A footpad lesion test to demonstrate the pathogenicity of Escherichia coli strains

Ann. Microbiol. ( Insl. Pasteur ) 1984, 135 B, 311-321

A FOOTPAD TO D E M O N S T R A T E

LESION THE

OF E S C H E R I C H I A

TEST

PATHOGENICITY

COLI

STRAINS

by D. Buzoni-Gatel, J. De Rycke and N. Bosseray Slalfon de Palhologie de la Reproduction, INRA, 37380 Nouzilly, Monnaie (France)

SUMMARY A virulence test based on the ability of E. coli strains to cause lesion in the footpads of mice was devised to demonstrate their pathogenicity. Virulence was expressed by a scale from 0 to 7 according to the importance of the lesions. Among 54 strains tested, those inducing death after intraperitoneal inoculation caused severe (edema or necrosis (score >4) in footpads. Three other virulence tests (virulence measured in pregnant mice, in adult mice intraperitoneally challenged and in baby mice after oral inoculation) were used to study seven of the most characteristic strains. Good correlation was observed: strains pathogenic in the footpad lesion test were pathogenic in at least one of the other tests. KEY-WORDS: Escherichia coli, Pathogenicity, Footpad; Mouse, Test.

Different types of virulent Escherichia coli have been identified as being responsible for diseases in young calves: in addition to enterotoxigenic E. coli the virulence of which is restricted to the digestive tract, there exists a group of E. coli strains isolated from eases of septicaemia or enteritis which has a high virulence for mice after intraperitoneal (i. p.) inoculation [6]. Several tests have been developed in mice to assess systemic virulence, including intraperitoneal (i. p.) [11], intravenous (i. v.) [19] and footpad inoculations [12]. But none of these tests has been used to study the mechanisms of the pathogenicity of bacteria. The purpose of our investigation was to devise a virulence test sensitive and :rapid enough to screen virulent strains of E. coli on the basis of their capacity to induce lesions in the footpad of mice after subcutaneous Manuscrit re~u le 16 avril 1984, accept~ le 15 n o v e m b r e 1984.

312

D. BUZONI-GATEL AND COLL.

(s. c.) inoculation, and t h e n to use this t e s t a n d o t h e r v i r u l e n c e t e s t s to s t u d y t h e p a t h o g e n i c i t y m e c h a n i s m s of t h e t e s t e d strains. W e s h o w e d t h a t strains causing lesions in t h e f o o t p a d are l e t h a l in t h e s t a n d a r d v i r u l e n c e t e s t of i. p. inoculation. T h e r e f o r e , our f o o t p a d lesion t e s t could be used as a sensitive v i r u l e n c e test. Using this f o o t p a d lesion t e s t and o t h e r v i r u l e n c e tests, we t r i e d to u n d e r s t a n d t h e p a t h o g e n i c p r o p e r t i e s of some v i r u l e n t E . coli strains.

MATERIALS

AND

METHODS

Strains.

Fifty-four strains were tested. Forty-four were isolated from faeces of diarrhoeic calves, three from cases of septicaemia (two strains, V2019 and R15 carrying the O78:K80 serotype, and one strain, SsVIR carrying the VIR plasmid [18] and five from healthy calves. One E. coli K12 strain was used as a non-pathogenic control strain. Mice.

OF1 outbred mice (Iffa-Credo, Saint-Germain-Sur-l'Arbresle, France) were born under controlled conditions (temperature 21 ~ C, relative humidity 60%, sterile filtered air, water and sterilized food ad libitum). Experiments. a) Footpad lesion test. Preparation o/live challenge. - - Bacteria were grown overnight either in trypticase soya broth (TSB; Biom~rieux, Marcy-l'Etoile, France) or on Minca agar medium [10] and diluted with saline water. Inoculated doses were approximately 0.8 to 2 • 10 s bacteria per mouse. Preparation o/ somcaled lysales. - - Roux flasks of M9 minimal medium [1] were inoculated with 5 ml of a bacteria culture grown overnight in TSB. After incubation (18 h, 37 ~ C), cells were collected in 5 ml of saline water, sonicated (Branson sonicator 6) and centrifuged (10,000 g, 25 rain). Supernatants were membrane-filtered (0.22 ~m). Preparation o / a m m o n i u m sulphate ( A S ) precipitates. - - The precipitates from the supernatant of sonicated bacteria were formed at 4 ~ C (30 rain) and collected at the following concentrations: 50, 35 and 20% AS concentration. The precipitates were then pelletted (10,000 g, 25 min), resuspended in 1 ml of sterile saline buffer, kept overnight at 40 C and then inoculated. Inoculation. - - Five female mice (6 to 7 weeks old) were inoculated in the hind footpad (0.05 ml) either with the live challenge (about 1 • 108 bacteria/mouse) or with sonicated lysate (treated with AS or untreated).

