Salmonella spp. H antigen. O antigen

Transcription

1 Salmonellosis

2 Salmonella spp. H antigen O antigen!more than 2000 serovar! Gram negative, rod shape,non spore forming bacteria! Facultative anaerobes!optimum growth at 37 C and grow well at 43 C!Motile (except S. gallinarum and S. pullorum)!cause Salmonellosis in humans and animals (broad host ranges)! Worldwide distribution

3 Salmonella spp! Widespread occurrence of Salmonellosis in animals, especially in poultry and swine! Environmental sources of the organism include water, soil, insects, factory surfaces, kitchen surfaces, animal feces, raw meats, raw poultry, and raw seafoods! S. typhi and the paratyphoid bacteria are normally caused septicemia and produce typhoid or typhoidlike fever in humans!other forms of Salmonellosis generally produce milder symptoms.

4 Salmonellosis!Salmonella sp. are second only to Campylobacter as a cause of bacterial diarrhea illness in the United States!It is estimated that from 2 to 4 million cases of Salmonellosis occur in the U.S. annually!salmonella enteritidis has ranked first or second in frequency of isolation from humans since 1988 and accounted for 21% of reported isolates in 1993.! Each year, an average of 55 outbreaks of SE infections are reported to CDC! approximately 11% of patients are hospitalized, and 0.3% die!salmonella typhimurium and Salmonella dublin

5 ! Unique characteristic Salmonella spp.!mesophiles (Temperature 7 C - 45 C)!Catalase positive and oxidase negative!infective dose: high infective dose (>10 5 CFU/ g)!lower infective doses for young infants and adult in poor health!bacteria can survive but not grow!not grow if water activity less than 0.95!Not grow if ph less than 4.6!Heat sensitive: Die if temperature more than 60 C

6 !Foodborne infection Salmonellosis! Incubation period: 6-36 hrs (usually hrs)!duration of illness: 1-7 days!clinical signs! fever! Acute gastroenteritis: Nausea, Vomit and diarrhea! Abdominal pain, abdominal cramps! Dehydration in severe cases (infant or elderly)! Arthritic symptoms may follow 3-4 weeks after onset of acute symptoms!low fatality rate! S. enteritidis fatality rate 0.3%

7 Salmonellosis!Foodborne infection!colonization of bacteria in the intestine!distal small intestine and colon!competitive with normal flora (produce organic acid)!invasion of the intestinal epithelium!invade villous tip of the ileum and colon!penetrate brush borders and enter cell!stimulate inflammatory response!stimulation of fluid secretion into intestinal lumen with the consequent watery diarrhea!intestinal mucosa releases prostaglandin which activate adenylate cyclase!release vasoactive substances that increase permeability of mucosal vessel

8 Salmonella spp.. outbreaks!most common: raw meats, poultry, eggs,! Less common: milk and dairy products, fish, shrimp, vegetable! Yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, and chocolate! High fat content! Various Salmonella species have long been isolated from the outside of egg shells. The present situation with S. enteritidis is complicated by the presence of the organism inside the egg, in the yolk.! Vertical transmission, i.e., deposition of the organisms in the yolk by an infected layer hen prior to shell deposition. Foods other than eggs have also caused outbreaks of S. enteritidis disease

9 Salmonella spp.. outbreak! 1985 Salmonellosis outbreak! involving 16,000 confirmed cases in 6 states! low fat and whole milk from one Chicago dairy.!salmonellosis in restaurant! S. enteritidis was isolated from employees and patrons of three restaurants of a chain in Maryland! At least 71 illnesses resulting in 17 hospitalizations! Scrambled eggs from a breakfast bar were epidemiologically implicated in this outbreak and in possibly one other of the three restaurants! The plasmid profiles of isolates from patients all three restaurants matched.

