ANNALS OF CLIN ICAL AND LABORATORY SCIENCE, Vol. 6, No. 5

Copyright © 1976, Institute for Clinical Science

Laboratory Diagnosis of Foodborne D iseasesH E N R Y L. KAZAL, M .D.

D e p a r tm e n t o f P a th o lo g y , St. V in c e n t’s H o s p i ta l a n d M ed ic a l C e n te r o f N e w York,

N e w York, N Y 10011

A BSTRACTM any bac te ria l sp ec ie s are re sp o n sib le for spo rad ic cases an d ou tb reaks

o f foodborne in tox ication an d in fection . T h e foodborne d iseases are c las­sified on th e basis o f th e p a tho gen e tic m echan ism s in vo lved in to four categories: p re fo rm ed toxin, en te ro to x in fo rm ed in th e co lo n ized sm all in ­te s tin e , m ucosal in vasio n (enteroco litis) and m ucosal invasion w ith b ac ­te rem ia . Invasive an d tox igen ic strains o f en te ro p a th o g en ic E scherich ia co li a re d isc u sse d . In v ivo te s t sy stem s for th e id e n tif ic a tio n o f e n ­tero tox igen ic organism s an d tissu e cu ltu re assays for th e h ea t-lab ile e n ­tero toxin o f E. co li are describ ed .

C u rren t laboratory m eth ods for th e d iagnosis o f food bo rn e d iseases o f m ajor p u b lic h ea lth in te re s t are rev iew ed —botu lism , staphylococcal in ­to x ic a t io n , C lo s t r id iu m p e r f r in g e n s e n t e r i t i s , s a lm o n e l lo s is , e n ­te ro p a th o g en ic E. co li in fection , V ibrio pa ra h a em o ly ticu s in fec tion an d B acillu s cereus en te ritis .

T he ro le o f th e lab o ra to ry in th e ep id em io lo g ic su rv e illan ce an d in v e s ti­gation o f foodborne d iseases is em ph asized .

D iseases re su ltin g from th e con sum p­tio n o f foods m ay b e c lassified as in toxica­tions o r in fec tio n s .46 F o o d b o rn e in tox ica­tion s are cau sed b y n a tu ra lly occu rring p la n t an d anim al po isons, b y chem icals d e l ib e ra te ly o r a c c id e n ta l ly a d d e d to food, o r by toxic m etab o lic p rod ucts of b ac teria , fungi an d a lgae fo rm ed in food p rio r to in gestion (exotoxins). F o o db orn e in fections are cau sed b y th e in g estio n o f m icroorgan ism s w h ich in d u ce a reac tio n in ho st tissu e by p e n e tra tio n in to th e in ­tes tin a l m ucosa or by p ro d u c tio n o f en- tero toxins w ith in th e lu m en o f th e col­o n ized bow el.

M any b ac te ria l spec ies have b e e n im ­p lica te d in foodborne d iseases (tab le I). B ryan6 has reco g n ized th re e categories:(1) d iseases o f con tem porary im p ortance ,(2 ) d iseases u su a lly tran sm itted by o th e r m eans b u t som etim es foodborne a n d (3) d iseases in w h ich p ro o f o f transm issio n is inconclusive . T h e p u rp o se o f th is p ap e r is to d iscuss th e ro le o f th e labora to ry in th e in vestiga tion o f bac te ria l food bo rn e d iseases o f p rim ary p u b lic h e a lth in te r ­est.

In tab le I I are sum m arized da ta from th e la te s t an n u a l rep o rt on food bo rn e d isease ou tb reak s o f p rov en e tio logy for th e year

3 8 1

3 8 2 KAZAL

TABLE I

Bacteria Implicated in Foodborne Diseases

Foodborne Sometimes Foodborne* Probably Foodbornet

Salmonella enteritidis Shigella spp. Streptococcus faecalis,(Multiple serotypes) Escherichia coli faecium (Enterococci)

Salmonella typhi (Enteropathogenic) Proteus spp.Staphylococcus aureus Streptococcus pyogenes Providencia spp.Clostridium botulinum (Group A, G) Klebsiella spp.Clostridium perfringens Vibrio cholerae Citrobacter freundiiVibrio parahaemolyticus Brucella spp. Enterobacter spp.Bacillus cereus (Raw milk, cheese) Edwardsiella tardaSalmonella arizonae iYersinia enterocolitica Pseudomonas aeruginosaPseudomonas cocovenenans §Mycobacterium tuberculosis, bovis Aeromonas spp.

(Bongkrek poisoning) (Raw milk) Bacillus subtilisProteus spp. §Corynebacterium diphtheriae Vibrio fetus

(Scombroid fish poisoning) (Raw milk)§Francisella tularensis

(Rabbit meat) §Streptobacillus moniliformis

(Raw milk) iBacillus anthracis

(Meat, sausage)

Non-agglutinable vibrios Listeria monocytogenes

*Usually transmitted by other means. §Rarely reported.tAssociated with disease, but proof of transmission by foods is inconclusive.

e n c e a sim ilar illness , u su a lly g astro in tes­tina l, a fte r ingestion o f a com m on food w h ich ep idem io lo g ic analysis im p lica tes as a sou rce o f il ln e ss .58 B otulism is an ex­cep tio n in th a t ev en a s ing le case is co n sid ­e re d an ou tb reak . M any o u tb reak s are n o t reco g n ized an d m any o thers are n o t p ro p ­e rly in v es tig a ted o r rep o rted . U n d e rre ­p o rtin g also exists w ith sporad ic cases o f g a s tro e n te r it is o r s in g le co m p la in ts o f “ food po iso n in g ” w h ich are eas ily over­looked . T he p rob lem can b e p e rce iv ed b y com p aring th e N ational R esearch C ouncil estim ate o f tw o m illion cases of salm onel­losis annually in the U nited States w ith the ac tua l rep o rt o f abo u t 20 ,000 cases an n u ­a lly an d th e 1973 figure o f 2,462 ou tb reak c a se s .67

I n a d d i t io n to th e 127 la b o ra to ry - con firm ed ou tb reaks in 1973, th e re w ere 180 ou tb reak s o f u n d e te rm in e d e tio logy id e n tif ie d on th e basis o f ep idem io lo g ic e v id e n c e . E p id e m io lo g ic c la ss ifica tio n u tiliz e s th e in cub a tio n p erio d to classify su ch o u tb reaks as p rob ab ly chem ical (less th an o n e hour), p rob ab ly S tap hy lo coccu s a u re u s (1 to 7 h o u rs ) , p ro b a b ly C los-

1973 p ro v id ed by th e C e n te r for D isease C o n tro l .36 T h e se statistics re la te to only one facet o f th e m ajor p rob lem o f bac teria l foodborne d iseases, th e foodborne d isease outbreak. An outbreak is defined as an inci­den t in w hich tw o or m ore persons experi-

TABLE II

Confirmed Foodborne Disease Outbreaks 1973 (CDC)

Outbreaks CasesEtiology Number % Number %

BacterialSalmonellaStaphylococcus aureus Clostridium botulinum Clostridium perfringens ShigellaVibrio parahaemolyticus Bacillus cereus Streptococcus GP. A Brucella melitensis

332010981111

26.015.67.97.16.30.80.80.80.8

2,4621,272

311,4241,388

22

2504

31.916.5 0.4

18.5 18.00.030.033.20.1

Subtotal 84 66.1 6,835 88.6

Viral 5 3.9 425 5.5Parasitic 10 7.9 59 0.8Chemical (Scombroid 12/28) 28 22.1 392 5.1

Subtotal 43 33.9 876 11.4

Total known etiology 127 100.0 7,711 100.0

Note: No laboratory confirmation in 180 additional outbreaks.

LABORATORY DIAGNOSIS O F FOODBORNE DISEASES 3 8 3

tr id iu m perfr in g en s (8 to 14 hours) and p rob ab ly Sa lm onellae o r o th e r in fectious agen t (more than 14 hours). O n this basis 76 percen t o f these 180 outbreaks w ere attri­b u te d toS . aureus an d C . p erfr in g en s . In a ­b ility o f con ven tio na l laborato ry p roce­d u res to d e te c t agen ts such as halop h ilic v ibrios o r v iru ses is no d o u b t a factor in th e fa ilu re to id en tify p a tho gen s in th e se ou t­breaks. F a ilu re to te s t for staphy lococcal en tero to x in in su sp ec t foods cou ld b e a factor w h en food cu ltu re s are n eg ative ow ing to th e h ea t o f co o k in g .68 L ack o f co m p le te laborato ry analysis m ay re su lt in u n d e r re p o r t in g o f C. p e r fr in g e n s o u t­b reaks since th e p ro ced u res re q u ire d for iso la tion from food a n d feces an d for id e n ­tifica tio n an d se ro ty p in g are re la tiv e ly so p h istica ted .36

R elatively few bacteria l sp ec ies are im ­p lic a te d in foodborne d isease ou tb reaks. T h e tw o im p ortan t b ac te ria l in toxications a re s ta p h y lo c o c c a l in to x ic a t io n a n d bo tu lism . T h e tw o lead in g in fec tion s are salm onellosis an d C. p erfr in g en s in fec­tion . A lthough scom bro id p o iso n in g is no t o f m ajor im p ortance , th e toxic sub stances fo rm ed by th e b reak d o w n o f h is tid in e in fishflesh by P roteus spp . an d o th e r b ac te ria acco u n ted for 12 ou tb reaks an d 326 cases in 1973. O n e o u tb reak in 1973, ow ing to com m ercially can n ed tu n a , in v o lv ed 232 cases in four sta tes .40 T h e first k now n ou t­break in N ew York City, ow ing to raw tuna fish, involved four adults and was reported

in July 1975.66 A lthough statistically o f lit­tle significance, Vibrio parahaem olyticus, Bacillus cereus and E scherichia coli are w e ll-d o c u m e n te d c a u se s o f fo o d b o rn e disease outbreaks and only thorough inves­tigation o f all ou tb reak s w ill rev ea l th e ir tru e sign ificance. R ecen t in te re s t in th e ro le o f E. coli in h u m an ad u lt as w ell as in fan t d ia rrh ea l d isease has led to th e d e ­ve lo p m en t o f n ew assay m eth ods forE .c o li e n te ro to x in s .29,33,48' 51 T h e se n e w te c h ­n iq u es sh o u ld p rov e he lp fu l in assessing th e ro le o f th is o rgan ism as an en tero patho - gen and in d e lin ea tin g th e re la tive im por­ta n c e o f e n te ro to x ig e n ic an d in v as iv e stra in s .16