ADso CFU i.p. i.v.

= = ---

a b o r t i v e d o s e 50 %. colony-forming unit. intraperitoneal(ly). intravenous(ly).

LDso s.c. PIDs0 TSA

: -: =

l e t h a l d o s e 50 %. subcutaneous(ly). p l a c e n t a i n f e c t i v e d o s e 50 %. trypticase soya agar.

E. C O L I V I R U L E N C E

T E S T IN MOUSE FOOTPADS

313

Measure o/virulence. - - After inoculation, lesions caused in the footpad were examined daily. The intensity of lesions was estimated with the use of a scale from 0 to 7 (fig. 1). Scores 0 to 3 were given to footpads slightly enlarged by cedema. From score 4 upwards, necrosis was sometimes observed, cedema became more and more serious and therefore the footpads were more and more distorted. Score 7 meant t h a t the footpad was completely distorted or necrosed. In all cases, the score took into account both cedema and necrosis.

0

1

2

3

4

5

6

7

FIG. 1. - - Scale established alter [oolpad s. c. inoculation. F o o t p a d s. c. i n o c u l a t i o n of 1 • 10 s E. coli led t o l o c a l a e d e m a a n d / o r n e c r o s i s . In o r d e r t o q u a n t i f y t h e i m p o r t a n c e of t h e s e l e s i o n s , a 0 t o 7 s c a l e w a s e s t a b l i s h e d , w i t h s c o r e 0 a t t r i b u t e d t o t h e limb without lesion, and score 7 attributed to the footpad showing necrosis or enlarged aedema with complete deformation.

b) Abortion and placental colonization in pregnant mice. Mice were mated when 7-9 weeks old. Day one of pregnancy was the day on which the vaginal plug was observed. Mice were inoculated (0.2 ml) in one of the tail veins on day 15 of pregnancy. Five mice were used for each strain and each dose. The mice were killed on day 18 of pregnancy. Preparation o/ inoculum. - - The strains were spread on broth and incubated (6 h, 37 ~ C). Then these strains were spread on trypticase soya agar (TSA; Biomerieux, Marcy-1 Etoile, France) slanted and incubated (18 h, 37 ~ C). Bacteria were collected and diluted with the appropriate volume of saline according to optical density (600 nm, Beckman B). Three ten-fold challenge doses were compared (103, 104, 105, or 104, 105, 10 ~ bacteria/mouse). They were chosen according to previous results (lethal dose 50% (LDso) and infecting dose 50%) obtained on non-pregnant mice. Abortions. - - Abortions, i. e. expulsion of foetuses between days 15 and 18 of pregnancy, were recorded. Doses which caused abortion to 50% of the mice (ADso) were determined by the Reed and Muench method [16]. Necropsg. - - This was performed as already described [2]. Mice were killed on day 18 of pregnancy and uteruses were aseptically removed. Uteruses of mice which had aborted discarded. Placenta discs (placentas) of pregnant mice were separated from foetuses. Placentas were individually stored at --20 ~ C until the bacteria were counted, and then individually homogenized in 0.2 ml of saline and spread on one TSA plate. Results were expressed in the form of the dose infecting 50% of the placentas (PlDso) determined by the Reed and Muench method [16].

c) Morlalit!t and spleen colonization o / a d u l t mice. Mice 6 to 7-weeks old were inoculated (0.3 ml) i. p. with live bacteria grown overnight in TSB and diluted in saline.

