BT9 Notes for AB2 Salmonella Lecture (M Stevens) PDF

Lecture title: Salmonella Lecturer: Professor Mark Stevens Learning outcomes To know how Salmonella are detected, classified & traced To know the diseases caused by Salmonella enterica serovars in domestic animals & humans To understand the role of host- & serovar-specific factors in the outc...

Lecture title: Salmonella Lecturer: Professor Mark Stevens Learning outcomes To know how Salmonella are detected, classified & traced To know the diseases caused by Salmonella enterica serovars in domestic animals & humans To understand the role of host- & serovar-specific factors in the outcome of infection To know that some serovars are a significant public health concern & understand how zoonotic infections are controlled To know the mechanisms underlying Salmonella pathogenesis To understand how these differ from strategies used by other enteric pathogens to produce disease Salmonella nomenclature Currently there are 2 species; S. enterica and S. bongori. The main interest is with the sub- species of S. enterica: enterica, diarizonae, salamae, houtenae, arizonae, indica. The key bacteria of interest are then serovars of Salmonella enterica sub-species enterica: e.g. Typhimurium, Dublin and Enteritidis. So short cut to: S. Typhimurium, S. Dublin and S. Enteritidis. Officially serovars are written with a capital letter and not in italics (as not species). These serovars are traditionally identified by their O and H antigens (i.e. by ‘serum’ recognition, hence serovar/serotype). Salmonella detection Enrichment culture based on unique aspects of Salmonella metabolism (e.g. ability to use tetrathionate as an electron acceptor in respiration) can be used to select Salmonella from samples containing other enteric bacteria. Chromogenic media can be used to detect Salmonella-specific activities (e.g. H2S production on XLD agar). In addition to serotyping, analysis of phage and antibiotic sensitivity or genome structure by pulsed-field gene electrophoresis, multi-locus sequencing typing and other methods can be used to identify sources of infection and monitor trends. Salmonella infections Salmonella strains are found in the intestines of a wide variety of animals, including fish, reptiles, birds and mammals. In most mammals, Salmonella strains are not present as commensals but can be shed asymptomatically in long-term ‘carrier states’ that follow acute infection. This is even more likely with birds and reptiles from which asymptomatic shedding can be a health risk to owners. The main disease syndromes associated with Salmonella infections are: 1. Enteritis; watery, foul-smelling, sometimes mucus containing and bloody diarrhoea. This leads to severe dehydration, electrolyte loss and acid/base imbalance. 2. Septicaemia; fever, loss of appetite, depression, reduced milk production in cows. Infections are severe and acute, sometimes fatal. This is a major issue for large poultry units. 3. Abortion; very rare but low level infections can be ‘activated’ during pregnancy leading to loss of the foetus. Salmonella serotypes can be broadly grouped into those generally specific to a host and those capable of causing infections across a range of animal species. In healthy outbred adults of a 1 given host, host-specific strains tend to cause more serious systemic disease, whereas the non-host-adapted (ubiquitous) strains tend to be restricted to the gut and may cause gastroenteritis. The non-adapted strains often cause zoonotic infections in humans. Host-adapted Non-adapted Cattle S. Dublin S. Typhimurium Sheep S. Montevedio S. Typhimurium S. Abortosuis S. Derby Pigs S. Choleraesuis S. Typhimurium Poultry S. Pullorum S. Enteritidis S. Gallinarum S. Typhimurium Human S. Typhi S. Typhimurium S. Paratyphi S. Enteriditis Zoonotic Disease As can be seen from the list above, the main issue of human infection from animals arises with S. Typhimurium and S. Enteritidis, in particular arising from poultry and pigs. These serovars make up about two thirds of Salmonella cases in the UK, other serovars that are emerging include: S. Virchow and S. Hadar. These are non-adapted strains that generally cause gastroenteritis, whereas the animal-specific infections (like typhoid fever in humans) cause systemic and potentially lethal infections. Salmonella infections in poultry The majority of infections are asymptomatic. Host-specific avian disease caused by S. Pullorum and S. Gallinarum is rare in the UK but can have devastating consequences, with mortality rates in flocks up to 100%. These diseases have been largely eliminated by serology testing and slaughter. Vertical infection is possible, with ovary infection leading to yolk sac infection and disease in the hatching chick. The same route accounts for infection of eggs by S. Enteritidis, though use of inactivated and live-attenuated vaccines in layers and broiler breeders under The 2 Lion Code has substantially reduced this. The Lion Code also includes measures to improve on- farm biosecurity and best-before dates. Salmonella infections in pigs In addition to poultry, pigs are considered an important source of food-borne Salmonella, and