial infections in mature chickens. The objective of this study was to investigate the effect of CpG ODN on Escherichia coli septicemia in neonatal broiler chicks. Two-day-old chicks, or embryonated eggs that had been incubated for 18 or 19 days, received 50 microg CpG ODN. Three days after exposure to CpG ODN, a virulent isolate of E. coli was inoculated subcutaneously in the neck of each bird. Birds were examined for 7 days post-E. coli challenge and dinical, pathologic, and bacteriologic assessments were conducted. The control group of birds that received no CpG ODN had a survival rate of 0% to 20%. In contrast, groups that received CpG ODN, either by intramuscular or in ovo routes, had significantly higher survival rates (P < 0.0001). Bacterial counts in air sacs were significantly lower when birds or embryos were treated with CpG ODN as compared with controls. A dose as low as 10 microg of CpG ODN, administered intramuscularly, was able to protect birds significantly against E. coli challenge. Formulation of CpG ODN with 30% Emulsigen did not enhance the protection. This study demonstrates that CpG ODN has systemic protective effects in broiler chicks against E. coli infections. This is the first time that CpG ODN has been demonstrated to have an immunoprotective effect against a bacterial infection in chicks following in ovo delivery.

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel(®) immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.

Antibody responses in adult and neonatal BALB/c mice to immunization with novel Bordetella pertussis vaccine formulations.

A balanced or Th-1 type immune response is required for effective clearance of many pathogens such as Bordetella pertussis, the causative agent of whooping cough. Since current acellular pertussis vaccines induce limited Th-1 type immune responses, novel vaccine formulations are needed to induce protective immunity in the infant in the earliest stages of life. Here, we developed a novel vaccine platform consisting of genetically detoxified pertussis toxoid (PTd) with multiple adjuvant components including CpG oligodeoxynucleotides, polyphosphazenes, and cationic innate defence regulator peptides. Co-formulation with these immunomodulators increased the serum IgG2a and IgG1 antibody titres in adult mice when compared to immunization with each of the selected adjuvants or immunization with PTd antigen alone. When used in combination, these adjuvants were able to induce a superior IgG2a response in both adult and neonatal mice, when compared to antigen alone or commercial vaccines. The increased response observed when using this adjuvant formulation was also initiated earlier and, moreover, was maintained over a period of greater than 22 months. The adjuvant platform also showed an ability to induce an immune response in a greater number of mice as compared to antigen alone. This suggests that this uniquely adjuvanted vaccine induces a stronger and more balanced immune response with an earlier onset of this response than vaccination with PTd antigen alone.

Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice

Type III secreted proteins from Escherichia coli O157:H7 are involved in the attachment of the organism to mammalian cells and have been shown to be effective vaccine components capable of reducing colonization of cattle by the organism. In the current study, we used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against EspA and Tir were also monitored. Subcutaneous immunization of mice with type III secreted proteins induced significant EspA- and Tir-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant EspA- and Tir-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Only mice that were immunized intranasally with formulations containing mucosal adjuvants, either cholera toxin or CpG-containing oligonucleotides, showed decreased E. coli O157:H7 shedding following experimental infection. Mice immunized subcutaneously with type III secreted proteins did not shed E. coli in feces. These results demonstrate the potential for the use of type III secreted proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7.

Protection of Neonatal Broiler Chicks Against Salmonella Typhimurium Septicemia by DNA Containing CpG Motifs

Abstract 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 µg of CpG-ODN, 50 µg of non-CpG-ODN, or saline. Four days after exposure to CpG-ODN or day 2 posthatch, 1 × 106 or 1 × 107 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. Protección de pollos de engorde recién nacidos contra septicemia causada por Salmonella Typhimurium mediante ADN con motivos CpG. Se ha demostrado que los oligodeoxinucleótidos que contienen motivos CpG (por sus siglas en Inglés CpG-ODN), estimulan el sistema inmune innato contra una variedad de infecciones bacterianas y de protozoarios en varias especies de vertebrados. El objetivo de este estudio fue investigar el efecto inmunoestimulatorio de CpG-ODN contra Salmonella Typhimurium en pollos de engorde recién nacidos. Pollitos de engorde de un día de edad o huevos fértiles en el día 18 de la etapa embrionaria, recibieron 50 µg de CpG-ODN, 50 µg de oligodeoxinucleótidos sin motivos CpG (No-CpG-ODN) o solución salina. Cuatro días después de la exposición a los CpG-ODN o dos días después del nacimiento, las aves se inocularon subcutáneamente en el cuello con 1 × 106 o 1 × 107 unidades formadoras de colonias de un aislamiento virulento de Salmonella Typhimurium. Posterior al desafío, las aves se observaron durante 10 días para la aparición de signos clínicos, alteraciones patológicas, aislamiento bacteriano de los sacos aéreos y mortalidad. La tasa de supervivencia posterior a la infección con Salmonella Typhimurium en los grupos que recibieron No-CpG-ODN o solución salina fue de un 40 a 45%. En contraste, las aves que recibieron CpG-ODN mostraron una tasa de supervivencia significativamente mas alta de 80 a 85% (P < 0.0001). En comparación con los grupos que recibieron No-CpG-ODN o solución salina, las cargas bacterianas y la patología de las aves que recibieron CpG-ODN fueron bajas. Las unidades formadoras de colonia de Salmonella Typhimurium en sangre periférica fueron significativamente menores en las aves tratadas con CpG-ODN que en las aves tratadas con solución salina. Esta es la primera vez que se demuestra que los CpG-ODN administrados in ovo tienen un efecto contra una infección bacteriana intracelular en pollos de engorde recién nacidos. Abbreviations: CCS = cumulative clinical score; cfu = colony-forming units; CpG-ODN = cytosine-phosphodiester-guanine oligodeoxynucleotides; IFN-γ = interferon gamma; IL = interleukin; NK = natural killer cells; PAMPs = pathogen-associated molecular patterns; TLR = Toll-like receptor; TSIA = triple-sugar iron agar