Official editorial secretary: W. A. de Haan, D.V.S.
Veterinary editor: L. S. B. G. H. Harmsen
Office: Rubenslaan 123, Utrecht, The Netherlands,
Tel. (030) 1 14 13

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DIERGENEESKUNDE

Ir. H. J. Bannenberg; Coöp. Zuivelvereniging „Campina"; Eindhoven, The
Netherlands.

Prof. Dr. J. Boogaerdt; Melkcontrole-Station V.V.Z.M.; Laan van Meerder-
voort 18, \'s-Gravenhage, The Netherlands.

Dr. H. ter Borg; Provinciale Gezondheidsdienst voor Dieren; Zaagmulderweg 1,
Groningen, The Netherlands.

Dr, O. B r a 11 i e; Statens Hovedlab. for Mastitis; Daelenenggt 20, Oslo, Norge.

Dr. D. H. J. Brus; Provinciale Gezondheidsdienst voor Dieren; Rechterstraat 80,
Boxtel, The Netherlands.

Ir. C. H, G a z e m i e r; Instituut voor Veeteeltkundig Onderzoek „Schoonoord",
Driebergseweg lOd, Zeist, The Netherlands.

Prof, Dr. K. Dedié; Staatl. Tierärztliches Untersuchungsamt; Lowenbreitestrasse
20, Aulendorf (Württ.), Deutschland.

Mr. J. B. van Dij k; Centraal Diergeneeskundig Instituut; Prof. Poelslaan, Rotter-
dam, The Netherlands.

Mr. R. G. Dijkstra; Provinciale Gezondheidsdienst voor Vee; Kruisstraat,
Leeuwarden, The Netherlands.

Dr. S. J. Edwards; Agricultural Research Council, Institute for Research on
Animal Diseases; Compton, England.

Dr. A. Florent; Institut National de Recherches Vétérinaires; Groeselenbcrg 99,
Uccle-Bruxelles 18, België.

Mr. G, Goedbloed; Provinciale Gezondheidsdienst voor Dieren; Kazernestraat
6, Gouda, The Netherlands.

Mr. Toimi Hämmäinen; College of Veterinary Medicine ; Hämeentie 57, Hel-
sinki, Finland.

Dr. F. H. J. J aarts veld; Provinciale Gezondheidsdienst voor Dieren; Rechter-
straat 80, Boxtel, The Netherlands.

Mr. J. S. v. d. Kamp; Provinciale Gezondheidsdienst voor Dieren; Zaagmulder-
weg 1, Groningen, The Netherlands.

Mr, M, Karsemeijer; President Royal Netherlands Veterinary Association;
Ambonstraat 12, Alphen a/d Rijn, The Netherlands.

Dr. K auk er; Staad. Veterinäruntersuchungsamt; Druseltalstr, 61, 35 Kassel,
Deutschland.

Mr, J, H. Kerkhof; Stichting Regionaal Orgaan; Hcuvelplein 276, Breda, The
Netherlands.

Dr. G. Kiel we in; Staatl, Tierärzdiches Untersuchungsambt; Löwenbreitestrasse
20, 796 Aulendorf (Württ.), Deutschland.

Mr. R. G. Kin g will; the National Institute for Research in Dairying; Shinfield,
Reading, England.

Mr. H, Koster; Institut für Lebensmittelkunde und Milchhygiene; Hans-Böckler-
Allee 16, Hannover, Deutschland.

Dr. Kraus; Institut für Lebensmittelkunde und Milchhygiene; Hans-Böckler-Allee
16, Harmover, Deutschland.

Ir. A. E. K r o m w ij k; Stichting Regionaal Orgaan; Coomhertstraat 5, \'s-Hertogen-
bosch, The Netherlands.

Mr. J. C. A. V. d. Maas; Provinciale Gezondheidsdienst voor Dieren; Landbouw-
huis, Alkmaar, The Netherlands.

Dr. E. Malling Olsen; Veterinaerdirektoratet; Nyropsgade 37, Kobenhavn,
V., Danmark.

Dr. Mempel; Tiergesundheitsamt der Landwirtschaftskammer Westfalen-Lippe;
von Esmarch-Str. 12, (21a) Münster (Westf.), Deutschland.

Mr. J. Mol; Melkcontrolebureau Meba; Vondelstraat 50-52, Amsterdam, The
Netherlands.

Mr. G. H. Overgoor; Provinciale Gezondheidsdienst voor Dieren; „Klein Rozen-
daal", Rozendaal (Geld.), The Netherlands.

Mr. J. C. Peijnenburg; Stichting Regionaal Orgaan; Godsweerdersingel 16,
Roermond, The Netherlands.

Dr. Flöge; Landwirtschaftskammer Weser-Ems, Tiergesundheitsamt; Mars-la-Tour-
Str. 1, 29 Oldenburg, Deutschland.

Mr. R. Post; Provinciale Gezondheidsdienst voor Dieren; Veemarkt 10, Zwolle,
The Netherlands.

Dr. O. R i c h t e r; Rindergesundheitsdienst; Haydnstrasse 11, München 15, Deutsch-
land.

Mr. J. de Rooy; Instituut voor Veeteeltkundig Onderzoek „Schoonoord"; Drie-
bergseweg 10a, Zeist, The Netherlands.

Mr. J. M. F. Saes; Provinciale Gezondheidsdienst voor Dieren; „Sonnenhuys",
Heythuysen (Limb.), The Netherlands.

Dr. A. Scheiner; Landwirtschaftskammer Hannover, Tiergesundheitsamt;
Vahrenwalderstrasse 133, Hannover, Deutschland.

Mr. P. S j o 11 e m a; Provinciale Gezondheidsdienst voor Dieren; Kruisstraat 43,
Leeuwarden, The Netherlands.

Mr. Ivar Engan Skei; Norske Melkeprodusenters Landsforbund; Bredgaten
10, Oslo, Norge.

Prof. Dr. Th. Stegeng a; Landbouwhogeschool; Duivendaal 5, Wageningen, The
Netherlands.

Dr. J. T e s i n k; Provinciale Gezondheidsdienst voor Dieren; Evertsenstraat 11,
Goes, The Netherlands.

Dr. K. T i 1 g n e r; Institut für Tiergesundheit; Güterbergstrasse 77, 23 Kiel,
Deutschland.

Dr. N. Tuyttens; Provinciaal Laboratorium voor Veeziektenbestrijding; Steen-
weg op Deinze, Drongen, België.

Mr. J. U w 1 a n d; Provinciale Gezondheidsdienst voor Dieren; Kazernestr. 6, Gouda,
The Netherlands.

Prof. M. Vandeplassche; Rijksuniversiteit Gent, Diergeneeskunde; Casino-
plein 21, Gent, België.

