Cytokines are defined as regulatory peptides, with molecular weights typically less than 30 kDa, that act as extracellular signals between cells of the immune system (Eckmann and Kagnoff, 2001). Cytokines can be produced by nearly every cell type and have pleiotrophic effects involving regulating and eliciting immune responses (Kaiser, 2008). Many of the identified mammalian cytokines are present in avian species, although fewer cytokines have been identified in birds compared to mammals (Kaiser et al., 2005). Avian cytokines typically have 25-35% amino acid identity to
mammalian orthologues (Weining et al., 1998, Schneider et al., 2000, Degen et al., 2004). Currently, genes for 35 cytokines have been identified in the chicken genome including, 23 interleukins (IL), 8 type I interferons (IFN), IFN- γ, granulocyte macrophage colony-stimulating factor (GM-CSF), and 2
transforming growth factors (TGFs) (Kaiser et al., 2005). Chemokines have more restricted effects on the immune response compared to cytokines, being responsible for regulating circulation of immune cells and recruiting them to sites of inflammation (Kaiser, 2008). Chemokines can be divided into four groups based on the spacing of the first two cysteines at the amino termini including XC, CC, CXC and CX3C (Kaiser, 2008, Martins-Green, 2001, Hughes et al., 2007). A study on the chicken genome has identified 24 chemokines (Kaiser et al., 2005, Kaiser, 2008).
In mammals, cytokines and chemokines can be polarized functionally into type 1 or type 2 immune pathways. These responses are regulated by Th1 and Th2 cells. The chicken immune response differs from the mammalian immune response, as it lacks components of the Th2 response, including eosinophils and IgE (Kaiser et al., 2005). Additionally, a chicken orthologue for the Th2 cytokine IL-5 has been identified as a pseudogene (Avery et al., 2004). This suggests chickens may have a reduced requirement for Th2 cytokines, compared to mammals; however, a cluster of Th2 cytokines has been identified in chickens, encoding for IL-3, IL-4, IL-13 and GM-CSF (Avery
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et al., 2004). Chicken IL-4 and IL-13 expression in lymphoidal tissue has been shown to be similar to that in mammals and co-stimulates the
proliferation of B cells with CD154 (CD40) (Avery et al., 2004). However, IL-3 is expressed at high levels in lymphoidal and non-lymphoidal tissue
compared to mammalian species, suggesting it is differentially regulated in chickens (Avery et al., 2004).
Th1 cytokine responses in avian species have been characterised in more detail then Th2 responses. Early production of IL-12 and IL-18 drive Th1 inflammatory responses and production of IFN-γ (Eckmann and Kagnoff, 2001). IL-12 is a heterodimer, consisting of two subunits, p35 and p40 (Degen et al., 2004). Several lymphoidal cell lines including, HD11, CU91 (T cell derived) and DT-40 (B cell derived) produce IL-12 following stimulation with ligands such as LPS, CpG-ODN and chCD40 (Degen et al., 2004). Chicken IL-12 has been shown to stimulate significant proliferation of spleen cells and production of IFN-γ by T-cells (Degen et al., 2004). ChIL-18 exhibits about 30% sequence similarity to mammalian IL-18 (Schneider et al., 2000). ChIL-18 is important for systemic resistance to Salmonella infection
(Raupach et al., 2006). Macrophages from resistant chickens produce significantly greater IL-18 than those from susceptible ones (Wigley et al., 2006). This suggests that Salmonella-resistantmacrophages are more efficient at stimulating IFN-γ production and initiating the adaptive immune response (Wigley et al., 2006). Biologically activate IL-18 can stimulate primary chick spleen cells to produce a significantly increased amount of IFN- γ (Schneider et al., 2000).
A third lineage of Th cells has been identified, called Th17, which produces IL-17 under the influence of IL-23 (Kaiser, 2008). There are 6 members of the IL-17 family in humans, of which 4 of these have been identified in the
chicken genome (Kaiser et al., 2005). IL-17 is thought to contribute to inflammation in the gut, following S. Enteritidis infection of chickens (Crhanova et al., 2011).
