A modified infant mouse assay for bacterial enterotoxins

TRANSACTIONS

OF THE

ROYAL SOCIETYOF TROPICALMEDICINEAND HYGIENE,VOL. 77, No. 5, 699-701 (1983)

A modified

infant

mouse

assay for bacterial

699

enterotoxins

R. J. BERRY,* VALERIE BURKE AND MICHAEL GRACEY Gastroenterology and Nutrition ResearchUnit, PrincessMargaret Children’sMedical ResearchFoundation, and the Department of ChiM Health, University of WesternAustralia, Perth, WesternAustralia Summary

The reproducibility of the infant mouseassayfor Escherichia coli ST and heat-labileenterotoxinsof Aeromonas spp. is improvedif both intestinalweight to remainingbody weight ratio (IWRBW) and the amount of diarrhoeaproducedare consideredascriteria for classifyingenterotoxigenicstrains. Animalswith profusediarrhoeamay have IW/RBW ratiosbelowthe widely acceptedcritical value for a positive test. Usingpoolsof supernatantsfrom broth culturesof three different strainsof E. coli, 15%of ST producerswould have beenregardedasnegativeusingIW/RBW ratio asthe only criterion of a positive test. In testing singlesupernatants,25%of ST producingE. coli would not have been correctly classifiedusingIWRBW alone. A scoringsystemwhich incorporatesIW/RBW ratios and the amount of diarrhoeaproducedimprovesthe usefulness of the test by allowingclear separationof positive and negativestrains.The scoringsystemis alsoapplicableto older mice for assayof E. coli ST so that a wider age range of mice can be usedallowing increaseduse of animal facilities. Introduction

The infant mouseassay(IMA) is usedwidely to detect heat-stabletoxin (ST) of Escherichiacoli. The toxin inducesintestinalfluid accumulationcausingan increasein intestinalweight to remainingbody weight (IWRBW) ratio; ratios>0*083are widely acceptedas positive(GIANNELLA, 1976).Alternatively, diarrhoea has been suggestedas the indicator of a positive response(MOONet al., 1978).We report a modification of this assayusing a combination of IWRBW ratio andthe degreeof diarrhoeainduced. This simple modificationincreasesthe reproducibility of the assay and allows detection of E. coli ST from single or, pooled culture supernatants.Toxigenic strains of Aeromunasspp. are important causesof childhood gastro-enteritis (GRACEY et al., 1982) and their enterotoxins are also detectableusing this method. The method, as described, will help facilitate epidemiologicalstudiesof the role of enterotoxigenic bacteriain studiesof diarrhoealdiseases in developing countries. Materials

and Methods

Bacteria

Reference strains of E. coii known to produce LT, ST, LT and ST or neither toxin were kindly given by Dr. Harley Moon and Dr. R. Bradley Sack. Other strains of E. coli were isolated during studies of enteric pathogens in Aboriginal communities in the tropical north of Western Australia from August 1979 to May 1980 and in a study of malnourished infants and young children with diarrhoea in Jakarta in September 1979. Faecal specimens were transported in Carey-Blair medium and subcultured on to MacConkey agar plates within 48 hours of collection. Lactose fermenting and non-lactose fermenting colonies picked from MacConkey plates were stored at room temperature in maintenance medium consisting of 5 g sodium chloride, 2.5 g Difco Bacto peptone 0118 (Difco Laboratories, Detroit, Michigan, USA), 2.5 g Oxoid Peptone L34 (Oxoid Ltd., Basingstoke, England), 134 ml, 0.15 M NarHP04, 66 ml 0.15 M KHrP04, and distilled water up to one litre at pH 7.

Preparation

of crude

culture

supernatants

Trypticase soy broth with yeast extract (TSB-YE) consisted of 17 g tryptone L42 (Oxoid Ltd..,. Basingstoke,

England), 3 g phytone peptone 11906 (BBL, Cockeysville, Maryland, USA), 5 g sodium chloride, 25 g KrHPO,, 2.5 g glucose and disttlled water to one litre at pH 7.3 plus 0.6% yeast extract L21 (Oxoid Ltd., Basingstoke, England). Bacteria were inoculated into 25 ml Erlenmeyer flasks containing 5 ml of TSB-YE and incubated at 37°C for 24 hours in a G-24 environmental gyratory shaker (New Brunswick Scientific, Edison, New Jersey, USA) at 300 rpm. Broths were centrifuged (10,000 g at 4°C for 30 min) and supematants pooled into groups of three and stored at 4°C. Isolates were tested within two months of isolation. Mice Random bred Swiss albino mice two to six days old were inoculated using a gastric tube (external diameter 0.6 mm) with 0.1 ml of culture supematant containing 2 drops per ml of 2.5% pontamine sky blue dye (Searle, High Wycombe, Bucks. England). Mice were incubated in stainless steel trays divided by a metal insert into compartments, 3.5 cm’ and 7.5 cm deep. The bottom of each tray was completely covered with a single piece of blotting paper. Three or four mice were inoculated with each culture supematant then incubated, with not more than two mice per compartment, for three hours at 28°C and 39% relative humidity.

