Group members: Viruses and Some Other Proven and Suspected Foodborne Biohazards Groups 10B Name Matrix numbers NURSYAHIRAH BT MD SANI UK29035 NUR NATJWA FATIHAH BiNTI RAMLAN UK28984 SITI NORAIDA BINTI JASMANI UK30010 NURUL WAHIDATUL FATIMAH BINTI AZEMI UK24924

Viruses 4 types of incidence viruses in foods : Being obligate parasites - Do not grow on culture media as bacteria and fungi - Methods of its cultivation consists tissue culture and chick embryo technique Viruses do not replicate in foods - Their numbers may expected relatively low compared to bacteria - Extraction and concentration methods necessary for their recovery. Laboratory virological techniques are not practiced in many food microbiology laboratories Not all viruses of potential interest to food microbiologist can

Development of reverse transcription-polymerase chain reaction (RT- PCR) is a detection methodology. It has allowed direct detection of some foodborne viruses in oyster and clam tissue. This efficacy of RT-PCR technique to detect viruses in foods has been demonstrated by a number of researcher: - In 1 study, 4 concentration and extraction methods were compared for recovery of added astrovirus, hepatitis A and poliovirus from mussels, the glycine solutions and borate buffer methods were found to be best.

Process of RT-PCR

As stated by Cliver et al. noted that virtually any food can serve as a vehicle for virus transmission, and they have stressed the importance of anal-oral mode transmission especially for viral hepatitis of food origin. Nonintestinal bacteria of human origin are sometimes found in foods, the same may be true virus. But because of their tissue affinities , food would serve as vehicles only for intestinal or enteroviruses.

Incidence in Foods and Environment Common food source of gastroenteritis-causing viruses is shellfish. Crustaceans do not concentrate viruses, molluskan shellfish do because they are filter feeders. Shucked oyster artificially contaminated with 10^4 pfu of a poliovirus retained viruses during refrigeration for 30-90 days with a survival rate 10-13%.

Molluskan Shucked oyster Types of crustaceans species

Coliform index is proven value as an indicator of intestinal bacterial pathogens in waters It appears to be inadequate for enteroviruses, which more resistant to adverse environmental conditions than bacterial pathogens. In a study, enteroviruses were found 35% of time in waters that met acceptable standards for shellfish harvesting. Investigators concluded that coliform standard for waters does not reflects the presence of virus.

With capacity of certain viruses to persist in foods, it shown that enteroviruses persisted in ground beef up to 8 days at 23C or 24C. They were not affected by the growth of spoilage bacteria In study in 14 vegetables samples for existence naturally occurring viruses, none were found. But when coxsackievirus B5 inoculated onto vegetables it did survive at 4C for 5 days. Latter investigators in US examined 60 samples of market foods but were unable to detect any. They concluded that viruses in the US food supply are very low.

Coxsackievirus B5 Enteroviruses species and pathogenesis

Distruction in Foods The survival of hog cholera (HCV) and African swine fever viruses (ASFV) in processed meat was studied by McKercher et al. From partly cooked canned ham were prepared, virus was not recovered. They recovered after brining but not heating. HCV were survived when the addition of curing ingredients and retained viability even after 22 days.

The boiling of crabs was found sufficient to kill 99.9% of poliovirus 1 and rotavirus and echovirus were destroyed within 8 minutes. African swine fever viruses Hog cholera (HCV)

Hepatitis A Virus Prior to 1990s, there were more documented outbreaks of hepatitis A traced to foods than any other viral infections. The virus belongs to family Picomaviridae as do polio, echo and coxsackie viruses and all have single-stranded RNA. Fecal-oral route is the mode of transmission. Raw or partially cooked shellfish from polluted water

Largest foodborne outbreak of hepatitis A recorded in US occurred in November 2003. Vehicle foods that caused this foodborne was imported green onions (scallions) served by fast food restaurant chain. In 2001, an outbreak of >46 cases occurred in Massachusetts. It associated with the consumptions of sandwiches. 3 death600 victims

Noroviruses Former Norwalk, Norwalk-like and small-round- structured viruses (SRSV) have been placed in this group as genus Norovirus of human caliciviruses (HuCV). Norovirus in 2 genogroups: - I - II