Mr. D. J. V e r v o O r n. Ministerie van Landbouw en Visserij, \'s-Gravenhage, The
Netherlands.

Mr. J. v. d. Vlerk; Provinciale Gezondheidsdienst voor Dieren; Groningerstraat
107a, Assen, The Netherlands.

Mr J H. P. Ver we ij; Provinciale Gezondheidsdienst voor Dieren; „Klein Rosen-
dael", Rozendaal (Gld.), The Netheriands.

Fräulein Weigth; Rinderklinik, Tierärztliche Hochschule, Hannover, Deutsch-
land.

Dr. D. M. Z u ij d a m; Landbouwschap; Raamweg 28, \'s-Gravenhage, The Nether-
lands.

Dr. Zettel; Staatl. Vet. Untersuchungsamt; Druseltalstr. 61, 35 Kassel, Deutsch-
land.

Dr. Th. S. Zwanenburg; Ministerie van Landbouw en Visserij; \'s-Gravenhage,
The Netherlands.

Namens het Bestuur van de Provinciale Ge-
zondheidsdienst voor Dieren in Noord-Bra-
bant heet ik U hedenmorgen van harte wel-
\' .^jjH^V kom.

stellen het bijzonder op prijs dat U in zo\'n
grote getale gehoor hebt willen geven aan
onze uitnodiging om dit mastitiscongres, op
internationaal niveau, te komen bijwonen.
Tezamen kunnen we nu van gedachten wisselen omtrent deze dierziekte-
bestrijding van de toekomst.

Het spijt me dat de schohng, die ik in mijn jeugd heb meegemaakt, niet
van dien aard is, dat ik hier in de diverse talen van de landen waarvan
hier vertegenwoordigers aanwezig zijn, een welkom kan toespreken. De
schuld hiervan is bij mijzelf gelegen, de last hiervan zal ik dan ook door
dit leven moeten dragen. Ik zou dan ook Dr. B r u s, die straks het congres
pl openen, willen vragen, om ook in een andere taal, b.v. de Engelse taal,
ik leef n.1. niet in de veronderstelling dat de heer Brus ook de Scandina-
vische talen stuk voor stuk onder de knie gekregen heeft, deze woorden in
het kort te vertalen, opdat het welkom dat ik U van ganser harte toeroep
namens het Bestuur ook als zodanig moge klinken in voor U allen begrij-
pelijke woorden.

Het mastitis-vraagstuk waarvoor wij deze dagen op het congres gesteld
worden laat ons, als praktische veehouders, niet onberoerd.
Wij hebben in de loop der jaren, wat deze ziekte betreft, alle mogelijke
narigheden ondervonden. Wij kennen moeilijkheden bij de melkwinning,
bij de melkbehandeling en ook bij het houden van vee als zodanig, alle
zaken die stuk voor stuk van invloed zijn op de portemonnaie van de vee-
houder. Vandaar dan ook dat \'t vanzelfsprekend is, dat hetgeen in de na-
oorlogse jaren op dit terrein door U aan onderzoek is verricht, dank ver-
dient.

Wanneer wij dan hier, voor wat betreft de Gezondheidsdienst voor Dieren
in Noord-Brabant, er in geslaagd zijn — ons richtende op het onderzoek
op de ontstekingscellen welke in de melk aanwezig kunnen zijn — een
bruikbare methode te vinden, dan verheugen wij ons erop U deze manier
van onderzoek te kunnen demonstreren. Mogelijk kan deze methode, door
er een beetje aan te schaven, gericht worden op de praktijk van de zuivel
en op de dagelijkse gang van zaken op de boerderij. Een bruikbare en niet

1 J. van Doren; vice-voorzitter van de Provinciale Gezondheidsdienst voor Dieren
in Noord-Brabant; Rechterstraat 80, Boxtel.

kostbare methode voor het aantonen van deze ontstekingscellen in de melk
zou een basis kunnen vormen voor de bestrijding van de mastitis als zo-
danig.

Wanneer wij hier hedenmorgen dan theoretische beschouwingen zullen
aanhoren betreffende het mastitis-vraagstuk in z\'n diverse vormen, wanneer
in de namiddag demonstraties zullen volgen op het laboratorium en deze
zullen worden toegeUcht door mensen uit de praktijk zowel van Gezond-
heidsdienst als uit de zuivelindustrie, zijn wij daarover verheugd.
Wanneer wij dan morgen, zowel op de boerderij als op de zuivelfabriek,
praktijkdemonstraties krijgen, dan hoop ik, dat hetgeen zowel vandaag als
morgen hier gegeven zal worden, voor U aanleiding zal vormen, U intens
op de problemen te werpen en in de discussie het zover te brengen dat dit
mastitiscongres vruchten mag afwerpen.

Ik hoop dat deze samenkomst datgene voor U allen zal brengen wat U
ervan verwacht en dat het voor ons als veehouders zal mogen geven, in de
naaste toekomst, een voor de praktijk bruikbare onderzoekmethode en be-
strijdingsvorm die de nadelen van deze uierontstekingen zoveel mogelijk
zullen gaan voorkomen en beperken.

You were addressed by Mr. J. van Doren, vice-president of the Board
of the Animal Health Service in the province of North Brabant. He re-
gretted not being able to welcome you in your own language. Mr. van
Doren is a live-stock owner and a leading figure in various organisations.
He is also chairman of the Board of the Co-operative Dairy Works „Cam-
pina" which we shall visit tomorrow.

The chairman of our committee, Dr. C. J. v a n M e e 1, died two months
ago. He was extremely interested in everything concerned with the stamp-
ing out of animal diseases. In the parliament of our country, he cham-
pioned the cause of live-stock owners.

During his last weeks of life in hospital, he spoke of this congress and
we consider it a matter of great regret that it was not given to him to be
here to-day.

Ladies and Gentlemen, it was in February 1963 that we started to con-
template a mastids congress. At that time, we received a letter from Dr.
Schalm, in which he wrote that he was planning to visit Europe in the
autumn of that year.

At the same time, a number of foreign visitors from the Scandinavian
countries and Germany were our guests in Boxtel. As we discussed pro-
blems relating to the diagnosis and treatment of mastitis, it became ap-
parent that it would be desirable to develop methods of investigation and
treatment as similar to one another as possible in various countries.
The stamping out of tuberculosis of cattle and brucellosis has almost been
completed in the north-west part of Europe.

The Scandinavian countries were the first to conclude their eradication
programme. They also were the first to study the possibilities of mass
control of mastitis. The methods adopted in the stamping out of tuber-
culosis and brucellosis, consisting in eradication of the bacteria causing
these diseases, are not suited for mass control of mastitis.

