Colonization of the Bovine Teat Duct Following Inoculation with Staphylococcus Aureus and Escherichia Coli

Br. vet. } . ( 979 ),135,149

COLONIZATION OFTHE BOVINE TEAT DUCT FOLLOWING INOCULATION WITH STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI By A. J. BRAMLEY, J. S. KING, T. M. HIGGS AND F. K. NEAVE

National Institute for Research in Dairying, Shinfield, Reading RG2 9AT

SUMMARY

The ability of Staphylococcus aureus and Escherichia coli to colonize the bovine teat duct was examined in a series of experiments in which varying numbers of the organisms were inoculated either at the teat orifice or 2 mm into the teat duct. Inoculation with 20 cfu of Staph. aureus failed to produce colonization but placing 200 or 2000 cfu either at the orifice or 2 mm into the duct produced colonization in 60 to 72% and 92% of teats respectively. The number of Staph. aureus recoverable from the teat apex of quarters increased steadily with time and was still rising after 19 days. Intramammary infection was an infrequent consequence of colonization in these shon duration experiments, only four infections resulting from a total of 78 colonizations. Contrastingly, inoculation of the duct with up to 43 x 10 6 cfu of E. coli resulted in only a transient colonization which seldom persisted for more than three or four days. The significance of these observations is discussed in relation to the pathogenesis of udder disease due to E. coli and Staph. aureus. I NTROD UCTION

The teat duct is both the route by which pathogens penetrate the udder to cause intramammary infection and the udder's main defence against udder disease (Reiter & Bramley, 1975). The teats of cows on farms are regularly contaminated with bacteria, often in large numbers, yet, infection occurs infrequently (Report, 1964). However, when small numbers of Staph. aureus or E. coli are placed into the teat sinus beyond the teat duct intramammary infection usually occurs (Newbould & Neave, 1965a; Bramley, 1976). Data from many countries indicate that Staph. aureus remains probably the most common cause of bovine udder disease (Neave, Dodd & Kingwill, 1966 ; McDonald, 1969, Mylrea et al. , 1977) although rates of new infection with this organism can be controlled by the application of post-milking teat disinfection (Neave, Dodd &

150

BRITISH VETERINARY JOURNAL, 135, 2

Kingwill, 1966; McDonald, 1969; Wessen &: Schultz, 1970). In addition, there is concern that udder disease due to Gram negative organisms, particularly E. coli and Klebsiella pneumoniae, is increasing. Nevertheless rates of new infection with coli forms are much lower than those with Staph. aureus, despite the high frequency with which teat ends become contaminated with coliforms, particularly under winter housing conditions (Bramley &: Neave, 1975) and the fact that post-milking teat disinfection does not reduce rates of udder disease with coliform bacteria (Kingwill et al., 1970). These differences between the pathogens relate in part to the different sources of the bacteria within the dairy herd (Bramley, 1974) but may also reflect fundamental diHerences in their abilities to colonize and/or penetrate the teat duct of the lactating cow. Whilst some studies of the behaviour of Staph. aureus in the duct indicate that it can readily colonize the teat orifice (Newbould &: Neave, 1965b; Forbes, 1968) little information is available on the behaviour of E. coli, although McDonald &: Packer (I 968 ) did investigate the effect of dipping teats in a culture of Aerobacter aerogenes and found little evidence of duct colonization. Consequently the objectives of this work were to compare the results of teat duct or orifice inoculation with varying numbers of Staph. aureus or E. coli and to determine how this might relate to the pathogenesis of udder disease caused by these organisms. MATERIALS AND METHODS

Five experiments were performed in which varying numbers of S. aureus or E. coli were placed either at the orifice or 2 mm into the teat duct. Subsequently measurements were made to determine whether bacterial growth or colonization occurred in the ducts. The methods common to all experiments are described below followed by details and results of the individual experiments.