Genogroups I Genogroups II Former Norwalk Former Snow Mounain viruses Similarities They are unenveloped ssRNA with a diameter of 27-40 nm Their genome consists of 7300 to 8300 base pairs

Norwalk virus was first recognized in a school outbreak in Norwalk, Ohio in 1968. Water was suspected but not proven as the source. It is the most prevalent of the noroviruses in foods. The virus is more resistant to destruction by chlorine than other enteric viruses. About 3.75 ppm chlorine in drinking water failed to inactivate the virus. Whereas poliovirus type 1

Noroviruses Poliovirus

Norovirus are now the leading cause of gastroenteritis in US with an estimated 23 millions cases per year During fall 2001, a rare waterborne outbreak of norovirus gastroenteritis occurred in state of Wyoming. There were around 84 victims. Ground water was contaminated with sewage. It was the source of human HuCV

Rotaviruses First demonstration of the virus in 1973 in Australia. First propagated in laboratory in 1981. 6 groups been identified. 3 known to be infectious for human. Group A: - Among infants - Young children throughout the world.

Group B: - Diarrhea in adults (been seen only in China) Rotaviruses belong to family Reoviridae. Characteristics: - 70nm in diameter - Nonenveloped - Contain double stranded RNA(dsRNA) Fecal oral route is the primary mode of transmission

Noroviruses Vs. Rotaviruses Noroviruses Rotaviruses They are unenveloped ssRNA with a diameter of 27-40 nm Their genome consists of 7300 to 8300 base pairs - Nonenveloped - 70nm in diameter - Contain double stranded RNA(dsRNA)

Peak season for infection occurs during winter months. Most susceptible are children between ages of 6 months and 2 years. Incubation period for rotavirus gastroenteritis is 2 days.

Symptoms: - Vomiting for 3 days accompanied by water diarrhea for 3-8 days - Often abdominal pain - Fever The host cell receptor protein for rotavirus serves as -adrenergic receptor. Once inside cells, they transported to lysosomes where uncoating occurs.

Rotaviruses

Yellow pigmented Enterobacter cloceae cause of neonatal necrotizing enterocolitis,neonetal meningtis and sepsis Although it considered to be an opportunistic pathogen- some strains produce enterotoxin-lethal to suckling mice Vehicles milk formulas - infant formulas contain Citrobacter freundii cause neonatal infection From 18 studies, 4 of them stated that E. sakazakii strains produce enterotoxin All 18 isolates were lethal to suckling mice at 10^8 cfu/mouse and 2 were lethal by peroral route Suckling mice- potential virulence is manifested by monolayers of CHO, Vero and Y-1 adrenal cells. Infant mortality rates 40%-60% In belgium 2 infants died- from unused prepared formula &

Studies of enteric bacteria From 141 powdered milk formulas 25% Pantoea agglomerans 21% E.cloaceae 14% E.sakazakii studies of 120 dried infant formulas in Canada - 6.7% contain E.sakazakii ISOLATED Test minimum growh T : 5.5- 8 C Max growth T : 41 -45C No growth at 4 C THERMAL RESISTANCE of E.sakazakii >most Gram negative bacteria Stationary phase of E.sakazakii is more resistant to osmotic and dry stress than E.coli and some other bacteria.

POISONING What is scombroid poisoning? Foodborne illness/allergic resulting from a contaminated fish that contain high amount of histamine (eg: tuna, mackerel etc..etc..etc) How histamine produced?? Does anyone here know? - Bacterial decarboxylation of generally large amount of histidine in musle (reported caused by sailfish non-scombroid) - May also from product which is organoleptically unacceptable contracted from both fresh and organoleptically spoiled fish.