L aboratory d e te rm in a tio n s p lay a m ajor ro le in th e su rv e illan ce aspects o f food­bo rn e d iseases. S urveillance invo lves th e d e te rm in a tio n o f th e in c id e n c e o f o r­gan ism s in foods, th e s tu d y o f factors th a t le a d to b a c te r ia l c o n ta m in a tio n , su rv iv a l a n d m u l t ip l ic a t io n , a n d th e e p id e m io lo g ic in v e s tig a tio n o f la b o ra ­to ry -co n firm ed sp o rad ic cases an d o u t­breaks. K now ledge o f th e natu ral h is to ry o f th e foodborne d isease en titie s is u sed in such in vestiga tions. C erta in bac te ria te n d to b e a s so c ia te d w ith p a r t ic u la r types o f foods: staphylococcus w ith ham , c u s ta rd s , c ream f ill in g s , p o ta to sa lad ; C lo s tr id iu m b o tu lin u m w ith im p rop erly p re se rv ed low ac id veg e tab les; C. per­fr in g e n s w ith slow ly-cooled coo ked m ea t

TABLE III

Bacterial Foodborne "Gastroenteritis"

Incubation (Hours) DurationPathogen Average Range Symptoms (Days)

Staphylococcus aureus 2 - 4 Clostridium botulinum 18 - 36

Clostridium perfringens 12 Bacillus cereus 8 - 1 6

Salmonellae 12 - 36

Vibrio parahaemolyticus 15 - 24

7 Nausea, vomiting, cramps, diarrhea 1 - 2192 Nausea, vomiting, abdominal pain, diarrhea Fatal

(early in one third) 3 - 1 024 Acute abdominal pain, diarrhea 118 Nausea, cramps, diarrhea, vomiting 1 or less5 Nausea, vomiting 1 or less72 Diarrhea, abdominal pain, fever, chills, Days - weeks

vomiting4 - 9 6 Diarrhea, cramps, nausea, vomiting, 1 - 1 0

fever, chills (Median 3)

21.5

5

3 8 4 KAZAL

TABLE IV

Pathogenetic Mechanisms in Bacterial Foodbome Diseases

Disease Organism

Foodbome intoxications (preformed toxin)Staphylococcal Staphylococcus aureusintoxication

Botulism Clostridium botulinumScombroid poisoning Proteus spp.

Bongkrek poisoning Pseudomonas cocovenenans

Enterotoxic enteropathies (enterotoxin formation in colonizedVibrio cholerae Escherichia coli

enterotoxigenic strains

Clostridium perfringens type A

Bacillus cereus

Shigella dysenteriae type 1LSalmonella enteritidis

CholeraEnteropathogenic Escherichia coli

infection Clostridium perfringens enteritis

Bacillus cereus enteritis

Shigellosis Mucosal invasion (enterocolitis)

S almone1losis

Shigellosis

EnteropathogenicEscherichia coli infection

Enteritis necroticans (Pig-bel)

Systemic infection (bacteremia) Salmonellosis typhoid fever

Salmonellosisparatyphoid fever

Brucellosis

UndeterminedVibrio parahaemolÿticus infection

Non-agglutinable Vibrio infection

Arizona infection

Heat-stable enterötoxins A, D (also B,C, E, F and others)

Heat-labile toxin A, B, E, (F)Histamine, saurine, formed in fishflesh

(tuna, mackeral, bonito)Bongkrek acid and toxoflavin formed in poorly fermented coconut cakes (Central Indonesia)

small bowel)Heat-labile toxin (CT)Heat-labile toxin (LT)Heat-stable toxin (ST)

Enterotoxin formation during sporulation in bowel

Toxin probably formed in bowel;? preformed in food

Invasive; role of enterotoxin secondary

Over 1,600 serotypes (typhimurium, newport, enteritidis, infantis, heidelberg, agona, saint-paul, etc.)

Shigella sonnei, flexneri, boydii, dysenteriae

Escherichia coli invasive strains

Clostridium perfringens type C

Salmonella typhi

Salmonella enteritidis ser. paratyphi A, B, C; ser. sendai

Brucellamelitensis, abortus, suis

Vibrio parahaemolÿticus

Vibrio enteritides

Salmonella arizonae

New Guinea pork feasts, gangrene of small bowel

Invasion and/or enterotoxin (?)

Resembles Vibrio cholera but noagglutination by cholera polyvalent antiserum

an d gravy; sa lm o n e llae w ith eggs an d poultry ; V. p a ra h a em o ly ticu s w ith sea­food; B. cereus w ith custards, cereal prod­ucts, p u d d in g s an d sauces. F ood h an d ­lers are lik e ly to b e im p lica ted in th e tran sm iss io n o f s tap h y lo co ccu s , sa lm o­n e llae an d sh ige llae .

T he c lin ica l fea tu res o f th e illn ess are h e lp fu l in th e in v estig a tio n (table III). T h e in cu b a tio n p e rio d an d th e du ra tion o f e a c h d is e a s e can b e re la te d to th e p a th o g en e tic m echan ism in vo lved (table

IV). A very short in cub atio n p erio d (1 to 7 hours) suggests staphylococcal in toxica­tion ; th e p re fo rm ed toxin acts qu ick ly to cau se a d isease o f sho rt du ra tion . T h e e n ­te r o to x in o f C. p e r f r in g e n s m u s t b e fo rm ed by sp o ru la tio n o f th e in g e s te d v e g e ta tiv e ce lls in th e sm all in te s tin e . T h e in cu b a tio n p erio d is longer, averag ­in g 12 hours, b u t again th e d u ra tio n is short. Invasive bac te ria re q u ire tim e to p e n e tra te th e m ucosa and in d u ce an in ­flam m atory reac tio n w h ich takes tim e to

LABORATORY DIAGNOSIS OF FOODBORNE DISEASES

TABLE VLaboratory Confirmation - Foodbome Outbreak

3 8 5

Patient Food Environ-C = Culture Handler mentT = Toxin test Reservoir Food Stool Vomitus Blood Stool Swab

Preformed ToxinStaphylococcus Man (skin, nares) C+T C c (Skin, nares) CBotulism Soil, water C+T C+T C+T T -

Enterotoxic EnteropathyClostridium perfringens Animal, man (feces) C C - c cEscherichia coli Man (feces) C C - C -

Bacillus cereus Soil, dust C C - - -

Mucosal InvasionSalmonella Animal, man (feces) C C - C cShigella Man (feces) C C - c -

Escherichia coli Man (feces) C C - c -

Systemic InfectionTyphoid, paratyphoid Man (feces, urine) C c - C (Agg) c -

UndeterminedVibrio parahaemolyticus Seawater, marine life C c c - c

reso lve. T h e in cu b a tio n p e rio d o f salm o­n e l la in fe c tio n s m ay b e as lo n g as 72 h o urs an d th e d u ra tio n sev e ra l w eek s. D e sp ite th e se corre la tions, th e re is con­sid e rab le ov erlap in th e range o f in cu b a ­tion perio ds so th a t th is on e item o f in ­form ation m ay b e m islead ing .

In th e in vestiga tion o f an ou tb reak , th e laborato ry aids th e ep id em io lo g is t by v e r­ifying a te n ta tiv e d iagnosis, trac in g th e sou rce o f con tam in atio n an d id en tify in g con tribu to ry factors. In ad d itio n to iso la t­ing th e m icroorganism , it m ay b e n ece s­sary to d e te rm in e th e n u m b e r p e r gram o f food, to ascerta in d e fin itiv e ty pes o f iso­la te s from so u rce , v e h ic le a n d v ic tim s (se ro ty p es , p h ag e ty p e s , co lic in ty p es) an d to d e te rm in e th e ty p e o f to x in in cases o f staphy lococcus in tox ication and b o tu lism .7 A s in g le d e fin itiv e ty p e recov­e re d from su sp ec t food an d from p a tien ts in crim inates th e food. A sing le d e fin itiv e ty p e re c o v e re d from su sp e c t food, p a ­tien ts an d p o ss ib le ca rrie r in c rim ina tes th e carrier, b u t on ly if h e has n o t e a ten th e su sp ec t food. P ro cedu res for lab o ra­to ry con firm ation o f th e im p o rtan t d is­eases are show n in tab le V. D efin itiv e ty p in g p ro ced u res cu rren tly ava ilab le are lis ted in tab le VI.

B otulismC lo s tr id iu m b o tu lin u m is an anae rob ic

gram -positive bac illu s w id e ly d is tr ib u te d in n a tu re in soil an d in m arine e n v iro n ­m ents. H ea t re s is tan t spores p re se n t in im p ro p e rly can n ed foods g erm in a te and m u ltip ly w ith th e e lab o ra tio n o f a h ea t lab ile p ro te in neu ro to x in w h ich has b e e n c a lle d th e “ m o st p o iso n o u s p o is o n .”39 S even ty pes o f C. b o tu lin u m have b e e n

TABLE VI

Definitive Typing Procedures for Bacterial Foodbome Pathogens

Pathogen Definitive Typing

Staphylococcus aureus Phage typingEnterotoxin testing

Clostridium botulinum Toxin testing (mouseneutralization)

Clostridium perfringens SerotypingVibrio parahaemolyticus SerotypingSalmonella typhimurium Phage typingSalmonella typhi Phage typingSalmonella paratyphi B Phage typingSalmonella, other SerotypingShigella SerotypingShigella sonnei Colicin typing

Phage typing

Single definitive type from suspect food andpatients incriminates the food.

Single definitive type from suspect food, patientsand carrier incriminates a carrier (if he hasnot eaten the suspect food).