The question may be asked whether the pure "microbe-hunter" will be
the front-line soldier in this field. Investigators in the various countries
do not speak the same language; this is true both in the literal and in
the figurative sense of the word but it also applies to the different me-
thods of invesdgadon. It therefore is not easy to understand one another.
We hope that this congress will help investigators to understand each
other in the fields of diagnosis and treatment of mastitis or, better still,
in that of the prevention of bovine mastitis.

I therefore am glad to be in a position to welcome all of you here in
North Brabant and I shall start with those guests who have come from
afar.

I am very glad to see you here. Dr. Schalm. As I said, the news of
your coming to Europe marked the beginning of our plans for the con-

1 D. H. J. Brus D.V.Sc.; Director of the Provincial Animal Health Service in North
Brabant; Rechterstraat 80, Boxtel, The Netherlands.

gress. Your method of identifying cells in the milk rather than bacteria
made it possible to carry out mass investigations. To start an organized
campaign against a disease, methods of mass investigation are essential
and these methods should be simple, reliable and inexpensive.

Dr. Funke of Sweden, heartily welcome to North Brabant.
Norway is represented here by Dr. A. B r a 11 i e, Dr. A. Kyrkjebö
and Mr. Ivar Engan Skei. Welcome to our country.
We have always regarded Denmark as an example of tuberculosis and
brucellosis control. It was Dr. Livoni of Denmark, who made efforts to
diagnose and cure mastitis in the field. I regret that he was not able to
attend and therefore I am doubly glad to see you here, Dr. Klastrup
and Dr. M a 11 i n Olsen.

Dr. Hämmäinen, your country, Finland, has always enjoyed a re-
putation of sympathy in the Netherlands. A small country with great
people.

For Great Britain, Dr. Edwards and Mr. K i n g w i 11 are attending
this congress. I am sure that there are good contacts between investiga-
tors of your country and those of countries in the north-west part of the
continent. I hope that there will be more contacts in the fields of prac-
tical organization and control of animal diseases.

From Belgium, we have Prof. Vanderplassche of the State Uni-
versity of Ghent. The co-operation between the Sterility Committee of
your university and workers in this field in The Netherlands is not a
sterile one. I am glad that you are willing to help us by conducting dis-
cussions to-day.

Dr. Florent of the State Serum Institute in Ukkel, welcome here.
Dr. Vereertbruggen and Dr. Tuyttens also are guests from
Belgium. Dr. Tuyttens, I regard you as a representative of the very
young Health Services in Belgium. We have been in very close contact
with you and some of your fellow-workers in our laboratory. I am sure
that we shall work together in the future.

A country in which work on mastitis has been done for a long time and
in many organizations, is Germany.

Es freut mich sehr, dass Sie heute in so grosser Zahl hier sind.
Von den deutschen Tiergesundheitsämtern darf ich begrüssen: Dr.
T i 1 g n e r aus Kiel, Dr. M e m p e 1 aus Münster, Dr. Richter aus
München, Dr. Scheiner aus Hannover. Dr. Schiel aus Oldenburg
hat auch eingeschrieben, ist aber leider durch Krankheit verhindert. Es
freut mich Sie, Dr. P 1 ö g e, an Stelle Dr. Schieis begrüssen zu können.
Sie, Dr. Tilgner, sind bereit als Diskussionsleiter aufzutreten.
Von den Staatlichen Tierärztlichen Untersuchungsämtern und Universi-
täten Deutschlands sind unsere Gäste : Dr. K a u k e r aus Kassel, Dr.
K i e 1 w e i n aus Aulendorf, Dr. Kraus aus Hannover, Prof. Dédié
aus Aulendorf und Dr. Zettel aus Kassel.

Es freut mich sehr, zwei junge Gelehrten in unserer Mitte zu haben,
Fräulein Herrguth und Herrn Köster, die ihre Doktorarbeit auf
dem Gebiete der Mastitis machen wollen. Wir sind sehr darauf gespannt,
was ihre Forschungen uns bringen werden.

lieh wilkommen. Sie sind die weiblichen Stimmen in unserem Chor. Ich
bin davon überzeugt, dass sie leise aber hell ertönen werden.

And now I come to the guests from our country, The Netherlands.
The members of our Board and of our own Mastitis Committee are at
home here. This also is true of the directors and bacteriologists of the
other Provincial Health Services.

A special word of welcome to the representative of the Ministry of Agri-
culture and Fisheries, Mr. Vervoorn.

From the Central Veterinary Research Institute, our fellow veterinarian
Van D ij k will be present during these days.

From the Faculty of Veterinary Medicine of the State University of
Utrecht, we see Mr. Nooitgedagt and, from the Agricultural Col-
lege of Wageningen, Dr. Schipper.

Of the members of the Central Mastitis Committee, Mr. Mol, Director
of the Milk Inspection Station, Amsterdam, is attending the congress.
Of the Higher Dairy School, we see Mr. K r o m w ij k here.
Mr. K e r k h o f and Mr. P e ij n e n b u r g are members of the Regional
Milk Hygiene Organs. We have been working together for the past two
years. I feel sure that your work and ours will be on a common basis
in the future. I am very grateful to your Foundation for the material
received for this congress.

I should like to say the same to Mr. Bannenberg of the Co-opera-
tive Dairy Works "Campina".

Mr. d e R o o ij of the Dairy Research Institute. We have been working
together for two years in order to determine the relationship between bo-
vine mastitis and the management of cattle by live-stock owners to the
best of our ability. The initial results are encouraging but more investi-
gations will have to be carried out.

A special word of welcome for Mr. K a r s e m e ij e r of the Royal Ne-
therlands Veterinary Association. Veterinary practitioners and the Health
Services are successfully co-operating in tuberculosis and brucellosis con-
trol. I am sure that we shall need one another in the control of other
diseases in the future.

And, last but not least, I welcome the secretary of the Animal Health
Committee, Dr. Z u ij d a m.

I hope to find the opportunity of addressing a special word to the various
speakers when they start to read their papers.

1 shall conclude by expressing the wish that anyone who speaks, may do
so slowly and will say very wise things in simple words such as those
which you normally use when you are a child of about twelve. It is not
easy to speak a foreign language correctly. I should say that, in this
case, it is more important to speak clearly than to speak correctly. I hope
that we may have a successful congress but also that we will have pleasant
days.

Dr. Brus: May I introduce to you Prof. A.
van der Schaaf, professor in bacteriology of the
Veterinary Faculty of the State University of
Utrecht.

After he got his degree he worked at the
Institute of Preventive Medical Science in
Leyden. Later he was in Indonesia at the State
Serum Institute in Bogor and also professor of
the Veterinary School.

After the war he became a bacteriologist at the
Animal Health Service in Friesland, the oldest
service in The Netherlands. Since 1955 he is
professor in Utrecht.

He was the promotor of the thesis by Dr.
Jaartsveld on the Brabant Mastitis Reaction.
I am glad. Prof, van der Schaaf, to have the
opportunity to ask you to speak.