ETHIOLOGICAL OF HISTAMINE-ASSOCIATED (SCOMBROID) POISONING Bacteria associated : 1. Morganella spp. - M. organii all starins produce histame >5000ppm - Isolate from temperature-abused albacore produced 5253ppm in tuna fish ( 25C) and 2769 ppm (15 C) - 4C no growth or production of histamine 2. P. phosphoreum produces histamine at fresh water Common member of the biota of pigs Significant pathogen Aligator Fish Turtles Frogs Snails Human Effect : i. diarrhea ii. Endocarditis iii. Meningtis iv. Soft tisue infection v. bacteremia Associated with diarrhea

Aeromonas Virulent strains of A.hydrophila produce 52 kDa single polypeptide possesses enterotoxic, cytotoxic and hemolytic activities Multifunctional molecules displays immunological cross reactivity with cholera toxin InvestigatorsIt is actually resembles aerolysin Aerolysin pore/ channel forming toxin kill cells by formring discrete Channel created by oligomerization of toxin molecule Cytotonic activity associated with A.hydrophila toxin induced rounding and steroidgenesis in Y-1 adernal cells Positive respons in the rabbit ileal loop, suckling mouse and CHO assays

A.hydrophila Was isolated from various sources 69% from 96 produce cytotoxins Isolated from diarrheal disease victims- 80% from 40 toxigenic Only 41% non-diarrheal positive for cytotoxin production Most enterotoxigenic starins are VP (Voges-Proskeur test) & haemolyisn positive and arbinose negative produce postive responses in suckling mouse, Y-1 aderenal cells assay and rabbit ileal loop assays FROM 147 isolates from patient of diarrhea, 91% were enterotoxigenic 70% of 94 enviromental strains produced hemolysis of rabbit red blood cells From 116 isolates Chesapeake Bay 71% were toxic by the Y-1 adrenal cell assay, Toxicity correlated with lysine decarboxylase and VP reactions Another study- 48 of 51 cultures from humans, animals, water and sewage produce positive responses in rabbot ileal loop assays with or more cells, and cell-free extracts from all were loop positive

A.hydrophila Isolation from meat and meat products Shown to possess biochem markers that are generaly associated with toxic strains of other species Mouse median lethal dose being log 8-9 cfu for most strains tested. Latter investigation suggested the possibility immunosuppressive states are important factors in food-associated infections by this organism-explain the difficulty of establishing this organism as the sole etiological agent of foodborne gastroenteritis

A.hydrophila GROWTH TEMPERATURE HABITAT 7 of 13 growth at 0-5C 4 of 13 growth at 10 C 1 at minimum 15 C Psychrotrophs had optimum growth between 15 C and 20 C growth T for some strains Optimum (35 C ) Max( 40-45 C) Was found in all 12 of 147 lotic and lentic habitats Hypersaline lakes and geothermal spring did not yield the organism Water- may contain until 9000/ml Ecological study in Chesapeake Bay Water column - < 0.3/1-5 x /ml Sediment about 4.6 x /ml Presence of A.hydrophila supported by Total bacterial counts Aerobic bacterial counts Viable bacterial counts Heterotrophic bacterial counts Inversely related Dissolved in Oxygen and salinity

Plesiomonas P. shigelloides found in water surface Soil Aquatic animals (fish,shellfish,etc) Terrestrial meat animals Content of G+C DNA differs from A.hydrophila 51% (Plesiomonas) 58-62% (A.hydrophila) Isolated from diarrhea patient Associated with general infection Produce heat stable enterotoxin Produce serogroup 0:17 strains react with Shigella group D Study of 16 strains of P.shigelloides from human with intestinal illness Did not always bind Congo red Non-invasive in Hep-2 cells Did not produce Shiga-like toxin on Vero cells Low-level cytolysin was produce consistently-mean for outbread Swiss mice was 3.5 x cfu No production of heat stable enterotoxin for 16 strains Conclusion : organism possess low pathogenic potential

P. shigelloides Recovered from stools of 6 diarrheal patients believe to be the etiological agent althought salmonella also recovered from 2 patients. Two outbreaks of acute diarrheal disease occurred in Osaka THE ONLY bacterial pathogen recovered was P. shigelloides . 1973 outbreaks : 978 from 2141 persons became ill symptoms lasted 2-3 days 88% diarrhea 82% abdominal pain 22% fever 13 % headache : from 124 stools examined 21 yields P. shigelloides 017:H2 same serovar was recovered from tap water - 1974 outbreaks : 24 from 35 persons Ill with similar symptom to those noted before : P. shigelloides 024:H5 recovered from 3 of 8 stools virtually in pure culture recovered from 39% of 342 water and mud sample, aquatic and newts A patient contracted gastroenteritis take 1 tablet of trimethoprim- sulfadiazine P.shigelloides recovered from blood She passed up to 10 water stools daily 39 C Noted that some previous patiens were immunocompromised or presented with similar conditions Isolated strains reacts with S.dysenteriae serotype 7 antiserum, placing it in O group 22 of P.shigelloides

