Suppression of heat-stable enterotoxin production by Yersinia spp. in milk

Veterinary Micro biology, 11 ( 1986 ) 163--172 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

163

SUPPRESSION OF HEAT-STABLE ENTEROTOXIN PRODUCTION BY Y E R S I N I A SPP. IN M I L K

tl. FUKUSHIMA', M. TSUBOKURA2, K. OTSUKP and Y. KAWAOKA2 'Pubh'c Health Institute of Shirnane Prefecture, Nishi-Hamamdo, Matsue, Shimane 690-01 (Japan) ~Department of Veterinary Microbiology, Faculty of Agriculture, Tottori University, Tottori 680 (Japan) (Accepted for publication 11 June 1985)

ABSTRACT

Fukushima, H., Tsubokura, M., Otsuki, K. and Kawaoka, Y., 1986. Suppression of heat-stable enterotoxin production by Yersinia spp. in milk. Vet. Microbiol., 11: 163--172. One of 16 raw milk isolates of Yeninia enterocolitica and Y. intermedia produced heat-stable enterotoxin (ST) in milk at 25'C but not at 4'C after 21 days of incubation. A catabolite repression of ST synthesis by the lactose-fermenting strain of Y. enterocolitica was observed when 4.6% lactose was added to trypticase soy broth. However, the lactose-fermenting strain was killed by acid produced by lactose fermentation in milk and did not produce ST in milk with the pH adjusted to neutrality. This study suggested that lactose and fat in milk are not the fundamental inhibitors of ST synthesis by Y. enterocohtica and that repression of ST synthesis may be related to other components.

INTRODUCTION Yersinia enterocolitica causes infection in humans, either in sporadic or e p i d e m i c f o r m (Zen-Yoji, 1981). T h e t e n t h o u t b r e a k caused by Y. enterocolitica 03 in J a p a n o c c u r r e d in S h i m a n e P r e f e c t u r e in J u n e 1984 and over 150 children a t t e n d i n g five schools were affected. A l t h o u g h epidemiologically it was s u s p e c t e d t h a t the o u t b r e a k was due to f o o d - b o r n e infection, the s o u r c e and m o d e o f i n f e c t i o n r e m a i n e d u n k n o w n . F o u r m a j o r o u t b r e a k s in which milk was implicated have been r e p o r t e d in the United States {Black et al., 1 9 7 8 ; S h a y e g a n i et al., 1 9 8 3 ; T a c k e t et al., 1984} and J a p a n ( M a r u y a m a , 1982). O f the three o u t b r e a k s in the

United States, one was caused by chocolate milk contaminated with Y. enterocolitica 08 (Black et al., 1978), another by either powdered milk, a milk dispenser, or turkey c h o w mein contaminated with Y. enterocolitica 08 (Shayegani et al., 1983), and a third by pasteurized milk contaminated with Y. enterocolitica 013 and 018 (Tacket et al., 1984). O n e outbreak

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164 which occurred in Okinawa Prefecture, Japan, in April 1980, affected 11.9% of 8835 children attending primary and junior high schools and was caused by Y. enterocolitica 03 transmitted by pasteurized milk (Maruyama, 1982). Fukushima et al. {1984) isolated Yersinia from pooled raw milk samples in Japan and there are also other reports of the isolation of Yersinia from raw and pasteurized milk and milk products (Schiemann, 1978; Schiemann and Toma, 1978; Hughes, 1979, 1980; Mollaret et al., 1979; Toma et al., 1979; Shayegani et al., 1981; Vidon and Delmas, 1981). Although Yersinia is capable of heat-stable enterotoxin (ST) production (Pai et al., 1978; Boyce et al., 1979; Kapperud and Langeland, 1981), the clinical significance of ST remains uncertain. Francis et al. (1980) suggested that milk is a good growth medium for Y. enterocolitica but a poor medium for ST production. Although Olsvik and Kapperud (1982) reported that the ST production of Y. enterocolitica seemed to be totally suppressed in milk, the reason for this is unclear. In the present communication suppression of ST production by Y. enterocolitica and Y. intermedia in milk at 4°C and 25~C is described. MATERIALS AND METHODS

Bacterial strains

Twelve isolates of Y. enterocolitica biovar 1 and four of Y. intermedia, which ferment L-rhamnose, raffinose, and mehbiose, were obtained from raw milk (Fukushima et al., 1984). One isolate of Y. enterocolitica biovar 4 serovar 03 (our laboratory No. Pa5353) came from a patient with gastrointestinal illness, in Matsue, Japan. All isolates were stored on nutrient agar at 4°C. Cultivation media

