Factors Affecting the Frequency of Isolating Streptococcus Agalactiae from Herd Milk Supplies and the Control of the Organism in the Dairy Herd

Hr. vel . } . ( 1979), 135, 11 9

FACTORS AFFECTING THE FREQUENCY OF ISOLATING STREPTOCOCCUS AGALACTIAE FROM HERD MILK SUPPLIES AND THE CONTROL OF THE ORGANISM IN THE DAIRY HERD By]. K. L. PEARSON , D. A. POLLOCKANOD. O. GREER

Government oj Northern Ireland Department of Agriculture, Veterinary Research Laboratories Stormont, Belfast, Northern Ireland

SUMMARY

The prevalence of Str. agalactiae throughout the world is summarized in relation to the need for adequate monitoring systems which might be helpful to those who wish to initiate control systems. While some countries have made considerable progress towards eradicating the organism from dairy herds, others have appeared to make none at all. Fifty-one herds comprising 862 cows were studied in Northern Ireland, the incidence of Str. agalactiae being monitored by monthly culturing of the bulk milk supply for a full year. The frequency of isolations per month varied from 0 to 12 and was correlated with the percentage of infections and latent infections in cows and quarters using strict techniques and definitions. 3400 quarter milk samples were cultured, the overall number of cows infected being 27% and quarters II · 7%. The inclusion of Str. agalactiae 'isolates' increased these percentages to.33 and 16% respectively. On statistical analysis better correlations were obtained using infections alone than when isolations from quarter samples were included, the results being very highly signihcant (P
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INTRODUCTION

In two previous papers (Greer & Pearson, 1973; Pearson, Greer & Pollock, 1976), the incidence of Streptococcus agalactiae in the national dairy herd in Northern Ireland was studied through the bulk-supply monitoring systems and found to be well over 30%. Comment was made on the observations of others throughout the world on its prevalence elsewhere; techniques for isolating and identifying the organism, the losses incurred in the dairy industry and the economical advantages likely to be achieved in e radicating the infection from herds. Despite the variety of cultural methods used for monitoring the organism in different countries, the uniformity of interest is still widespread. . Recent papers refer to the problem as it still exists in the United States Uasper, 1973; McDonald, 1973; Newbould, 1975; and Smith & Ward, 1975), the 20% cow incidence in some parts of West Germany (Hahn et at., 1974), the quarter incidence in Bulgaria of 45% (Kolev, Pavlov & Venev, 1974) and the cow incidence of 16% in Czechoslovakia (Havelka, 197 5) . Milojevic (1974) found Str. agalactiae still to be the predominant udder pathogen in Yugoslavia while Plommet & Le Loudec ( 1975) in France feel that little real progress is being achieved there in eradicating the organism from the national herd. In other parts of the world the occurrence of the organism where studied is still important, J. C. Ballek (1977 , personal communication) referring in one survey to a 47 % herd incidence in Victoria, Australia. Mylrea et al., (1977) found 28 out of 35 herds in New South Wales to have Str. agalactiae carriers, although the incidence of quarters infected was only 4·9%; while in Colombia in South America H. Lorbacher de Ruiz ( 1977 , personal communication) recorded that 30% of clinical and sub-clinical cases were due to Str. agalactiae. In the Republic of Ireland P. F. O'Reilly ( 1976, personal communication) quotes that Nyhan found 56% of herds to be infected in one part ofthe country in 1964, while 10 years later the incidence in a survey near Dublin was vel-y much lower ( 14% of clinical cow samples), presumably resulting from control measures taken. In an earlier survey in New Zealand (Brookbanks, 1966) a cow incidence of nearly 16% was recorded , while more recently in Italy (Valenti & Caldora, 1974) 54 streptococcal strains isolated from 36% of cows on a number of family farms were identif-ied as Str. agalactiae. D. J. Watson (1976, personal communication) and Pearson et al., (1976) refer to the continuing problem in the United Kingdom as do several unauthored surveys; while G. Grootenhuis (1976, personal communication ) records that the incidence in the Netherlands had dropped from 6·9% to 5·4% between 1973 and 1975. The Scandinavian countries appear to have made most progress in eradicating the organism, Bakken ( 1975) referring to its dramatic decrease in Norway between 1937 and 197 2, while Bratlie (1975 ) and R0n (1976 ) state that the incidence in recent surveys was as low as I· 5%. I n Denmark, Olsen (1975 ) states that group B streptococci account lor only I % of all infections associated with mastitis, while the Str. agalactiae isolation rate in Finland (Koiranen, 1976) in a surveyof3708 cows was just over 2%. Some of the reasons for the lack of progress in eradicating Str. agalactiae from regions and countries are obvious, but perhaps the most important is that few workers