10 Bacterial plasmid from food sample Plasmid profiles A B Outbreak plasmid A B Outbreak Cut with restriction enzymes plasmid

11 Salmonella spp.. outbreak! 1984 Salmonellosis outbreak! 186 cases of Salmonellosis (S. enteritidis) were reported on 29 flights to the United States on a single international airline! An estimated 2,747 passengers were affected overall! No specific food item was implicated, but food ordered from the first class menu was strongly associated with disease!1994 S. typhimurium outbreak,! Wisconsin: 17 patients with acute gastrointestinal illness characterized by diarrhea and abdominal cramps! At least 14 patients reported having eaten raw ground beef that was either plain or seasoned with onions and an herb mix during the 72 hours before illness onset! Stool samples for culture were obtained from 11 patients; Salmonella serotype Typhimurium was isolated from seven specimens.

12 Salmonella spp.. outbreak! The CDC estimates that 75% of those outbreaks are associated with the consumption of raw or inadequately cooked Grade A whole shell eggs! The U.S. Department of Agriculture published Regulations on February 16, 1990, in the Federal Register establishing a mandatory testing program for egg-producing breeder flocks and commercial flocks implicated in causing human illnesses!this testing should lead to a reduction in cases of gastroenteritis caused by the consumption of Grade A whole shell eggs.

13 Salmonellosis outbreak study Outbreak of Salmonella enteritidis Associated with Nationally Distributed Ice Cream Products! From September 19 through October 10, 1994,! a total of 80 confirmed cases of Salmonella enteritidis (SE) infection were reported! Cases were characterized by diarrhea, abdominal cramps, and fever! Recent increases in SE cases also were reported from South Dakota (14 cases during September 6-October 7, compared with 20 cases during all of 1993) and Wisconsin (48 cases during September 6- October 7, compared with 187 during all of 1993).! This report summarizes preliminary findings from the outbreak investigation.

14 Salmonellosis outbreak study!epidemiological investigation! to assess potential risk factors for infection! the department of health conducted a case-control study of 15 cases and 15 age- and neighborhood-matched controls.! A case was defined as culture-confirmed SE in a person with onset of illness during September. Eleven case-patients (73%) and two controls (13%) reported consumption of Schw an's ice cream within 5 days of illness onset for case-patients and a similar period for controls! Odds ratio= 10.0; 95% confidence interval=

15 Salmonellosis outbreak study!epidemiological investigation! Press releases informing the public of this problem! Advised persons who had been ill since September 1 and who had consumed Schwan's ice cream to contact the health department! During October 8-11, a total of 2014 persons who had consumed suspected products and had been ill with diarrhea contacted the DH by telephone! Samples of ice cream from households of ill persons grew SE! These products had production dates in August and September! The implicated products are distributed nationwide, primarily by direct delivery to homes, and are sold only under the Schwan's label! Investigations to examine the extent and causes of the outbreak are under way.

16 Salmonellosis outbreak study!s. enteritidis contamination in ice-cream Plasmid!Plasmid profile!copy number!cut with restriction enzyme!etc!compare isolates from patients, ice-cream, raweggs Cut with restriction enzymes PatientsI ce-cream Egg

17 !Recall product Salmonellosis outbreak study! On October 7, the company voluntarily stopped distribution and production at the Marshall plant pending further findings from these investigations! Product recall! Find source of contamination! Source of contamination?!check HACCP (critical control point)!pasteurization? Milk/Ingredient!Transportation? Milk/Ice-cream!Milk was pasteurized at one place and transported to ice-cream plant by truck

18 Salmonellosis outbreak study! Source of contamination?!milk was pasteurized at one place and transported to ice-cream plant by truck!truck tank clean?!sanitation before and after transportation!what is the source of S. enteritidis?!some trucks also transport liquid egg!cracking of tank seal!se survived from sanitation!contamination of pasteurized milk!contamination of S. enteritidis in ice-cream!source of S. enteritidis is liquid egg

19 Role of Veterinarian!Salmonella free farms! Prevent horizontal and vertical transmission!slaughter house hygiene! Meat hygiene and inspection! Meat quality&trade barrier!outbreak investigation! Department of health! Diagnosis and researches!education

20 Salmonella spp.!control of Salmonella spp. infection!education: Good hygiene practices!consumer!food handler!reduce incidence of microorganism at farm level and raw food!reduce incidence of microorganism at processing plant!proper storage!temperature < 5 C : not grow!proper food handling!heat treatment: Cooking temperature

