Wolfgang Köster

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.

Salmonella enterica Serovar Enteritidis Pathogenicity Island 1 Is Not Essential for but Facilitates Rapid Systemic Spread in Chickens

ABSTRACT Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both ΔSPI-1 and ΔinvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and ΔSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or ΔSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the ΔSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.

Salmonella enterica Serovar Enteritidis tatB and tatC Mutants Are Impaired in Caco-2 Cell Invasion In Vitro and Show Reduced Systemic Spread in Chickens

ABSTRACT 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 ΔtatB 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.

Salmonella enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. For the purposes of our studies we used a chicken isolate of S. Enteritidis (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen-free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type strain Sal18: Sal18 attTn7 : : tet and Sal18 attTn7 : : cat, while the other two groups received the wild-type strain (Sal18 attTn7 : : tet) and one of two mutant strains. From this study, we concluded that S. Enteritidis strains deficient in the SPI-1 and SPI-2 systems were outcompeted by the wild-type strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains: the wild-type strain, and three other strains lacking either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge, we observed a reduced systemic spread of the SPI-2 mutants, but by day 3, the systemic distribution levels of the mutants matched that of the wild-type strain. Based on these two studies, we conclude that the S. Enteritidis SPI-2 T3SS facilitates invasion and systemic spread in chickens, although alternative mechanisms for these processes appear to exist.

Evaluation of Salmonella enterica serovar Enteritidis pathogenicity island-1 proteins as vaccine candidates against S. Enteritidis challenge in chickens

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of gastrointestinal disease in humans worldwide, which mainly results from the consumption of contaminated poultry meat and eggs. Vaccination of chickens is an important strategy to lower the prevalence of Salmonella in poultry flocks. The S. Enteritidis type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island-1 (SPI-1) is an important virulence factor that plays a role in invasion and systemic spread in chickens. In this manuscript, we evaluated the efficacy of SPI-1 proteins as vaccine candidates for protection against S. Enteritidis oral challenge. Our results demonstrate for the first time that SPI-1 T3SS proteins elicit antigen specific IgG antibody responses in chickens. In one study we show that vaccination with the aforementioned proteins reduces the levels of S. Enteritidis in the liver, but not in the spleen and cecal contents of chickens. However, a second study shows that vaccination of hens with SPI-1 proteins using a seeder model of infection does not affect the levels of S. Enteritidis in the cecal contents or internal organs of progeny obtained from these hens. Hence, the SPI-1 proteins, in conjunction with other proteins, may form important components of subunit vaccines used for protection against colonization by S. Enteritidis in poultry.

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

SUMMARY. 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 &Dgr;motAB mutant. The motAB genes code for the flagellar motor, which enables the rotation of the flagellum. The nonmotile &Dgr;motAB 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 &Dgr;motAB 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 &Dgr;motAB 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 &Dgr;motAB mutant was also tested for its colonization potential in a 1-day-old chicken model. The nonmotile C. jejuni &Dgr;motAB mutant was not able to colonize any birds at days 3 and 7, suggesting that motility is essential for C. jejuni colonization. RESUMEN. Nota de Investigación—Papel del gene motAB en la adherencia e internalización de Campylobacter jejuni en células Caco-2 polarizadas y en su colonización cecal. Campylobacter jejuni, una bacteria gram negativa móvil que se encuentran comúnmente en el tracto gastrointestinal de pollo, es una de las principales causas de gastroenteritis bacteriana en los seres humanos en todo el mundo. Un flagelo intacto y funcional es importante para la virulencia y la colonización por C. jejuni. Para entender el papel de la motilidad de C. jejuni en la adherencia e internalización en células Caco-2 polarizadas y en la colonización cecal en pollos, se construyó una mutante de C. jejuni denominada NCTC11168 V1 &Dgr;motAB. Los genes motAB codifican para el motor flagelar, que permite la rotación del flagelo. La mutante no móvil &Dgr;motAB expresaba un flagelo completo, lo que permitió diferenciar entre las funciones de los flagelos completos y la movilidad en la capacidad de C. jejuni para colonizar. Para estudiar la capacidad de adherencia y la invasividad de la mutante de C. jejuni denominada &Dgr;motAB se optó por utilizar células Caco-2 polarizadas, que se cree que son más representativas de la arquitectura de las células intestinales y de su función in vivo. Aunque la mutante de C. jejuni &Dgr;motAB mostró una adherencia significativamente mejor a las células Caco-2 que la bacteria de tipo silvestre, se observó una reducción significativa en la capacidad de invadir las células. En este estudio se obtuvo evidencia de que la rotación flagelar desencadena la invasión por C. jejuni en células Caco-2 polarizadas y se cree que C. jejuni es propulsada hacia dentro de las células con una rotación con perforación. La mutante &Dgr;motAB también se analizó en su potencial de colonizar en un modelo de pollos de un día de edad. La mutante de C. jejuni no motil &Dgr;motAB no fue capaz de colonizar en ninguna de las aves a los días 3 y 7, lo que sugiere que la motilidad es esencial para la colonización C. jejuni.

