Jourtiul oJ' Applied Bucteriologji 1976, 41, 31-45

An Investigation of Calf Carcass Contamination by Escherichia c d i from the Gut Contents at Slaughter

KATHERINE HOWE, A. H. LINTON

Department of Bacteriology, The Medical School, University of Bristol, Bristol BS8 1 TD, England

AND A. D. OSBORNE

School of Veterinary Medicine, Langford House, Langford, Bristol BS 18 7DU, England

Received 19 November 1975 and accepted 12 March 1976

The degree of carcass contamination at slaughter of three groups of calves (a total of 36 animals) was studied and the 0-antigen types of Escherichiu coli found on the carcass surface and in rectal contents were determined. I n one-third of the animals E. coli strains found on the surface of a carcass belonged to the same 0-serotype as those found in the rectal sample of the same calf. Nearly all the 0-serotypes found on the carcasses were also found in the rectal con- tents of at least one calf, showing that cross-contamination of carcasses had occurred. The E. coli isolated from the rectal contents were all resistant to at least one antibacterial agent.

LARGE NUMBERS of antibiotic resistant organisms are frequently found in the gut flora of calves (Guinee 1972; Smith & Halls 1966; Howe et al. 1976). These resistant organisms may contaminate the carcass at slaughter and consequently comprise a route for the potential transmission of R factors from animals to man. Shooter et al. (1970) reported that an 0 8 strain of Escherichia coli found in the rectum of one bullock could also be isolated from the carcasses of three other bullocks slaughtered consecutively. Therefore, E. coli, originating from calves, may contribute to the flow of plasmids from animals to man. Workers involved in their slaughter are exposed to possible risk of in- fection by resistant organisms. This possibility is supported by implication since Linton et al. ( 1972) showed that rural communities harbour significantly greater numbers of resistant organisms than an urban community less exposed to the animal population.

In contrast to calves reared on a milk substitute diet for veal, those reared on a nor- mal diet (hay, concentrates, etc.) to adult cattle for beef harbour very few resistant coliform bacilli by six months of age (Larsen & Larsen 1972; Wierup 1975: Howe et al. 1976). Veal carcasses are therefore the only ones within this meat animal likely to be contaminated with resistant strains.

The work reported here was an attempt to establish how many resistant organisms contaminate the carcass surface during the slaughter of calves and how frequently E. coli of the same 0-serotype found in gut contents could also be isolated from the car- cass surface of the same or other animals at slaughter.

1371

38 K. HOWE. A. H. LINTON A N D A. D. OSBORNE

Materials and Methods

Origin of animals

An investigation to determine carcass contamination at slaughter was carried out at a small slaughter-house mainly concerned with the preparation of veal calves. Animals for slaughter originated from two sources. Most were reared in a typical veal-calf rais- ing building near the slaughter house and kept in individual pens arranged in two rows. each of 22 animals, for 16 weeks. They were fed a milk substitute diet. During the first 7 weeks the starter diet was supplemented with selenium (0.045 parts/106). zinc bacitracin (50 parts/106), nitrovin (500 parts/106), furazolidone (50 parts/106) and aureomycin ( I O S 1 5 0 parts/106). A finisher ration, fed from 8-14 weeks, incorporated selenium (0.045 parts/106), zinc bacitracin (50 parts/106) and nitrovin (25 parts/106). These animals were brought into the slaughterhouse one hour before slaughter. Animals from the other veal-raising unit arrived on the day before slaughter. They were brought by lorry and kept overnight in a small covered lairage. No information was available regarding the use of antibiotics in these animals.

Procedures for sampling

Examination of faeces prior to slaughter

One group of animals, being raised at the slaughterhouse unit, was sampled on three occasions prior to slaughter. Faecal samples were collected in plastic sleeves. A medical swab was dipped in the sample, plated directly onto Bile Lactose Agar without salt (BLA; Oxoid CM7b) and spread to obtain discreet colonies. Ten colonies were picked from each culture plate and identified biochemically by an Eijkmann test at 44 OC and an indole test at 44OC (Anon. 1956; Anon. 1969). All biochemically confirmed E. coli were 0-serotyped by an agglutination test using a microtitre technique and their an- tibiotic sensitivity or resistance patterns determined, according to methods previously described (Howe 8c Linton 1976).