All strains were tested for ST production in two different media at 4°C and 25°C; (i) trypticase soy broth (TSB, B B L Laboratory) containing 0.6% yeast extract (TSB-YE), (ii) whole milk pasteurized at 140°C for 3 s and packed under safety conditions (Meiji Milk Products Co. Ltd., whole milk containing 8.1% non-fat solids, 2.9% protein, 3.4% fat, 4.6% lactose, 0.7% ash). Y. enterocolitica biovar 1 serovar 013.7 (lactose fermentor, lipase positive; laboratory No. RM44), Y. intermedia serovar 014 (non-lactose fermentor, lipase negative; laboratory No. RM17) and Y. enterocolitica biovar 4 serovar 03 (non-lactose fermentor, lipase negative; laboratory No. Pa5353) were tested for growth, pH and ST production in seven media. These media included media (i) and (ii) above, and (iii) whole milk sterihzed at 121°C for 10 rain (WM-10), {iv) skimmed milk (Difco Laboratory) sterilized at 121°C for 10 rain, (v) T S B with butter (TSB-B) with a fat concentration

165

of 3.4%, equal to that of whole milk, (vi)T S B with lactose (TSB-L) filtered through a 0.45 u m filter (lactose was added to T S B to obtain a final milk lactose concentration equal to that of whole milk, 4.6%) and (vii) T S B containing 4.6% glucose (TSB-G) filteredthrough a 0.45 # m filter.

Conditions for growth, pH and ST production I02-~ and l0 g viable cells were inoculated into each medium (I0 ml) in culture tubes (2.5 mm × 150 ram), placed in a shaker with the tubes inclined at nearly 30° and incubated at 4°C or 25°C for 21 days with constant shaking (200 times min-'). The cultures in TSB-YE and whole milk of 16 milk strains were used for assaying ST after 2 and 21 days of incubation. The cultures of RM17, RM44 and Pa5353 strains were used for determination of growth, pH and ST production. Sampling from the culture tubes was performed at days 2, 3 and later intervals at 4°C and 25°C. Cell numbers ml-' were determined by an aerobic plate count (APC) of colonies on trypticase soy agar. A portion of the culture was tested for pH, the remainder was centrifuged (10,000 × g, 20 rain), and the supernatant was assayed for ST in infant mice. The mouse assay procedure, ori~nally described for Escherichia coli (Dean et al., 1972), can also be used for assaying the enterotoxigenicity of Y. enterocolitica in milk (Francis et al., 1980; Olsvik and Kapperud, 1982). A gut/intestine-less carcass weight (G/CW) ratio of ;~ 0.090 is generally accepted as a positive test for ST.

ST production in TSB-L and whole milk with the pH adjusted to neutrality RM44 was cultured in TSB-YE and TSB-L at 25~C for 54 h and in whole milk at 25°C for 117 h. The pH of TSB-L and whole milk was adjusted to > 7.0 by the addition of 1 M NaOH to these media, when the pH was < 7.0. Growth, pH and ST production were tested at each sampling time. RESULTS

ST production in whole milk and TSB- YE ST was not detected in whole milk at 4°C and 25°C after 2 and 21 days of incubation, except for one strain of Y. enterocolitica biovar 1 serovar 013.7 (RM89) which showed a G/CW ratio of 0.091 at 25°C after 21 days of incubation. However, all strains of Y. enterocolitica and one strain of Y. intermedia produced ST in TSB-YE at 25°C after 2 days of incubation, and 8 strains of Y. enterocolitica produced ST in TSB-YE at 4°C after 21 days of incubation but not within 2 days of incubation (Table 1).

166 TABLE

I

Production of heat.stablc milk at 4. and 25°C

-.

cntcrotorin

--

Strain no

-

SeroyIT (bwvu’

by milk strams of GlCi

r.iob

--

-

.-.

--..

Yenin&

organirmr

--

Yxh-o~‘coRM258 RM339 RM 42 RMl87 HM21.1 HM 4.1 Rhl 89 RM152 RM230 RM329 HM346 RM347 Pa5353 I’ mtrrmrdvr HM 17 HM37 1 RM284 RYPXH

Effect

and whole

_-..

