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

Abstract

Salmonella enterica subspecies enterica infection remains a serious problem in a wide range of animals and in man. Poultry-derived food is the main source of human infection with the non-host-adapted serovars while fowl typhoid and pullorum disease are important diseases of poultry. We have assessed cecal colonization and immune responses of newly hatched and older chickens to Salmonella serotypes Enteritidis, Infantis, Gallinarum and Pullorum. S. Enteritidis and S. Infantis colonized the ceca more efficiently than S. Gallinarum and S. Pullorum. Salmonella infection was also associated with increased staining for B-lymphocytes and macrophages in the cecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds stimulated the expression of CXCLi1 and CXCLi2 chemokines in the cecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the cecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes.

Introduction

Salmonella enterica subspecies enterica (S. enterica) remains a major bacterial disease affecting a wide range of hosts and of food-borne significance. From the point of view of infection biology, the 2463 serovars of S. enterica [1] fall into two broad classes. A small number of serovars produce typhoid-like infection in a restricted number of host species. These pathogens include S. Typhi in man, S. Dublin in cattle, S. Choleraesuis in pigs and S. Gallinarum and S. Pullorum in poultry. These serovars are transmitted via the fecal–oral route but colonize the gut poorly and invade with bacterial multiplication in the spleen, liver and other organs. They re-enter the gut in the later stages of disease but do not colonize the gut in the absence of clinical disease and, therefore, rarely enter the human food chain [2], [3]. S. Gallinarum and S. Pullorum are serologically identical but differ in their virulence characteristics. S. Gallinarum produces fowl typhoid, a severe systemic disease with high mortality in birds of all ages and which, although it has been largely eliminated from those countries which intensified their industries decades ago and which are able to control the poultry house environment, is still a major problem in those countries which have high ambient temperatures and where environmental control is problematic [4]. S. Pullorum produces pullorum disease characterized by high mortality only in very young birds and followed by persistent disease-free infection in convalescent birds leading to vertical transmission at onset of lay [5], [6].

The second class contains the vast majority of the remaining serovars which are capable of infecting a wide range of hosts, including humans. The serotypes of this group rarely produce systemic disease in normal healthy, adult animals but colonize the gut without disease and are thus able to enter the human food chain producing food-poisoning [3]. This group comprises the non-host-adapted serotypes, such as S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo and many others. Serovars, such as S. Infantis and S. Hadar, which are becoming more prevalent in many European countries, are characterized by good colonization ability and low virulence for chickens [7]. Consumption of contaminated poultry-derived food, including meat and eggs, is considered as the main source of human gastroenteritis [8], [9], [10]. S. Enteritidis and S. Typhimurium are anomalous since they belong to both classes in being associated with most cases of human gastroenteritis but also in producing typical typhoid in mice. S. Enteritidis has been a major cause of this world-wide zoonosis for the last 2 decades but is under control in many countries which have introduced comprehensive hygienic measures and control policies [10], [11]. This has been possible as a result of its relatively high invasiveness which has allowed the development of serological surveillance and the development of live S. Enteritidis and S. Typhimurium vaccines [12], [13], [14]. Another live vaccine, which was produced initially for vaccination against S. Gallinarum, is the semi-rough strain S. Gallinarum 9R, which shows some degree of cross protection against S. Enteritidis infection without spread to the egg contents of the vaccinated flocks [15], [16], [17].

The intestinal epithelium is a physical, physiological and immunological barrier against enteric pathogens. For many years, the innate immune system was seen as a scavenger system which is responsible for combating the invading pathogens. However, it is now clearly evident that innate effector cells mediate a specific immune response, of controlling and regulating importance, which directs the further adaptive immune response [18]. Indeed, the initial (innate) response dictates the nature of the adaptive response which may vary according to serotype [19], which has been also seen in poultry [20], [21]. This complex interplay between the innate and adaptive immune responses is important for clearance of Salmonella. However, previous studies have shown that cellular immune responses are much more important for tissue clearance of Salmonella in mice [22] and also appear to be essential to clearance from the gut in poultry [23]. Changes in cytokine and chemokine gene expression following Salmonella infection have been studied in vitro following infection of epithelial cells and monocytes [24], [25], [26] and in the avian host [27], [28], [29], [30]. However, the role of the caecal tonsils in the development of local and systemic immune responses to Salmonella is still not well-characterized. This organ is important as it seems likely that it has a major controlling influence on entry of bacterial and other pathogens into the ceca. In mammals, there is evidence that Salmonella can invade the specialized epithelial cells, microfold (M) cells, that are present on the epithelial lining of the gut-associated lymphoid tissues (GALT), such as the Peyer's patches, which sample and transport the luminal antigens into the sub-epithelial lymphoid tissues [31]. In poultry, the molecular basis underlying Salmonella invasion and pathogenesis is unclear. However, it is suggested that the systemic Salmonella serovar, S. Gallinarum, also displays such tropism to lymphoid tissues, such as Peyer's patches and cecal tonsils, and can cross the gut during the early stages of fowl typhoid and enter systemic sites via enterocytes and the intestinal lymphoid tissues [32].

The development of more rational approaches to vaccination will require a better understanding of the pathogen and host immune responses. Therefore, in this study we investigated the changes in cellular composition and cytokine and chemokine expression in the cecal tonsil of chickens following infection with Salmonella serovars known to have different biological and pathological characteristics to determine how far the immune response to these pathogens is associated with the differences in the infection biology. The gene expression profile of the spleen was also studied in response to Salmonella infection. From the above introduction it is clear that S. Gallinarum (invasive, producing typhoid, non-colonizing), S. Pullorum (invasive, typhoid in very young birds, non-colonizing), S. Enteritidis (invasive but non-typhoidal, colonizing) and S. Infantis (poorly invasive, no disease in chickens, colonizing) represent different virulence characteristics within those Salmonella enteric serovars that infect poultry. Representative strains whose biology has been studied previously were therefore to be used.