3 8 6 KAZAL

TABLE VII Laboratory Diagnosis - Botulism

Identify toxin A, B, E, F (mouse-toxin neutralization test)

Identify Clostridium botulinum (smear and culture)

A. Patient serum (pretreatment or convalescent)Feces (especially if serum is negative)Gastric contents, vomitusAutopsy (blood# gastrointestinal contents., liver)

B. Food extractsPlainTrypsinized (activation of some types B, E, F)

C. Rapid in vitro assays less sensitive: radioimmunoassay,immunodiffusion, passive hemagglutination, etc.

A. Specimens:Food extractsGastric contents, vomitusFecesAutopsy

B. Enrichment culture (CM-glucose-starch broth)Untreated inoculumTreated inoculum (spore selection)

AlcoholHeat

C. Anaerobic incubation (30° C, 3 to 5 days)Gram stainSubculture to BAP and egg yolk agar for isolation and identification

Toxin test (culture supernate; Clostridial isolate)Perform electromyography - (facilitation

of muscle action potential during rapid repetitive stimulation).Botulism - positive; Guillain-Barre - negative.

reco g n ized (types A to G). E ach e lab o ­rates an im m unolog ically d is tin c t toxin. T ypes A and B cause m ost rep o rted o u t­b reaks, an d h o m e-can n ed v eg e tab les are th e m o st co m m o n ly im p lic a te d foods. W ith o n e e x c e p tio n , a ll r e p o r te d o u t­b reak s o f ty p e E b o tu lism h av e b e e n traced to fish o r fish p roducts. O n ly on e ou tb reak ow ing to ty pe F has b e e n re ­p o rted in th is country . T ypes C and D p ro d u c e d isea se a lm o st ex c lu s iv e ly in anim als. A bou t 10 p e rc e n t o f all rep o rted ou tb reak s w ere cau sed by com m ercially p r e p a r e d f o o d s .41 In 6 8 8 f o o d b o rn e bo tu lism ou tb reak s know n to th e C e n te r for D isease C ontro l from 1899 to 1973, th e re w e re 1,784 cases w ith 978 d e a th s .41 In re c e n t years, th e case fatality ra te has fallen from above 60 p e rc e n t to ab o u t 23 p e rcen t. In 1974, seven o f 30 rep o rted cases w ere fa ta l.62

T h e in c u b a tio n p e r io d is u su a lly 18 to 36 hours b u t w ith a range o f tw o hours to e ig h t days. N ausea, vom iting , ab d o m i­nal cram ps an d d iarrhea , w ith o u t fever,

n o t in fre q u e n tly occu r first, s im u la tin g o th e r foo d b o rn e d iseases . S ym m etrica l neu ro lo g ic m an ifesta tions d ev e lo p w ith ­ou t senso ry chan ges or a lte ra tion o f m en ­ta l status. T he d iagnosis sh o u ld b e con­s id e re d in p a tien ts w ith acu te b ila te ra l c ran ia l ne rve im p airm en t an d sym m etri­cal d e sc e n d in g w eak n ess o r p ara ly sis . Com m on neurologic symptoms are b lurred v ision , d ip lo p ia , d y sarth ria , dy sp h ag ia , d y sp h o n ia a n d g e n e ra l iz e d w e a k n e ss . In v o lv em en t o f th e m uscles o f resp ira tio n m ay follow. T h e d isease m ay b e fatal in th re e to te n days.

In su sp ec ted cases, an a ttem p t is m ade to dem o nstra te b o th th e so lu b le tox in and th e organism (table V II). M aterials sus­p e c te d o f con ta in in g toxin m ust b e h an ­d led only by experienced personnel since m in u te q u an titie s ab so rb ed th ro u g h th e ey e or a b reak in th e skin or a cq u ired by in g estio n or in h a la tio n m ay b e fatal. Im ­m un iza tion w ith bo tu lin a l toxoid is rec ­om m end ed .

T h e m ost e ffective p ro ced u re for con­

LABORATORY DIAGNOSIS OF FOODBORNE DISEASES 3 8 7

firm ing th e d iagnosis is id en tifica tio n o f th e sp ec ific b o tu lin a l tox in in th e p a ­t ie n t’s p re tre a tm e n t serum . T ox in tests can also b e p erfo rm ed on feces, gastric con ten ts an d au top sy specim ens. F ecal sam ples sh o u ld b e te s te d rou tine ly , par­ticu la rly w h e n no food is av a ilab le for te s tin g or w h e n toxin can n o t b e d e tec ted in th e se ru m .41 A rep o rt o f d e tec tio n of ty pe B tox in in seru m and stool spec i­m ens 28 days a fte r o n se t o f illness in a p a tie n t w ith a p o sitive stool cu ltu re for C. b o tu lin u m ty p e B suggests th a t toxin m ay b e p ro d u ced in v ivo .61 A lthough th e re is no ex p e rim en ta l ev id en ce to show th a t th e organ ism can m u ltip ly in th e hu m an in te s tin a l tra c t, a tte m p ts a t lab o ra to ry c o n firm a tio n e v e n la te in th e c lin ic a l cou rse ap p ea r w arran ted . All specim ens te s te d for tox in sho u ld b e h e ld a t 4° u n til e x a m in e d to a v o id lo ss o f th e th e r - m o lab ile toxin.

An in d ire c t laborato ry d iagnosis can be m ade by dem o nstra tion o f th e toxin in extracts o f food .59 A hom ogenate o f 50 g of fo o d m a d e in a c h i l l e d m o r ta r w ith g e la tin -p h o sp h a te b u ffe r is re frig e ra ted 12 to 18 hours to p rom ote toxin ex trac­tion . A fter cen trifu ga tion in th e cold , p a rt o f th e su p e rn a tan t is try p sin ized to e n ­h an ce th e toxicity o f som e ty p e B, E and F toxins. A m ix tu re o f n in e parts o f food ex tract an d one p a rt o f try p sin so lu tion ad ju sted to p H 6.0 to 6.2 is in cu b a ted at 37° for 45 m in u tes.

E xtracts o f feces (50 g) and gastric con­ten ts (50 ml) each m ixed w ith eq u a l parts o f th e bu ffe r are p rocessed in th e sam e m an n er a ltho ugh om itting tryp sin iza tio n sin ce th e toxin has a lready b e e n activated b y p ro teo ly tic in te s tin a l enzym es. Serum do es n o t req u ire extraction o r tryp sin iza ­tion.

T h e stan dard p ro ced u re for toxin te s t­in g is still th e m ouse tox in -neu tra lization te s t .20 F o u r pairs o f 15 to 20 g “ p ro tec ted ” m ice are in jec te d I.P . w ith 0.5 m l vo l­um es o f te s t m ix ture (0.3 m l o f each o f th e

specific antitoxins A, B, E and F m ixed w ith 1.2 m l o f sp ec im en ex tract o r p a tien t serum ). T h ese four tu b es are in cu b a ted at 37° for 30 m in befo re in jection . “ U npro­te c te d ” m ice rece iv e sp ec im en w ith o u t a d d e d an tito x in . An a d d itio n a l p a ir o f con tro l m ice rece iv es sp ec im e n ex trac t h e a te d at 100° for te n m in u tes. T h e m ice are o b se rv ed for four days for ruffing of th e fur, lab o red b rea th in g , lim b w eakness an d to ta l para ly sis . I f tox in is p re se n t, o n ly th e m ice g iven h e a ted ex tract and o n e o f th e p ro tec tiv e an titox ins w ill sur­v ive. Incon c lu sive resu lts occur ow in g to n o n b ac te ria l le tha l factors (dea th o f all m ice) o r h igh con cen tra tion s o f o n e o r a com b ina tio n o f toxins. In e ith e r case th e te s t is rep e a te d w ith a d ilu te d extract.

C u ltu res o f su sp ec t food, gastric con­te n t an d fecal susp ensio ns are m a d e 20 a l­th o u g h f in d in g th e o rgan ism do es n o t p ro v e th a t toxin has b e e n form ed. S e lec ­tion o f spores b y k illing n o n sp o rin g b ac­te r ia can b e acco m p lish ed by tre a tm e n t of th e su sp en sio n at room tem p e ra tu re w ith 100 p e rc e n t e thano l. A lternatively , tu b es o f c h o p p e d m e a t m e d iu m in o c u la te d w ith th e su sp ensio n are h e a te d p rio r to a n a e ro b ic in c u b a tio n , u s in g d i f f e re n t te m p e ra tu re s since spores vary in th e ir re sis tan ce to heat. T yp e E spores are sus­c e p tib le to heat. P art o f th e sp ec im e n is a lw ays c u ltu re d w ith o u t p r io r h e a tin g since on ly veg e ta tive C. b o tu lin u m cells o r h e a t sensitiv e spores m ay b e p resen t. All cu ltu res are in cu b a ted at 30°; h ig h e r tem p e ra tu re s m ay p re v e n t toxin p ro d u c ­tion by som e types. C u ltu res sho w ing gas an d tu rb id ity are m ouse te s te d in th re e to five days, u s in g su p e rn a tan t flu id . S ub­cu ltu re s to b loo d agar an d egg yoke agar a re u se d to iso late co lon ies w h ich , w h en p ic k e d to c h o p p e d m e a t m e d iu m , a re id e n tif ie d b y cu ltu ra l an d b io ch em ica l p ro ced u res an d are again te s te d for toxin type.

E lec tro m y og rap hy is a v a lu ab le a id in d if fe re n tia tin g b o tu lism from a ty p ic a l

3 8 8 KAZAL

G uilla in -B arre syndrom e, th e en tity m ost o ften con fu sed w ith bo tu lism in cases re ­p o rted to th e C e n te r for D isease C o n­tro l .60 In a p a t ie n t w ith b o tu lism , th e a m p litu d e o f th e m u sc le ac tio n p o te n ­tial is u su a lly d im in ish ed in resp o n se to a s in g le sup ram ax im al n e rv e s tim u lu s; how ever, p a ired stim u li d e liv e re d a t an in te rva l o f 2 .5 m illiseco nds an d rep e titiv e supram axim al s tim u la tion at ra tes o f 20 to 50 p e r secon d re su lt in facilita tion (au- g u m en ta tio n ) o f th e ac tio n p o te n tia l .59 T h ese find ings m ay n o t b e p re se n t early in th e illness and m ay n o t b e p re s e n t in all m uscle groups. T h ey are no t specific for bo tu lism since th ey have b e e n no ted in p a tien ts w ith th e E a to n -L am b ert syn­drom e.