Bovine tuberculosis in The Netherlands and most neighbouring countries
has already been eradicated and, in addition, the anti-brucellosis campaign
will be completed in the near future. Therefore the period that veterinary
health services shall have time to occupy themselves with mastitis as a herd
problem is rapidly approaching.

Mastitis causes considerable losses to farmers. This is not a new situation
but this disease does seem to occur more frequently than formerly notwith-
standing the fact that the arsenal of therapeutic drugs has grown enor-
mously.

Mastitis as a herdproblem is the subject of this symposium and we are here
to learn from each other about the extent of the problem, about the diag-
nostic laboratory methods now available and thirdly about the most pro-
mising therapeutic and preventive measures.

According to Little and Plastridge, bovine mastitis is an inflam-
mation of the mammary gland which is characterized by tissue changes,
which lead to changes in the secretory product of the tissue, whether the
etiology be of an infectious, chemical, or thermal nature or result from an
injury. Of those four causal factors the first and the last one are the most
considerable, for chemical substances and overheating or chilling can very
seldom be considered as important etiological factors. Concerning the in-
fectious agents and injuries perhaps most veterinarians think that for the
aetiology of mastitis in milkcattle germs are of much more importance
than injurious causes.

Recent investigations of Stuart and coworkers in the Central Vete-
rinary Laboratory at Weybridge indicate that the Mycoplasma bovigenita-
lium also has to be considered as a primary cause of mastitis. This agent

1 Prof. A. van der Schaaf, professor at the State University of Utrecht; Biltstraat
172, Utrecht, The Netherlands.

is not filtrable, but as with viruses, it is very difficuU to show its presence
by microscopical and cultural methods and we do not know how frequently
it occurs in The Netherlands. Nevertheless doubts have to be raised about
Streptococci, Staphylococci, Corynehacterium pyogenes, E. coli, Klebsiella
and Pseudomonas aeruginosa as primary causes of inflammatory conditions
in the cow\'s udder. i J^

Injuries and perhaps viruses come first as etiological factors. When these
factors have done their job as pathfinders the different microbes, which
can be considered as more or less ubiquitary, can invade the mucous mem-
brane of the duct system and later on also the alveolar tissue.
It is of course not necessary that the injuries are of a violent character.
On the contrary the slow acting and insidious momenturns of disfunction-
ing milking machines and handmilkers are of more importance. Predis-
posing hereditary factors are also to be considered. There are for example
heavy milking, long teats, pendulant udder, inequal lactation of hind and
front-quarters and hormonal disbalance. Environmental factors as faulty
construction of barns, short and narrow stalls, lack of bedding and a warm,
humid atmosphere can be predisposing as well as for injuries as for bacte-
rial infections. It will always be very difficult to unravel which is the real
primary factor which causes the mastitis.

In agreement with the supposition that injuries form the primary causes
of mastitis are the many subclinical cases of so called traumatic mastitis
with more than 1 million leucocytes in 1 milliliter milk but without the
wellknown pathogenic microbes.

This does not mean that bacteria have no significance for the pathogenesis
of mastitis. On the contrary the bacteria are secondary invaders of the
damaged tissue. They can affect the wounded or inflamed mucous mem-
branes of the streak canal, the teat cistern or lactiferous sinus so seriously
that an entire disfunction of the milkproducting acini follows.
The germs which can multiply in milk and form exo- or endotoxins are
of course of most importance for a control program, but it would be a blun-
der when the combat against bovine mastitis would start with the syste-
matic application of antibacterial therapeutics. The primary task will be
to discover the herds with a mastitis-problem and then to find out which
injuries predispose for subsequent infections by streptococci, the staphylo-
cocci and eventually yet other germs such as PPLO\'s.

Dr. Jaartsveld has done a good job in studying the methods which
would be useful to apply as screen tests and which have to indicate the
problem herds. He made the conclusion that the Schalm- or Galifornian
Mastitis Test, the C.M.T., was the most reliable one, and finally he modi-
fied the test from a plate test to a tube test. He completed the possibility
for mass-application of the test by introducing an objective method for
measuring the increase of the viscosity of mastitis milks caused by adding
a 2% solution of sodium laurylsulphate. He then designed a capillary-tube
of a fixed length and diameter which was fused to a funnel and showed
that a flowtime through the capillary in more than 10 seconds of a quan-
tity of 0,6 ml milk mixed with 0,4 ml reagent was an indication that the
sample contained more than 500.000 cells per ml. For cattle in full lacta-
tion this may be considered as an indication of mastitis. In problem herds
there are of course more than 1 cow with mastitis and so, as with the ring-

test in brucellosis, the viscosity-test can be used for screening samples of
canmilk.

I had the honour and the pleasure to be promotor of Dr. Jaartsveld
and as godfather to the child of his brain I gave the test the name of
Brabantse Masdtis Reactie or B.M.R.

A godfather usually gets also the possibility to contribute to a prosperous
development of his godchild and therefore with the help of Mrs. Kramer,
my technical assistant, we attempted some trials for improvement. We had
no success with our attempts to conserve the milks in such a way that the
tests could be postponed for 24 or 48 hours after sampling.
But is was shown that with milk samples stored at 6° Celcius the addition
of sodiumhydroxide to the reagent in such a quantity (0,6 ml 10% NaOH
per 40 ml 2% sodiumlaurylsulphate) that the pH rose to 12, the results of
the test were the same on the first as on the second day after collecting the
samples.

We also paid attention to the further standardisation of the test and we
could demonstrate that is was not conect to carry out the B.M.R. when the
milk and the reagent were cold. After storing in a refrigator the samples
can be put in the incubator for about half an hour and then the reagent
with a temperature between 20 and 30° C. can be added. If the milk or
the reagent are too cold the intensity of the viscosity is diminished. This
would result in wrong readings.

Pending these trials it could also be demonstrated that with samples which
contained more than 1 million of cells per milliliter the flowtime was not
exactly the same as when the test was repeated. The differences in results
are caused by slimy clumps of leucocytes which can obliterate the capil-
lary tube from time to time. These clumps can be invisible to the naked eye.
When the number of leucocytes is less than 500.000 the differences in flow-
time are negligible. For practical circumstances this means that a sample
which gives three dots in the administration of the flowtime occasionally
can also get four dots. The next tables illustrate all those observations in
detail.

For the routine of the screening of thousands of milksamples, received
daily from the dairies, these differences in flowtime are of no practical sig-
nificance.

It is my opinion that the B.M.R. can be very useful for those animal health
services and milkcontrolstations which have to assist the farmers commu-
nity to increase the quantity and improve the quality of their milk without
extension of labour and costs. I hope that this symposium will introduce
new ways for the anti-mastitis campaigns which would serve this purpose.

