TADD, A. D. & HURST, A. (1961). J. uppZ. Bact. 24 (2), 222-228.

THE EFFECT OF FEEDING COLICINOGENIC ESCHERICHIA COLI ON THE INTESTINAL E. COLI

OF EARLY WEANED PIGS

RUMM H

BY A. D. TADD AND A. HURST TJnilezw Research I,aboratory. Rhnrnbrook, Bedford

' : The Eacherichio coli contents of samples of faeces from 24 early weaned pigs were estimated qualitatively and quant,itatively in two experiments. At weaning (10 days of age) the E. coli content was about, 108/g of faeces. Haemolytic IC. coli, often pathogenic serotypes, emerged at 12-20 days of age and then tended to disappear at 30- 40 days of age. Colicinogenic E. coli, isolated from the faeces of pigs from another herd, were grown in a 50 1. batch fermenter and added to the diet of the early weaned pigs, from weaning and until the pigs were 40 days old. Within a few days of feeding the colicinogenic E. coli, colicinogenic types formed 20-50yo of the coli-aerogenes flora of the faeces. The feeding and establishment of colicinogenic E. coZi did not suppress the haemolytic E. coli and did not affect the incidence of mortalit,y among the pigs.

PREVIOUS WORK established a connection between oedema disease in young pigs and the presence of haemolytic strains of Escherichin coli (Lemcke, Rellis & Hirsch, 1957). This agrees with the observations of other workers (Schofield & Davis, 1955; Gregory, 1955; Timoney, 1956: Sojka, Erskine & Lloyd, 1957). Young pigs seemed most susceptible to the disease 7-14 days after weaning (Schofield & Nielson, 1955; Lloyd, in the discussion to the paper by Timoney, 1957).

Lemcke & Hurst (1961) observed that the dominant serotypes in early weaned pigs can suddenly change. The symptoms characteristic of oedema disease may no longer be in evidence but scouring then usually becomes prevalent. Pigs may, there- fore, have different dominant serotypes of haemolytic E. coli and different disease symptoms, the most obvious of which is scouring.

In attempts to find a practical remedy for scouring the feeding of' strains of' 144'. coli not usually associated with the disease has been tried. It was hoped that these would displace the strains causing disease. Colicinogenic E. coli were used in preference to other strains because i t seemed that they would displace the dominant types in the gut more readily: also, the colieinogenic property could be used as a marker to follow the passage of such organisms through the animal.

Colicinogenic strains of li:. coli produce antibiotics termed colicines ; considerable literature has accumulated during the last 60 years on the inhibitory phenomena associated with E. coli (we Fredericq, 1948). The antibiotics are readily produced on agar but culturing on cellophane is required for their production in liquid media. Different strains produce various antibiotics all of which are selective and inhibit only Gram-negative organisms. particularly other strains of E. coli. Methods for

Colicinogenic E. coli i n the pig gut 223

isolating these strains and assaying the antibiotic were reported by Heatley & Florey (1946).

The experimental work is described in two parts : the techniques used for following the disease, and the application of colicinogenic E . coli.

MATERIALS AND METHODS dsseasment of discaw. Samples of faeces were taken daily whenrver possible.

About 1 g was accurately weighed into a sterile stainless steel tube containing 6 stain- less steel ball bearings of 0.25 in. diam. A volume of quarter-strength Ringer's solution equivalent to 9 times the weight of faeces was added, and the tube vigorously shaken for 2 min. The 10-1 dilution thus produced was usually of suitable consistency for pipetting, and further decimal dilutions in quarter-strength Ringer's solution were prepared from it in the normal manner. Dilutions in thr range of to lo-* were plated on penicillin-blood agar using a volumetric loop (Berridge, 1954) and an IJ-shaped stainless steel spreader. The medium was prepared as follows: a layer of nutrient agar containing benzylpenicillin (Crystapen, Glaxo Ltd.) a t a concentration of 0.1 i.u./ml was poured into a Petri dish and allowed to solidify. This was covered with a thin layer of nutrient agar containing 5% of oxalatecl horse blood (Burroughs Wellcome Ltd.) and 0.1 i.u. of penicillin/ml: when solid the poured plates were dried at 45".

Isolated colonies of coli-aerogenes organisms were usually produced by this method. The concentration of penicillin was sufficient to suppress the growth of lactobacilli and streptococci, and drying the surface of the medium prevented confluent growth. Zones of haemolysis were more clearly visible in the thin layer of blood agar than in plates containing a thicker, single layer ; this made differential counting of haemolytjic+ and nonhaemolytic colonies a simple and rapid process.