Animals and milking procedure Experiments were carried out on lactating Friesian cows during the months of May, June and July. All animals were free from intramammary infection at the start of each experiment as determined by examination of milk samples taken aseptically at weekly intervals before the experiment (Neave &: Oliver, 1962). Cows with teat blemishes and with moderate or severe erosion of the teat orifice were rejected . Throughout the experiments the cows were at pasture and were milked twice daily in a tandem milking parlour using machines which were pasteurized with water at 85°C before milking each cow. The animals were prepared for milking by udder washing with warm running water and drying with paper towels. No post-milking teat dip was used during the experimental periods with the exception of one period in Experiment 1. Cultures Staph. aureus strain 'm' NCDO 1499 (Sharpe, Neave &: Reiter, 1962). Staph. aureus strain 'F' NCI?O 1496 (Sharpe, Neave &: Reiter, 1962). A lyophilized culture was subcultured in nutrient broth, grown for 18 h at 37°C, cooled to 4°C and counted following dilution in half strength broth (t LB).

BOVINE TEAT DUCT COLONIZATION

151

Inoculation suspensions were prepared by diluting the original culture with skim milk to give concentrations of 20, 200 or 2000 colony forming units (cfu)/O ·OO 1 ml. E. coli strain P4 NCDO 2070 (Bramley, 1976). The organism was subcultured in dextrose lemco broth and incubated for 6 h at 37°C on the day of inoculation when it was centrifuged and the pellet resuspended in 10 ml of 1 LB + 10% glycerol. This suspension was either inoculated undiluted or after further dilution in 1 LB .

lrwculation method Inoculation was performed using an 'Agla' micrometer syringe modified as described by Hibbitt &Jones (1967). The collar on the inoculating needle was fixed to allow inoculation of the lower 2 mm of the duct when placed against the teat orifice. When inoculation of the orifice only was required the tip of the needle was not inserted but allowed to touch the wall of the teat orifice. The volume of inoculum delivered in each case was 0·001 ml. Recovery oj te Jt organiJms from teatJ The number of test organisms recoverable from the teat apex was examined at regular intervals after the initial inoculation by swabbing the teat apices b efore milking using cotton wool swabs wound onto wooden applicator sticks and moistened with 1 LB. Each swab was held at right angles to the teat and rubbed across the teat apex 10 times, then placed in a 1 oz universal bottle containing 2 ml of t LB (E. coli experiments) or skim milk (Staph. aureUJ experiments). About 30 min after collection swabs were refrigerated and examined within 3 h . The swab bottles were shaken for 10 to 15 sec on a vortex mixer. 0 · 1 ml samples of undiluted material and of appropriate dilutions in 1 LB were plated on calf blood agar (BA) (Staph . aureUJ experiments), or MacConkey agar (E. coli experiments). After overnight incubation at 37°C colonies typical of the inoculated strain were counted . Staph. aureUJ 'm' was distinguished from other strains by its characteristic very wide zone of a-haemolysis when grown on BA. It is also resistant to 1·5 i.u. penicillin and 2 Ilg ampicillin when tested with sensitivity discs (Oxoid) produces a clear zone of haemolysis on 8% salt blood agar (SBA) (Neave & Oliver, 1962) and does not produce acid in litmus milk. Strain ' F' was distinguished by having a narrow zone of ahaemolysis on BA and no a-haemolysis on SBA. It was found to be sensitive to penicillin and ampicillin and produced acid in litmus milk. A minimum of one coliform colony/plate was tested for agglutination with a specific '0' rabbit antiserum raised against E. coli N . EXPERIMENT 1

The effect 0/ inoculating either the teat orifice or the lower 2 mm 0/ the teat duct with 2000 cfu Staph. aureUJ 'm' Eight cows were used. Two teats of each animal were inoculated following an afternoon milking by placing 2000 cfu of Staph. aureUJ 'm' 2 mm into the duct, and the other two teats were inoculated with 2000 cfu at the orifice. Treatments were applied to either the right or left side pairs of teats in each cow, and randomized between cows.