scoIingof test

Mice were killed by cervical dislocation and IW/RBW ratio was calculated. During initial surveys a ratio 20.08 was considered positive. Diarrhoea was graded on a scale from 0 to 4+ by comparison with a reference standard (BURKE et al., 1981), related to the intensity of blue staining of blotting paper covering the floor of the tray. An IW/RBW ratio of 40.07 scored 0,0.07 to 0.79 scored 1,0*080 to 0.89 scored 2, 0.090 to 0.099 scored 3 and
700

MODIFIED

INFANT

MOUSE

ASSAY

Assay conditions for E. coli ST E. coli strainH-10407(humanLT+/ST+) wasusedto

investigate factorsimportantin the assay.In’theseexperimentsmethods wereunchanaed extent that culturesunernatantswereusedwithintwohoursof-preparation andserial two-fold dilutions were comparative purposes.

used to quantitate The

end

point

ST production was defined

FOR

BACTERIAL

8 I

AAAA l *e AAAA a

scores

were

compared

using

the

Wilcoxon

~~0.05

as the

l e

A

Rank-Sum testandKurskal-Wallis testfor categorical data with

l ee

:

Statistical methods mouse

E.coli A A.hydrophila

l

for

as the

reciprocal of thehighestdilutionwhichproducedanIMS of at least2. Osmolalities of brothsandculturesupernatants weremeasured by freezingpoint depression with a Halbxnikro osmometer (Knauerand Co., West Berlin). Infant

ENTEROTOXINS

significance

level

(LEHMANN,

1975).

A

Results

0

Culture supernatantswere pooled into groups of three and componentstestedassingleculture supernatantsif there wasdiarrhoeaor if the IW/RBW ratio exceeded0.07. Using these criteria, 48 of the 760 original poolswere retestedassinglecultures. 27 of the 48 pools retested yielded 61 single culture supernatantspositivefor ST. Four of these27positive pools, which containednine of the 61 ST-producing E. coli, had IW/RBW ratios <0*08 but were retested becauseof the presenceof diarrhoea. Culture supernatantsof other Enterobacteriaceae including Salmonella, Shigella, Proteus, Klebsiella, Enterobacter, Citrobacter, Arizona and Serratia aswell as Pseudomutt+ which were isolated from these surveys, were mcluded in the original pools and constitutedlessthan 10%of the total strainstested. No pooled or single broth supernatant from this group producedsignificantdiarrhoeaor an IW/RBW ratio >0*08. Relationship

between diavhoea

and ZWIRBW

There was a statisticallv sitmificant (~<0*05) inverse relationship between diarrhoea“scores’ and IWRBW ratios. Positiveswith 4+ ‘diarrhoeahad a mean IWlRBW ratio of 0.076 + 0.003; positives without diarrhoeahada meanIW/RBW ratio of 0.112 + O-007.33 ST positive assayshad IW/RBW ratios lessthan the widely acceptedlower limit of 0.083. Infant

mouse score

The IMS, which incorporatesboth diarrhoeaand IW/RBW, wasretrospectivelyappliedto all assaysof pooledand singlebroths. All positive pools had an IMS of 3 or moreandall the positivesinglebrothshad an IMS of 2 or more. On confirmatory retesting of singlebroths, all five which had an IMS of 2 had highervalueswhile the one broth with an IMS of one retestedaszero. No ST negativesinglebroth had an IMS one and no ST positivesinglebroth and an IMS lessthan 2. Aeromonas

Culture supernatantsfrom 103strainsof Aeromonas from Australia. India and Bannladeshwere t&ted in the IMA under the samecklitions; 76 producedan IMS of two or more. Fig. 1 showsthat this activity was heat-labile, being abolished by heatingat 56°Cfor 10min, whereasthe activity of E. coli ST wasunaffectedby heatingat 100°Cfor 30min. SDD.

unheated

4i%&?t% heated

Fig. 1. Effect on IMS of heating culture supematsnts of E. (100% for 30 min.) and A. hydrophiln (56°C for 10 min.).

co/i

Assay conditions for E. coli ST

Age of mice did not significantly affect IMS but two-day-old mice produced the highest IW/RBW ratiosand the leastdiarrhoea,while six-day-oldmice produced a low IWRBW ratio and the most diarrhoea. During initial evaluationof possibleculture media, Casaminoyeast extract broth had an osmolality in excessof 500mmol/l, and false positives occurred whensterilebroth or the referencepanelof E. coli was testedwith incubationof miceat 28°Cand 37°C.The osmolality of TSB and TSB-YE was less than 400 mmol/l, and false negativesor positivesdid not occur with the referencepanel. H-10407 grown in TSB-YE wasoften positive to a higher dilution than TSB (32V. 16), but this differencewasnot signiknt. H-10407culture in TSB-YE at 300 rpm produced higher scores than when cultured at 150 rpm (~~0.05) but both were positive to a dilution of 32. An incubationtemperatureof 37°Cfor 3 hourswas unsatisfactory since mice often died under these conditionswhen inoculatedwith ST-containingTSBYE, and at this temperaturediarrhoeawascommon usingTSB-YE and TSB brothswhich did not contain ST. H-10407grown in TSB-YE at 300 rpm produced positive IMAs to dilutions of 16 and 32 when mice wereincubatedat 25°Cand28°Crespectively,but this difference was not significant. The infant mouseassayis sensitiveand reliablefor detection of E. coli ST or heat-labile toxins of Aeromows SDD. if both IW/RBW ratio and the presenceof hiarrhoea are consideredin classifying toxigenic strains.Usingthe IWRBW ratio alonemay miss positive strains which causeprofuse diarrhoea becausethe fluid lossreducesIWRBW ratios which are significantly lower in testsshowing4+ diarrhoea than in those without diarrhoea. In our study 15% (9/61) of ST-producing E. coli would have been missedduring initial screeningof pooled supernatantsif diarrhoea had not been recorded. During initial retestingof supernatantpools