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and authorities have systematically monitored its incidence and made any constructive effort to reduce it. The level of infection varies considerably from herd to herd and one of the objects of the present work is to relate the occurrence of StT. agalactiae in cows and quarters to the frequency with which the organism can be isolated from the herd bulk supply when it is monitored culturally on a regular basis. The advantages of any such monitoring system would be helpful to institutions anxious to identify infected herds and enab le them to concentrate advisory effort on those most heavily infected. The reasons for the variation between herd bulk milk isolation frequency and the levels of infection in cows in different herds are discussed, particularly with relevance to the success or failure of any eradication programme. MATERIALS AND METHODS

Herds and cows studied Fifty-one herds were used for the survey, the average number of cows milking at the time of study being 17. Cow numbers ranged from four to 49, 14 herds having less than IO cows, 23 herds between 10 and 20 and the remaining 14 herds having over 20 cows each. The total number of milking cows examined was 862 and excluding quarters which were blind, milk samples for culture were taken from 3400 milking quarters. Milk samples tested Forty-three of the herds supplied milk in churns and the pooled supply was tested monthly from a I ml sample taken from every can submitted to the creamery for a fu ll year. The remaining eight herds used a bulk tank system for collection and for a full year a 10 ml representative sample was taken monthly for cu lture in the same way. The s'a mpling techniques are described in previous papers (Greer & Pearson, 197 3; Pearson et al., 1976) and a minimum of 10 months herd supply was used for analysis. Forty-four herds were sampled on 12 successive months, five on II months and two on 10 months. Cultural methods For the identification of StT. agalactiae in the herd milk supply the cultural techniques are fully described in the two previous papers mentioned above, and are based on the inoculation of the O· 05 ml volume of milk plated on modified Edwards medium (Oxoid) containing 5% bovine blood, the selection of light blue colonies, the use of CAMP reaction as a guideline and final checking against Lancefield group B serum by the gel precipitation technique. The frequency of isolation of Str. agalactiae from each herd was recorded as a percentage of positive isolates made in relation to the number of monthly tests performed . It must be emphasized that the identification of the organism at this stage is classified by us simp ly as isolation of the organism, while the term infection as discussed by us applies only where cow and quarter samples are under study (Pearson & Greer, 1974). Cellular response is taken into account in identifying infection and involves the

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use of a seven-second CMT bench test in conjunction with the degree of isolation of the organism from 0 ·05 ml of milk from each quarter. The methods for taking and culturing quarter milk samples from the 862 cows involved in the present study are outlined in the 1974 paper, the cows in each herd being sampled only once during the year of the trial. 3400 quarter milk samp les were examined and we have adhered strictly to the 1974 definitions of ' infection' and 'isolation' (or latent infection as some people may call the latter) for the purposes of correlation and discussion. The limitations orany test applied to a single milk sample for identifying infection are fully appreciated by us, but for practical and economic reasons we were obliged to sample the cows in each herd on only one occasion.

Other tests The annual mean cell count of each herd for the 51 herds was available through routine electronic somatic cell counts and is referred to as relevant. The overall arithmetic mean count of the 5 1 herds was 700 000 cells per m l, the range varying from 238 000 to 1 284 000 cells per ml.