---;c

TSB.YE

Whole milk

---24

-__ 2 -

-

__

-_

2.5 cc

--

in T8B.YE

21 -

-

TSB.YE

21

_-

21

2

-

-

Whole milk -21

--

05 27(l) 05 27( 1) 0631(l) 07 n (1) 078 (1) 0137(l) 013.7(l) 0137(l) 013 7(l) 013 7( 1) O137rl) 0137(l) 03 (4)

0.141 0.116 0 099 0.11; 0 132 0 157 0 132 0 124 0 142 0.125 0 143 0 129 0.145

riF KT ST ST NT ST S’I NT NT s T N’I NT ST

0 070 0.061 0 064 0.058 0.059 0 064 0 066 0 060 0 070 0.065 0 062 0.065 0 063

0’060 0.056 0 078 0.086 0 069 0.085 0.091 o.oi9 0 066 o.ol3.5 0.074 0 078 0.065

0.065 0.054 0.059 0 OS8 0.066 0.066 0 062 0 061 0.056 0 059 0 055 0056 0 059

0.137 0.060 0 164 0 133 0 098 0 05; 0.180 0.130 0 116 0 on3 O.OH5 0.149 0 065

0 067 0 072 0.077 0.083 0 07H 0.062 0 083 0.089 0.087 0.07H 0.074 0 077 0 05:

0 1.I 022

0 062 0 058

0 066 0 062

0 061 0 Otil

0 C63 0 060

0 OGl 0.053

0.065 0.062

0.070 0 059

0 124 0 059 -

N’I 0 055 -

0 061 0 OiG 0 OGI 0 055 ._ -

0 060 0 06.1 --.

0 058 0.054 . ._

0 064 0 058

OUT’ OU’I

.._

-

_I_

-

‘_

of lactose on ST production

.

at 25°C

Examination of the effect of lactose on growth, pH and ST production was performed in TSB-YE, TSB-L and whole milk using lactose-fermenting (RM44) and non-lactose-fermenting (RM17 and Pa5353) Yeninia. These three strains increased to > 10’ APC ml’ ’ in three different media by the third day. The lactose-fermenting Yersiniu lowered the pH in TSB-L and whole milk to < 5.0 on the second to third days and APC ml-’ to < lo> on Days 64, and raised the pH and APC ml-’ of TSB-L to > 7.0 and > lo9 on Day 12 but did not raise the pH and APC ml-’ of whole milk after Day 12. The APC and pH of the TSB-YE, however, remained at > 106 ml ’ and > 8.0, respectively. ST was not produced in whole milk but was produced in ‘I’SB-I,, which showed a G/CW ratio of 0.096 after 21 days of incubation (Fig. 1). When the pH was adjusted to neutrality, ST was produced in TSB-L, though not in whole milk, after 33 h of incubation,

167

9

o~

5

3'

1~,

a,

Plo

2

o.

~0. 0.050

O

3

6

9

12

15 18 210

3 6 9 IP 15 18 21 O Days of Incubation

3

6

12

9

15 18 ?1

Fig. 1. Viable cell c o u n t s (log;0 aerobic plate c o u n t (APC)), pH values and g u t / i n t e s t i n e less c ~ c ~ , ~ weight ( G / ~ / ) ratio o b t a i n e d w h e n l a c t o s e - f e r m e n t o r ( R M 4 4 ) and nonlactose-fermentors

(RM17

and Pa5353)

were cultured at 25:'C in T S B - Y E

(e), T S B - L

( " ) , TSB-G (-',) and w h o l e milk (s).

7

5

•~

a.

< i

t.

i

1

I

1

i

o. 1 3C

C .OgO -, ~.3

- - = - = =. = =_=.__ : - = ~ / = _ _ - = _ : / , z ~ (}. Ct~O

,

0

17

?4

,

315

.

•

4~ b4 0

~4ours of

~

12

P4

,

36

4~

60

72

s~

.

96

~S.---~

120

incubation

Fig. 2. Viable cell c o u n t s , pH and G/CW/ ratio o b t a i n e d w h e n l a c t o s e - f e r m e n t o r ( R M 4 4 ) was cultured at 2,5°C in TSB-YE (=), TSB-L ( . ) , and WM-10 {/,) and in TSB-L (o) and WM-10 ( - ) a d j u s t e d the pH to 7.0 by IM NaOH ( i ) .