Representative haemolytic colonies were picked from the incubated plates and investigated in more detail. Biochemical tests of the cob-aerogenes isolates (Report, 1956) showed them to be invariably E . coli. Further characterization was by slide agglutination with specific antisera prepared against strains isolated from the pig herd in previous experiments. The serological tests used are described in detail by Lemclre & Hurst (1961).

Selection of colicinogenic E. coli. A modification of the method of Heatley & Florey ( I 946) was used. Nutrient agar plates were dried and then inoculated with cultures of E. coli isolated from samples of faeces. These samples were obtained from a herd in which antibiotic feeding was not used and which was free from scouring. Two strains of suspected colicine producer8 were streaked on each plate in such a manner that two straight lines of confluent growth about 2 in. apart were produced. After overnight incubatioii a t 37", the cultures were killed with chloroform vapour. This was done by placing discs of filter paper soaked in chloroform in the lids of the Petri dishes a t room temperature for 30 min; the paper was then removed and the plat,es incubated with the lids raised for a few hours a t 37" to drive off residual chloroform.

The test organisms, haemolytic E. coli of serotypes E4 and E57 (Sojka et al. 1957), w r e next strealwd across the plates to produce lines of confluent growth at, right

224 A . D . T a d d a n d A . Hurst

angles to the killed cultures. After incubation for a further 24 hr, the plates were examined for zones of inhibition adjacent to the streaks of colicinogenic E. coli.

The colicine producers which caused the most marked inhibition of both pathogenic serotypes were selected and further tested by a new screening test, providing condi- tions more akin to those in the gut of the pig. MacConkey's broth, which contains bile salts and has a slightly alkaline pH, was used to simulate two of the features of small intestine contents; 0-5:/, of trypsin (B.D.H.) and lo:/, of horse serum were also added. Tubes of this medium were simultaneously inoculated with equal volumes of overnight cultures of two organisms. One was a nonhaemolytic, colicino- genic strain of E. coli and the other a serotype E57 haemolytic strain of E. coli (at that time the predominant pathogen in the pig herd). After inoculation, the surface of the broth was sealed with a layer of sterile petroleum jelly to create anaerobic conditions, thus further simulating conditions in the intestine.

After incubation for 24 hr a t 37", counts on blood agar were done on the contents of the tubes. The lack of haemolysis round colonies of colicinogenic organisms distinguished them from the haemolytic colonies of the test serotype.

Strain C18, which was used in the feeding experiments, was one of 3 strains of colicinogenic E. coli which appeared in pure culture after incubation for 24 hr. having achieved romplete dominance over the haemolytic serotype E57.

Detection of eolicinogenic E. coli after feeding. The penicillin blood agar plates prepared for the assessment of the disease were inoculated on the surface with decimal clilutions of faeces. After differential counting, the haemolytic and non- haernolytic colonies were killed with chloroform vapour and then sprayed with a thick suspension of colirine sensitive E. coli. The plates werp incubated for a further 24 hr a t 37", and re-examined. Zones of inhibition were seen round colicinogenic colonies whereas the indicator organisms grew up to the edges of thr noncolicinogenic (including haemolytic) colonies.

Feeding trials. Twelve piglets were used in each of two feeding experiments, being allocated to the trial immediately after weaning at 10 or 11 days of age. In each of the experiments two groups of 6 pigs were used, one group to act as control. The groups were halanced with respect to sex, weight and litter

For usr as a feed supplement, E . coli C18 was grown in a stirred and aerated 60 1. batch frrmenter a t 37" for one day. The medium contained 1 of peptorie and 1 yo of glucose and was buffered with Na,HPO, and NaH,PO,. The cells were harvested in a Sharples supercentrifuge, from which they were recovered as a paste containing 709/, of moisture. This paste was mixed with an equal weight of 0.1% peptone water to produce a creamy slurry. This mas incorporated uniformly into an antibiotic free proprietary brand of pig meal; loo/, (w/w) of slurry was used in the first experi- ment and 20yo in the second.

RESULTS At weaning, when the pigs were aged 10-12 days, the faeces generally had a high coli-aerogenes count of about I 08-1 OS/g, but haemolytic E. coli were not detected (Fig. 1) . A few days after weaning (12-20 days of age) haemolytic E . coli, often of serotypes E4 and E57. appeared and sometimes became the major component of the

Colicinogenic E. coli in the pig gut 2 2 5

coli-aerogenes flora. If the young pigs survived this critical phase the haemolytic E. coli declined and with the method used could no longer be detected when the pigs were 30-40 days old ; the total coli-aerogenes population also declined.

Fig. 1 shows the pattern of development of coli-aerogenes flora characteristic of scouring troubles and gut oedema in our herd. The emergence and disappearance of haemolytic E. coli as part of the total E. coli population was considered to be the most reliable indication of potential disease ; successful treatments should alter this pattern

00

90

BO

70

60

50

40

30

20

10

Fig. I . The total 4 . coli and the percentage of haemolytir E . col i in faeces samples from early wearied pigs. Full circles, haemolytic E. coli; open circles, colmerogenes bacteria.