21 Isolation of Salmonella from food!at least 4 days to isolate Salmonella from food! 5 steps to isolate Salmonella! Nonselective pre-enrichment! Selective enrichment! Selective plating! Biochemical confirmation! Serology confirmation

22 Isolation of Salmonella from food! Nonselective pre-enrichment! 25 g of sample ml of Lactose broth! Incubate 24 hrs at 35 C! Purpose: to facilitate the isolation! Lactose broth, nutrient broth and tryptic soy broth (TSB)! to give organisms that may have been injured in food processing steps a chance to repair any injuries before being exposed to selective agent

23 Isolation of Salmonella from food!selective enrichment! portion of nonselective broth is transferred to selective enrichment (1 ml of Lactose broth to SC and TT)! two selective enrichment broth!selenite Cystine (SC)!Tetrathionate (TT)! Incubate 35 C 24 hrs

24 Isolation of Salmonella from food! Purpose: to facilitate the isolation! Selenite Cystine (SC)!Selenite: Reduces the growth of fecal coliforms and enterococci! Tetrathionate (TT)!Tetrathionate: Inhibits or kills coliforms and enhances the growths of S. typhi and S. paratyphi!iodine and Brilliant green: Inhibits non-salmonellae

25 Isolation of Salmonella from food!selective plating! Streak for isolation from the SC and TT broth onto 3 agar media!xld agar!he agar!bs agar! Incubate 35 C 24 hrs! Purpose: to obtain isolated colonies of Salmonella and to differentiate Salmonella from other organisms! Salmonella not ferment lactose! Salmonella produce hydrogen sulfide

26 Isolation of Salmonella from food! XLD agar (Xylose lysine desoxycholate)! Desoxycholate: selective for enteric organism! Xylose: fermented rapidly to acid by most enteric organisms! Lysine: selective for salmonella! Salmonella red colonies with or without black centers (Coliforms develop as yellow colonies)! HE agar (Hektoen enteric agar)! Bromthymal blue and fuschin: Indicators of lactose fermentation! Thiosulfate and ferric ammonium citrate: Hydrogen sulfide organisms! Salmonella blue-green colony (Coliforms develop pink to orange colony

27 Isolation of Salmonella from food! BS agar (Bismuth sulfite agar)! Highly selective! Use to isolate S. typhi from feces and sewage! Bismuth sulfite and brilliant green: Inhibits gram-positive and coliform organisms! Salmonella brown to black color with metallic sheen (non-salmonella develop brown green colonies)! Other selective plating media! BGA (Brilliant green agar)! SS (Salmonella Shigella agar)

28 Isolation of Salmonella from food! Biochemical confirmation! Select one typical colony from XLD, HE, BS! Inoculate (Streak and stab technique)!triple sugar iron (TSI)!Lysine iron agar (LI A)! Incubate at 35 C 24 hrs

29 Isolation of Salmonella from food! Purpose: non selective differential media! TSI (Triple sugar iron agar): Fermentation of dextrose, lactose and sucrose is detected by conversion of phenol red indicator! Salmonella acid butt (yellow) and alkaline surface (red)! Purpose: non selective differential media! LIA (Lysine iron agar): Dextrose is rapidly fermented by enteric organisms and Bromcresol purple as indicator! Salmonella decarboxylate lysine change ph purple color both butt and surface! LIA: Hydrogen sulfide detection

30 Isolation of Salmonella from food TSI Organisms Slant Surface Gas H2S E. coli A A + - S. enteritidis K A + + LAI Organisms Slant Surface Gas H2S E. coli K K - - S. enteritidis K K + +

31 Isolation of Salmonella from food!serological confirmation! Inoculate growth from slant into BHI broth! Incubate at 35 C 6 hrs! 0.5 ml of sample ml of Salmonella polyvalent antiserum (One loopful for slide agglutination)! Incubate at C! Observe every 15 mins! Results: Positive Agglutination! Purpose: Use to confirm that Salmonella has been isolated or to determine what species of Salmonella is present

32 Non selective pre-enrichment Selective enrichment Sample 25 ml Lactose broth 225 ml 37 C 24 hrs SC TT 37 C 24 hrs Biochemical confirmation Selective plating TSI LI A 37 C 24 hrs XLD HE BS