168

and the A P C in both media rose to 109 ml' (Fig. 2). Non-lactose fermentors in three different media (TSB-YE, TSB-L and whole milk) maintained the A P C at > 1(I' ml-' and the p H at > 5.0 after 21 days of incubation, and ST was produced by Pa5353 after 2 days of incubation in T S B - Y E and TSB-L but not in whole milk (Fig. I). Examination of the effect of glucose on growth, p H and ST production was performed in TSB-G with strains RM44, R M 1 7 and Pa5353. After the three strains increased to > 109 A P C ml -~ in TSB-G on Day 2, the p H dropped to < 5.0 on Day 2 and the A P C fell to < 10' ml '' on Days 6--9. Strains R M 4 4 , R M 1 7 and Pa5353 did not produce ST (Fig. 1).

Effect of fat on the ST production at 25°C The effect of fat on growth, p H and ST production was examined in TSB-B, skimmed milk and whole milk, using the lipase-positive and lactosefermenting strain (RM44) and lipase-negative and non-lactose-fermenting strains ( R M 1 7 and Pa5353). All three strains increased in number to > 109 A P C ml ~ on Day 3 in three different media (TSB-B, skimmed milk and whole milk). In the skimmed milk and whole milk cultured lipase-positive strain, the pH dropped to < 5.0 after 3 days of incubation and A P C to 102--10~ ml-' after 12 days of incubation. However, with the lipase-positive strain cultured in TSB-B, the p H was maintained at > 6.0 and the A P C at 11

¢.

a. <

3

b--o

<1 !

9

]

7 ..Y.

f

3

0,210

2 o. 1to L

0 0

0.130

0.090

0.050 0

3

6

t;ays of

~

12

lb

18

21

Incubation

Fig. 3. Viable cell c o u n t s , pH and G/CW ratio o b t a i n e d w h e n lactose-fermentor (RM 4 4 ) was cultured at 25°(? in TSB-B (o), s k i m m e d milk (~) and w h o l e milk (:.).

169 > 10s ml-'. ST was produced after 2 days of incubation in TSB-B but not in skimmed or whole milk (Fig. 3). Both lipase-negative strains maintained the A P C at > 10 ~ m l " a n d t h e p H a t > 5.0 for 21 days in three different media (TSB-B, skimmed milk and whole milk). Pa5353 produced ST in TSB-B after 3 days of incubation and in skimmed milk after 21 days of incubation but not in whole milk.

Effect of refrigeration temperature on growth and ST production The effects of refrigeration temperature (4°C) on growth, pH and ST production in TSB-YE, TSB-L and WM-10 media by RM14, RM44 and Pa5353 strains were examined. All three strains slowly increased to 109 APC ml-' in TSB-YE and TSB-L by Day 12 and in whole milk by Day 15. The growth curve and the change in pH were much the same with all three strains, except for the lactose fermentor which lowered the pH in TSB-L to < 5.0 after 15 days of incubation (Fig. 4). RM44 and Pa5353 strains did not produce ST in TSB-YE and whole milk into which l 0 s cells had been inoculated. These slzains did not produce ST in three different media held at 4°C for 21 days.

7'~ =

~Y

/

e~ 1 9

5 3

.o

0.17C

L

O. 130

o

0.090

~9

w

0.050

o

3

~

Days o f

Fig.

l

~ 1~ ~

-

==~=

~8 ~1

incubation

4. V i a b l e c e l l c o u n t s , p H a n d G / C W r a t i o o b t a i n e d w h e n l a c t o s e - f e r m e n t e r ( R M 4 4 ) w a s c u l t u r e d at 4°C in T S B - Y E (o l 0 s, • 1 0 ' c e l l s i n o c u l a t e d i n t o t h e m e d i u m ) , TSB-I,(,)andWM-10(-).