314

D. BUZONI-GATEL AND COLL.

Mortality o[ mice. - - Six mice received 3 to 6 x 107 bacteria. Deaths were recorded for l0 days. Lethal dose 50% (LDso). - - The LDso was determined by the method of moving averages [13]. Splenic colonization. - - Mice were killed 24 h after the inoculation of about one LDso. Five mice were used for each strain. Spleens were removed and stored at --20 ~ C. Splenic colonization was assessed by plating dilutions of spleen homogenates on TSA. The degree of infection was the average loglo number of bacteria per infected spleen.

d) Lethality ]or baby mice. Randomization o/baby mice. - - Newborn mice born on the same day from different mothers were pooled and then each mother was individually caged and given l0 baby mice. Inoculation. - - Baby mice 2 to 3-days old were orally inoculated (intubation 0.1 ml) with l0 s E. coli from a bacteria broth (TSB culture grown overnight). Ten litters (100 baby mice) were inoculated with each strain. Deaths of baby mice were recorded daily for 14 days.

RESULTS Footpad lesion lest.

Four groups could be constituted with the 54 tested strains (fig. 2). The strains of group I induced irreversible necrosis; the strains of group II induced severe injuries, with the average score superior to 4 until day 7 p. i., after which the mice progressively recovered; on d a y 10 p. i., however, the lesions were still obvious; the strains of group I I I caused lesions which sometimes reached score 4, but by d a y 10 recovery was certain. In group IV, the strains did not affect the footpad. Since the importance of the lesion increased until day 5 p. i., we chose this d a y to attribute scores in the following study. On d a y 5 p. i., score 4 obviously appeared as the limit of pathogenicity. This score separated the pathogenic strains (groups I and II) from the nonpathogenic strains (groups I I I and IV). Relationship between tests of le/halilg and of footpad lesions.

The 54 E. coli strains were studied with respect to their lethal properties for mice inoculated i. p. with 3 to 6 x 107 bacteria; 26 of these strains did not induce death and were then labelled ~(non-lethal )). The 28 others were r lethal ~, as most inoculated mice died within 48 to 72 h. Strains of the lethal group, as determined after i. p. inoculation, caused death in 31% of mice after footpad inoculation. Fifty-seven percent of the mice inoculated with the lethal strains were scored 5 to 7, while scores 0 to 4 were given to 90% of the mice after inoculation of any ~ non-lethal ~ strains (table I). Six strains representative of the lethal group (JD9, CIP 125:80, JD7, DM2/2, V003 and V2019) and one belonging to the ~ non-lethal ~ group

E. COLI VIRULENCE TEST IN MOUSE FOOTPADS

of,

315

group I

~6 m.

aofa

~4

group

II

0

,of 9

~2

group

III

group

IV

o

:

~

:

~

--..~

mean o~ []

days after the inoculation

FIG. 2. - - Groups of 5 mice were inoculated with each of lhe 54 E. eoli strains in one [ootpad. E v e r y d a y a score was a t t r i b u t e d to each footpad. E a c h dot was t h e d a i l y a v e r a g e of t h e scores for each strain. F o u r g r o u p s of s t r a i n s could be described (o, A, A, and []).

TABLE I. - - Results in the footpad lesion test in relation to lethality by i. p. administration for 54 strains of << E. coli >>. N u m b e r of mice per class in f o o t p a d cedema t e s t (z) D i s t r i b u t i o n of s t r a i n s in t h e l e t h a l i t y assay (1): 26 non-lethal strains : LS.I.1 Others Total 28 lethal strains : JD9 CIP 125:80 JD7 DM2/2 V003 V2029 Others Total

0 to 4

5 to 7

5 117

0 13

0 0

122

13

0

0 0 0 0 0 0 17

5 0 0 5 2 0 66

0 5 5 0 3 5 24

17

78

42

0) 3 to 6 • 107 CFU. (~) Classes defined as in figure 1; i • 108 CFU i n o c u l a t e d to 4 to 5 mice per strain.

Death

316

D. BUZONI-GATEL AND COLL.

TABLE I I. - -

Footpad test

R e l a t i o n s h i p b e t w e e n different v i r u l e n c e tests.