RESULTS

Frequency of isolation of Str. agalactiae from herd supply The results in Table 1 show the distribution of the 51 herds studied in relation to the month ly frequency of a to 12 isolations. Cow and quarter incidence of Str. agalactiae Infections. These were based on the isolation of at least 26 colonies of Str. agalactiae fi-om o· 05 ml of quarter secretion and the presence of a CMT reaction of two, three or greater. A total number of 233 cows (27 ·03%) of the 862 cows tested were found to have one or more quarters infected and the distribution in the 5 1 herds varied from zero to IS 7·5% of the animals tested. Infected quarters were of course more numerous in the overall su rvey, but the percentage of the quarters tested (excluding 48 quarters which were blind ) was lower; TABLE I FREQ.UENCY OF STR. AGALACTIAE ISOLATIONS FROM MONTHLY HERD SUPPLIES OF 51 HERDS OVER A PERIOD OF ONE YEAR

No. of month ly isolations

0

No. of herds tested

5

2

I~

3

4

5

6

3

4

6

4~

* Including seven herds tested only on 10 or II months .

5"

8

9

10

II

12

5~

6·

4·

4

3

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398 (II· 71 %) of the 3400 secretions being positive. On a percentage basis, these also varied from zero to 87 ·5%. Isolations. A Str. agalactiae isolation (or latent infection) was recorded if any degree of bacterial isolation from 0 ·05 ml of quarter secretion was concurrently associated with a low grade or nil CMT reaction (see Materials and Methods). This resulted in the identification of a further 54 cows found, on the day of visits, to be excreting Str. agalactiae and the identification of an additional 149 quarters excreting the organism. The total percentage of cows excreting the organism was thus 33 ·3% (287 of 862 tested) and the total percentage of quarters was 16·1% (547 of3400). Clearly the potential danger of Str. agalactiae carriers in herds cannot thus be based on strong detectable cellular reactions alone and is presumably linked to the highly contagious nature of the organism and its epidemiology, whatever its classification as an obligatory pathogen may be. The 149 quarters found to be excreting the organism with low grade or negative CMT reactions were probably newly infected, or the pathogen was present only in the streak canal, teat sinus or the lower cistern area of the mammary gland. A breakdown of the infections and isolations in cows and quarters is shown in Table II, the results bei ng based on the total number of cows tested in each monthly

isolation category. Infections in cows ranged from 3 to 4.7% while infections plus isolations ranged from 9 to 56%. Q.uarter infections varied from I to 24% while the addition of isolations increased the range from 3 to 31%. The increased trend of infections and isolations was expected and is clearly

TABLE II MONTHLY ISOLATION OF STR. AGALACTIAE FROM HERD MILK IN RELATION TO THE COW AND Q.UARTER INCIDENCE IN 51 HERDS

Bulk milk isolation frequency ofStr. agalactiae

No . of

Cows

Quarters

0 I 2 3 4 5 6 7 8 9 10 11 12

67 35 24 31 63 78 93 120 52 152 68 44 35

261 138 94 123 250 308 368 469 205 603 267 174 140

Totals

862

3400

Str. agalactiae ,injections No. (%) Cows

Quarters

5tr. agalactiae infections & isolates (",0) No. Cows

Quarters

3(1· I) 13(9 .4) 6(6· 4) 11(8 ·9) 11(4·4) 41(13 ·3) 46(12 ,5) 25(5 ·3) 24.(1 I · 7) 95(15· 7) 54(20·2) 36(20· 7) 33(23·6)

6(8·9) 9(3·4) 1'1(31·4) 21( 15·2) 7(7.4 ) 5(20.8) 10(32.2) 16( 13.0 ) 9(14·3) 14(5·6) 32(41·0) 56(18 ·2) 31(33·3) 63(17· J) 34(28·3) 50(10· 7) 17(32· 7) 37(18·0) 57(37 ·5) 110(18 ·2) 38(55·9) 82(30· 7) 21(47·7) 43(24· 7) 16(45· 7) 39(27·8)

233(27·0)398(1 I· 7)

287 (33·3)547(16· J)

2(3·0) 9(25 · 7) 5(20.8) 8(25.8) 6(9·5) 24(30·8) 26(27 ·9) 21(17·5) 15(28 ·8) 50(32·9) .32(47 ·0) 20(45·4) 15(42·8)

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illustrated in the results as a whole, despite the fact that few herds were used in the lower range of isolation category. The single herds studied in one and two-month ly isolations showed cow infections of 26% and 21 % respectively, but the quarter infections of 9% and 6% improve the correlation. In contrast the nine herds with four and seven-monthly isolations showed relatively low levels of infection, particularly in the quarters which had percentages of four and five respectively. In the latter group however the inclusion of Str. agalactiae isolations doubles the percentage of positive quarters . In the five herds culturally negative through 12 months examination of creamery samples, two herds were positive when the cows were examined. One herd had 12 milking animals at the time of visit; two animals and three quarters were infected, but the number of latent infections increased the positive cows and quarters to five (41 · 7%) and eight ( 16 · 7%) respectively. The second herd of nine animals had only one cow with one quarter culturally positive, but no cellular reaction accompanied it. The first of these creamery negative herds was poorly managed and cows were bought at random from markets, a feature common to many of the other positive herds studied in the survey.