170 DISCUSSION Pal et al. (1978) studied the enterotoxigenicity of many strains of Y. enterocolitica derived from humans, animals, food, milk and water and found that 14 out of 44 st~'ains from raw milk produced ST in media contaming 2% casamino acids, 1% yeast extract, and 0.4% glucose. Y. enterocolitica and Y. enterocolitica-like organisms derived from raw milk, wildliving small mammals and water, produced ST at 4°C after cultivation in TSB-YE for 7 days (Kapperud and Langeland, 1981) and in TSB for 15 days (Francis et al., 1980). Francis et al. (1980) found that three of 36 stratus obtained from raw milk produced ST in milk at 25°C but not at 4°C. Conversely, Olsvik and Kapperud (1982) reported that ST of Y. enterocolitica was not detected in milk cultured at 4°C and 22~C by the infant mouse test. Thus, it is suggested that milk is a good medium for the propagation of Y. enterocolitica, but not for ST production (Francis et al., 1980; Olsvik and Kapperud, 1982). Kapperud and Langeland (1981) reported that ST production by Y. enterocolitica has so far only been detected under experimental conditions with efficient aeration and agitation of the culture, and that those conditions are vastly different from those in stored foods. Thus, it is suggested that milk would be a good culture medium for the growth of Y. enterocolitica but not necessarily for the production of ST. However, the question of why ST production by Yersinia is suppressed in milk has remained unclear. Alderete and Robertson (1977) reported that glucose inhibits the production of ST by enterotoxigenic E. coil and suggested that glucose serves as a catabolite repressor of ST synthesis. Boyce et al. (1979) indicated that glucose may repress Yersinia enterotoxin synthesis. Okamoto et al. {1980) reported that repression of ST synthesis by Y. enterocolitica was not observed when 0.4% glucose was added to the medium. In our study, when 4.6% glucose was added to TSB (TSB-G), Yersinia preferentially utilized the glucose and the growth of these organisms was initiated on Day 3. A catabolite repression of ST synthesis by Yersinia in TSB-G was observed. Milk isolates of Yersinia organisms did not produce ST in sterilized milk with 4.6% lactose at 4°C and 25°C in 21 days, except for one lactose fermentor which showed a marginal ratio of 0.091 at 25°C after 21 days of incubation. A catabolite repression of ST synthesis by a lactose-fermenting Y. enterocolitica was observed when 4.6% lactose was added to TSB (TSBL), but this was not observed with non-lactose fermentors. The lactose fermentor lowered the pH of TSB-L to < 0.5 on Day 2 and the APC of TSB-L to 10 ~ ml j from 10 '° ml 1 on Day 6. When the pH in TSB-L was adjusted to neutrality, the APC of this strain rose to 10 ~ ml ~ and ST was produced. Boyce et al. (1979) reported that the ST of Y. enterocolitica is acid stable. ST synthesis by the non-lactose-fermenting strains of Y. enterocolitica (Pa5353) appeared unrelated to the existence of lactose in TSB. These findings suggest that the lactose fermentor was killed by the acid

171 produced by lactose fermentation, S T produced in T S B - L was not inactivated by a p H < 5.0 and that lactose serves as a catabolite repressor of S T synthesis in TSB-L, but only for lactose fermentors. The lactose fermentor produced S T in T S B - L after 21 days of incubation, suggesting that it survived by utilizing other components in TSB-L and S T was thus produced. Lactose fermentors cultured in milk, which included 2.6% casein and isoelectrically precipitated at < p H 4.6, lowered the p H in whole milk to < 4.6 on Day 6. Milk sediments adhered to the tube wall at the surface and the A P C decreased to 104 ml -z on the sixth day. The enterotoxigenic lactose fermentors could not produce S T in milk with the p H adjusted to neutrality, and enterotoxigenic non-lactose ferrnentors could not produce ST in milk. Olsvik and Kapperud (1982) reported that ST produced in T S B - Y E were still detectable after blending with milk or sodium casemate. These findings suggest that lactose-fermenting organisms were killed by the acid produced by the fermentation of lactose, and that the nutrient for growth of the

organisms was improved by the precipitation of casein at low pH (< 4.6). Thus, it is indicated that ST produced in milk was not inactivated by milk components. A detectable amount of ST was either not produced in milk, or the ST production by enterotoxigenic Yersinia in milk was inhibited by components other than the lactose. Moreover ST production by a lipase-positive strain (Y. enterocolitica biovar 1 serovar 013.7, RM44) was observed in TSB with butter (TSB-B) but not in skimmed milk. However, ST production by a lipase-negative strain (Y. enterocolitica biovar 4 serovar 03, Pa5353) was observed in TSB-B, but less than that by RM44, and in skimmed milk after 21 days of incubation. These findings suggest that ST production is not inhibited by fat in the milk but that a very small amount of ST produced in milk by lipase-negative Yersinia organisms may escape detection by being fat soluble. The present study shows that the lactose and fat of milk are not the fundamental inhibitors of ST synthesis and that the repression of ST synthesis in milk may be related to other components. ST synthesis by enterotoxigenic strains may be repressed at refrigeration temperature, since ST production by enterotoxigenic strains was negative at 4°C in TSB-YE into which 10 I or 10 ~ cells of each organism were first inoculated.