In the first experiment in which colicinogenic organisms were given in the diet, the mortality rate was high. Nine of the 12 experimental pigs died, 5 from the control and 4 from the test group and, as only 3 pigs survived, proper bacteriological assessment was impossible. Postmortem examination Rhowed that the deaths were associated with E . coli serotype E.57 in both control and treated groups. Feeding with colicinogenic strains apparently had no effect, although these organiams were detected in the faeces of every pig of the test group, often in large numbers.

2 2 6 A . D . Tadd and A. Hurst

The mortality rate in the second experiment was low and only one pig in the control group died. The results of the bacteriological examination are shown in Fig. 2 , in which the percentage of haemolytic E. coli in the faeces of the control group is compared with that of the test group. It can be seen that feeding with colicinogenic E . coli did not affect the emergence or dominance of haemolytic E. coli.

U

x 0

.- I - E

2

Age (days)

Fig. 2. The emergence of haemolytic E. coli in faeces samples from two groups of early weaned pigs. Full circles. control group; open circles, group fed with colicinogenic E. coli.

Fig. 3 gives the results of the examination of samples of faeces obtained from the test group. Colicinogenic strains were established a t the same time as the haemolytic E. coli emerged and when the pigs were about 20 days old these organisms formed about half of the coli-aerogencs flora of the faeces. In spite of this, however, the haemolytic flora, which consisted mostly of serotypes E4 and E57, predominated and multiplied to give very high numbers. When the pigs were about 30 days old a new factor(s) became operative which depressed both the haemolytic and the colicinogenic populations. The decline of the total coli-aerogencs population a t this stagc is also shown in Fig. I .

Fig. 3.

Colicinogenic E. coli in the pig gut

t Age (days) I The emergence of haemolytic and colicinogenic E. coli in faeces samples from a group of early weaned pigs fed on a diet containing colicinogenic E. coli. Full circles, colicinogenic E. coli; open circles, hmmolgtic E. coli.

227

DISCUSSION During the growth of young pigs the dominant strain of B. coli in the intestine may become displaced by other strains. For example, Lemcke & Hurst (1961) injected into sows specific vaccines which, it was hoped, would give rise to sufficient antibody in the colostrum to protect young pigs against scouring. Although high antibody titres were obtained, scouring and mortality were not affected, because between the times of vaccination and farrowing the dominant strains changed throughout the herd. Sears, James, Saloum, Brownlee BE Lamoreiix (1956) observed in man and dogs that, whereas one serotype of E. coli always dominated, a second type persisted in small numbers. For no apparent reason the dominant strain would suddenly decline and the strain which hitherto occurred only in small numbers would become dominant. Eventually this process would be repeated and a new dominant strain would emerge. These workers fed dogs with cultures of known types of E . coli but were unable to change this pattern. An understanding of the causes of the dominance and later disappearance of a given strain would be of help in attempts to control scouring conditions in animals.

It is possible that antagonistic relationships among the flora in t,he intestine could account for these phenomena, and it was therefore logical to test the colicines

228 A . D . Tadd and A . Hunt

produced by E. coli previously isolated from the intestine. Two experiments in which these organisms were fed and established in the pigs showed that the dominant haemolytic strains could not be suppressed by this means.

Without a programme of slaughter, it is impossible to say how many of the colicinogenic organisms became established in different, parts of the intestine. This is rather important because, although they appeared in the faeces, the numbers of colicinogenic organisms could have remained low in the small intestine, which may well be the site of multiplication of haemolytic E. coli. It would be necessary to carry out slaughtering experiments before i t could definitely be concluded that colicines are not one of the factors responsible for the successive dominance of strains.

This possibility is strengthened by the discrepancy between the in vitro and in vivo results. Feeding and establishment of colicinogenic E. coli did not affect the dominance of haemolytic E. coli strain E57. I n vitro, however, under anaerobic conditions and in the presence of trypsin and bile salts, colicinogenic E. coli C18 became completely dominant over haemolytic strain E57 in mixed culture incubated overnight. Since these conditions resemble those in vivo i t may be that the organisms contained in the diet, do not become established in the correct portion of the gut, or do not produce colicine.

The authors wish to thank the Directors of Unilever Ltd. for permission to publish this paper and their colleagues in the Animal Nutrition Division who collaborated SO willingly.

The present address of A. D. Tadd is Cyanamid of Great Britain Ltd., Lederle Laboratories Division, Gosport, Hants.

A. Hurst was formerly known as A. Hirsch.

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(Received 9 June, 1961)