Statistical analysis The results were correlated statistically from the data of the 51 herds as a whole, and Table III shows the regression equations and correlation coefficients . The most relevant factors considered in the fo ur correlations were (1) the percentage of quarters infected, (2) the percentage of quarters with infection plus those with latent infection (isolates), (3) the percentage of cows infected and (4) the percentage of cows infected plus those with latent infection . The percentages of cows infected are approximately double the quarter percentages and this 2 : 1 ratio applies whether the excretion factor is based on an accompanying TABLE III SUMMA RY OF CORRELATION DATA FROM 5 1 HERDS ON THE STATISTICAL RELATIONSHIP BETWEEN THE FREQUENCY OF HERD ISO LATI ONS AND THE P ERCENTAGES OF COWS AND Q UA RTERS EXCRETING OR INF ECTED WITH STR. AGALACTIAE

Tests compared y

X

N

Regression formula

r

I. % f s infected

V % isolation

51

2. %fs infec ted plus latent infections 3. % cows infected

V % isolation

51

Y=0· 1916X + 2·0802 ±0·0442· Y=0 ·2 17 6X+4·828 ±0·0588 "

0·526 (P
V % is.olation

51

V % isolation

51

Y=0 ·35 1 X+8 ·5046 ± 0·0923 " Y=0·3303X+ 15·549 ±0·1041"

0·4776 (P
4. % cows infected plus latent infections

• Standard error of regression coefficient.

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cellular reaction or not. The frequency of isolating Str. agalactiae on a monthly basis from the 5 I herds forms the X value of the equation while the four variable factors above contribute in each case to the Y value. Better relationships were thus found when only infections were taken into account, with both cows and quarters the results being very highly significant (P < 0 ·00 1). When isolations were included the correlation coefficients dropped marginally, in the quarters still being significant at the 0 ·001 level, but in respect of cows the signifi cance was on ly 0·01.

DISCUSSION

It seems strange that nearly 30 years after the development of intramammary penicillin formulae we are still studying the worldwide distribution of Streptococcus agalactiae in dairy herds and the economic problems associated with it. Apart from the Scandinavian countries however, there appears to be very little room for complacency in relation to its prevalence elsewhere and few countries or states appear to have achieved such a dramatic lowering of incidence as the Danes or Norwegians. I n 1973 and 1976 (Greer & Pearson; Pearson, Greer & Pollock) we were as surprised as anyone to find in random surveys in Northern Ireland that the prevalence in both small and large herds was over 35%, and the findings of the present work strongly suggest that even this was an underestimate. The testing of the herd milk supp ly each month for one year gave us these figures, but it is clear from our quarter and cow studies that some positive herds will not be detected by cu lturing samp les from the herd supply. Postle (I 968 ) gave a useful review of literature on the subject and in one experimental herd found that it was necessary to have at least 5% of quarters excreting Str. agalactiae before the organism could be isolated from the bulk milk supply. Kirkbride, Shave & Blades (1972) observed that if the quarter incidence was below 10%, the screening of bulk-tank supplies would frequently not detect the organism, while J. C. Ballek (I 97 7, personal communication) suggests that some herds in Victoria would be missed by bulk-tank cu lture in view of the very low prevalence, e.g. 2% of quarters infected with Str. agalactiae. . Postle dicussed some of the reasons why his 5% can be taken on ly as an arbitrary figure and we would stress this point, as there is no such thing as a standard cow or quarter, and that many variab le factors, including the stabi li ty of the herd, wi ll inHuence the result obtained in any specific situation. Probably the most important factor is the cultural method used - both for cow samples or herd supply - and, although enrichment techniques may reveal more positive samples (Postle, 1968 ; Weisner & Hubler, 197 5), the advantages may be diminished particularly with herd samp les in view of the heavy initial contamination of the supp ly with other organisms. Ward et al. , (1969), Ward, Postle & Berman (1970), Kirkbride et al., (1972), Greer & Pearson (I973) and Smith & Ward (1975) discuss the pros and cons of enrichment methods, but in the present work we saw littl e merit in using them and relied more on the precise identification of Str. agalactiae by sero logical methods as the final indicator. Few workers appear to have adopted serological procedures in work of this kind and we have already pointed out that it is totally