S ev era l in v itro assays for b o tu lin a l toxin have b e e n stu d ied . Jo h n so n e t al37 u til iz e d b o th h e m a g g lu tin a tio n in h ib i­tion an d passive h em ag g lu tin a tio n for th e rap id an d specific d e tec tio n o f b o tu lin a l toxins an d p re se n te d ev id en ce th a t th e se sero logical p ro ced u res are ap p licab le to th e d e tec tio n of b o tu lin a l toxins in food. V erm ilyea et a l55 d e te c te d toxins o f C. b o tu lin u m in foods by m eans o f a d o u b le g e l-d iffu sio n s lid e te s t . M e s tra n d re a 43 te s te d a m icrocap illary tu b e d iffusion sys­tem w hich could be u sed as a rapid screen­in g m e th o d for d e te c tio n o f b o tu lin a l tox in in foods. T h e rad io im m u n o assay te c h n iq u e for ty pe A toxin d e v e lo p ed by B oroff and S h u -C h en 4 is p rom ising . I t app ears th a t n o n e o f th e in v itro m ethods cu rren tly availab le is as sen sitiv e as the m ouse n eu tra liza tio n t e s t 59

Staphylococcal IntoxicationS ta p h y lo c o c c a l fo o d in to x ic a t io n

ran k ed secon d am ong re p o rte d bac teria l fo o d b o rn e o u tb re a k s a n d f o u r th in n u m b er o f cases in 1973. T h e d isease is an in tox ication ow ing to th e in g estio n o f a p ro te in exotoxin (en tero toxin) form ed by ce rta in strains o f S. aureus in con tam i­n a te d foods su c h as co o k e d h am an d

o th e r m ea t p rod uc ts, p o u ltry and d re ss­in gs, sau ces a n d g rav ies , p o ta to sa lad an d cream -filled pastry . M ost likely to be in vo lved are cooked h ig h p ro te in foods w h ich are re la tiv e ly free from con tam ina­tio n by o th e r bac te ria . S taphylococcus is n o t a strong co m p etito r and its g row th is lim ited by th e m icroorgan ism s p re se n t in m ost raw an d fe rm en ted foods. W h en th e food is co n tam in a ted by h a n d lin g an d in ­ad eq u a te ly re frig era ted for e ig h t hours or m ore, staphy lococci m ay b e found in ex­cess o f 500,000 organ ism s p e r gram o f food. T h e a n te rio r n ares o f m an is th e m ain rese rv o ir and u p to 50 p e rc e n t o f f o o d h a n d le r s m ay b e c a r r i e r s .63 T h e source o f food con tam in atio n is usually from su p e rf ic ia l sk in in fec tio n s , e s p e ­cia lly o f han ds, arm s an d face or from th e nose by h an d carriage o r nasal d rop le ts .

E n te ro tox in is form ed, w ith rare excep­tion , by coagulase po sitive staphylococci (S. a u r e u s ) . A b o u t 50 p e r c e n t o f th e strains have th is capability . F iv e antigen- ica lly d is tin c t ty p e s o f en te ro to x in are know n (A-E). P u rifica tion o f th e se toxins has m ade it p o ss ib le to p rep a re specific an titox ins an d th us to ap p ly serological p r o c e d u r e s in th e id e n t i f i c a t io n a n d quan tifica tion o f th e to x in s .25 E n tero tox in A is m ost com m only im p lica ted in food- b o rn e ou tb reak s w ith en te ro to x in D nex t in o rd e r o f f req u en cy .25 In d iv id u a l cu l­tu re s o f S. aureus m ay p ro d u ce toxin o f m ore th an on e type . T h e en tero tox ins are re s is ta n t to h e a t (100° for 30 m in), a l­th ou gh th e th e rm al sensitiv ity varies w ith th e ty pe , an d are no t read ily destro y ed at o rd inary cooking tem p era tu res .

I n g e s te d e n te ro to x in is a b s o rb e d rap id ly from th e in te s tin e an d its em etic e ffect app ears to b e d u e to s tim u la tion of cen te rs in th e cen tra l nervous system . I t is p ro b ab le th a t h u m ans re sp o n d to as li t­tle as o n e m eg o f en te ro to x in A. A fter a b r ie f in c u b a tio n p e r io d (one to sev en hours), th e re is an a b ru p t o n se t o f nausea, v o m it in g , a b d o m in a l c ra m p s a n d

LABOI^ATORY DIAGNOSIS OF FOODBORNE DISEASES 3 8 9

d iarrhea . T h e attack su b sid es in one to tw o days.

T h e d iagnosis o f an o u tb reak is b ased on a com p atib le c lin ica l p ic tu re , th e iso ­la tion o f S. aureus o f a s ing le p h ag e ty pe from an im p lica ted food and in th e feces an d vom itus o f p a tien ts an d th e id en tifi­ca tion o f en te ro to x in in th e im p lica ted food. An organ ism o f th e sam e ph age type m ay b e reco v e red from a food h an d le r (tab le V III).

A varie ty o f cu ltu re m ed ia m ay b e used . T h e se tak e ad v an tag e o f th e fo llow ing charac teristics o f S. a u re u s : (1) a to le r­ance of h ig h sa lt co n cen tra tio n s (7.5 to 10 p e rc e n t N aC l), (2) th e d e v e lo p m e n t o f a c lea r zone o f p ro teo ly sis a roun d colon ies on agar co n ta in in g eg g yoke em u lsio n (becom ing o p aq u e on lo n g er in cub a tio n ow in g to lip ase activity), (3) th e ab ility to aerob ica lly re d u c e a te l lu r ite salt to e le ­m en ta l te llu riu m w ith th e form ation of b lack co lon ies, (4) th e ab ility to u tilize m ann ito l as th e so le o r m ajor sou rce of carbon (in a m ed iu m w ith a p H in d ica to r m ann ito l functions as a d iffe ren tia l agent) an d (5) th e ab ility to g row in th e p resen ce o f a n t ib io t ic s w h ic h in h ib i t o th e r o r­gan ism s (polym yxin, neom ycin).

T h e m ed ia can b e d iv id ed in to th ree types: (1) basic p la tin g (b lood agar, s taph 110 agar, m ann ito l sa lt agar), (2 ) se lective e n r ich m en t (ch o p p ed m ea t 10 p e rc e n t salt b ro th , tryp ticase soy 10 p e rc e n t sa lt broth) an d (3) se lec tive p la tin g (staph 1 1 0 - egg yoke agar, te llu r ite - po lym yxin - egg yo ke agar, B aird -P arker agar).

S pecim ens for gram sta in an d cu ltu re in c lu d e vom itus an d feces from patien ts, nasal sw abs an d pus from skin lesions of su sp ec t carriers an d food sam ples. Serial d ilu tio n s o f food h o m ogen ate are inocu­la t e d in to s e le c t iv e e n r ic h m e n t a n d se lec tiv e p la tin g m edia . C oun ts o f typical co lon ies on th e agar p la tes allow calcu la­tion of th e n u m b e r o f p re su m p tiv e S. au­reus p e r gram o f food. S pec im en s o th e r th an food are p la te d on th e basic m ed ia

TABLE VIIILaboratory Diagnosis - Staphylococcal Intoxication

Isolate organismFrom patient, carrier

From food homogenate

Characterize all isolates

Detection of Enterotoxin

Gram stain (vomitus/ feces)

Plate on basic media Gram stainMake serial dilutions Inoculate selective

enrichment media Inoculate selective plating media

Calculate Staphylococcus aureus per gram

Gram stain Coagulase Mannitol Antibiogram Phage type Toxin production Serotype (difficult)

in food (A, B, C, D, E)Precipitin tests

(specific antisera)

Other tests

Single gel - diffusion tube test

Double gel - diffusion tube test

Double gel - diffusion slide test (Casman and Bennett - microslide gel diffusion test)

Feeding (young Rhesus monkeys)

Radioimmunoassay Reversed passive

hemagglutination Immunof1uorescent methods Other

fo r re c o v e ry o f is o la te d c o lo n ie s . A ll staphy lococcal iso la tes are charac te rized by m orphology an d gram stain , coagulase te s t and m ann ito l ferm en tation .

I t is c ritica l for ep id em io lo g ic p u rp o ses to c h a ra c te r iz e th e iso la te s fu r th e r by p h a g e ty p in g . I t is a lso im p o r ta n t to exam ine c u ltu re filtra tes for e v id en ce o f p ro d u c tio n o f toxin o f th e sam e ty p e as th a t reco v e red from food sam ples. A n tib io tic s u s c e p t ib i l i ty p a t te rn s (a n tib io g ra m s) m ay b e h e lp fu l i f th e organism s are phage no n ty p ab le . S ero ty p in g is too com plex for p ractical u se b u t is o f p o ten tia l v a lu e as a h igh ly s tab le m ark er system w h ich w o u ld allow th e ty p in g o f m ost hum an stra in s o f stap hy loco cci.11

E ntero to x in can b e d e te c te d by b io as­say an d sero logical assay m ethods. O w ing to th e sm all am ounts o f toxin in con tam i­n a ted food an d its rap id abso rp tio n in th e in te s tin e , it is n o t p o ss ib le to d e te c t toxin in vom itus, feces an d serum . O n ly su sp ec t foods an d c u ltu re filtra tes are assayed .

3 9 0 KAZAL

T h e m ost com m on b io logical assay p ro ­c e d u re s are th e in tra p e r ito n e a l o r in ­travenous in jec tion o f cats an d k itten s and th e feed in g o f young rhesus m onkeys. T h e sensitiv ity o f th e m on key to th e em etic effect o f th e toxin p ara lle ls th a t o f hum ans and m akes th is th e m ore re liab le test. At­tem p ts to d ev e lo p o th e r assay m ethods h a v e b e e n u n s u c c e s s f u l . B io a ssa y is n e e d e d in th e c o n tin u in g search for new en tero tox ins.

Sero logic assays are m ore sensitiv e and less exp ensive . G el-d iffusion te ch n iq u es are m ost w id e ly u sed . H a ll e t a l34 com ­pared th re e such te ch n iq u e s u sing spe­cific an tise ra to en tero to x in s A and B to d e tec t en te ro to x in in Culture su p ern a tan t flu ids an d food extracts. In th e s in g le gel- d iffusion tu b e test, food ex tract is lay ered d irec tly ov er an agar co lum n con tain ing an tiserum . A n tigen d iffuses th ro ug h the agar an d form s a b a n d o f p rec ip ita te w h ich m oves dow n th e agar co lum n. Q u an tita ­tion is p o ss ib le b eca u se th e len g th o f the p rec ip ita te b an d can b e co rre la ted w ith th e co n cen tra tio n o f a n tig en p resen t.

T h e d o u b le gel-d iffusion tu b e te s t em ­ploys a bo ttom agar layer con ta in in g an ­tiseru m , a m id d le lay er o f agar free from reagen ts an d an u p p e r lay er w h ich con­tains an tig en . As th e tw o reag en ts d iffuse th ro ug h th e n eu tra l zone, a b an d o f p re ­c ip ita te form s w h e re th e y m e e t in op ­tim um p roportions. T h is m e th o d can be u sed to d e te c t im p u ritie s in en tero tox in p rep a ra tio n s . T h e d o u b le ge l-d iffu sion slide te s t is a s lide m odification o f th e O u ch te rlo n y p ro c e d u re .12' 56 A n tiserum is p laced in th e cen tra l w ell o f th e agar and su sp ec t toxin w ith kn ow n toxin controls are p laced in th e p e rip h e ra l w ells. T he m eth o d w as ad o p ted an d d ev e lo p ed by C asm an and B e n n e tt .9,10 T h is m icroslide gel d o u b le d iffusion te s t is th e p roced ure of cho ice b eca u se i t p rov id es confirm ation o f th e spec ific ity o f th e p rec ip ita te in ex­am in ing extracts o f foods w h ich m ay pro­du ce nonspecific p rec ip ita tes .