Nu de rundertuberculose in verscheidene landen is uitgeroeid en dit ook voor
abortus-Bang praktisch het geval is, begint de tijd te naderen, dat meer aandacht
besteed kan viiorden aan de mastitis als koppelziekte. Men krijgt de indruk, dat,
hoewel de therapeutische mogelijkheden groter zijn geworden, het aantal probleem-
bedrijven toch stijgt.

Bij de aetioiogie van deze ziekte moet men denken aan verwondingen, infecties en
verder aan chemische en thermische invloeden. De beide eerste zullen de belang-
rijkste zijn. AU infecticuze oorzaken dient men, naast bacteriën, ook aan Mycoplasma
bouigenitalium te denken.

Spreker wijst er op, dat zelfs ontstoken kwartieren die melk met meer dan een
miljoen ontstekingscellen per cm® bevatten, bacteriologisch steriel kunnen zijn. Men
moet bij mastitis, als probleem, denken aan verwondingen en mogelijk ook aan
virussen als primaire oorzaken. Bacteriën spelen meer een rol als parasieten, die na-
dien het verzwakte weefsel binnendringen. De bacteriën dient men als gelegenheids-
parasieten en niet als primaire oorzaken te beschouwen.

De verwondingen behoeven maar uiterst klein te zijn. De verwondingen zijn niet
alleen de directe oorzaken van uierontsteking, maar ook de toestand van het uier
of de bouw van uier en tepels of de verhouding tussen voor- en achterkwarticrcn
enz. kunnen een rol spelen. De bouw en het klimaat van de stal kunnen ook belangrijk
zijn. Het starten van een georganiseerde bestrijding van mastitis als stalprobleem op
basis van behandelingen met antibiotica, zou spreker een blunder vinden. Men moet
op deze bedrijven de diepere oorzaken waardoor vermindering van weerstand in
het uier optreden, gaan opzoeken en deze zoveel mogelijk wegnemen. Het zoeken naar
en het behandelen van de begeleidende bacteriën is van aanvullende betekenis.
De Brabantse Mastitis Reactie is een geschikte methode om, via onderzoek van bus-
monsters, de probleembedrijven op te zoeken. Het is gebleken, dat, bij celhoudcndc
melkmonsters, de doorstroomtijd met het ouder worden van dc mclkmonsters terug-
loopt.

Tezamen met Mevr. Kramer heeft spreker gevonden, dat indien de testvloeistof op
Ph 12 wordt gebracht dit effect grotendeels geneutraliseerd kan worden. Verder
blijkt dat de B.M.R. het best bij een temperatuur van 20 ä 30° C kan geschieden.
Bij lagere temperaturen wordt de behandelde melk minder visceus. Bij melk met
vrij veel cellen kunnen de doorstroomcapillairen wel eens door klompjes slijm ver-
stopt raken, waardoor cen te hoog aantal cellen wordt aangegeven.
Spreker vindt de B.M.R. een bruikbare methode voor Gezondheidsdiensten en Melk-
controle-stations. Deze toch moeten de veehouders helpen een zo goed mogelijke
melk tegen een zo laag mogelijke kostprijs te produceren.

Dr. Brus: Dr. Schalm; As I told you today,
your coming to Europe was the inspiration for
this congress.

I am glad we already had a little talk in Han-
nover. I am sure that the mastitis problems in
the U.S.A. and specially in California with the
big dairy-herds can differ from the problems
in some parts of Europe.

While we talk about mastitis and too many
cells in milk you are already going a step
further and thinking about the risks of too
few cells in the milk.

It may seem that my lecture does not fit into the general trend of this con-
ference but I am of the opinion that our experience with coliform mastitis
may be of special interest to you. This form of mastitis could become a
greater problem in the future when the more common forms of mastitis
have been brought under control.

In the U.S.A. a number of dairy herds located at Universities and research
centres have been subjected to rigid programs of control of streptococcaj
and staphylococcal mammary infections. These control programs have fre-
quently employed extensive treatment of the infected mammary glands
with antibiotic and therapeutic agents. It seems rather strange that at
least six of such herds have been reported to have experienced considerable
trouble from acute coliform mastitis after the more common forms of udder
infection were brought under control. This would suggest the possibihty that
a too vigorous removal of the more common bacterial flora of the bovine
mammary gland may lead to the opportunity for the bacteria of the soil
and the environment to establish themselves in mammary glands and pro-
duce mastitis.

I would like to discuss with you some of my own experiences in this regard.
The dairy herd in question had the best management among all the dairy
herds we studied. The cows came into the barn only to be milked. Be-
tween milkings they were outside on cement. A portion of this area was
bedded with straw and overhead shelter was provided.
In two years, from 1943 to 1945, Streptococcus agalactiae infection was re-
duced from 35% to 0%. This was accomplished before antibiotics were
available so the elimination of the infection was accomplished by segrega-
tion of infected cows and eventual slaughter. Then, in 1945, a program to
reduce staphylococcus mammary infection was started using segregation
and intramammary therapy with antibiotics and chemotherapeutic agents.
At the beginning of this phase of the work, about 50% of the cows were

infected with pathogenic staphylococci (toxin producers and coagulase posi-
tive strains) although clinical mastitis was not a serious problem. The older
cows with chronic staphylococcal infections did not respond well to treat-
ment and, therefore, many of these cows remained infected until removed
from the herd. However, by segregation of younger cows and early treat-
ment of new infections, the incidence of staphylococcal mammary infec-
tions became reduced from 50% to 27% in a period of 4 years.

It was at this time that acute coliform mastitis made its appearance in the
herd. During the first year, and before streptomycin was available to us,
10% of the herd experienced acute coliform mastitis. This meant 20 cases
in one year. Three of these cows died and many others had to be destroyed.
This severe coliform mastitis coming upon a very excellent mastitis control
program was entirely unexpected and caused us to wonder if we had been
responsible by distorting the more natural bacterial flora of mammary
glands.

In 1959, we were able to begin experiments into the pathogenesis of coli-
form mastitis. The following is a brief summary of the most significant
findings. In the original herd, several coliform types were involved, includ-
ing both Escherichia coli and Aerohacter aerogenes. We choose the latter
organism for our experimental trials for it is easily identified by simple cul-
ture media and therefore we could be more certain that is was the expe-
rimental strain when reisolated from milk of inoculated cows.
In the beginning we had to answer the question relative to number of germs
and number of inoculations required to induce acute coliform mastitis. We
know now that when a lactating gland is secreting leukocyte-free milk as
few as 5 to 10 A. aerogenes germs can produce acute mastitis within 24
flours after their introduction via the "streak" canal. Normally, however,
we employed larger doses to study the pathogenesis of the disease. For
example one cow received 100 A. aerogenes into one quarter, 10,000 germs
into another, and 1,000,000 germs into a third quarter. The fourth quarter
was left as a control. From experiments of this type we demonstrated that
the incubation period between introduction of the germs into glands se-
creting leukocyte-free milk and first clinical signs of mastitis is determined
by dose-size. With less than 50 germs, it takes about 20 hours; with
1,000,000 germs, clinical signs of peracute mastitis develop 5 to 6 hours.