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unreliable to base identification on CAMP positive reactors which do not split aesculin. The terms 'infection' or 'isolation' are also two variable factors which should be clearly defined. In the case of a herd milk supply there is no dispute about terminology and if Str. agaiactiae is found to be present culturally it is simply an isolate. In quarters and cows however a finer degree of assessment is required (see IDF Bulletin, 1971) and only where a cellular reaction accompanies the isolation should the term infection be used. In the present paper we have adopted the standards laid down by us in 1974 (Pearson & Greer) and if a strong CMT reaction did not accompa ny Str. agalactiae isolation from quarter samples, the term isolation or 'latent' infection was used. Klastrup (1975) also favours this interpretation. This resulted in over 20% more cows and 30% more quarters being found to be excreting Str. agalactiae in addition to the 27% of cows and 11· 7% of quarters classified as being infected. Thus in a situation where no enrichment techniques were applied we de tected an embarrassingly high percentage of carrier cows and quarters, and were obliged to seek a relationship with bulk-tank Str. agalactiae isolates on the basis of the two parameters ( I ) infection and (2) isolates plus infection, in view of the fact that both make a positive contribution to the herd milk supply. Justification for using our strict definition of Str. agalactiae infection in this study is shown by the fact that better correlations were obtained when quarter and cow inlections only were included in the equations. Both were very highly significant in the 5 1 herds as shown in Table III while the inclusion of latent infections lowered the relationship with the frequency of Str. agalactiae isolation from herd supplies. The cow relationship in fact dropped to 0 ·4128 which is significant only at the 0 ·01 level. There are many initial reasons why few workers will agree on the percentage of quarters or cows required to be excreting Str. agalactiae in any herd before the organism can be found in the bulk milk supply. Postle (1968) opts for 5% of quarters; Kirkbride et al., (1972) say that it would be necessary to have at least 10%. Clearly the presence of latent infections as found in this study must be one of the most unstabilizing factors in trials carried out over a prolonged period. Havelka ( 1974 ) discussed the epidemiology of Str. agalactiae in herds and his work suggests that temporary 'infection' of quarters must be very prevalent. This is borne out by more recent work carried out by G . Grootenhuis (1976, personal communication) in Holland , by Koiranen (1976) in Finland and by Mylrea et ai. , (1977) in Australia. The presence of Str. agaiactiae - positive quarters in the absence of cell elevation is given special consideration by these workers in relatiON to methods of detecting and co ntrolling the organism. The present work indicates that even the monthly testing of herd supplies will miss some positive herds and confirms our suspicions of 1973 (Greer & Pearson) that the low-incidence herd will not be detected even by frequent testing. We would not agree with Skardova (1975) who found that bulk-tank testing was of little value - even in herds with a relatively high cow incidence. In Denmark the annual testing of the herd supply is co mbined with the follow-up cultural testing of all cows in positive herds (jensen, 1976) under Government legislation, but as nearly 98% of Danish herds are free from Str. agalactiae in any case the limited value of bulk-supply culture may not be of much relevance. The cultural examination of cows or quarters is the only reliable