D e te c tio n o f traces o f en te ro to x in in food n ecess ita tes co n cen tra tin g th e food ex tract to as sm all a vo lum e as possib le . T h e slide te s t req u ire s on ly 0.02 to 0.025 m l o f an tig en an d an tise rum . T he m e th o d em ploys ex traction o f th e en tero to x in w ith 0.2 M N aC l fo llow ed by se lec tiv e ab so rp ­tion by m eans o f a carboxym ethy l c e l­lu lo se co lum n an d con cen tra tion o f th e e lu a te by d ia lysis. T h e ex tract from 100 g o f food is co n cen tra ted to 0.2 ml. T h e te s t is sen sitiv e an d specific. I t is capab le of d e te c tin g as little as 0.003 /¿g o f en te ro ­toxin A p e r g o f food ,57 less than the am ount w h ic h is like ly to cause h u m an illness.

M any o th e r sero logical m ethods have b e e n d e sc r ib e d in c lu d in g th ose b a sed on h e m a g g lu tin a tio n - in h ib it io n , im m u n o - f lu o re scen t d e tec tio n , rev e rsed passive h e m a g g lu t in a t io n , r e v e r s e d im m u n o - o sm o p h o resis an d so lid -p h ase rad io im ­m unoassay . Jo h nso n e t a l38 u tilized th e so lid -ph ase rad io im m unoassay p ro ced u re for th e d e te c tio n o f en tero tox ins A an d B in food. T h e m eth o d has h ig h sensitiv ity an d req u ire s m in im al p rep ara tio n o f food extracts for analysis.Clostridium Perfringens Enteritis

B oth raw foods and cooked foods (m eat, m ea t p ie s , s tew , gravy) are freq u en tly con ­tam in a ted w ith C. perfring ens w h ich is p re se n t in h u m an and anim al feces, soil, d u s t a n d sew ag e .8 T he cooking process d riv es off d isso lv ed oxygen and th e re s is ­ta n t sp o res are ac tiv a ted (h ea t shock). Slow co o lin g p rov id es th e p ro p e r te m p e r­a tu re for germ ina tio n and m u ltip lica tio n to th e lev e l o f 108 to 109 veg e ta tive cells p e r gram o f food. T he in g es ted veg eta tive cells sp o ru la te in th e sm all in te s tin e . An en tero to x in , fo rm ed only by spo ru la ting cells , is re le a se d on com p le tion o f spo ru ­lation . A fter an in cub a tio n p e rio d o f ab o u t 12 hours, p ro fuse d ia rrhea a n d abdom inal cram ps are n o ted w ith o u t nausea , vom it­ing o r fever. Sym ptom s usu a lly su b sid e w ith in a day. T h e feces con ta in 105 to 108

LABORATORY DIAGNOSIS OF FOODBORNE DISEASES 3 9 1

organism s p e r gram w ith m any spores, far above th e norm al le v e l8 o f 102 to 104. A carrie r sta te m ay p e rs is t for w eeks. D iag­nosis is b a sed on c lin ica l an d e p id em io ­logic ev id en ce su p p o rted by iso la tion o f th e sam e sero ty pe from feces and sus­p e c te d food.

S m ears o f food sam ples an d feces u su ­ally show th ick gram -positive rods. E n- dospo res m ay b e se e n in food sm ears. T he anae rob ic te c h n iq u e s u se d in cu ltu rin g n e e d n o t b e s trin gen t. Q u an tita tiv e p la te counts a re m ad e on food sam ples using a se lec tive m ed ium co n ta in in g sulfite and egg yoke em u lsio n p lu s sub stances w h ich lim it th e grow th o f o th e f organism s. T he re d u c tio n o f su lfite a llow s reco v e ry o f b lack co lon ies w h ich alsQ show an o p aqu e w h ite halo ow ing to a lte ra tio n o f th e egg yoke em u lsio n by th e bac te ria l enzym e Ie c i th in a s e C (a lp h a to x in ) . S h a h id i- F erg u so n perfrin gen s (SFP) agar con tains kanam ycin an d po lym yxin B .50 A sim ilar selective m edium is tryptose-sulfite-cyclo- se rin e (TSC) agar .35

B lack co lo n ie s a re c o u n te d an d th e co u n t ad ju s ted a fte r th e ratio o f C. per­fr in g e n s is d e te rm in e d in a rep resen ta tiv e sam pling o f th e se co lon ies. Iden tifica tion is b ased on m orpho log ical an d b io ch em i­cal characteristics. T he organ ism is a gram positive bacillu s w ith b lu n t end s. Spores are no t u su ally p ro d u ced in th e m ed ia used . A lactose-m otility m ed iu m is u sed to dem o nstra te lack o f m otility an d th e p ro­du ction o f acid an d gas from lactose. N i­tra te red u c tio n has also b e e n u sed as a confirm atory test.

S ince C. p erfr in g en s is no rm ally p re s ­e n t in feces, th e p u rp o se o f stool c u ltu re is to ascerta in th e sero type p re se n t in th e p a tien t. F ecal sam ples are in o cu la ted in to tw o tu b es o f th io g ly co lla te b ro th , one of w h ich is h ea ted at 80° for 15 m in u tes. Both tu b es are in cu b a ted at 46° for four hours an d th e n su b cu ltu red to b loo d and egg yoke agar p la tes. C o lon ies show ing a d o u ­b le zone o f hem olysis on anaerob ic sh eep

b loo d agar an d an o p aq u e halo on egg yoke agar a re id en tified as C. p erfr in g en s by th e te c h n iq u e s g iven p rev iously . In ­h ib itio n o f Iec ith in ase p ro d u c tio n can b e d em o n stra ted by coating h a lf o f an egg yoke agar p la te w ith ty p e A an titox in . S ub cu ltu re on th is “half-an titox in” p la te w ill grow , b u t h a lo form ation w ill b e p re ­v en te d on th e an titox in side.

A t le a s t th re e d iffe re n t iso la te s from food an d th re e from feces are se ro ty p ed by slide agg lu tina tion . At p re sen t, 87 specific an tise ra are availab le . In a com m on source o u tb reak a s ing le sero ty pe iso la ted from su sp ec t food can b e reco v ered from th e stools o f an ap p rec iab le n u m b er o f p a ­tien ts.

T h e p ro te in en te ro to x in re sp o n sib le for C. p erfr in g en s e n te ritis causes “ food p o i­son in g” sym ptom s in m onkeys an d h u ­m ans, induces- flu id accum ula tion in li­ga ted ilea l loops o f rabbits and p rod uces ery th em a in th e sk in o f rabb its and g u in ea p i g s .44 S u c h b io lo g ic a l te s ts a re n o t specific an d a re no t su ited for ro u tin e use. T h e p ro te in en te ro to x in has b e e n p u rified an d specific an tise ra have b e e n m a d e .26 In one s tu d y ,26 rev e rsed passive hem ag g lu ti­na tio n w as th e m ost sensitiv e test, fol­lo w ed by m icroslide gel d iffusion, s in g le gel an d e lec tro im m uno d iffusion, gu in ea p ig skin test, m ou se le tha lity te s t a n d rab ­b it ilea l loop test. T h e rev e rsed passive h e m a g g lu tin a tio n te s t can b e u se d for m ass sc re en in g o f en tero to x in p ro d u c in g C. p er fr in g e n s in foods, h u m an stools, an im al in te s tin a l co n ten t, e n v iro n m e n t and food po iso n in g ou tbreaks.

S alm onellosisS alm onella in fec tions are tran sm itted

ch iefly from anim als to m an an d on ly oc­casionally from m an to m an. T he p resen ce o f th e se bac te ria in th e in te s tin a l trac t o f m any spec ies o f dom estic and w ild an i­mals leads to fecal con tam ination o f a va­rie ty o f foods, e sp ec ia lly m eat, p o u ltry and eggs o r egg p rod ucts. A ny food p ro d u c t is a

3 9 2 KAZAL

p o ten tia l sou rce of h u m an in fection . Sal- m o n e llae are th e le ad in g cause o f co n ­firm ed b ac te ria l foodborne d isease ou t­b re a k s . In te rm s o f re p o r te d sp o rad ic cases and estim ates o f the extent of unrec­ognized cases the disease is a major p u b ­lic h e a lth p ro b lem . All m em b ers o f th e genus Sa lm onella are p a th o g en ic for m an a n d /o r an im a ls . O n ly a b o u t 50 o f th e know n 1700 sero types occur com m only .

T h ese organism s in vad e th e in testina l m ucosa by p en e tra tin g th e ep ith e liu m . A few sero types, ad ap ted to th e h u m an host, th en in v ad e th e b loo dstream an d cause e n te ric fever (typho id and p a ra ty p h o id fever) o r a sep ticem ic syndrom e. In th e g re a t m a jo rity o f ca se s , h o s t d e fe n se s localize th e in fec tion to th e m ucosa o f the sm all b o w e l an d colon re su ltin g in “gas­t ro e n te r i t i s .” D ia rrh e a an d ab d o m in a l pa in b eg in ab ru p tly after an in cub a tio n p e rio d o f 5 to 72 hours, to g e th e r w ith fever and , freq u en tly , n au sea and vom iting.

L aboratory d iagnosis is b ased on cu l­tu re s o f stools or rec ta l sw abs, su sp ec t foods an d en v iro n m en ta l sw abs. T h e p ro ­c e d u re s e m p lo y e d a re s im ila r fo r a ll spec im ens since q u an tita tio n p roced ures are n o t g en era lly u se d to d e te c t salmo- ne llae in foods. B lood, u rin e an d o ther bo dy flu ids m ay y ie ld positive cu ltu res in th e system ic sa lm onelloses; a fourfold rise in agg lu tin in s against th e som atic or O an tig ens m ay b e d em o n stra ted in acu te an d co n v a lescen t ph ase specim ens.

A 25 g sam ple o f food (or sw abs o f food and food p ro cess in g e q u ip m en t) is p u t in to s e le c t iv e e n r ic h m e n t b ro th (se len ite -cystine b ro th an d te tra th io n a te b rillian t g reen b ro th). T h e cu ltu res are in ­cu b a ted for 24 to 48 hours. S ince food- m anufac tu ring p rocesses such as d ry in g or freez in g m ay dam age th e organism s so th a t th ey cann o t surv ive in th e se slightly toxic m ed ia , a p re -en rich m en t cu ltu re o f lactose o r n u tr ie n t b ro th , using 100 g of food sam ple p e r lite r, is also u se d to e n ­hance grow th p rio r to en rich m en t. F rom

e n r ic h m e n t b ro th th e cu ltu re is s treaked o n s e le c t iv e , d i f f e r e n t ia l m e d ia ,— b r illia n t g re e n agar an d b ism u th su lfite agar. A fter 24 hours in cub atio n , su sp ec t c o lo n ie s a re in o c u la te d in to a t r ip le ­sugar-iron agar slant. S lants w h ich show a ty p ica l sa lm o n e lla reac tio n (red s lan t and y e llo w b u tt w ith b lack en in g an d gas) are te s te d fu rth e r w ith som atic (O) an d flagel­lar (H) an tisera . B iochem ical te s ts are p e r­fo rm ed to confirm po sitive cu ltu res .