Heat-killed A. aerogenes, at a dose level of 1 million dead germs, injected
into normal lactating glands stimulated a leukocytosis of several million cells
per ml. of foremilk followed by a rapid return to normal. With the same
number of live A. aerogenes, the leukocyte numbers reached 80 millions
or more per ml. of foremilk. The glands became swollen and there were
solid particles in the inilk. During this period the milkweight (total milk)
dropped about 50%.

However, if the inflammatory response was successful in completely des-
troying all Aerohacter aerogenes germs, the gland returned to normal with-
in 7 to 14 days. On the other hand, if the initial inflammatory response
was delayed for just a few hours, the cow became very sick and sometimes
died. This was explained in that the coli-bacteria multiply rapidly in leuko-
cyte-free milk and if there is a delay in the leukocyte response the bacterial
population becomes very large. Finally, when the leukocytes do enter the
gland and destroy the bacteria, a large quantity of endotoxin is released,

making the cow very sick. The endotoxin may damage the leukocyte-pro-
ducing centres in the bone marrow and then a persistent leukopenia deve-
lops.

Another possibility in coliform mastitis, observed in our trials, was a too
rapid fall in leukocyte numbers, after the initial inflammatory response and
before all Aerobacter aerogenes were destroyed. When this happened, a
second attack of acute mastitis developed. In some glands a chronic infec-
tion resulted and then acute mastids appeared whenever the leukocyte num-
bers fell below 200,000 per millilitre of foremilk.

Thus, in our experience, coliform mastitis is a disease resulting from rapid
multiplication of coli-bacteria in leukocyte-free milk. It appears, therefore,
to be a disease of the normal lactating gland that has become acciden-
tally invaded by coliform bacteria. The cow becomes sick from the endo-
toxin released when the leukocytes of the initial inflammatory response
destroy the bacterial population.

In one experiment, after 500,000 Aerobacter aerogenes had been intro-
duced into a normal lactating gland, we took 10 ml. of milk from the
gland in 3 hours and again in 6 hours after the inoculation. The milk was
centrifuged and smears were made of the sediment and stained by Wright\'s
method (blood stain). Phagocytosis was demonstrated at the 3rd hour when
bacteria were numerous but leukocytes were just beginning to enter the
milk. By the 6th hour there were many leukocytes but the bacteria were
too few for any evidence of phagocytosis to be seen. When the same num-
bers of Aerobacter aerogenes were introduced into lactadng glands having
leukocytes already in the milk from a pre-existing mild mastitis, the ino-
culated bacteria were prevented from rapid multiplication for the leuko-
cytes began to destroy the bacteria immediately. Some increase in cell num-
bers could be demonstrated for next few milkings in such glands but the
germs could not be reisolated and no signs of clinical mastitis developed.
Mammary quarters producing milk with cell counts of 300,000 to 500,000
per ml. of foremilk were found to have a high degree of protection against
experimental coliform mastitis. This level of cells in the milk would be
representative of a mild inflammatory reaction and such milk would have
a score of 10 seconds on the B.M.R. test for mastitis.

Now that there is great interest in mastitis control in many countries of
the world, it is important to realize that coliform mastids is a disease of
the normal leukocyte-free mammary gland. Young cows have a good pro-
tection against coli-bacteria entering the mammary gland because the
"streak" canal functions as an effective barrier. However, as a cow grows
older and the "streak" canal of the teat becomes more patent, the acci-
dental entrance of coliform bacteria into the mammary gland is more likely
to take place. These older cows need the protection against coliform bac-
teria given by pre-existing leukocytes in the milk. Thus, if we insist in mas-
titis control that mammary glands of all cows must be free of all evidence
of even mild inflammation, then it is possible and highly probable that the
incidence of acute coliform mastitis will increase.

The cows used in our experimental trials did not develop any immunity to
coliform mastitis. The cows were used many times in the experimental pro-
duction of acute mastitis. The only protection against the disease appeared
to be pre-existing leukocytes in the milk.

My story was not intended to disturb your considerations to improve milk
quality by bringing mastitis under control. However, it was intended to
serve as a warning and at the same time to encourage some tolerance for
low level leukocyte counts especially in the milk of the older cows. Cell
counts of 300,000 to 500,000 per ml. of foremilk will give a high degree
of protection against the development of acute coliform mastitis in the
event of accidental entrance of coli-bacteria into such mammary glands.

Op bedrijven waar het gelukt is mastitiden, waarbij Streptokokken en stafylokokken
cen rol spelen, tot een minimum terug te brengen, kunnen plotseling ernstige vormen
van coli-mastitiden bij de runderen optreden.

De sanering van deze bedrijven op eerstgenoemde mastitiden bij de runderen vond
in de oorlog.sjaren plaats zonder daarbij gebruik te maken van antibiotica. Door
hygiënische maatregelen en zonodig door ruiming van moeilijk te genezen dieren
werd Streptococcus agalactiae uitgeroeid. In 1945 werden ook de stafylokokken
aangepakt door middel van isolatie en behandeling van de runderen met antibiotica
en chemotherapeutica.

In het begin waren 50% van deze dieren stafylokokkendragcrs zonder dat deze
alle aan klinische mastitis leden. Bij de oudere koeien was het succes gering, doch bij
de jongere dieren daalde het percentage dragers in 4 jaren van 50% tot 27%.
Vanaf dat moment werd de coli-mastitis bij dc runderen een probleem. Het eerste
jaar leden 20 dieren hieraan (10%), waarvan er 3 stierven. In 1959 begonnen
onze experimenten.

Wij vonden in de koppel Escherichia coli- en Aerobacter aerogenes mastitiden. We
gebruikten alleen Aerobacter aerogenes bij onze experimenten en constateerden dat
5 ä 10 bacteriën voldoende waren om in een lactcrend kwartier, dat leucocyten-vrije
melk produceert, binnen 24 uur coli-mastitis te doen ontstaan. Verder bleek ons, dat
de snelheid van optreden van dc ontstekingsverschijnselen afhankelijk was van het
aantal ingebrachte bacteriën, mits de melk vrij was van leucocyten. Met 50 kiemen
duurde het 20 uur en met een miljoen kiemen slechts 5 ä 6 uur, voordat cen klinisch
waarneembare mastitis ontstond.

