Salmonella food poisoning ( Salmonella enterica subs enterica )

1. Coloured in by selecting area , and format>autoshape>fill . NB >Line could have been selected to give a border, instead. Salmonella food poisoning(Salmonella enterica subs enterica) An example of possible style. Best viewed in the “slide show” mode in power point, press escape to get out of presentation mode November 2001

2. Background: Salmonella is a Gram negative motile rod. The species are distinguished as : Serotypes . Over 2000 serotypes are known. The serotypes in the past have been given species status, (cf. E coli and see Panel 1). A few of are specific to one or two hosts causing systemic diseases (severe), eg S typhi and S paratyphi in humans S gallinorum and S pullorum in chickens S dublin in cattle S cholerae-suis in pigs S typhimurium and S enteritidis in mice Note that S typhimurium and S enteritidis also cause typhoid-like disease in humans (systemic) and in poultry (esp new chicks or stressed laying hens) . Incidence of Salmonella food poisoning Belongs to infectious type ie Live organisms needed. Salmonella enteritidis became a nuisance in mid 1980s through vertical and horizontal transmission in large poultry farms world-wide. (O,Brien 1990 and Davies et al 1997) Background and trends of disease • Panel 1: New naming system for SalmonellaWorld Health Organisation (WHO) Collaborating Centre for Research on Salmonella at the Institut Pasteur, Paris • Based on DNA hybridisation studies : serotypes belong to one group i.e. a single species, like E coli serotyping • Thus S enteritidis PT4 should be S enterica subsp. enterica serotype Enteritidis PT4 . • Much better?!Some serious pathogens have kept their names (and species): eg Salmonella typhi and Salmonella paratyphi Caused persistent infections due to carry-over on fomites (See Fig 1 for trends of disease incidence). Outbreaks have been mainly associated with poultry products :chicken/turkey and hens' eggs. In France : 50% of food poisoning is due to Salmonella enteritidis and typhimurium ( = Salmonella enterica subsp. Enterica) Barrbezange et al 2000,

3. Transmission: Source Chicken carcass • From fig 1 it can be seen that there was an increase in Salmonella food poisoning in the 1980’s, but as a result of control measures (slaughter of infected flocks) we now have a decline in disease incidence. • Transmission • Salmonella is persistent in the environment of breeders, layers, and broilers with an ease of the faecal-oral route. Thus there is a large reservoir of chronic carriers. Note that mice can transmit their strains to chickens and then on to us. It is Also transmitted to humans via eggs. Uyttendale et al 1998, reports in Belgium, incidence of Salmonella on poultry products as % contaminated: • Year 1993 1994 1995 1996 • % contaminated 19.4 24.1 21.9 36.7 Fig 1 Trends in Salmonella food poisoning in England and Wales (PHLS 2001)

4. Control of the disease in hens, are based on controlling the gut flora (panel 2) 1. competitive exclusion Infection of newly hatched chicks with Salmonella causes massive gut colonisation and excretion in faeces. Later the chick's gut flora becomes more complex and helps to exclude Salmonella (shorter subsequent infections with fewer orgs excreted). Prophylaxis achieved feeding newly hatched chicks with microbes from gut flora to encourage rapid "adult" flora to be achieved. 2. AntibioticsNot recommended because of spread of antibiotic resistant strains to human pathogens 3. Vaccines using host-specific serotypes give good immunity. live strains (attenuated) avirulent strains (must be immunogenic and non-pathogenic) and genetically stable are used, as killed vaccines are not as affective. Interestingly, administration of live vaccines may encourage change in gut flora to that of the adult chick (as well as immunity to the Salmonella used) (Zhang-Barber et al 1999)) Vaccines against non-host specific strains (eg food poisoning) still "empirical”. There has been some success eg with S typhimurium Combinations Vaccine and competitive exclusion found advantageous (Methner et al 1999) Transmission Chicken Carcass: Control • Panel 2: Control methods for Salmonella in Hens and their eggs • Competitive exclusion • Antibiotics • Vaccines • Combinations

5. Eggs as a source of Salmonella Food poisoning • Source: Eggs From a Recent 1997 statistical study in Wales (Hayes et al 1999) • Pandemic of Salmonella enteritidis (PT4) in late 1980s due to hen's eggs, lead to • Compulsory testing of laying flocks • Slaughter of infected flocks • In 1991 0.2-1.6 % of eggs contaminated with Salmonella in England and Wales • Northern Ireland, about the same 0.43% over a period 1996-7 • Visible egg contamination and cracks not a reliable indicator of contamination • 27 million eggs eaten in UK each day, at 1% contamination would have 270,0000 contaminated eggs per day consumed! • Contamination cannot be predicted by appearance of eggs • Food poisoning from eggs is associated with raw and undercooked egg and mayonnaise sandwiches.Contamination in two ways (eg see Himanthongkham et al 1999): • i) Penetration of egg from outside shell. Shell membrane keratin and mucin) inhibits multiplication • ii)From poultry with systemic disease of Salmonella • Government advice: • Pasteurised eggs should be used by catering industry (but in fact they are not often used by home caterers!) • National recommendation is to wash hands after handling shell eggs. • Refrigerate eggs for storage (Salmonella cannot grow below 8°C)