C o m p le te sero ty p in g is essen tia l. T he sam e se ro ty pe sh o u ld b e id en tified in p a ­tien ts an d in th e in c rim in a ted food. In ad d itio n phage ty p in g is ava ilab le for Sa l­m o ne lla ty p h i , Sa lm onella p a ra ty p h i B an d S a lm one lla ty p h im u r iu m .

F eca l spec im ens are p ro cessed b y in ­o cu la tin g e n ric h m e n t b ro th an d by p la tin g d i r e c t ly on s e le c t iv e a n d d if f e re n tia l m edia . M acC onkey o r E o sin m e th y len e b lu e agar are u se d to g e th e r w ith an ad d i­tional differential agar such as xylose lysine desoxycholate (XLD) or H ektoen en teric (H E ). B isciello and S ch rad e2 ev a lu a ted H E agar in c o n ju n c tio n w ith b r i l l ia n t g reen , b ism u th su lfite an d S alm onella- S higella agar for the detection o f salmo- n e lla e in foods an d feeds. H E agar w as foun d to b e m ore e ffic ien t th an th e o th e r m ed ia for recovery of sa lm onellae in con­ta m in a te d sam p les . T h is m e d iu m th u s app ears to b e va luab le in th e iso la tion o f sa lm on ellae from b o th c lin ica l an d non- c lin ica l spec im ens.

Enteropathogenic Escherichia Coli Infection

C erta in strains o fE . coli have for m any years b e e n associa ted w ith acu te d ia rrhea in in fan ts. T w en ty d is tin c t an tig en ic ty pes are now recogn ized as “ en te ro p a th o g en ic E . co li” (E E C ). R ecen tly , d ia rrh ea l d is­ease has b e e n a ttrib u ted to E . coli strains o th e r th an th e se classic sero types. Som e o f th e se are sero log ically ty pab le w h ile som e are n o n -ty p ab le . T w o p a th o g en ic m echanism s are now know n to b e involved

LABORATORY DIAGNOSIS OF FOODBORNE DISEASES 393

in E . co li en te ric in fec tion . Som e strains are invasive , p e n e tra tin g th e m ucosa o f t e r m in a l i le u m a n d c o lo n to c a u s e d ia rrh ea l d isease sim ila r to sa lm onellosis o r s h ig e l lo s is . O th e r s tr a in s a re e n ­tero tox igen ic , p ro d u c in g toxins in th e col­o n ized sm all in te s tin e . P ro d u c tio n o f a h ea t-lab ile toxin (LT) is assoc ia ted w ith a cho lera-like d isease . A hea t-s tab le toxin (ST) is a know n cause o f an im al d ia rrh ea l d isease an d p ro b ab ly has a sim ilar effect in m a n . B o th in v a s iv e a n d to x ig e n ic s tra in s h av e b e e n in c rim in a ted n o t o n ly in p ed ia tric d ia rrh ea33 b u t also in fo o d b o m e d isea se ou tb reak s in a d u lts .6 T ox igen ic stra in s have b e e n im p lica ted in trav e le rs ’ d ia rrh e a .30,51 T h e re la tive im p ortance o f in vasiv e an d toxigenic strains has y e t to b e d e te rm in ed .

T h e s tan d ard m e th o d for th e id en tific a ­tio n o f E E C is sero typing . S ero ty p in g m ay b e u se fu l in id en tify in g c e rta in strains o f invasive E. coli b u t it is in a d eq u a te as a m e a n s o f id e n t i f y in g to x ig e n ic s tra in s .15,19 T oxin p ro d u c tio n is co n tro lled b y p la sm id s (ex trach ro m o so m al D N A ) and not by surface antigens. T h ere are tw o p lasm ids, one cod in g for L T an d ST, th e o th e r for ST only. P lasm ids a re tran sfe ra ­b le from one stra in to an o th er, an d it is lik e ly th a t any sero ty pe can acq u ire th e ab ility to form toxins. O th e r lab o ra to ry tests for in vasiv eness an d tox igen ic ity w ill b e d iscussed .

E . c o l i is u s u a lly t r a n s m it te d from person -to -perso n , b u t it m ay also b e a ir­bo rne, w aterborne or foodbom e .6 T he res­e rv o ir is th e stool o f in fe c te d h u m an s. A sy m p to m a tic co lo n ic c a rrie rs o f to x i­genic strains are know n and food handlers m ay b e re sp o n s ib le for o u tb reak s , b u t th e p re sen ce o f a to x igen ic s tra in is u su ­a lly a sso c ia ted w ith i l ln e s s .64 A coffee su b s titu te , salm on and c h ee se are l is te d as veh ic le s in vo lved in o u tb reak s .6 A m ajor com m on sou rce o u tb reak in la te 1971, ow in g to ch ee se im p orted from F ran ce , in v o lv ed 387 p erso n s in 13 s ta tes an d th e

D is tric t o f C o lum bia . E . coli se rogroup 0:124 w as iso la ted from feces o f ill p a ­tien ts as w ell as from th e c h e e se s .5 In one group o f 28 p a tien ts , th e organ ism w as show n to b e invasive an d som e o f th e se p a tien ts h ad a d y sen te ry -like sy n d ro m e .54

S in c e b o th in v a s iv e a n d to x ig e n ic strains are invo lved , it is d ifficu lt to d e ­lin ea te a s ing le c lin ica l p a tte rn fo rE . coli fo o d b o m e in fec tion . A m ed ia n in cu b a ­tion p e rio d o f 10 to 12 hours w ith a range o f 5 to 48 h o u rs6 cou ld re p re se n t e ith e r invasive o r toxigenic d isease . Sym ptom s o f m alaise , head ach e , fev e r an d ch ills to ­g e th e r w ith vom iting , d ia rrh ea and ab­dom inal p a in sug gest an invasive p roc­ess. T h e ex p ec ted p a tte rn in en tero to x in d isease is w atery d ia rrh ea las tin g several days o r lo n g er w ith little o r no abd om in al cram ps an d fever.

F eces or rec ta l sw abs from p a tien ts are c u ltu red u s in g b loo d agar an d s tan dard p la tin g and se lec tive e n r ic h m e n t m edia. T h e ab sen ce o f th e m ore eas ily reco g ­n iz e d sa lm onellae an d sh ig e llae an d th e p re sen ce o f a s ign ifican t n u m b e r o fE . coli in cu ltu re s o f su sp ec t food is an in d ica tio n for fu rth e r stu dy o f E . co li iso la tes from b o th s o u rc e s . I s o la te s a re id e n t i f i e d b ioch em ica lly . T en o r m ore co lon ies from b lood agar p la tes are te s te d w ith O an d OB an tisera . F u r th e r se ro ty p in g is ca rried ou t b y w e l l - s ta n d a r d iz e d p r o c e d u r e s .21 F u rth e r te s tin g to id en tify in vasiv e o r tox­igenic s tra in s has in th e p a s t d e p e n d e d o n in v iv o t e s t s y s te m s n o t w id e ly availab le ,—th e S eren y te s t for in vasiv e­n ess arid an im a l in te s tin a l m o d e ls for toxin p ro d u c tio n . S im p le r rap id ti s sue cu l­tu re assay system s have b e e n recen tly d e ­v e lo p ed a n d have b e e n u se d a t th e C en te r for D isease C ontro l in th e in v estig a tio n o f an o u tb re a k o f e n te ro to x ig e n ic E . co li d ia rrh ea l illness at C ra te r L ake N ational P ark .64

T h e gu in ea-p ig -con ju nctiv itis m o d e l o f S e ren y 49 w h ich w as d e v e lo p e d for th e study o f Sh igella strains has b e e n used

394 KAZAL

Enterotoxin Assays (Cell-free Culture Supernatants)TABLE XX

Adenyl Cyclase ActivatorsCholera (CT) E. coli Clost. Shig. B.

Assay Injection Site Positive E. coli (LT) (ST) perf. dys. cereus

Rabbit Ligated loopileal loop

Infant Stomachrabbit*

Rabbit skin Skin (I.V.Evans blue)

Infant mouse StomachY-l Adrenal Tissue culture

tumorChinese Tissue culture

hamster ovary Hela Tissue culture

Fluid accumulation +loop

Fluid accumulation +gut

Induration# discoloration + (vascular permeability)

Fluid accumulation gut Cell rounding +Increased steroidogenesis Cell elongation +

+ + + +

Cell detachment (%) Low Low High High

*Test material is culture, not cell-free supernatant.

w ith E. coli strains as an assay for tissu e in v asio n . In o c u la tio n is m ad e by loop from an o v ern ig h t b loo d agar c u ltu re in to th e con junctival sac. T h e anim als are o b ­se rv ed daily for four days for ev id en ce o f k e ra to c o n ju n c tiv it is ( red n ess a n d pus) w h ich d ev elo ps on ly w ith invasive b ac te ­rial strains.

A n im al in te s tin a l m o d e ls hav e b e e n u se d to dem o nstra te en tero tox ins o f sev­e ra l e n te r ic p a th o g e n s (tab le IX). E n ­tero tox ins s tim u la te sec re tio n o f in te s tin a l f lu id an d e lec tro ly tes. C h o lera toxin (CT) a n d E . coli h ea t-lab ile toxin (LT) activate ad en y l cyclase w h ich is m em brane b o u n d in in te s tin a l ep ith e lia l cells re su ltin g in th e con versio n o f in trace llu la r A TP to cyc­lic A M P. T h e cy c lic A M P c a u se s th e m ucosal ce ll to sec re te w ate r an d e lec tro ­ly te s .3 E. coli h ea t-s tab le toxin (ST) and th e en te ro to x in s o f C. p e r fr in g en s and Sh ig ella d ysen teria e type 1 do n o t in te rac t w ith th e adeny l-cyclase cyclic AM P sys­tem so th a t m ore th an one b ioch em ica l pa thw ay m ay b e in vo lved in f lu id p ro d u c ­tion by in te s tin a l tissue.