14 14

16 16

Teat duct Teat apex

14

I (J)

168 196

13 14

152 114

II

No. C.M. + ve

2

No. C.M. + ve

0

No. qtrs becoming infected (clinical)

G.M. = geometric mean numb er of Staph. aureus 'm '/ positive swabs.

No. teats becoming colonized

No. teats

Site of inoculation

218 1020

No. C .M . + ve

14 12

6

14 14

1658 4404

No. C .M. + ve

14 14

4672 10644

No . C.M . + ve

7

Days following inoculation

Pre-milking swabs

J

ORGANISMS (EXPT. j)

13 17420 13 45986

No. C.M. + ve

10

C.M.

451420 644240

No. + ve

13 13

postmilking swabs

10

THE RECOVERY OF STAPH . AUREUS ' m' BY SWABS FROM TEAT APICES FOLLOW ING INOCU LAT IO N OF THE APEX OR TEAT D UCT WIT H 2000

TABLE I

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BOVINE TEAT DUCT COLONIZATION

153

A single swab was taken daily for seven days from the teat apices of all the cows at an afternoon milking prior to udder preparation for milking. After afternoon milking on the tenth day all animals returned to a full hygiene milking routine which included udder washing in chlorinated water (600 parts/l06 avo chlorine) and teat dipping after milking in sodium hypochlorite (40000 parts/10 6 av.cl). On the thirteenth day swabs were again taken before afternoon milking followed by aseptic quarter samples.

Results Examination of the swabs (Table I) showed that multiplication of Staph. aureus had occurred at the teat apex in 28 of the 32 inoculated teats, similar numbers of apices being positive for both inoculation treatments. The recovery after 24 h was low, the largest number of organisms per swab being 1340, but the numbers increased steadily with time and after 10 days the geometric m ean Staph. aureus count for all positive quarters was 28300 with seven swabs yielding > 200000. Apex swabs taken immediately after milking on this occasion (the 10th day after infusion) gave a much higher recovery of Staph. aureus than the pre-milking swabs and had a GM of 539280 cfu/swab. Examination of apex swabs and aseptic quarter samples on the 13th day after inocu lation (three days after the commencement of teat dipping), revealed only two cows sti ll yielding Staph. aureus. In one cow, although the apex swabs were all negative, one quarter showed signs of clinical mastitis (clots and discoloured secretion) and the aseptic milk samples yielded 3600 cfu Staph. aureus 'm' /ml. This was considered an intramammary infection. In a second cow although two quarters yielded low numbers of Staph. aureus 'm' on the apex swabs (60 and 520 cfu), subsequent apex swabs were negative, and examination of the aseptic quarter samples revealed no bacteria and no rise in somatic cell count. These two quarters were considered uninfected . All three quarters had been originally inoculated at the teat apex. EXPER I MENT 2

A comparison of the inoculation of the teat orifice with 20 or 2000 cfu Staph. aureus 'm' Because inoculation of the orifice with 2000 cfu usually resulted in duct colonization in Experiment 1 the effect of a much smaller inoculum, 20 cfu, was investigated. The experiment was conducted using nine cows, six of which had been used in Experiment I. Both inoculum levels were applied to the teat orifice. The comparison was made within cows in three animals, i.e. right V. left side teats and between cows in the other six animals. This was to check on the possibility that pathogens transferred between the teats of a cow during milking' might influence the results of the experiments. Results None of the quarters inoculated with 20 cfu became colonized in 14 days, and no Staph. aureus were recovered from any swabs taken of teat apices on this treatment, even following enrichment of swabs at 37 0 C for 16 h (Table II). Inoculation with 2000 cfu showed a very similar result to that achieved with the

TABLE II

No. teats becoming colonized

12 0

No. teats

18 18

2000 20

0 0

No. qtrs becoming infected (clinical)

0 0

No . C.M. +ve

3 0

36

No. C.M. + ve

2

G.M. = geometric mean number of Staph , aureus 'm'/positive swabs. cfu=colony forming units.