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method of ensuring that the organism is absent from a herd, but the economics of carrying out this procedure may not always be acceptable, even if facilities and manpower are available. A final comment in relation to herd detection methods is merited in view of the numerous factors which might influence or interfere with a positive result. We listed a number of these in a previous paper (Greer & Pearson, 1973) in relation to the cow itself, e.g. high yielding newly infected quarters, low yielding atrophied quarters both of which would be classified as infected but with grossly differing outputs of viable Str. agalactiae. Dry cows not under therapy might also be missed if not tested, so called blind quarters might have positive secretions; teat sores may be carrying the infection, but probably most important would be the purchase of infected stock (McDonald, 1973) a practice still common in many parts of the world. The ultimate aim of this work study and that of many others is to give veterinarians and other ancillary authorities the objective of eradicating Str. agalactiae from herds and countries, but the evidence at the beginning of this paper clearly illustrates that little progress has been made in many countries in this respect. The economical advantages of eradication have been realistically shown by Natzke et al. (1972) and many others throughout the years, but in practice the high hopes of Edwards (l962),Jasper (1973) and Newbould (1975) have still to be realized. Yet it is 45 years since Minett et al., (1933) illustrated how it could be achieved -long before the penicillin era. The absence of uniform and regular monitoring systems for determining the national incidence of Str. agalactiae, except perhaps in the Scandanavian countries, has given many of us a sense of false security and a new drive is required to detect and deal with the organism. Emphasis of the risk to humans varies from one country to another and is obviously greater where a high proportion of the country's milk is consumed without pasteurisation (McDonald, 1973; Hahn et at., 1974; Weisner & Hubler, 1975 ). Many herds in Europe are still hand milked and the spread of the organism and human risk are greater than where milking machines are used (Plommet & Le Loudec, 1975). Therapy has contributed considerably but the costs of total herd blitzing with intramammary products are not widely accepted, nor should drugs alone be considered a panacea (Uvarov, 1970). Few workers have obtained roO% clearance of the organism with one course of therapy alone (johnston, 1975; plommet & Le Loudec, 1975 ; Christie & Strom, 1976) and with the well-tried control system based on dry cow therapy and teat dipping only partial control has been achieved, despite the statements of Kingwill (1973) and Eberhart (1973) that the system is highly successful. Even when Str. agalactiae is completely eradicated from a herd some workers have observed the embarrassing emergence of other pathogens in its place (Bakken, 1975; Plommet & Le Loudec, 1975; & Pearson, 1977). These are usually coagulase-positive staphylococci. The problem of developing L forms of Str. agalactiae was also noted by Wilson et at. (197 1). A reduction of infection can be obtained with any piecemeal approach but it is stressed by many workers (McDonald, 1973; Olsen, 1975; Plommet & Le Loudec, 1975; Smith & Ward, 1975; Jensen, 1976; D. J. Watson 1977, personal communication) that follow-up visits and further cow tests are essential. Initial culling may be

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required (Milojevic, 1974; Natzke & Everett, 1975 ) and the presence of very high cell counts in herds may be a useful pointer to identifying herds if it is considered worthwhile concentrating contro l measures on those most seriously affected (Pearson et ai., 1976; R.J. Eberhart, 1977, personal communication). The intensive use of laboratory services in conjunction with cow sampling and field visits is expensive; but it is considered essential by Olsen (1975 ) and he illustrates the eco nomical advantages of the Danish system on a cost-benefit basis. In Norway, R0n (1976) also emphasizes the value of laboratories towards reducing Str. agalactiae infections although Gudding (1976) has some reservations about the large number of milk samples which are taken by field workers without adequate co-ordination of efTort . On the other hand, the laboratory service is given little part to play in the control system o utlined by NIRD and CVL in England, although Dodd (1973 ) does make reference to the need for laboratory support if a Str. agalactiae problem is found to exist. Few approaches will result in the eradication of Str. agalactiae until the husbandry and ma nagement of each infected herd is closely studied. Some factors in relation to this have already been discussed, the most important of which are the age of stock, the milk producers attitude to various forms of chemotherapy, laboratory use and manpower availability. Financial considerations may neutralize any initial effort to promote a well co-ordinated contro l programme, but some progress will be made if an improvement in milking routine and hygiene is instituted and loopholes for reinfection can be diminished. Only then is it worthwhile introducing a more intensive diagnostic and therape~tic system which has a high measure of co-ordination and application which will not be abandoned. ACKN OWLEDGEM ENTS

We are gratefu l to the management and staff at all creamery depots participating in the survey, to the Milk Marketing Board for Northern Ireland for cell count results and Mrs Elizabeth Bayliss for her technical assistance.

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