R.

J. BERRY

as single samples, 25% (15/61) would have been missed using IW/RBW ratios alone. We found it impossible toYevaluatediarrhoea on the blotting paper without using a gastric tube to inoculate the mice because leakage of blue dye occurred from the transcutaneous injection site, which is used by many workers, and obscured the presence of diarrhoea. Diarrhoea has not previously been recorded in reports recommending IW/RBW ration as the index of response in the infant mouse assay for E. coli ST while MOON et al. (1978)recommendthat diarrhoea in miceincubatedat 37°CshouldreplaceIW/RBW in discriminating ST producing strains. GIANNELLA (1976) found decreasedIW/RBW ratios in mice incubated at 37”C, and in sometests the ratio was lower than the critical value of 0.083. Howeverhe did not record the presenceof diarrhoeain theseanimals. MOON et al. (1978) reported diarrhoea in mice incubatedat 3O”C,with micemorethan four daysold having diarrhoeaafter shorterincubationsthan younger mice. In our experience six-day-old mice had lower IW/RBW ratios and more diarrhoea than two-day-old mice after incubation at 28°C but using the IMS which incorporatesIW/RBW and diarrhoea avoidsfalsenegativesin older mice. The suitability of micefrom a wider agerangecontributesto better use of animal facilities. Infant mousescoreswerehigher when brothswere shakenat 300 rpm but the addition of 0.6% yeast extract to TSB. and incubationsof mice at 28°Cdid not affect the assay significantly. Casaminoyeast extract broth was an unsuitable culture medium becauseits high osmolality produced false positive results. Although we pooled only three culture supernatants instead of the five reported by BYERS & DUPONT(1979), our data con&m that pooling is satisfactoryin screeningfor ST producing E. coli if both IW/RBW and diarrhoea are measured.The sensitivity of the method was shown by dilution experiments in which ST containing broths were positive in dilutions of one in 8 to one in 128. The infant mousescore discriminated very well betweenST positive and negativestrainson repeated testing. All ST producing E. coli and all enterotoxigenie strains of Aemnonus spp. had scoresof 2 or more when tested in single broth cultures and no negative strain scored more than one.

et

701

al.

The assayconditionsdescribedand, in particular, the useof both diarrhoeaand IWiRBW asindicesof responsemakes the infant mousetest reproduciblein screeningAer-s spp. and pooled samplesfrom strainsof E. coli for enterotoxins.Thesemodifications shouldfacilitate epidemiologicalstudiesof the role of thesetoxigenic organismsin diarrhoea. Acknowledgements

We thank Harlev Moon and Bradlev Sack for kindly supplyingreferencestrainsof roxigenic E. co/i. The assisiante of Doctors Suharyono and Sunoto in the Department of Child Health!Universityof Indonesia and Dr. Randolph Spargo andSisters JoanHayandJosephine Seraphun of the Western Australian Community and Child Health Services for assistance in the collection of specimens is gratefully acknowledged. Sally Dalton-Morgan provided excellent technical issistance. The work was supported by a grant from the TVW Telethon Foundation, Perth, Western Australia and the Wellcome Trust, London. We are also grateful to Airlines of Western Australia for help with travel. References

Burke, V., Robinson, J,, Berry, R. J. & Gracey, M. (1981). Detection of enterotoxins of Aeromonas hydrophila by a suckling-mouse test. Journal of Medical MicrobioloB, 14, 401-408. Byers, P. A. & Du Pant, H. L. (1979). Pooling method for screening large numbers of Escherichia coli for production of heat-stable enterotoxin, and its application in field studies. 3aumalof Clinical Microbiology, 9, 541-543. Giannella, R. A. (1976). Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infection and Immunity, 14, 95-99. Gracey, M., Burke, V. & Robinson, J. (1982). Aeromonasassociated gastroenteritis. Lancer, ii, 1304-1306. Lehmann, E. L. (1975). Nonparametrics: Statistical methods based on ranks. San Francisco: Holden-Day, pp. 5-23 and 210-219. Moon, H. W., Fung, P. Y., Whipp, S. C. & Isaacson, R. E. (1978). Effects of age and ambient temperature on the response of infant mice to heat-stable enterotoxin of Escherichia coli: assaymodification. Infection and Immunizy, 20, 36-39.

Accepted for publication

20th February,

1983.