(cfu)

Inoculum

9 0

204

No. C.M. + ve

4

9

11

14

12 0

3894

()

12

4382

12 0

9542

12 16543 0

No. C.M. No. C.M . No. C.M . No . C.M. + ve + ve + ve + ve

7

Days fo llowing irwculation

O R GAN ISMS (EXPT. 2)

THE RECOVERY OF STAPH. AUREUS ' m' BY SWABS FROM TEAT APICES FOLLOWING INOCULATION OF THE TEAT APEX W IT H 20 O R 2000

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12 12

No. teats inocUlated

8

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No . teats becoming rolonized

1(0) 0

2 (0) 0

No. qtn becoming injected (clinical)

eli.! = co lony fd rining units. G.M . = geom etric mean orStaph. aureUJ/positive swab.

200 200

2000 2000

(cju)

Inoculum 5

8

8

II 10

881 200

2794 1i20 8

8

II 10

1145 1020

70378 9360

No. a.M. No. G.M. + ve + ve

2

8 7

10

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No. +ve a .M .

24400 39400

294940 14400

8

7 6

8

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No. +ve

Days followihg inoculation

35100 223340

1467600 1600000

G.M.

14

7 6

10 8

No . +ve

453000 2416000

1549400 3310000

G.M.

19

THE RECOVERY OF STAPH . AUREUS AFTER INO CULATION OF T HE TEAT APEX WITH T WO LEV ELS OF TWO STRAINS OF STAPH. AUREUS (EX!'T.3)

TABLE III

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156

BRITISH VETERINARY JOURNAL, 135,2

same treatment in Experiment 1. Twelve of the 18 inoculated quarters (66%) became colonized and the numbers of Staph. aureus recovered from the swabs increased steadily with time (Table 11). Aseptic quarter samples collected at the end of the experiment did not reveal any evidence of intramammary infection with Staph. aureus. EXPERIMENT 3

A comparison of the effect of orifice inoculation with 200 or 2000 cfu of two strains of Staph. aureus Twelve cows were used to compare levels of inoculum of 200 and 2000 cfu of two strains of Staph. aureus (' m' and 'F'). The experi~ental conditions were as described for Experiment 1 and the comparisons were made between the quarters of each cow using a factorial design. Results In this experiment Treatment 1, 2000 cfu 'm', produced a higher proportion of colonized teats (92%) than in either of the two previous experiments while strain 'F' resulted in 83% of teats colonized (cf. Experiment 1 - 82% colonizations with 2000 'm'). Inoculation with 200 cfu resulted in a lower number of colonizations (66%) for both strains (Table III). The smaller inoculum not only reduced the number of teats colonized but also the numbers of bacteria recoverable from the colonized teats throughout the experiment. Aseptically drawn milk samples taken at the end of the experiment revealed two quarters of one cow and one quarter of a second cow to be infected with Staph. aureus 'm'. All three infections were in teats inoculated with strain 'm', two with 2000 cfu and one 200 cfu. EXPERIMENT 4

An examination of the effect of teat duct inoculation with large numbers of E. coli strain P4 Th is experiment was designed to investigate the extent of teat canal colonization or penetration with E. coli P4 using similar techniques to the earlier experiments with Staph. aureus. In addition, by using the reaming technique of Murphy (1959), the possibility that streak canal keratin inhibited the growth of E. coli was investigated. The size of inoculum (2 ·5x 10 6 cfu) of E. coli P4 was much higher than that used for experiments for Staph. aureus because of some preliminary evidence which suggested that the teat canal was not readily colonized by E. coli (Bramley, 1974). The treatments applied to the four teats of each cow were: I ) reaming of the whole length of the teat duct followed by inoculation. 2) reaming of the lower 3 mm of the teat duct followed by inoculation. 3) E. coli inoculation with no pre-treatment. 4) the fourth teat was left uninoculated as a control. To determine if and when penetration of the teat duct by E. coli occurred, foremilk samp les were collected without cleaning the teat with alcohol at each milking following inoculation and the cell count measured using a Coulter counter. Teat-end swabs were taken not only before but also after milking.