6. An outbreak of suspected Salmonella food poisoning was investigated in a small seaside town. It was noted that some cockles had been caught and sold locally. Could these be the source of infection? Cockles are filter feeders. Thus they will filter out Salmonella and other coliforms from polluted water. Owner of a shop had S enteritidis PT19 in stool sample (but showed no symptoms) (so did her 8-year-old son, but with symptoms) No positive cultures from seas or new cockles! Cockles in UK are depurated (stored in irradiated water (UV) for 42h) to rid them of pathogenic bacteria (not viruses) Can be heat treated as an alternative 90C for 90s (eliminates viruses) Should be harvested from a classified area of sea bed, these were not. A member of the public had collected and boiled cockles, (no official record of this) Cooled them in running water while de-shelling. stored overnight in fridge. sold them to fish and chip shop, transporting them in a cool box mostly refrigerated, some displayed at room temperature during shop hours How was this found out? Where did the disease originate? The seller of the cockles or the fish and ship shop owner?NB Med micro students would need to supply the answer to this in this assignment (the actual text came from a problem in Food Microbiology module!)Also note that sometimes I have changed the font size from 12 to 14, for effect Case Study: Cockles (Greenwood et al 1998) Salmonella enteritidis PT4

7. Course of disease Rapid onset: 8-48 hours Duration: 2-5 days Symptoms : Nausea, vomiting, diarrhoea; low-grade fever. PathogenicityMechanisms best understood with S typhimurium infection of mice as a model for a systemic (eg via S typhi) in humans. The stages are. transport to gut wall adhesion to epithelial cells invasion of host cell destruction by host defence system Endotoxin production and symptoms recovery . Generally antibiotics are not needed. The patient recovers naturally Host defence systems to be overcome by Salmonella low pH in stomach peristalsis in intestine (sweep bacteria away?) Normal intestinal flora (competition for nutrients, inhibitors) Mucus Endotoxin Lipopolysaccharide (LPS) complex of outer envelope of Gram negative microbes such as E coli Shigella and Salmonella. Injection of killed Salmonella or purified LPS cause reactions such as: Fever Altered blood cell counts Intravascular coagulation Hypotension (drop in blood pressure) Shock Death And interestingly : Tumour necrosis The Lipopolysaccharide in fact has two components : Core (R) polysaccharide which is a short chain of sugars and the O antigen , and a side chain of 3-5 sugars attached to the R component. Toxicity associated with lipid A component and immunogenicity with the polysaccharide ( O antigenicity Pathogenicity and Course of disease

8. Pathogenicity • Pathogenicity islands Marcus et al ( 2000) • Recent work has shown that some pathogenicity genes are held not on the main chromosome but on pieces of DNA afloat in the cells: pathogenicity islands • At least 5 Salmonella pathogenicity islands (SPI 1-5) on chromosome associated with virulence of the organism. Some of the virulence genes are also on (conserved) plasmids. SP1 most characterised • How does Salmonella influence the host cell? Type III secretion system used to deliver virulence factors into host cells and alter host cell signalling mechanisms. SPI 1 and SPI 2 code for these activities: they are contact dependent (is SP 3 also?) • Products of SPI 1 enter M-cells of epithelium in gut and cause penetration of epithelium. Needle - like projections spanning host cell membrane and bacterial membrane have been seen. Is this how further gene products are injected (= translocation)? The M-cell membrane becomes ruffled..full of small finger-like projections during this attack phase. The bacterial cells are then enclosed in a vacuoles (macropinosomes). gene products are needed to force the host cell to do this. The cells are thought to be killed in these vacuoles whereupon endotoxins are release (left) and the disease symptoms occur.

9. Conclusions • It is evident that food poisoning by salmonella spp is still common throughout Europe, although the incidence of cases in England and Wales has been declining over recent years. The major sources are poultry meat and eggs. Recent step taken to prevent the young chicks from becoming infected with Salmonella on farms, eg attempts to control the gut flora by earlier colonisation of benign organisms. Alternatively, vaccination of hens against Salmonella is having some effect. • Great strides are being made in the understanding of the pathogenicity of the organisms, particularly with respect to the initial stages of cell adhesion. Pathogenicity island have been identified and their gene products are in the process of been identified. Will this lead to new vaccines preventing attachment of Salmonella to epithelial cells? • The disease is rarely fatal in humans however,and once contracted is over quickly. Treatment of patients is at present rare, and is done so with antibiotics. Certainly improved food hygiene (especially concerning egg handling) would be affective in reducing the incidence of the disease.

10. References • Barrbezange et al 2000, FEMS letters, 192,101-106 • Davies et al 1997, Veterinary microbiology58277-293*) • Greenwood et al 1998, Comm Dis and Public H1 35-37 • Hayes et al 1999:, Commun. Dis. Public. Health 2(1), 66-67*) • Himanthongkham et al 1999 J Food Microbiol 49161-167) • Marcus et al ( 2000), Int J Food Microbiol49 35-42*, • O,Brien 1990 World's poultry Science J, 46, 119-124, • PHLS 2001,disease facts, WEB site, www.phls.co.uk • Uyttendale et al 1998, Int J Food Microbiol. 40 1-8*, • Zhang-Barber et al 1999 Vaccine17 2538-2545*