Examination of rectal contents and carcasses at slaughter

At slaughter faecal material was collected in plastic sleeves directly from the rectum at evisceration. Decimal dilutions were prepared in saline (0.85%) from weighed amounts and plated onto BLA and Bile Lactose Agar plates containing 25 pg/ml tetracycline (BLA + T). Presumptive coliform counts were determined after incubation at 37 OC for 24 h. Each carcass was sampled by rubbing a large, sterile alginate swab, moistened by dipping in 0.1% peptone-saline over a specified area of 9 .6 cm2. The same area was then rubbed by a dry swab. The area was defined by a sterile metal template. Three different sites on each carcass were sampled. The two swabs from each area were placed in the same universal bottle containing 9 ml of 0.1% peptone-saline and transported to the laboratory. After each sample had been agitated on a Vortex

CALF CARCASS CONTAMINATION BY E. COLI 39

agitator for 2 min, decimal dilutions were prepared in saline and known volumes plated onto BLA and BLA + T. Presumptive coliform counts were determined after overnight incubation at 37OC. Up to ten colonies were taken from the culture plates exhibiting discrete colonies from both rectal samples and carcass swabbings. All biochemically confirmed E. coli .were O-serotyped.

Three groups of calves were studied during three separate visits within a 3-month period. In the first group the faeces of 22 calves were sampled on three occasions 38, 30 and 10 days prior to the date of slaughter. These calves were reared at the veal- producing unit near the abattoir. The 22 calves were penned separately in a single row on one side of the calf house. On the day of slaughter carcasses of 12 of the 22 animals, reared to full weight, were sampled on the right hand side. Samples of rectal contents were collected, and the pleural cavity, the neck region and the hindquarters swabbed.

The second and third groups of calves were sampled only on the day of slaughter. In both groups rectal contents and three carcass sites were examined. The second group comprised eight calves reared at the abattoir unit and six ‘bobby’ calves (animals not more than 2 weeks old) recently purchased but surplus to their requirements. All these animals were sampled on the left hand side only. The third group of ten calves, reared elsewhere and brought into the abattoir for slaughter, were studied to compare groups of calves of different origin and rearing. Of these, five were sampled on the left hand side and five were sampled on both left and right hand sides of each carcass.

Results

The results of the presumptive coliform counts for all carcass swabbings and rectal contents. sampled on the day of slaughter, are given in Table 1. By these samplings it was possible to determine whether one side of the carcass was more contaminated than the other and also to compare contamination of small carcasses (‘bobby’ calves) with those of typical veal animals. Of the total of 123 carcass swabbings 46 (37.4%) did not yield coliform bacilli. Where contamination occurred it was generally heavier on the in- side of the carcass (pleural cavity) compared with the outside (neck and hindquarter): this is in agreement with work reported by Walton (1970). In contrast to findings in the pig (Linton. unpublished) the low surface contamination experienced with calves may be a result of the removal of the heavily contaminated hide leaving behind a relatively uncontaminated surface.

There appears to be no significant difference between the contamination of the right and left hand sides of the veal carcasses within the group studied (Nos 42-46) but the group of six ’bobby’ calves was generally more contaminated. This may be a direct con- sequence of size and handling, the smaller animal being more difficult to eviscerate without contaminating the body cavities.

The variations in the presumptive coliform counts on the carcass sites between the three groups may be due to undefined causes prevailing on different days of slaughter; in general, however. the counts in the hindquarter (uppermost as the carcass is hung) were lower than those from the other two sites.

40 K. HOWE, A. H. LINTON AND A. D. OSBORNE

Faecal presumptive coliform counts revealed very consistant results between in- dividual full term calves and between these and ‘bobby’ calves indicating that large numbers of coliforms persisted throughout the life of the calf fed a veal diet. The counts on BLA + T indicate almost all isolates were tetracycline resistant.