BOVINE TEAT DUCT COLONIZATION

157

Results Despite the use of a large inoculum (12500 times the lowest Staph. aureus challenge to produce colonization), multiplication of the E. coli at the teat apex was only detected in five of the 24 inoculated quarters. In the remaining quarters generally low numbers of E. coli were recovered for a maximum of three days following inoculation . In one of the five quarters showing some multiplication there was a rise in the foremi lk cell count to 25 x 10 6 /ml and clinical signs of mastitis after six days indicating penetration of the duct. Antibiotic therapy was then administered. This quarter had had the entire teat duct reamed prior to inoculation (Table IV). A second of these five quarters, in which the lower 3 mm of the duct had been reamed, showed a cell count rise from 0·25 x 10 6 to 2 X J06 cells/ml between the second and third milkings fo llowing inoculation but the count had subsided to 0· 1 x 10 6 /ml by the fifth milking. No clinical signs were detected and the cell counts of the foremilk from the other quarters in the animal remained relatively unchanged. It is possible that penetration occurred and was followed by spontaneous elimination of the organism by the defence mechanisms TABLE IV THE RECOVERY OF E. COLI P4 AFTER INOCULATING REAMED AND UN REAMED TEAT DUcrS WITH 2·5x 10' CFU ORGANISMS (EXPT. 4)

Treatments (each 8 quarters)

Time of swabbing"

Days after inoculation 2

No. C.M. No. C.M. No. C.M. + ve + ve + ve

Whole duct reamed

AM I 2 PM I 2

Orifice only reamed

AM I 2

No reaming

AM I 2

PM I 2

PM I 2 Un inocu lated control

AM I 2 PM I 2

No. C.M. +ve

8 8 6 4

4990 212 191 58

5 3 3 3

752 1986 162 274

3 3 2 2

458 475 210 429

2 2 2 0

8 8 4 4

1823 74 22 7 60

5 2 3

200 49 74 60

2 I 0 2

249 160 0 63

8 7 4 3

1359 133 7I 58

2

805 145 705 86

120 160 80 20

I 0 2

20 50 40

4 2

3 0 0 0 0

0 0 0 0

1* = before mi lking ; 2* = after mi lking. cfu= colo ny fo rming units. C.M . = geometric mea n number of E. coli P4/positive swabs.

6

5

4

J

27 129 475 30 0

No . C.M. No. C.M. + ve + ve

I 2

80 226

30 60

I I 2

360 440 I 666 · 820

7800 1600

100 1550

I 2

220 110 180 160

220 320

1800 1900

0 0 0 0

0 0

0 0

158

BRITISH VETERINARY JOURNAL, 135,2

of the gland. Swabs from the teat end of this quarter became negative six days after inoculation and the most persistently positive quarter of the five had become negative after eight days. E. coLi P4 was recovered in small numbers of four swabs taken from the uninoculated control teats during the first two milkings following inoculation. These were presumably cross contaminations but the effect was slight, the maximum number of organisms recovered/swab being 80 cfu. There was no evidence from this experiment that reaming of part or the entire teat duct had any influence on the multiplication or survival of E. coLi in the teat duct, although both quarters showing penetration had been given a pre-inoculation reaming treatment. EXPERIMENT 5