T h e ra b b it ilea l loop assay23 d e tec ts C T and E. coli L T and ST as w ell as o ther en tero toxins. C ell-free su p ern a tan t from an E . coli cu ltu re is in jec ted in to an ex­

te rio rized an d lig a ted loop o f bo w el. To d e te c t LT, th e ab d o m en is c lo sed for 18 hours. T h e loop is th e n rem o ved , th e flu id co n te n t w ith d raw n an d m ea su re d a n d th e len g th o f th e emjpty seg m en t d e te rm in ed . V olum e p e r le n g th ratio (m l p e r cm ) is d e te rm in ed . M axim al values w ith E . coli ST d ev e lo p m ore qu ick ly so th a t a six-hour te s t is also used .

T h e in fan t-rab b it assay 31 d e te c ts C T an d b o th E . coli toxins an d m ay b e m ore se n s itiv e th e n th e p re v io u s te s t. T en - day-o ld rabb its are in fec ted w ith w hole live organism s by in tragastric in jection . T h e anim als are k illed a fte r sev en hours. U sing th e en tire bo w el from py lo rus to rectum , th e ratio o f in te s tin a l flu id to total in te s tin a l w e ig h t (m l:gm o f w h o le bow el) is d e te rm in ed . A ratio above 0.18 is “ po si­tive.” T he infant-m ouse assay14 detects E. coli ST. A 24-hour culture supernatan t is m ixed w ith a b lue dye (as a m arker) and in jec te d in to th e stom ach o f o n e to four day-o ld m ice. A fter four hours, th e m ice are k illed an d th e ratio o f in te s tin a l to total rem aining body w eigh t is determ ined. A ratio o f m oré th an 0.09 is co n sid e red “p o sitive .”

T h e rab b it skin te s t22' 24 d e tec ts C T and E . coli L T b u t n o t ST. C ell-free cu ltu re

LABORATORY DIAGNOSIS OF FOODBORNE DISEASES 395

su p e rn a ta n t is in je c te d i.d. E van s b lu e dy e is in jec te d i.v. 18 hours la ter. In a po sitiv e te s t, th e in jec te d toxin causes in ­c reased cap illa ry p e rm eab ility at th e skin site w h ich b eco m es in d u ra te d an d b lu ish . T h e te s t is a t le a s t as se n s itiv e as th e rab b it- ilea l-lo o p te s t an d m u ltip le tests can b e do n e us in g one rabb it.

T w o n ew tissu e c u ltu re assays th a t d e ­te c t E . coli L T b u t n o t ST offer h o p e of r a p id , s e n s i t iv e , c o n v e n ie n t m e th o d s su itab le for ep idem olog ic investigations. B oth o f th e se assays are b ased on m or­pho log ic chan ges in th e cells w h ich m ay r e p re s e n t a re sp o n se to s tim u la tio n of ad en y l cyclase activ ity b y th e toxin.

D o n ta 17 u se d m onolayer cu ltu res o f a p e rm a n e n t ad ren a l tu m o r tissue ce ll line , Y -l. T ox in acts on th e cells in d u c in g an in c rease in th e o u tp u t o f ce rta in stero ids. A t th e sam e tim e th e re is d is tin c t change in m orpho logy o f th e f la tten ed cells w h ich undergo rounding. W ith this Y-l adrenal tum or cell system , enterotoxin can b e d e ­tec ted directly in fecal and o ther in testinal m ate ria l o f an im als .18 T h e system has also b e e n u se d to d e te rm in e th e lev el o f an ­tib o d ie s to E. coli L T in th e serum s o f v o lu n te e r s .19 A m in ic u l tu re a d a p ta tio n m akes it p o ss ib le to te s t several h u n d re d E. co li cu ltu re s p e r day for L T p rod uc tion w ith a re su lt o b ta in ab le w ith in tw o to th re e days o f stool co llec tio n .47 T h e ad­ren a l ce ll assay w ill d e te c t 0.2 m icrogram o f c ru d e E. coli toxin p e r m l o r 5 ng o f p u rif ie d ch o le rag en p e r ml.

A seco n d assay, ad ap ted by G u erran t,32 u tilizes th e C h in ese h am ste r ovary ce ll line . A ce ll-free su p e rn a tan t o f a 48-hour E . co li c u ltu re is in cu b a ted w ith fresh ly su sp e n d e d cells an d causes ce llu la r e lo n ­gation o f u p to 50 p e rc e n t a fte r a 24 -hour in cu b a tio n . T h e p ercen tag e o f cells th a t e lo n g a te is d e te rm in e d b y th e co n cen tra ­tion o f toxin. T h is assay w as u sed recen tly in a stu dy o f acu te p ed ia tric d ia rrh ea in w h ich th e m ajority o f p a tien ts w ere show n to have toxigenic L T iso lates o f E . coli.33

Vibrio Parahaemolyticus Infection

F rom 1969 to 1974, th e re w ere 14 o u t­b reak s (1,202 cases) associa ted w ith con ­tam in a ted seafood w h ich w ere a ttrib u ted to V ib r io p a r a h a e m o ly t ic u s . T h e f irs t labora to ry -confirm ed ou tb reak in M ary­land in 1971, ow ing to steam ed clam s, in ­volved over 305 p eo p le .13 Infection is fre­q u e n t in Japan ow ing to the ingestion of raw fish. Tw o large outbreaks occurred in 1975 on cruise ships sailing from Florida. O n e o u tb reak on an in te rn a tio n a l p as­senger flight has b een reported .45

T his m arine b ac te riu m is u b iq u ito u s in coastal w aters d u rin g w arm w ea th e r an d is a sso c ia te d w ith m arin e life . A few o r­ganism s in im p ro p erly h an d le d raw fish and seafoods, or in cooked foods in contact w ith th em , can in crease to th e in fective d o se o f 106 to 109 organism s in th re e to four hours s ince th e gen era tio n tim e is as sho rt as 10 to 12 m in u tes . A lthough large n u m b ers o f organism s are p re se n t in feces d u r in g th e i l ln e s s , p e r s o n - to - p e r s o n tran sm iss io n is u n kn ow n . An asym ptom a­tic ca rrie r s ta te has n o t b e e n d e lin e a te d a n d th is a re a r e q u ire s in v e s tig a t io n .52 C lin ica l ob serv a tio n suggests th a t th is e n ­te r ic in fec tion m ay b e b o th invasive and en tero to x ig en ic .

T h e usu a l in cub a tio n p erio d is 4 to 96 hours w ith a m ed ian in th e 15 to 24 h o u r range. W atery d ia rrh ea is acco m p an ied by a b d o m in a l c ra m p s , n a u s e a , v o m itin g , h ead ach e , fev e r an d ch ills. D u ra tio n o f illn ess ranges from severa l hours to over 10 days. T h e ou tb reak s are o f exp losive charac te r an d th e illn ess re sem b les sal­m o n e llo s is .1

T h e o rg a n is m is a f a c u l ta t iv e ly anaerob ic , fe rm en ta tiv e , ox idase-positive , ha lop h ilic , m o tile gram -negative rod. A sa lt co n cen tra tio n o f at le a s t 2 p e rc e n t m u st be p ro v id ed a n d it w ill to le ra te up to 9 p e rcen t. S trains from stool cu ltu re o f p a ­tien ts w ith gastro en te ritis spo t in o cu la ted on a specia l hu m an b loo d agar m ed iu m

3 9 6 KAZAL

(W agatsum a’s agar) p ro d u ce a zone o f b e ta h e m o ly s is ( th e K an ag aw a te s t) . S in ce strains from seafoods and seaw ate r do n o t show hem olysis, th is is reg a rd ed as a te s t for p o ten tia l patho gen ic ity .

F ec e s o r rec ta l sw abs, food hom oge- nates and en v iro n m en ta l sw abs are cu l­tu re d on m ed ia co n ta in in g 2 to 3 p e rc e n t N aC l for is o la tio n , id e n t if ic a tio n an d sero ty p in g o f th e organism . D ire c t exam i­natio n is n o t reco m m en d ed . I f th e labora­to ry is n o t a le r t to th e p o ss ib ility o f a ha lop h ilic o rgan ism , th e d iagnosis w ill b e m issed . S pec im en s are s treak ed on thio- s u l f a te - c i t r a te - b i le s a l t - s u c ro s e a g a r (TCBS) for se lec tiv e iso la tion . E n r ic h ­m en t is no t n ecessary in th e acu te s tages; 1 p e rc e n t p ep to n e w a te r con ta in in g 3 p e r­cen t N aC l in c u b a ted 18 hours at 35° can b e u s e d . B lu is h -g re e n n o n - s u c ro s e fe rm en tin g co lon ies on TC B S p la tes in ­cu b a ted o v ern ig h t can b e d iffe ren tia ted from th e ye llow co lon ies o f o th e r v ibrios. T h ree o r m ore su ch co lon ies are u se d for gram s ta in an d in o c u la tio n o f su cro se b ro th and a trip le -su gar-iro n agar slant, each w ith a d d ed N aC l. A se ries o f tu b es o f1 p e rc e n t p ep to n e b ro th w ith a range o f con cen tra tion o f N aC l from 0 to 11 p e rc e n t is in o cu la ted w ith o n e d rop o f a b ro th cu l­tu re . O rgan ism s w h ich grow on ly in 3, 7 an d 9 p e rc e n t N aC l an d w h ich p ro d u ce a lkaline slan t, ac id b u tt and no gas on T S I are te s te d fu rth e r to d iffe ren tia te th em from other vibrios. V. parahaem olyticus is readily differentiated from V. cholerae by failu re to fe rm en t sucrose an d fa ilu re to a g g lu tin a te in c h o le ra O g ro u p I a n ­tise ru m an d by N aC l req u irem en ts .

A p o s itiv e K anagaw a te s t, a sso c ia ted w ith the pathogenicity of the organism , is f req u en d y o f v a lue . T h e v a lu e o f sero ty p­ing rem ains u n c le a r b eca u se sero ty pes o f iso lates from food an d p a tien ts in an ou t­b re a k m ay n o t c o in c id e a n d s e v e ra l se ro ty p es hav e b e e n iso la ted from th e stool o f a s ing le p a tien t. C u rren tly , e lev en O and 57 K an tig ens are know n.

B acillus C ereus E n te ritisB a c illu s c e reu s w as re p o r te d as th e

cause of foodbom e disease outbreaks only e ig h t tim es b e tw e e n 1968 and 1974. B. cereus e n te ritis has b e e n m ore freq u en tly rep o rted in E u ro p e . O v er 20 years ago, H au g e d e sc r ib e d fo u r N o rw eg ian o u t­b reaks invo lv ing 600 perso n s ow ing to a van illa sa u c e .28 A fter an 8 to 16 h o u r in cu ­b a tio n p e rio d , w a te ry d ia rrh ea an d ab­dom inal cram ps w ith n au sea an d som e vom iting ap p ear. S h o rte r in cub a tio n p e ­riods o f one an d a h a lf to five hours w ith n a u s e a a n d v o m itin g r e s e m b l in g stap h y lo co cca l in to x ica tio n also occu r. T h e p a th o g e n e tic m ech an ism in vo lved m ay b e e n te ro to x in fo rm ation . An en- tero toxin cou ld b e p erfo rm ed in th e food v eh ic le in som e cases a n d form ed after c o lo n iz a tio n o f th e in te s t in e in o th e r cases, thus acco u n tin g for th e tw o sets o f in cu b a tio n perio ds.