Abortion and placental infection of p r e g n a n t mice, i.v. inoculation

Strains

Mean score (l)

AD~0

PIDso

1.S.I.1 JD9 CIP125:80 ,ID7 DM2/2 V003 \:2019

2 7 5.5 t 7 t 5.8 7 t t

>6.0 5.6 >5.9 4.9 4.2 4.0 <3.0

5.4 5.5 4.0 3.7 > 4 . 0 (4) > 4 . 0 (4) <3.0

Lethality and infection of adult mice i.p. inoculation

Lethality in b a b y mice (s)

LDso

Splenic infection (~-)

Number of dead

8.8 7.0 6.9 6.4 6.9 6.4 6.8

NT 2.8 2.1 3.7 3.3 3.9 5.6

19 57 20 84 76 86 64

(1) 1 x 10 s CFU, m a r k defined as in figure 1. (2) Splenic infection (loglo) on the 1st day after one LDso dose inoculation. (3) Oral inoculation of 100 b a b y mice with 1 • 108 CFU. (4) All mice challenged with more than 1 • 10 ~ CFU aborted. Only 10 % of the placentas were colonized in mice challenged with I x 104 CFU. t = death occurred. NT -- not tested.

(LS.I.1) were therefore chosen to be studied in complementary virulence tests.

Virulence measured in pregnant mice. PID~o measured the ability of strains to colonize placentas of pregnant mice. All tested strains were able to colonize the placentas, even the nonlethal control strain, but large differences were observed between them in two respects: PIDso values (table II) and average number of bacteria recovered per colonized placenta (not shown). Some needed a large challenge dose to infect 50% of the placentas (JD9 and CIP 125:80), while others (V2019, JD7) were easily able to multiply in the colonized placentas. When a strain multiplied heavily, it usually induced abortion (JD7 and V2019), but two strains (JD9, CIP 125:80) were able to colonize the placentas without being strongly abortive, as was the non-lethal control strain (LS.I.1). Two other strains (DM2/2 and V003) induced abortion, but surprisingly, placental colonization was restricted to some placentas in still pregnant mice.

Virulence for adult mice i. p. challenged. The LDso of every strain from the lethal group was low compared to that of strain LS.I.1 from the non-lethal group (table II). Since the virulence property of lethal strains may depend both on their ability to multiply in vivo and on their toxin production, we compared LDso to in vivo growth,

E. COLI VIRULENCE TEST IN MOUSE FOOTPADS

317

represented by the number of splenic bacteria, one day after one LDso was injected. This ability to multiply can be expressed by the difference between LDso and splenic bacteria enumeration one day after challenge. This number decreases at various rates. On this basis, two strains (JD9, CIP125:80) were quickly destroyed; three (JD7, DM2/2, V003) were more resistant and one (V2019) was very resistant.

Baby mice mortality after oral inoculation. Death occurred soon after the inoculation of JD7, JD9, DM2/2 and V003 (75 ~ of the deaths were recorded within less than 2.5 days), whereas death seemed more delayed with strain V2019 (75% of deaths occurred within 5.1 days). Some strains were very lethal, killing more than 70% of the baby mice in 14 days (JD7, V003, DM2/2 and V2019); others were less lethal (JD9) or not lethal at all (CIP 125:80, LS.I.1) (table II).

Toxicity of different bacterial fractions tested with the footpad lesion test. Previous tests were related to the growth and/or resistance of strains

in vivo. Some strains were able to grow in vivo but were not pathogenic in all tests. This suggested they lacked some virulence factors. Because the footpad lesion test was related more or less to other pathogenic tests, we looked at soluble substances capable of inducing (edema and/or necrosis. Four different patterns were observed (table III). Sonicated supernatants I I I . - - Toxicity of different fractions of strain t e s t e d in the model of subcutaneous footpad inoculation.

TABLE

R e s p o n s e in t h e f o o t p a d (*) Mean v a l u e of lesion i n t e n s i t y Fractions inoculated

Strains

Heated bacteria 1000 C, 30 rain

LS.I.1 JD9 CIP125:80 JD7 DM2/2 V003 V2019

0 0 0 0 0 0 0

AS US

US-20 ~ C

Heated US

0 0

0 NT

(} NT

t

t

t

7

5 3.2 0 NT

0 1.2 NT NT

tt t4 0

20 %

0 NT 3.3 t (**) 0 3 NT NT

35%

50%

0 NT

0 NT

t

t

3.6 5.2 NT NT

4 44 NT NT

(*) As defined in figure 1. (**) 2/5 d e a t h s were observed. US = u l t r a s o n i c a t e s u p e r n a t a n t . US-20 ~ C ~ US frozen at --20 ~ C. H e a t e d US = US h e a t e d a t 65 ~ C, 30 rain. AS ~ a m m o n i u m s u l p h a t e p r e c i p i t a t e of US a t different c o n c e n t r a t i o n s . NT ~ n o t tested; 4 = d e a t h d e l a y e d after 3 to 4 days; 44 ~ d e a t h w i t h i n 24 b.