The inocuLation ofincreased numbers ofE. coLi P4 into the teat duct After the failure to establish a persistent teat duct colonization with E. coLi in Experiment 4, Experiment 5 was designed to examine the effects of an even larger inoculum. A suspension of E . coLi was prepared producing an inoculum of 43 x 10 6 cfu, this together with dilutions containing 4·3 x 106 and 0·43 x 10 6 cfu/O·OO 1 ml were compared in eight cows using the method described in Experiment 4. In this experiment no teats were reamed before inoculation. ResuLts One animal died of causes unr.elated to the experiment 24 h after inoculation, thus reducing the number of quarters/treatment to seven. At this time the two teats inoculated with the higher levels of E. coLi were still positive for this animal but the results have not been included in Table V. During the first 24 h there were higher recoveries from the teats receiving the larger inocula but this was not maintained and was not evident from the third milking onwards. By the sixth milking after inoculation only six teats remained positive and these were equally distributed among me three levels of inoculum. At the fifteenth milking only two quarters remained positive, one having been inoculated with 4·3 x 10 6 cfu and the other with 0·43 x 10 6 cfu. The. former of these persisted until the eighteenth milking and the latter until the twenty-second milking. No clinical signs were detected in either of these quarters and no abnormal changes in foremilk cell count occurred . It· was observed that one quarter, apparently colonized with E. coLi P4, was found to be negative on swabs for the five successive occasions between the fourth and tenth milking after inoculation, the E. coli being re-isolated at the eleventh and fourteenth milkings (Table V). It remains unclear whether this was as a result of recontamination or due to a failure to isolate organisms from a colonized duct. No other quarter in this animal was positive during this period.

DISCUSSION

The results of the five experiments demonstrate clearly the mark.ed differences in the abilities of the strains of Staph. aureUJ and E. coLi testedi to colonize the teat duct.

TABLE V

PM 1 2

AMI 2

PM I 2

AM I 2

PM I 2

AM I 2

PM I 2

AM I 2

Time oj swabbing·

7 7 4 5

6 6 4 5

6 4 4 4

0 0 0 0

2760 666 160 918

2910 222 3113 142

1448 2477 118 932

3 4 3 4

3 2 1 2

2 3 3 3

0 0 0 0

294 512 81 681

280 2384 500 573

314 10146 306 11944

2

No. C.M. +ve

1

No. C.M. + ve

1· = before milking. 2· = after milking. cfu=colony forming units. G.M.=geometric mean number of E. coli P4/positive swabs.

Apex inoculated with 43 X 106 du E. coli

Apex inoculated with 4·3 x 10 6 c!iJ E. coli

Apex inoculated with 0·43 x 10 6 eft. E. coLi

Uninoculated control

Treatments (each 7 quarters) 4

2 3 1 2

0 2 0 2

I 2 I 2

0 0 0 0

2060 115 400 705

0 150 0 300

380 2207 360 18633

1 2 0 2

0 0 0

I 2

0 0 0 0

20 70 0 312

0 0 0 20

600 1300 80 204

No. C.M . No . C.M. + ve +ve

J

6

0

0 0

20 40

0 60

720 1800

0 1

0

0 0

0 40

0 340

20 66

No. C.M . No . C.M. +ve +ve

5

Days after inoculation 8

0 1

0 1

0 0

0 20

0 4020

1780 980

0 1

0 1

0 0

0 0

0 40

0 200

0 0

No. C.M. No. C.M. +ve +ve

7

THE RECOVERY OF E. CO LI P4 BY SWABS FROM THE TEAT APEX FOLLOWING INOCULATION WITH THREE LEV ELS OF CHALLENGE (EXPT. 5)

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BRITISH VETERINARY JOURNAL, 135,2