S. Typhimurium is the most common serovar isolated from pigs. Acute infections typically involve diarrhoea, vomiting and pyrexia but can be followed by a persistent carrier state. Swine typhoid/hog cholera caused by S. Choleraesuis has not been seen in the UK for many years, but remains common in some countries. A pressing need exists to reduce Salmonella in pigs to control zoonosis, possibly by vaccination. Such an approach would have to work alongside current meat juice ELISA testing under an existing Zoonoses Action Plan, leading to a call for DIVA vaccines, those that differentiate infected from vaccinated animals. Salmonella infections in cattle and sheep In the UK, serious host-adapted infections in cattle are caused by serovar Dublin, which can also be associated with enteritis along with serovars such as Newport and Typhimurium. In sheep the key subspecies is diarizonae. Infections during pregnancy can lead to abortion. Salmonella Pathogenesis For the majority of infections, the bacteria are ingested and are then capable of invading epithelial cells lining the gastrointestinal tract, particularly in the small intestine. So in contrast to most of the E. coli strains discussed in the last lecture, Salmonella strains induce their own uptake into eukaryotic cells. Initial contact with host cells is probably through flagella and fimbriae (type 1 fimbriae, SEF, PEF & LPF), but Salmonella strains express at least two type III protein secretion systems (T3SSs). One of these (T3SS-1) injects effector proteins that induce cytoskeletal changes resulting in bacterial invasion; the bacteria enter the cell inside a ‘Salmonella containing vacuole’ (SCV). Important effector proteins driving this uptake are SipA, SopB and SopE. Such proteins hijack cellular pathways, for example by mimicking the function of eukaryotic proteins. There are reports that Salmonella targets microfold (M)-cells for invasion, although the bacteria also invade via enterocytes. Once inside the vacuole, other proteins are secreted by a second type III secretion system (T3SS-2) into the cytoplasm of the eukaryotic cell. These prevent assembly of phagosome oxidase (inhibiting the oxidative burst) and alter the trafficking of the SCV to prevent fusion to lysosomes, therefore reducing the likelihood of the Salmonella being killed in the vacuole. The bacteria can replicate in the cells, eventually being released following cell death into the lamina 3 propria. Important effector proteins secreted by the second type III secretion system include SpiC, SseL and SifA. The cytoskeletal changes induced by the bacteria, interference with signalling pathways, disruption of tight junctions and the loss of epithelial cells all impact on barrier integrity and adsorptive function leading to diarrhoea. In addition, intracellular bacterial products will be recognised by cellular PRRs and once through the epithelial barrier, extracellular PRRs will recognise bacterial products such as LPS and flagella, both sets triggering inflammatory responses, leading to fluid and cellular influx contributing to diarrhoea. As with many pathogens the genes for virulence factors are clustered together on pathogenicity islands within the chromosome. Salmonella pathogenicity island 1 (SPI-1): is required for invasion of epithelial cells. This island encodes T3SS-1, which induces membrane ruffling, cytoskeletal rearrangements and bacterial uptake. Salmonella pathogenicity island 1 (SPI-2): is required for bacterial survival and multiplication within the phagosome. The genes for T3SS-2 are encoded on SPI-2 and are preferentially expressed in the host cell. Other important virulence factors include fimbrial adhesins (potentially 13 different types in some strains); flagella, iron acquisition systems and two-component signal transduction systems that inform the bacteria about their environment and therefore allow appropriate gene expression (e.g. of SPI-2 genes). The main difference between host-adapted and non-adapted serovars appears to be in how successfully (or not) the bacteria are cleared following this initial invasion though the epithelial barrier of the gastrointestinal tract. Serovars that elicit strong intestinal inflammatory responses tend to be controlled at the mucosal surface via recruitment of neutrophils whereas serovars associated with systemic disease tend not to cause enteritis and may disseminate by ‘stealth’. In S. Typhi and some S. Dublin strains, this is partly due to the Vi capsule, which masks underlying LPS from TLR4. Typhoid-associated serovars traffic in the blood and lymph to multiple organs in the animal (especially the spleen and liver). High bacterial loads at these sites and in the blood can exacerbate inflammatory responses, especially to LPS (toxin shock), which can be fatal. Salmonella as live oral vaccines Salmonella spp. elicit good humoral and cell-mediated responses. Attenuated strains are used that cannot grow properly in the host, e.g. by limiting aromatic amino acid biosynthesis. Such attenuated bacteria can also be engineered to express other antigens (from plasmids) to provide protection against other infectious agents in addition to Salmonella. 4