T h e o rg a n is m is a n a e ro b ic , gram - p o s i t iv e , s p o re - fo rm in g m o ti le ro d , closely re la ted to B acillu s an th rac is . I t is com m on in soil an d d u st, on vegeta tion and in m any raw an d p ro cessed foods. It is u su a lly c o n s id e re d n o n p a th o g e n ic for m an. A varie ty o f m o ist cooked p ro te in foods a llow p ro life ra tion o f th e organism w h e n im p ro p e rly re fr ig e ra te d . T y p ica l foods are cerea l p rod uc ts, custards, p u d ­d ing s, sauces an d m ea t loaf .6 F r ie d or b o iled rice has b e e n im p lica ted in a series o f re c e n t ou tb reaks in th e U n ited K ing­dom .42

Laboratory diagnosis requ ires the use of a selective m edium (such as phenol-red eg g y o k e p o ly m y x in agar) c o n ta in in g m an n ito l .53 C o lon ies su rro u n d ed by a halo o f p rec ip ita te ow ing to le c ith in a se form a­tion a n d show ing a re d back g ro u n d ow ing to in ab ility o f th e organ ism to d issim ila te m an n ito l a llow p re su m p tiv e id en tifica ­tion . P la tin g o f se ria l d ilu tio n s o f food ho m ogen ate is n ece ssa ry to en u m era te th e organism s b eca u se cou n ts as h igh as 106 organism s p e r gram m ay n o t be sig­

LABORATORY DIAGNOSIS O F FOODBORNE DISEASES 3 9 7

n i f ic a n t . C o n f irm a to ry t e s t s , c h ie f ly b ioch em ica l, are necessary . Stool sp ec i­m e n s s h o u ld b e c u l tu r e d s in c e h ig h cou n ts o f B. cereus p ro v id e su p p o rtiv e evidence. S tandard stool culture is done to rule o u t o ther en teric pathogens.

In a r e c e n t o u tb re a k in v o lv in g tw o p eo p le a n d traced to m ash ed p o ta to es ,65 B. cereus w as im p lica ted on th e basis o f sho rt in cu b a tio n p e rio d , du ra tion o f abo u t one day , a food co u n t o f 1.8 x 107 p e r g and d em o n stra tio n o f en te ro to x in p rod uc tion by th e iso la te . R ab b it ileal loop an d skin p e rm e a b ility te s ts w e re u tiliz e d . G latz an d G o ep fe rt27 re cen tly d e sc r ib ed a skin factor in c u ltu re filtrates o f B. cereus and found a co rre la tio n b e tw e e n loop -flu id in d u c in g a b i l i ty a n d sk in a c tiv ity in gu in ea p igs. T h e skin te s t can b e u se d to assess th e en tero to x ig en ic ity (for rabbits) o f iso la tes o f B. cereus.Sum m ary

T h e lab o ra to ry d iagnosis o f b ac te ria l food bo rn e d isease is an in teg ra l p a rt of ep id em io lo g ic su rve illan ce an d in vestiga­tio n . T h e iso la tio n o f a p a th o g e n in a spo rad ic case o f in fection w arran ts in v es­tiga tion to d e tec t th e source. C o llec tin g an d c o lla ting d ata in app aren tly u n re la ted cases m ay rev ea l u n su sp ec ted com m on sou rces o f in fec tio n an d lead to action w h ich can con tro l an ou tb reak and p re ­v e n t fu tu re ou tb reaks. In th e investiga tion o f o u tb reak s, th e goal o f laborato ry con ­firm ation is still n o t re ach ed in m any in ­stances. T h e ou tcom e d ep en d s on b o th in vestiga tive an d laborato ry capab ilities .

B ac te ria l fo o d b o rn e d isea se s are d i­v id e d in to in tox ications an d in fections. T h e p a th o g en e tic m echan ism s in vo lved are la rge ly u n d ers to o d and th e se d iseases can b e c lass ified on th is basis. T h e four m ajor ca teg o ries are p re fo rm ed toxins, e n ­tero tox in form ation in th e in fec ted (col­on ized) bo w el, m ucosal invasion a n d bac­terem ia . Sero logical m ethods w h ich m ay re p la c e th e s tan d a rd m ouse-tox in n e u ­

tra liza tio n te s t (botulism ) and th e m icro ­slide gel d iffusion te s t (staphylococcal in ­to x ic a tio n ) a re in th e d e v e lo p m e n ta l stages. T h e d ev e lo p m en t o f n ew tissu e cu ltu re assays for th e h ea t-lab ile tox in of en te ro p a th o g en ic E. coli o p en s th e w ay for p o ss ib le rap id m eth ods o f id en tifica ­tion o f o th e r en tero toxins. R ecovery o f th e causa tiv e o rgan ism s is a basic re q u ire ­m e n t for lab o ra to ry con firm ation in all c a te g o rie s a lth o u g h le a s t im p o rta n t in bo tu lism . A w areness o f invasive stra in s o f E. co li and in creased ab ility to id en tify th e to x in s o f e n te ro to x ig e n ic s tr a in s h as s tim u la te d in v es tig a tio n in th e a rea o f p ed ia tric an d ad u lt d ia rrh ea l d isease . T h e p a th o g en e tic m echan ism in som e in fec ­tion s (V. pa ra h a em o ly ticu s) rem ains to b e d e te rm in ed . W ith th is organ ism as w e ll as o thers, m ore th an one m echan ism m ay b e involved .*

L aborato ry p ro ced u res for th e d e fin i­tive ty p in g o f bac te ria such as toxin ty p in g an d ph age ty p in g are o f g rea t im p ortance in ep idem io lo g ic investigation . T h e d is ­covery o f n ew toxin types sho u ld re su lt in in c re a s e d e p id e m io lo g ic c a p a b il i t ie s . P urifica tion o f know n toxins such as C. p erfrin g en s en tero to x in paves th e w ay for th e d e v e lo p m e n t o f n e w s e ro lo g ic a l te ch n iq u es .A ckn o w led g m en t

A cknow ledgm ent is m ade to F ran k L . Bryan, Ph.D ., Chief, F oodborne D isease T rain ing, B ureau

o f T rain ing, C en ter for D isease Control, A tlanta, GA, for advice and assistance in the preparation of this paper.

R eferen ces1. BARKER, W. H.: Vibrio parahaem olyticus ou t­

breaks in th e U n ited States. L ancet 1 -.551-554,1974.

2. B i s c i e l l o , N. B ., J r . an d S c h r a d e , J . P.: E valuation o f H ektoen en te ric agar for th e d e ­

* An enterotoxic factor w ith p ro perties s im ilar to th e E. coli en terotoxins has b een iso la ted from cu l­tu res o f Salm onella (Koupal, L. R. and D eib e l, R. H.: Assay, characterization , and localization o f an e n ­terotoxin p roduced by Salm onella. Infect. Im m un. 11:14-22, 1975).

3 9 8 KAZAL

tec tio n of sa lm onella in foods an d feeds. J.A.O.A.C. 57:992-996, 1974.

3. B i t e n s k y , M. W., K e ik n s , J. J., and F r e e m a n , J.: C yclic adenosine m onophosphate and clin i­ca l m e d ic in e (P art I). A m er. J. M ed . Sci. 266:321-347, 1973.

4. B O R O F F , D . A. an d S h u - C h e n , G .: R adio­im m unoassay for type A toxin o f C lostrid ium botu linum . Appl. M icrobiol. 25 :545—549, 1973.

5. B r a c h m a n , P. S., T a y l o r , A., G a n g a r o s a , E. J., M e r s o n , M . H ., an d B a r k e r , W. H.: Food poisoning in the U.S.A. T h e M icrobiological Safety o f Food. H obbs, B . C. and C hristian , J. H.B., eds. N ew York, A cadem ic Press, p. 147,1973.

6. B r y a n , F. L.: D iseases T ran sm itted by Foods. C e n te r for D isease C on tro l P u b lica tio n No. 75-8237, pp . 2 -10, 1975.

7. B r y a n , F. L .: G uide for Investigating Food- b o rne D isease O utbreaks and A nalyzing Sur­v eillance D ata. C en te r for D isease C ontrol, 2nd Prin ting , 1975.

8. BRYAN, F. L.: W hat th e sanitarian sh o u ld know about C lo strid iu m perfringens foodbom e ill­ness. J. M ilk Food T echnol. 32:383-389, 1969.

9. G a s m a n , E . P. and B e n n e t t , R. W.: D etection o f staphylococcal en tero tox in in food. Appl. M icrobiol. 73:181-189, 1965.

10. C a s m a n , E. P., B e n n e t t , R. W ., D o r s e y , A.E ., and S t o n e , J. E .: T he m icroslide gel d ou­b le diffusion te s t for the d e tection and assay of staphylococcal enterotoxins. H ealth Lab. Serv. 6:185-198, 1969.

11. C O H E N , J. O.: Sero log ical ty p in g o f s tap h ­ylococci for ep idem iolog ic stud ies. R ecen t A d­vances in Staphylococcal R esearch. Ann. N.Y. Acad. Sci. 2 3 6 :4 8 5 ^ 9 4 , 1974.

12. C r o w l e , A. J.: A s im p l i f i e d m ic ro d o u b le d i f ­f u s io n a g a r p r e c ip i t i n t e c h n iq u e . J . L a b . C l in . M e d . 52:784-787, 1958.

13. D a d i s m a n , T. A., N e l s o n , R., M o l o n d a , J. R., and G a r b e r , H. J.: V ibrio parahaem olyticus gastro en te ritis in M aryland. I. C lin ical and ep id em io lo g ic aspects. A m er. J. E p id em io l. 96:414-426, 1973.

14. D e a n , A. G., C h i n g , Y. C ., W i l l i a m s , R. G., and HA RDEN, L . B .: T est for E scherich ia coli en terotoxin u sing infant m ice: app lication in a study o f d iarrhea in ch ild ren in H onolulu . J. Infect. D is. 725:407-411, 1972.

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19. D o n t a , S. T., S a c k , D . A., W a l l a c e , R. B., D u p o n t , H. L., and J a c k , R. B.: T issue-cu lture

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