Een vermeerdering van het aantal leucocyten in de melk is zowel met levende als
met dode bacteriën te bereiken. Door de aanwezigheid van cen groot aantal cellen
verdwenen de ingebrachte bacteriën soms snel, doch het duurde 7 ä 14 dagen voor
de veroorzaakte ontsteking genezen was. Vooral bij de dieren waar tijdens de besmet-
ting geen ontstekingsccllen in de melk voorkwamen, was de reactie van de uier t.o.v.
de ingebrachte bacteriën vertraagd. Deze koeien werden daardoor erg ziek en stierven
soms.

Wij nemen aan dat in leucocyten-vrije melk de bacteriën-vermccrdering snel verloopt
en tot een grote populatie komt. In kwartieren waarvan de melk tijdens de besmetting
300.000 ä 500.000 cellen per ml bevatte, had de besmetting met Aerobacter aerogenes
veel minder succcs. De aanwezige leucocyten elimineren blijkbaar de bacteriën. Naar
onze mening is de uier van dc jonge koe van nature door het enge tepel-kanaal
voldoende beschermd tegen infecties die van buiten af komen. Bij oudere koeien
zullen geheel of vrijwel geheel leucocyten-vrije kwartieren t.g.v. een besmetting
grotere kansen op acute ontstekingen hebben. Zijn in de melk echter cen matig aantal
leucocyten aanwezig, dan verminderen deze risico\'s sterk.

Men zal bij een mastitisbestrijding dus moeten vermijden het aantal ontstekings-
cellen tc ver terug te brengen.

following the lectures of Prof, Van der Schaaf and Prof, Dr, O, W. S c h a 1 m.
Question: Dr, D, M, Z u y d a m (The Hague, The Netherlands):

I would like to put to Prof, Van der Schaaf two questions.
He mentions the interesting paper of Stuart and S 1 a v i n about
P,?,L,0, infections in the udder of the cow, I am very much interested
in this question, because mycoplasmosis plays an important part in the
diseases of poultry. It is a base-infection of the respiratory tracts in
birds and it causes the mycoplasmosis-gallinarum or chronic respiratory
diseases.

First : do you think. Professor Van der Schaaf, mycoplasmosis or
P,P,L,0. infection in the udder of the cow may be the base of secondary
bacterial invasion?

The second point is: do you already consider making a mycoplasma- or
P.P.L.O. antigen for milk or milkserumtest in order to find the myco-
plasma infected herds?

Stuart and S1 a v i n of Weybridge have discovered that sometimes
P.P.L.O. is a cause of mastitis. It goes from one cow to another; there
are no bacteria. The difficulty for the diagnosis is that this micro-organism
— this P.P,L,0, — is very difficult to cultivate. We have tried to cultivate
it, but up till now we did not have any success; maybe in the future
we will have more success.

About the second question: you ask a serological test which would be of
help to detect the mycoplasmosis in the udders of the cows. I think it is
time to go on to find the actual causes, which producc mastitis in the
udders of cows. Maybe mycoplasmosis plays an important part, but up till
now we do not know.

The question you ask me, Dr. Zuydam, I will pass to all members of this
congress. Slavin and Stuart tried to cure the P.P.L.O, mastitis.
They found penicillin was useless, because P,P,L,0, is resistant against
penicillin. But also the tetracyclines had no result although the P,P,L,0.
are sensitive for tetracyclines. The infection does not come back in the
following lactiation period.

Some remarks about the results of Professor Van der Schaaf,
In Aulendorf Professor K i e 1 w e i n and I used a tube-test. This test
was more useful than the Schalm-test, We join your oponion about the
alkaline reaction of the reagent. In Aulendorf we used a Schalm-test
with a dedocyl-sulfate. This was buffered to pH 9, for chemical reasons.
The action of dedocyl and sodium laurylsulfate is best at pH 9,
We did some first experimental work about the strain of Miyagawanella
bovis, which we got from the Virus Institute of Tubbingen from Dr,
S t r a u b. We used the complement fixation-test with the milkserum
from mastitis cows in which we could not find bacteria in order to find
Miyagawanella-irdccûon!,. Up till now we are not sure we have found
these infections in cows. This work is sdll in progress.

As to the results of Prof. S c h a 1 m, I should like to say we are controlling
some herds very strictly for raw milk production. We are keeping the
total amounts of leucocytes very low. I think below 100.000 cells pro
m.l. We have few Staphylococci-inlections, the Staphylococci-inkctions
occur in about 10% of the cows. These herds are in very good state of
control, but we never had any form of coliform mastitis in these herds.
I think there must be other conditions, that will cause the coliform
mastitis.

The coliform mastitis we speak about is not common and only occurs in
these herds, in which the mastitis control is done for several years. The
cows have had in average about four lactation periods. The productive
cows stay longer in the herds.

We have the opinion, that especially in the older cows the coliform
mastitis occurs. I think the coli enters the udders by way of the teats.
We know nothing or little about the coh coming in the udder by way of the
bloodstream. The cohform mastitis most occurs in a short time after
refreshing, but that is not necessary.

As for the disinfection the teatcup of the machine was dipped between
every milking into a chlorine solution, also the teats were tipped in
chlorine. The sanitation was excellent.

We were dealing with an unusual situation. If all experiences are correct,
coli grows only in leucocyte- free milk. A leucocyte count of 300.000 to
500.000 leucocytes per milliliter, which is subnormal, is highly protective
against colimastitis.

Most cases of coliform mastitis occur some days after refreshing. As far as
I know we have the greatest amount of cells in the milk, some days after
refreshing and I therefore can not understand that in this period most
cases of coliform mastitis occur.

The normal cells in the colostrum of the cows are no leucocytes. When
I speak about leucocytes, I mean the polynuclear leucocytes. Colostrum
is rather low in the number of leucocytes. Maybe this is the solution of
the problem.

Dr. Br us: Mr. Kingwill, I was very glad to hear that you will address the
Conference on the Control of mastitis by hygiene.

From my co-worker Dr. Jaartsveld, I have already heard about your work and
I am very happy to welcome you here. ,

From the results of our work at Shinfield and other published data we
believe that there are good prospects of developing hygiene into an
effective means of controlling udder infection by the common pathogens.
This is not a new idea, but there is no proof that any hygiene system so
far developed is effective.

The value of hygiene systems is based on the hypothesis that infection
by the common pathogens is normally catised by organisms transmitted
during milking on hands, udder cloth and teat cup liners, and that this
transfer can be largely prevented. Research in our institute herd showed
that, using cows with Iiealthy teat skin, this transfer could, to a very
large degree, be prevented. To find out whether this result could be
achieved under a range of commercial conditions and to measure its
effect on new infection, a field trial was planned.

The aim of the experiment is to compare the incidence of new udder
infection when no special hygiene is practised with the incidence when
using the highest practical level of hygiene at milking time.