Growth P. shigelloides Obserbed at 10 C In 59% of 59 fish from Zaire water Organism content in River fish > lake fish Appear not to produce enterotoxin only 4 from 29 isolates produced +ve response in rabit ileal loops No record of foodborne cases but resulting in two outbreaks.

Bacteroides fragilis Obligately aerobic Gram ve produces an ileal loop- positive enterotoxin potential significance of foodborne disease associated with diarhhea 1984 enterotoxin first demonstrated 1987 enterotoxin strains of B. fragilis were first associated with human diarhhea. B. fragilis estimated to constitute between 1% - 2 % of human intestinal biota non-pore former, more sensitive to aerated environment than clostridia , yet has been recovered from municipal sewage can grow in the presence of 20% bile Produced as single chain MW 20KDa Differs from the classical bacterial enterotoxin belonging to class of zinc-binding metalloprotease, designated metzitcins Enterotoxins has wide range of protein substrates undergoes autodigestion Intestinal damage beliced to be due to its proteolytic action Elicits a positive response in ileal loops of lambs and others Etiological agents only 50% identified as foodborne outbreaks in US previously recognize agents need to be included B.fragilis, Klebsilla pneumoniae and Enterobacter clocae warrant more attention 2 organisms produce heat-stable enterotoxins that similar with E.coli Their potential in food noted

Erysipelothrix rhusiopathiae Phylogenetically closely related to Listeria cause disease in : Human erysipeloid Localized disease of hand and arms of handlers of fresh meat and fish- systematic involvements unknown Animal ( swine ) erysipelas Characterised by diamond-skin lesions similarities make it to be logic as foodborne pathogen It is facultative anaerobes catalase ve Oxidase -ve Produce Hydrogen Sulphide gas At least 23 serovars are known the only other species is E.tonsillarum (separated by habitat of porcine tounges and different serovars) STUDY 1982 135 sample consists of equal number of chicken, beef and pork. Found E.rhusiopathiae in: - 36% pork - 13% chicken - None in beef In a plant, 54% of pork loins +ve, many isolates possess mouse virulance 112 sample retail pork in japan - 34% contained this bacterium - 14 from 38 isolates represents serovars

Erysipelothrix rhusiopathiae STUDY MEAT SAMPLE in Japan Contain this organism: 44 % of 93 wild boar 50 % of deer Representing 13 serovars Chicken Sample in Japan (750 samples) - 15.7 % of skin samples - 59.2 % of 179 feather samples E.rhusiopathiae represents 273 of 297 isolates RemainderE.tonsillarum Other study of 153 chicken in Japan: 30% contain Erysipelothrix spp. with 65 of 67 is E. rhusiopathiae

Klebsilla pneumoniae Streptococcus iniae Symptoms felt 6 hours after consuming burger by a patient Organisms along with generic E.coli was isolated from leftover burger and patient blood Burger and blood match in cultural methods Strain of K. pneumoniae was LT+ and ST- Coliform in leftover hamburger was 3.0 x /g and 1.9 x /g in bun 6 human infections-traced to a fish product 1972- cause disease in Amazon dolphins 1986 cause disease in tilapia and trout at Israel - Taiwan & US was next 1991 first human case in Texas 1994 second in Otawwa 1995-1996 -4 human cases (Ontario, Canada) Organisms isolated from fish( tilapia from US fish farms) and patients Appears to be fish pathogen- cause disease in human -Ontario case organisms entered body through hand lesions Beta-hemolytic in sheep blood Produce fulminant soft tissue infections in human

Prion disease

Prion Are unique proteins that can convert other proteins into damaging ones by causing them to alter their shape. Normal cell prion protein (PrP) exists in the brain cell membrane. It carries out some vital functions and is then degraded by proteases. This normal PrP cell is protease sensitive so it does not accumulate.