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1.7
Salmonella Virchow
Since the 1990s, S. Virchow has increased in prevalence in the UK and is often reported as the third most frequent serovar isolated from human salmonellosis cases (Ispahani and Slack, 2000, Matheson et al., 2010). S. Virchow infection is most commonly associated with gastroenteritis in humans, but has been frequently associated with bacteraemia, especially in the immuno-compromised and in children (Matheson et al., 2010, Ispahani and Slack, 2000, Gulcan et al., 2012). In rare cases, S. Virchow has also been shown to cause severe complications with other illnesses, such as deep vein thrombosis and has been shown to cause invasive disease in otherwise healthy adults (Schifferdecker et al., 2009, Eckerle et al., 2010). The main source of S. Virchow infection in humans is thought to be poultry, however sporadic cases of S. Virchow from different sources have been reported (Bennett et al., 2003, Callaway et al., 2011, Sato et al., 2000). Five serovars including S. Virchow have been given priority in prevalence studies on farms by the EU, for control of entry into the food chain, due to their significant risk to public health (Arnold et al., 2010, Snow et al., 2008, Snow et al., 2007).
S. Virchow has a uniquely high prevalence and association with invasive disease in some countries. In Israel, S. Virchow is 1 of 3 serovars most commonly isolated from human patients and accounted for 15% of all stool and 22% of all blood isolates between 1997 and 2002 (Weinberger et al., 2006). Studies in Israel have found S. Virchow to be very invasive in children and the elderly and to have a high resistance to antibiotics (Weinberger and Keller, 2005, Weinberger et al., 2006, Weinberger et al., 2004). S. Virchow is also frequently isolated from humans in Switzerland, being ranked between the 4th and 8th most frequently isolated serovar between 2004 and 2009 (Bonalli et al., 2011). In other countries, including the United States, S. Virchow infection in humans is uncommon compared to other serovars,
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Clinical evidence from a combined dataset of 10 countries indicates that there is a high association between S. Virchow and invasive disease,
although invasiveness of Salmonella serovars varies from country to country (Langridge et al., 2009, Jones et al., 2008). The invasive index for S.
Virchow, based on the ratio of the number of blood culture positives divided by total isolations of each serovar, is 4.4, more than double that of S.
Enteritidis (1.8) and S. Typhimurium (1.6) that numerically cause the greatest burden of non-typhoidal salmonellosis (Langridge et al., 2009).
Increasing antimicrobial resistance of S. Virchow has been reported in previous studies. A study in France and Belgium found some S. Virchow isolates express the blaCTX-M-2 gene, which is located on a large conjugative plasmid (Bertrand et al., 2006). This gene confers resistance to ciprofloxacin, which is a fluoroquinolone used to treat invasive salmonellosis in immuno- compromised patients (Bertrand et al., 2006). A high incidence of resistance to nalidixic acid, a quinolone used to treat invasive salmonellosis, has also been reported in S. Virchow, with 90% of strains isolated in Israel between 1997 and 2004 conferring resistance (Solnik-Isaac et al., 2007). A study comparing antimicrobial resistance genes in S. Virchow, S. Enteritidis and S. Hadar strains, isolated from humans and food-producing animals in England and Wales found S. Virchow strains, isolated from humans, had a greater number of different resistance genes compared to the other 2 serovars (Hopkins et al., 2007). A wider study testing non-typhoidal Salmonella strains, isolated from 10 different European countries, for antimicrobial susceptibility showed that 73% of S. Virchow isolates were resistant to at least one antimicrobial and had an increased resistance to all of the antimicrobials tested, particularly nalidixic acid (Meakins et al., 2008). Multiple drug resistant (MDR) clonally related S. Virchow isolates, from humans, have been detected that express Salmonella genomic island I (variant SGI I-J3), which is largely responsible for the diversity of MDR S. Virchow isolates (Chu et al., 2012). A widespread increase in antimicrobial resistance of S. Virchow is a major concern to public health, as the serovar is associated with invasive disease and antimicrobials are used to treat invasive salmonellosis.
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