Colonization was readily achieved even with relatively low inocula (200 cfu) of either strain of Staph. aureus and was typified by a steadily increasing recovery of the organism from the teat apex swabs. Despite the high incidence of colonization only four intramammary infections due to Staph. aureus developed during the course of the experiments, confirming the observations of Forbes (1968 ) that colonization of the lower duct with staphylococci can persist for considerable periods in the lactating cow without intramammary infection. The rapid removal of colonization following the introduction of post-milking teat disinfection suggests that the colonizing organisms are restricted to a small portion of the duct, close to the teat orifice, where they are accessible to the disinfectant. The same observation has been made following examination of the removal of naturally occurring Staph. aureus colonization by teat disinfection (Neave, 1970). I n our experiments we were unable to detect any difference in the frequency of colonization following inoculation of the orifice and inoculation 2 mm into the duct. McDonald (1970) suggested that the distal 2 mm of the teat duct were of crucial importance in the prevention of intramammary infection. Prasad & Newbould (1968) reported a much greater frequency of intramammary infection when Staph. aureus were placed 4 mm into the duct compared with inoculation to only 3 mm. These observations and the apparently frequent localization of colonization at the teat orifice may indicate that the inhibitory properties of the duct are not uniform but increase towards the proximal end of the teat duct, thus effectively restricting colonization to its distal part. This hypothesis has been made by Milne (1978) who suggested that an inhibitory substance may be secreted from the region of Furstenberg's rosette. The findings of Neave et al. (1968) were that unmilked quarters were far more susceptible to infection than milked quarters, following external exposure of the teat to Staph. aureus. They also suggested that regular flushing of the teat duct at milking played a part in reducing the probability of growth through the duct, and restricting colonization to the teat orifice. In constrast E. coli inoculations, even with very large numbers of organisms (43 x 10 6 cfu) did not produce true colonization, although some teat ducts retained the organisms for several days . This supports the findings of McDonald & Packer ( 1968) who found no evidence of colonization when teats were dipped in a suspension of Aerobacter aerogenes. Those teats remaining positive showed a gradual decline in the numbers of E . coli isolated and they usually became negative within three days, a quite different result to that achieved with staphylococci. Penetration of the duct leading to a clinical case of mastitis occurred in one quarter and there was some indirect evidence that penetration followed by elimination within the gland may have occurred in a second quarter. Both of these quarters had been reamed prior to inoculation and this may have influenced penetration (Murphy, 1959); it has been well documented that many coliform infections of the udder are self-limiting (Murphy & Hanson, 1943; Schalm, Lasmanis & Carroll, 1964). No other effect of reaming was detectable either on the persistence or multiplication of the E. coli in the duct. This contrasts with Murphy's findings with Strep. agalactiae and also with those of Bramley (1974) who found reaming influenced both multiplication in and penetration of the duct of the unmilked animal by Klebsiella pneumoniae. However, as with Staph. aureus the evidence suggests that the milking process may be of major importance as a defence mechanism since Bramley & Neave (1975) produced several intramammary infections following

BOVINE TEAT DUCT COLONIZATION

161

inoculation of the duct of unmilked animals with various coliform organisms. I n the experiments with E. coli the opportunity was taken to examine the numbers of E. coli recoverable from teat apex swabs collected both before and after milking. There were several teats in Experiments 4 and 5 showing a pattern of low or negative recoveries of E. coli before milking followed by larger recoveries after milking. Whilst this might relate to the better contact made by the swab with the surface following the softening of the skin by the milking process, there is an alternative hypothesis. It is possible that the E. coli did not survive at the orifice but were lodged some distance into the duct and were brought to the orifice only during milking. The observations and findings from this series of experiments correlates well with the patterns of udder disease on farms . The importance of preventing teat duct co lonization in the control of Staph. aureus infection has been described and the value of teat disinfection in doing so demonstrated. In contrast teat disinfection is of little value in the control of coliform mastitis (Kingwill et al., 1970). These results indicate that one reason for this, in addition to the different sources of the organism, is that E. coli apparently does not readily produce colonization of the duct of the lactating cow. Bramley & Neave (1975) reported that with natural challenge the pattern of isolation of co liform bacteria from teat-end swabs suggested transient contamination rather than colonization. A similar conclusion was reached by Brander (1973 ) and Jasper & Dellinger (1975). From these observations and those of Murphy ( 1959) and McEwen & Cooper ( 1947) it appears that those organisms which readily colonize the teat duct, Strep. agalactiae, Staph. aureus and Corynebacterium bovis, are also responsible for most intramammary infections and from the reports of Thomas et al. (1972 ), Newbould & Neave (1965a) and others the act of milking is a major determining factor in the amount of penetration of the udder by organisms in the teat duct. If colonization of teat ducts with E. coli was common the recovery of this organism from milk samples would be frequent in view of its consistent presence in the environment of the udder. However, co li forms are very infrequently found in milk samples taken aseptically.