The pathogenic form(PrPSc )is distorted is resistant to proteases it accumulates in brain tissue and gives rise to diseases. It has been postulated > Distorted prion molecule act as template > Converts normal protein to distorted form. Normal protein (-helical form) takes on protease-resistant - flat form when it become pathogenic. Pathogenic forms tend to aggregate into amyloid fibrils where they cause nerve cell degeneration, it leads to clinical

Transmissible Spongiform Encephalopathies (TSEs) TSEs are a family of rare progressive neurodegenerative disorders that affect both humans and animals They are distinguished by long incubation periods, characteristic spongiform changes associated with neuronal loss, and a failure to induce inflammatory response

Bovine spongiform encephalopathy (BSE) BSE also known as mad cow disease. BSE is a progressive neurological disorder of cattle that results from infection by an unusual transmissible agent called a prion. First recognized in Great Britain in 1984. 1986 - Specifically diagnosed in cattle 1990 - 14,000 confirmed cases out of 10 million cattle had been recognized. 1993 - The epidemic seemed to peak around 1,000 new cases per week. February 1998 - Total of 172,324 cases were seen cattle in the United Kingdom. - Total of 600 cases were recorded in 8 countries outside U.K with 256 in Switzerland.

Since 1996 - 4.5 cows have been destroyed. Between 1986 and November 2003: 183,634 cattle were diagnosed with BSE in U.K 4,469 cattle were diagnosed in other 22 countries. Around 84 humans death were recorded in U.K in the year 2000. The first case confirmed in: Countries Year Japan September,2001 North Africa (cow in Alberta, Canada) May 20, 2003 United States (in Holstein downer cow in Mses December,2003

Around 35 million beef animals up to age 24 months are slaughtered annually in United States. With an additional 6 million of older dairy cows. Testing for prion proteins consists of: Immunohistochemistry method. Considered to be the gold standard Conformation-dependent immunoassay (CDI) ; develop in 2003. Screening test ; develop by the Bio-Rad Corporation (TeSeE). Herdchek BSE Antigen Test; develop by IDEXX Laboratories. 5 to 6 other post mortem test for central nervous system tissue of cattle. Normal cell brain Brain with BSE cell

Creutzfeldt-Jakob Diseases (CJD,vCJD) CJD is a degenerative neurological disorder that is incurable and invariably fatal. CJD is at times called a human form of mad cow disease. CJD normally appears in persons around age 60 or older. In March 1996, new variant of CJD (nvCJD, vCJD) was reported in the United Kingdom in a small group of people, whom were much younger than individuals with CJD. vCJD was found to afflict individuals in the late teens to the

It can be concluded that vCJD is the human equivalent BSE and the agents for BSE and vCJD appear to be the same. Between February 1994 and October 1995 10 persons in United Kingdom were found to have new variant form of CJD. 8 died. Most were under age 30. For the years 1995-1998 there were 39 cases of vCJD. 2003 around 150 vCJD cases were seen in humans throughout Europe. about one-third in the United Kingdom.

For 5 year period ; between 1991-1995 ; in the United States: 94 CJD deaths were recorded. 9 were below age 55. None conformed to vCJD. Over 85% of CJD patients die within one year of onset. The average annual death rate from CJD was 1.2/1 million population. Using a mouse assay, prions could not be detected in beef muscle and milk from infected cattle.

The latter investigator suggested that the brain tissue of a TSE infected cow can be expected to contain about 1011 prions per gram. The nerve tissue is ground with muscle tissue = 108 prions per gram or 1010 prions in a 100g portion. To effect 12D reduction, 22D is required.

Some of the times in minutes needed to achieve a 22D were calculate as follows:= D160 0 C = 1.0 D140 0 C = 11.0 D120 0 C = 110.15 It has been suggested that there is need for new processing or packaging technologies such as: high temperature short time (HTST) treatments be carried out in order to render products free of prions.

Chronic Wasting Diseases (CWD) This is a prion or TSE diseases first detected in captive mule deer in 1967 in the state of Colorado. Has been diagnosed in wild deer and elk at: Wyoming Colorado Nebraska Sasktchewan Province of Canada

This diseases appears to be transmitted through saliva or feces. It is estimated that 4-6 % of mule deer and