Immunization of chickens with Salmonella enterica subspecies enterica serovar Enteritidis pathogenicity island-2 proteins

Several structural components of the type III secretion systems (T3SS) encoded by Salmonella pathogenicity island (SPI)-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. In this study we evaluated whether chickens vaccinated with SPI-2 T3SS components could mount a significant humoral immune response (as measured by serum IgG titres) and whether these antibodies could be transferred to progeny (as measured by egg yolk IgG titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with SE. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgG that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered Salmonella enterica subspecies enterica serovar Enteritidis (SE) per bird in most situations.

Effect of the Salmonella Pathogenicity Island 2 Type III Secretion System on Salmonella Survival in Activated Chicken Macrophage-Like HD11 Cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment, as E. coli is effectively eliminated.

A global survey of bacterial type III secretion systems and their effectors

The type III secretion system (T3SS) is an important genetic determinant that mediates interactions between Gram-negative bacteria and their eukaryotic hosts. Our understanding of the T3SS continues to expand, yet the availability of new bacterial genomes prompts questions about its diversity, distribution and evolution. Through a comprehensive survey of ∼20xa0000 bacterial genomes, we identified 174 non-redundant T3SSs from 109 genera and 5 phyla. Many of the bacteria are environmental strains that have not been reported to interact with eukaryotic hosts, while several species groups carry multiple T3SSs. Four ultra-conserved Microsynteny Blocks (MSBs) were defined within the T3SSs, facilitating comprehensive clustering of the T3SSs into 13 major categories, and establishing the largest diversity of T3SSs to date. We subsequently extended our search to identify type III effectors, resulting in 8740 candidate effectors. Lastly, an analysis of the key transcriptional regulators and circuits for the T3SS families revealed that low-level T3SS regulators were more conserved than higher-level regulators. This comprehensive analysis of the T3SSs and their protein effectors provides new insight into the diversity of systems used to facilitate host-bacterial interactions.

The Salmonella Pathogenicity Island-1 and -2 Encoded Type III Secretion Systems

Salmonellae are motile, facultatively anaerobic, Gram-negative rods measuring 0.3-1.5 by 1.02.5 uf06dm in size. The genus Salmonella was named for Dr. Daniel Salmon, a veterinary bacteriologist at the United States Department of Agriculture (USDA) (Gast, 2003, Salyers & Whitt, 2002). The Salmonella species are closely related to Escherichia, Yersinia, and Shigella, and contain a circular chromosome approximately 4.7 Mbp in size with an overall GC content of 52% (Marcus, et al., 2000, Salyers & Whitt, 2002, Thomson, et al., 2008). The genus Salmonella lies within the kingdom Eubacteria, class Gammaproteobacteria, order Enterobacteriales, and family Enterobacteriaceae. Salmonella is divided into two species, Salmonella bongori and Salmonella enterica. Within Salmonella enterica there are 6 subspecies: salamae, arizonae, diarizonae, houtenae, indica, and enterica (Tindall, et al., 2005). These subspecies can be further classified into approximately 50 serogroups based on their lipopolysaccharide (LPS) O antigen component (Sabbagh, et al., 2010). Salmonella enterica subspecies enterica finds its niche in warm-blooded animals and is the primary species associated with human infections. S. bongori and other S. enterica subspecies are more commonly associated with cold-blooded animals, and in some cases can cause disease in these animals (Brenner, et al., 2000).