ACKNOWLEDGEM ENTS

The authors would like to thank Miss E. M. Hogben for her able technical assistance and Mr N. Jackson and his farm staff for their cooperation .

REFERENCES

BRAMLEY, A.j. (1974 ). Ph .D. thesis, University of Reading. BRAMLEY, A. J. ( I 976 ).joumal oj Dairy Research 43,205. BRAMLEY, A. j. & NEAVE, F. K. (1975). British Veterinaryjoumall31, 160. BRANDER , G. C. (I973). Veterinary Record 92, 50 I. FORB ES. D. ( 1968 }. joumal oj Dairy Research 35 , 399. HIBBIlT, K. G. &J ONES, G. W. (1967). Veterinary Record 80,270. JASP ER, D. E. & DELLINGER,j. D. (1975 ). Cornell Veterinarian65,380. KINGWILL, R. G., NEAVE, F. K., DODD, F. H., GRIFFIN, T . K., WESTGARTH, D. R. & WILSON, c. D. (1970). Veterinary Record 87,94 .

162

BRITISH VETERINARY JOURNAL, 135,2

McDONALD,]. S. (J 969l. journal of the American Veterinary Medical Association 155, 903. McDoNALD,]. S. (J970 ). Proceedings of the International Conference on Cattle Diseases, Philadelphia, Penn. Aug. 1970, p . 97 . McDoNALD, j. S. & PACK ER, A. R . ( 1968 ). Americanjournal of Veterinary Research 29, 1525. McEwEN, A . D . & COOPER, M. B. (1947) . Veterinary Record 59, 655 . MILNE, ]. R. (J978). Proceedings of the International Symposium on Machine Milking. 17th Annual Meeting of the National Mastitis Council Inc. Louisville, Kentucky, 1978, p. 57. MURPHY,]. M . (J 959 ). Cornell Veterinarian 49, 411. MURPHY,j. M. & HANSON,].]. (J943). Cornell Veterinarian 33, 61. MYLREA, P . ]., HOAR E, R. ]. T., COLQUHO UN, P ., LINKS, 1. ]., RICHARDS, R. ]. & BARTON, M .

(1977). Australian VeterinaryjoumaL53, 534. NEAVE, F. K. (1970) . In The Control of Bovine Mastitis, ed. F. H . Dodd & E. R. jackson, p. 55. British Cattle Veterinary Association. NEAVE, F. K., DODD, F. H. & KINGWILL, R. G. (J966). Veterinary Record 78, 521. NEAVE, F. K. & OLIVER,]. (J 962). journal of Dairy Research 29,79. NEAVE, F. K., OLIVER,]., DODD, F. H . & HIGGS, T. M. {I968l.journal of Dairy Rmarch35, 127 . NEWBOULD, F. H. S. & NEAVE, F. K. (1965a).journal of Dairy Rmarch32, 163. NEWBOULD, F. H . S. & NEAVE, F. K. (I965b).journal of Dairy Research 32, 171. PRASAD, L. B. M. & NEWBOULD, F. H. S. (1968). Canadian Veterinary journal 9, 107 . REITER, B. & BRAMLEY, A. ]. (I975). Proceedings of the Seminar on Mastitis Control, Reading. IDF Bulletin Document 85, p. 210. REPO RT (J 964). National Institute for Research In Dairying. Report 1964, p . 32 . SCHALM, O. W ., LASMANIS,]. & CARROLL, E.]. (1964) . American journal of Veterinary Research 25, 75. SHARPE, M. E., NEAVE, F. K. & REITER, B. (1962).journal of Applied Bacteriology 25, 403 . THOMAS, C . L., NEAVE, F. K., DODD, F. H . & HIGGS, T. M . (J972).journa! of Dairy Research 39, 113. WESSEN, D. P. & SCH ULTZ, W . D. (J970).journal of Dairy Science 53, 1391.

(Accepted for publication 5 September 1978)