REFERENCES Alderete, J. and Robertson, l).J.,1977. Repression of heat-stableenterotoxin synthesis in enterotoxigenicEscherichia coli. Infect.Immun., 17 :629--633. Black, RE., Jackson, R.J.,Tsai,T., Medvesky, M., Shayegani, M., Feeley,J.C.,MacLeod, K.I.E. and Wakelee, A.M., 1978. Epidemic Yersinia enteroeolitica infection due to contaminated chocolate milk. New Engl. J. Med., 298: 76--79. Boyce, J.M., Evance, D.J., Evance, DG. and DuPont, H.L., 1979. Production of heatstable, methanol-soluble enterotoxin by Yersima enterocolitico. Infect. Immun., 2,5: 532--537.

172 Dean, A.G., Ching, Y., Williams, R.G. and Harden, L.B., 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis., 125:407--411. Francis, D.W., Spaulding, P.L. and Lovett, J., 1980. Enterotoxin production and thermal resistance of Yersinia enterocolitica in milk. Appl. Environ, Microbial., 40: 174--176. Fukushima, H., Saito, K., Tsubokura, M., Otsuki, K. and Kawaoka, Y., 1984. Significance of milk as a source of infection for human ycrsiniosis. I. Incidence of Yersinia orRanisms in raw milk in Shimane prefecture, Japan Vet. Microbial., 9: 139--146. Hughes, D., 1979. Isolation of Yersinia enterocolitica from milk and a dairy farm in Australia. J. Appl. Bacterial., 46: 125--130. Hughes, D., 1980. Repeated isolation of Yersinia enterocolitica from pasteurized milk in a holding vat at a dairy factory. J. Appl. Bacterial., 48: 383--385. Kapperud, G. and Langeland, G., 1981. Enterotoxin production at refrigeration temperature by Yersinia enterocolitica and Yersinia enterocolitica.like bacteria. Current Microbial., 5 : 119--122. Maruyama, T., 1982. Yersiniosis as a zoonosis. J. Jpn. Vet. Meal. Assoc., 3 5 : 2 - - 8 (in Japanese). Mollaret, H.H, Bercovier, H. and Alonso, J.M., 1979. Summary of the data received at the WHO reference center for Yersinia enterocolitica. Contr. Microbial. [mmunol., 5: 174--184. Okamoto, K., Ichikawa, H., Kawamoto, Y., Miyama. A. and Yoshii, S., 1980. Heat-stable enterotoxin produced by Yersinia enterocolitica isolated from patients. Microbial. lmmunol., 24: 401--408. Olsvik, O. and Kapperud, G., 1982. Enterotoxin production in milk at 22 and 4 C by Escherwhia coil and Yersinia enterocoh'tica. Appl. Environ. Microbial., 43: 9 9 7 1000. Pal, C.H, Mor~, V. and Toma, S., 1978 Prevalence of enterotoxigenicity in human and nonhuman isolates of Yersinia enterocolitica. Infect. Immun., 22 : 334--338. Schiemann, D.A., 1978. Association of Yersini¢, enterocolitica with the manufacture of cheese and occurrence in pasteurized milk. Appl. Environ. Microbial., 36: 274--277. Schiemann, D.A. and Toma, S., 1978. Isolation of Yersinia enterocolitica from raw milk. Appl. Environ. Microbial., 35 : 54--58. Shayegani, M., DeForge, I., McGlynn, D.M. and Root, T., 1981. Characteristics of Yersinm enterocolitica and related species isolated from human, animal, and environmental sources. J. Clin. Microbial., 14: 304--312. Shayegani, M., Morse, D., DeForge, l., Root, T., Parsons, L.M. and Mavpin, P., 1983. Microbiology of a major food borne outbreak of gastroenteritis caused by Yersinia enterocolitica serogroup 0:8. J. Clin. Microbial., 17: 3,5--40. Tacket, C.O., Narain, J.R., Sattin, R., Lofgren, J.P., Konigsberg, C., Rendtorff, R.C., Rausa, A., Davis, R. and Cohen, M.L., 1984. A multistate outbreak of infections caused by Yersinia enterocolitica transmitted by pasteurized milk. J. Am. Med. Assoc., 251 : 483--486. Toms, S., Lafleur, L. and Deidrick, V.R., 1979. Canadian experience with Yersinia Pnterocolitica (1966--1977). Contr. Microbial. Immunol., 5: 1.14-.-149. Vidon, D.J.M. and Delmas, CL., 1981. Incidence of Yersinia enterocolitica in raw milk in Eastern France. Appl. Environ. Microbial.. 41 : 355--359. Zen-Yoji, H., 1981. Epidemiologic aspects of yersiniosis in Japan. In: E.J Bottone (Editor), Yersinia entert~colitica CRC Press Inc., Boca Raton FL, pp. 205--216.