An assessment of direct contamination of carcasses from the gut of the same animal was possible by comparing the 0-antigen types of E. coli found in the gut of each calf with those found on the carcass of the same animal. The isolation of a particular 0- serotype from each sample was recorded without specifying the number of isolates of that 0-serotype from the same sample. Of the 3 0 mature calves and the six ‘bobby’ calves studied evidence of direct carcass contamination by specific 0-serotypes from the rectal contents of the same animal occurred in 12 animals (33%). In seven animals a single 0-serotype was found common to both rectal contents and carcass; in five calves two 0-serotypes were common to both.

The number of calves in each group from which each 0-serotype was isolated are in- dicated in Table 2. The occurrence of the various 0-serotypes in the rectal contents and on the carcass surfaces for each group of calves are recorded. In the first group (Nos 1-22) the total number of calves from which an 0-serotype was isolated in at least one of the three faecal samples taken prior to slaughter are separately recorded. The results are divided between calves 1-10 and calves 11-22 since only the latter group were sampled additionally at slaughter. Within this group (calves 1 1-22), 18 different 0- serotypes were found on the carcasses, and of these 12 were present in the rectal con- tents of the group at the time of slaughter. If all four faecal examinations on the 12 calves are considered then 16 out of 18 0-serotypes were found in both sites. If the faecal examination of all 22 animals are considered (i.e. calves in the same house and during the same period) all 0-serotypes found on the carcasses of calves 11-22 were shown to be present on at least one occasion in the rectal contents of one or more calves in the same calf house.

No information was available on the 0-serotypes present in the rectal contents of the other groups of calves prior to slaughter. However, it was possible to determine the number of times the same 0-serotype found on a carcass was also present in the rectal contents on the day of slaughter for each group of calves (Table 2). Frequently the same 0-serotype was isolated from more than one group of calves but by summing the results for all groups it was found that of 7 1 separate isolations of various 0-serotypes from the rectal contents, 33 (46.5%) were also isolated from the carcasses of the various groups of calves.

In the final column of Table 2 the serotypes found throughout the three groups of calves are summarized. A total of 42 different 0-serotypes were found on at least one calf carcass and, of these, 33 (78.6) were also found in rectal contents of at least one calf. Of the nine 0-serotypes found only on carcasses, five were antibiotic sensitive. Since no antibiotic sensitive strains were found in the rectal contents this may indicate that these 0-types did not originate from the calf gut.

Since the number of colonies from each sample was limited to ten some minority strains of E. coli may have been missed. Observations in typing 100 colonies from the same sample indicate that over 90% of 0-serotypes in such a sample are found in the first ten colonies. This is in agreement with work reported by Vosti ef al. (1962) but it does indicate that a few other 0-serotypes may be present in very small numbers.