318

D. BUZONI-GATEL AND COLL.

produced (1) no lesion (LS.I.1, V2019, JD9), (2) immediate death (DM2/2, V003), (3) nervous troubles with paralysis of hind limb, tremor and then delayed death within 3 days (CIP 125:80), or (4) oedema and necrosis of the inoculated footpad (JDT). Freezing and heating of the supernatant suppressed some of these properties. Ammonium sulphate precipitates were still very toxic. It can be concluded that soluble toxic substances were produced in vitro by four strains. One was not stable at freezing (V003), two were destroyed at 65 ~ C (JDT, DM2/2) and the last (CIP 125:80) was stable at this temperature. DISCUSSION To prove the virulence of E. coli strains isolated from different pathological cases, simple, rapid tests are required. In order to study the virulence of 54 E. coli strains isolated from newborn calves, we assessed a virulence test based on the capacity of strains to induce lesions in the footpad of mice. This test was firmly correlated with the standard virulence test of i. p. inoculation: the strains which were lethal for i. p. inoculated mice caused obvious lesions in the footpad of mice after s. c. inoculation. We then compared the virulence of seven strains with our virulence test and with other tests. Six of these chosen strains were pathogenic after both i. p. and footpad inoculations and one of them was nonpathogenic. Placenta and spleen are the target organs from which bacteria can be isolated in infected organisms. Therefore, we tried to measure the virulence of the chosen strains through their ability to induce abortion, to colonize placentas and spleen and to cause death. With the footpad test, we looked for toxin released by sonicated bacteria. A clear correlation appeared between the footpad lesion test, the ability to colonize placenta and eventually to cause abortion, the ability to multiply (or to remain) in the spleen, and the ability to kill newborn mice. Nevertheless, these correlations were not absolute as the studied strains showed different pathogenic processes. Two strains (JD9 and V2019) did not synthesize toxic factors in our experimental conditions. The JD9 strain induced abortion, but the ADso and PIDso were rather high. This strain was moderately pathogenic for baby mice and, as it was rapidly cleared from the spleen after one LD~o inoculation by the i. p. route, it was not very resistant in vivo. The JD9 strain therefore shows moderate virulence properties. In contrast, the V2019 strain was highly pathogenic in all tests and heavily multiplied in vivo. Therefore, this strain shows septicaemic properties and, moreover, carries the classical serotype (078:K80) of septicaemic strains [15, 17]. Toxic factors could be extracted from four strains (JD7, DM2/2, V003 CIP 125:80). JDT, DM2/2 and V003 were rather similar even though the toxic processes involved were different. DM2/2 and V003 were closely related from the points of view of abortificient properties and high PID~o

E. COLI VIRULENCE TEST IN MOUSE FOOTPADS

319

(>4). Their abortive properties do not imply placenta colonization (research in progress). Their oral inoculation into baby mice soon caused death. They were fairly resistant in vivo. Their heat labile toxic factor could be responsible for the pathogenic process. As human E. coli producing haemolysin are known to be associated with pathogenic properties and necrotic activity [9, 12, 5], haemolysin synthesized by both strains could be responsible for death, abortion and necrosis. The JD7 strain was slightly different from the two strains described above since, although it induced abortion, it showed a rather low PID~o. Both the production of heat-labile toxin and the ability to multiply could be involved in its pathogenic processes. Strain CIP 125:80 was very different from the other three strains. Its heat-stable toxic factor could be labelled a neurotoxic factor, since paralysis, tremor and death delayed by 3-4 days were observed after footpad s. c. inoculation. It was not resistant in vivo and was not pathogenic for baby mice or pregnant mice even though it colonized the placentas. In contrast with the usual strain of E. coli, this strain synthetized urease [7]. A urease-producing strain has already been the subject of investigations of pathogenicity [3], but no nervous trouble was mentioned. At the moment, we do not know the relationship between urease production, neurotoxic factor production and the ability of this strain to produce cedema in the footpad of mice. We conclude that both properties, the ability to multiply in vivo and toxin production, are involved in the different tests and, in particular, are involved in footpad lesions. The exact pathological mechanisms implied in footpad lesions are not known, though the observations suggest immunological reactions, such as delayed hypersensitivity, antibody-mediated Arthus-like sensitivity or activation of the complement by the alternative pathway. Indeed, such immunological reactions have already been described for other pathological bacteria [4, 8, 141. In the footpad test, these immunological reactions could be joined to a direct toxic effect. We have shown that this footpad test is useful in analysing pathological properties. It therefore seems to be a valid test for diagnosis in E. coli pathological processes.