CALF CARCASS CONTAMINATION BY E. COLZ 41

TABLE 1 Presumptive coliform counts on faeces and carcasses at time of slaughter

Carcass surface? A 7 7

Rectal contents* Pleural cavity Neck Hindquarter Calf ( p A - x & &

reference BLA BLA + T BLA BLA + T BLA BLA+ BLA BLA +

11 12 13 14 15 16 17 18 19 20 21 22

23 24 25 26 27 28 29 30 31 32 33 34 3s 36

37 38 39 40 41 42 43 44 4s 46

42 43 44 45 46

9.1 8.7 9.2 8.0 8 .4 9.1 9 .3 8 .7 9.4 8.9 8.7 8 .0

8 .4 8.9 8 .9 9 .3 8 . 5 7.3 8 .2 8 . 5 9.3 9.3 9.2 8.8 9 .2 9 .9

8 .2 8 .3 8 .3 7.8 8 .3 8.2 8 .3 8.3 7.8 8.3

7.0 7.0 8 -3 8.4 8.3

9.1 8.7 9. I 7.9 7.4 8 .3 9. I 7.5 9.5 8.0 8.8 8. I

8 . 5 8.8 8.6 Y . 1 8.5 7.3 8.2 9.4 9.3 9.4 8.6 8.6 9. I 9 .9

8.2 8.4 8.3 7.7 8 .3 8.2 8 .4

7.7 8 .3

8.3

7 . I 7 . 1 8.4 8.4 8.7

1st group, right-hand side of carcass 2.2 - - 2.2 1.8 2.5 2.2

I ..5 2.2 1.5 2.5

-

2.0 I .8 2.0 2.4 2.2 2.3 1.8

-

- - - 2 . 5

2.7 2.0 1 .5 2.1 2.2

2.5 1.8

-

- -

- -

2nd group. left-hand side of carcass 4.2 3.5 2.2 2.9 3.1 5.3 5.3 1.5 2 . I 2.6 I .8 2.4 I .8 1 . 5 3.3 3.3 2.2 2.2 1.8 2.0 I .5 4.7 4.7 3.8 3.7 3.8 3.3 3.4 3.3 2.9 2.9 2.3 3.8 3.6 3.3 3.2 3.4

-

-

-

-

-

3rd group, left-hand side of carcass 2.0 2. I 2.1 I .5

- 2.4 2.3 1.5 2.1 1.8 1.5 2.1 2.3 - 1.5 1.8 2. I 2.5 1 .5 2.0 1.8

- 2.0 1.5 - 2.7 2.4

- - -

- -

- -

- - - - -

-

3rd group. right-hand side of carcass 2.3 1 . 5 1 .5

2.1 2.0 1.5 - 1.5 1.5 I .5 1 .5

I .5 2.3 - -

- -

- - -

BLA. bile lactose agar: BLA + T. bile lactose agar + 25 pg/ml tetracycline. Calf numbers 3 1-36 refer to 'bobby' calves.

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44 K. HOWE, A. H. LINTON AND A. D. OSBORNE

Discussion

These animals were reared on a diet which encourages a high proportion of coliforms in the gut flora. In one third of the animals studied evidence of direct carcass contamina- tion with E. coli from the gut of the same animals was indicated by the presence of the same 0-antigen type in both sites. However, if all the 0-serotypes found in the faeces of the calves slaughtered at the same time are considered, the proportion of carcasses con- taminated with 0-serotypes of faecal origin is much higher. There is, therefore, con- siderable evidence of carcass contamination by E. coli from the calf gut in this commer- cial abattoir.

The predominance of a particular 0-serotype may be judged by considering the number of calves from which it was isolated. In descending order of prevalence, from 56.5% to 10.9%, the following 0-serotypes were found: 013, 0107, 09, 04, 015, 020, 0 100,O 10 1.0 1 13, 023, 0 1 15 and 073. The 0-serotypes most predominant in the rectal contents of the calves in the abattoir survey did not closely match those most com- monly found in a previous study (Howe & Linton 1976). However, in a situation where animals are kept together in a single unit it is to be expected that 0-serotypes common and intrinsic to that environment will be most prevalent. A similar finding with pigs was reported by Craven & Barnum (1971).

Of the 57 0-serotypes isolated from the rectal contents in the slaughterhouse survey 13 had not been previously found in calves. Combining the results from the two previous surveys (Howe 8c Linton 1976; Howe el af. 1976) with those of the present work, 110 0-serotypes have now been isolated from calves; many of these have been found to harbour antibiotic-resistance plasmids. The observation that a very wide range of 0-serotypes in calves were antibiotic-resistant with evidence of plasmid carriage is very significant. This is much wider than that experienced in man and indicates that R factors have penetrated a wider range of 0-types in calves (Hartley el al. 1975).

Mews M. and B. Thorner and J. Morrison provided excellent co-operation at the calf rearing unit and the abattoir to whom we offer our grateful thanks. We are greatly in- debted to Mrs Helen Clements and her colleagues for their skilled technical assistance. This work was supported by grants from the Medical and Agricultural Research Councils.

References

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