RESUMg M I S E AU P O I N T D ' U N T E S T D E LI~SION D U C O U S S I N E T PLANTeklRE DE LA S O U R I S POUR METTRE

EN

] ' V I D E N C E L& V I R U L E N C E

D E S O U C H E S D E
Un test de virulence base sur la capacite des souches de E. coli i~ provoquer une lesion dans la patte de souris, a et6 mis au point pour mettre en evidence leur pouvoir pathog~ne. Cette virulence a ere mesuree grace a une echelle de 0 a 7 suivant l'importance de la lesion. Sur 54 souches testees, celles provoquant la mort apr~s inoculation intraperitoneale, ont cause un oed~me important ou une necrose (note >4) de la patte. Trois autres tests

320

D. BUZONI-GATEL AND COLL.

de virulence (virulence mesur~e chez les souris gestantes, chez les souris adultes non gravides aprON inoculation intrap~riton~ale et chez les souriceaux aprON inoculation orale) ont ~t~ utilis~s pour ~tudier 7 des souches les plus caract~ristiques. On a observ~ une bonne correlation entre les diff~rents tests : les souches pathog~nes dans le test de l~sion de la patte, se sont r~v~l~es pathog~nes dans au moans Fun des autres tests. 9~/~OTS-CL/~S : Escherichia coli, Pouvoir pathog~ne, Coussinet plantaire ; Souris, Test. ACKNOWLEDGEMENTS We thank Dr Renault (CRCB, Athis-Mons, France), Dr Sojka and Dr Morris (Central Veterinary Laboratory, New Haw, Weybridge, US), Dr Smith (Houghton Poultry Research Station, Huntington, UK) and Dr Verger (INRA, Nouzilly, France) for providing strains V2019, R15, S5VIR and V003, respectively, and Dr Plommet for his helpful criticisms.

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[13] MEYNELL, G. G. • MEYNELL, E., Quantitative aspects, the normal distribution. Estimation of the EDso by moving averages, in cc Theory and practice in experimental bacteriology )). 2nd ed. (p. 207-209). Cambridge University Press, Cambridge, 1970. [14] MITSUYAMA, K., NOMOTO, K. & TAKEY, K., Direct correlation between delayed footpad reaction and resistance to local bacterial infection. In/ect. Immun., 1982, 36, 72-79. [15] ORSKOV, F. & ORSKOV, I., Special E. coli serotypes from enteropathies in domestic animal and man. Zbl. Vet.-Med., 1979, 29 (suppl.), 7-14. [16] REED, L. J. ~r MUENCH,H., A simple method for estimating fifty percent end points. Amer. J. Hgg., 1938, 27, 493-497. [17] SMITH, H. W., Transmissible pathogenic characteristics of invasive strains of Escherichia coli. J. Amer. vet. reed. Ass., 1978, 173, 601-607. I18] SMITH, H. W., A search for transmissible pathogenic characters in invasive strains of Escherichia Coli : the discovery of a plasmid-controlled toxin and a plasmid-controlled lethal character closely associated or identical with colicin V. J. gen. Microbiol., 1974, 83, 95-111. [19] Van DEN BOSCH, J. F., DE GRAAFF, J. & MAcL~_REN, D. M., Virulence of Escherichia coli in experimental hematogenous pyelonephritis in mice. ln[ect. Immun., 1979, 25, 68-74.