The spread of bacteria by milking is very greatly reduced by the use of a
chemical disinfectant for hand and udder disinfection and the pasteuri-
sation of teat cup liners. A solution of Chlorhexidine digluconate (Hibitane;
100 p.p.m.) is used to disinfect the milker\'s hands between each cow and
for washing each cow\'s udder with cither an individual paper towel or
sterile cloth before milking. A pasteuriser (see below) circulates approxi-
mately 2 litres of water at 85° — 90° C through the teat cup liners
and milk tube for 5-8 seconds before each cow. Immediately after milking
the teats of each cow are dipped in a solution of Hibitane (5000 p.p.m.).
These methods are applied to each cow in the hygiene herds by the follo-
wing milking routine throughout the trial year.

1 F. K. Neavc, F. H. Dodd and R. G. Kingwill; Scientific Staff Members; National
Institute for Research in Dairying, Shinfield, Reading.

3. wash udder using paper towel or sterile cloth and disinfectant
solution (Hibitane; 100 p.p.m.);

4. milk cow with automatically pasteurised teat cup. (proceed with
work on other cows until milking complete);

5. dip both hands in disinfectant solution (Hibitane; 100 p.p.m.)
before machine stripping and removing teat cups;

4. no attempt to rinse or disinfect teat cups or hands between cows,
except to remove gross soiling.

Diagnosis of infection is based on the recovery of bacteria from milk
samples, physical appearance, Whiteside test reactions, and cell counts
when other tests are inconclusive. Two individual quarter milk samples
from each cow were examined at the start of the experiment or at calving,
and quarter samples from each cow are examined at four-monthly intervals
or at the end of lactation.

Confirmatory milk samples are examined when a change of infection is
indicated and repeated examinadon of samples made until the position
is defined. Strains of Staph, aureus are phage typed to aid the diagnosis
of new infection. Before therapy two milk samples are taken by farm staff,
and Institute staff collect post-treatment samples approximately 21 and
28 days after therapy to find out if the infection has been eliminated.

To measure the effectiveness of the hygiene routine or to indicate the
reasons for failure to prevent the spread of bacteria, swabs of each cow\'s
teats are examined at the start of the experiment and at four-monthly
intervals. At weekly intervals in each hygiene herd teat swabs have been
examined from a few random non-infected cows and swabs from teat
sores and milker\'s hands and teat cup liners after pasteurisation
In all herds teats were examined at the start of the experiment and at
4-monthly intervals of more frequently, and a record made of teat lesions
and orifice erosion.

No attempt was made to alter the general management or milking techni-
que in any herd, but details of management and changes were recorded. At
the start all milking machines were checked and corrected, if necessary,
to provide 15" Hg vacuum, effective pulsation and a controlled re.serve
of vacuum.

Antibiotic tlierapy is restricted to clinical cases of mastitis, and farmers do
not have details of sub-clinical infections. Veterinary surgeons examine
any severe cases of mastitis, but with the collaboration of all practitioners
a standard antibiotic therapy is available for cases where only clots in the
milk or mild induration is detected. In all cases the milker takes two milk
samples before inspection, and gives 1 tube of antibiotic per affected
quarter, repeated twice at 48-hour intervals.

Field staff visit each herd weekly to check the milking routine, collect data
and samples, and service the pasteurisers. Senior staff visit herds for all
teat examinations, to investigate problems and for general supervision.
Faults in equipment are corrected before the next milking.

specially designed punched card is used to record every item of in-
formation for each cow, and this provides a means of deciding when
confirmatory samples or swabs are required. Further cards are used to
record and analyse each infection and each course of therapy.
A weekly sampling programme is entered in each herd diary, and field
staff take the diary for each visit and record in it all observations.
Milkers are provided with a clinical record sheet to record mastitis and
antibiotic treatments.

In order to guarantee the pasteurisation of teat cups in the absence of super-
vision it was decided to make the pasteurisation equipment automatic,
fitting it as an integral part of the milking equipment (see below).
This decision was useful in that it meant that the experiment was testing
a practical process but it limited the experiment to parlour milked herds.
It was further decided that these milking parlours should be of the one
unit per stall type because of the long unit-idle time. These considerations
together with the difficulties of measuring residual effects with crossover
design led us to choose a design with the treatment comparison made
between herds.

Available data indicated that 9 herds per treatment were required for a
year to give an 80% chance of measuring a 40% reduction in the incidence
of new infection at the 5% level of significance. To limit costs and allow
for withdrawals the experiment is to be run over two years, commencing
with 7 herds on each roudne. Herds were selected according to the number
of cows, type and level of infection and cooperation of personnel. Before
the allocation of treatments all farmers agreed to accept either control or
hygiene routine. The only reasonably blocking criteria was the presence or
absence of Streptococcus agalactiae infection, and the experiment is a com-
pletely random design with random allocation of treatment to herds in
these two groups.

The pasteuriser consists of an electrically heated water bath containing
a basket to hold the teat cups, and a releaser. The position of the lid of
the bath governs the cycle of operation. When the lid is fully closed
milking vacuum is apphed to the teat cups. Raising the lid seals off the
milking vacuum and physically isolates the long milk tube from the
connection to the milk line. The teat cups may now be placed in the
basket with their mouths in hot water.

When the lid is lowered to a pre-determined mid-position vacuum is
applied via the releaser to the teat cups, and water is drawn through
the teat cups, clawpiece and milk tube.

When approximately 2 litres of water has entered the releaser a float
rises to displace a ball valve. Vacuum enters a diaphragm motor which
releases a spring catch, allowing the basket to rise. The teat cups are
lifted clear of the water, and can drain, and at the same time the vacuum
supply to the releaser is blanked off.

When the milker is ready, raising the lid to remove the cluster and fully
closing the lid resets the vacuum for milking and positions the basket for
the next cycle.

From Table V, which gives provisional results of new udder infections in
the first eight months of the trial, it is clear that there are uncontrolled
factors affecting the new infection rate within the two groups of herds that
have a greater influence than the experimental treatment. Nevertheless,
the total of new infections in the hygiene herds appears to have been
reduced by 50% compared with the control herds, and when cross-
infections are eliminated the reduction is 60%. This result is encouraging,
but it is obvious that in spite of hygiene, new infections occur frequently
in herds 1, 2 and 4.

Measurements of the pathogens on teat cup liners after pasteurisation, and
the outside of teat cups (indicating the level of hand contamination) show
that the hygiene routine is satisfactory in these respects. (Table III). The
more important criterion is the presence of staphylococci on the teats of
non-infected cows, and although Table II shows that these were reduced
by the routine the results have not been as good as were obtained in
the research herd.

The presence of staphylococci on teats must be partly due to the high
incidence of chapped teats in the hygiene herds (table IV), a result that
was not anticipated. Although it would appear impossible to have an
effective hygiene routine unless the skin of the cow\'s teats is healthy, the
presence of chaps in both hygiene and control herds is not closely related
to the incidence of new infection (see tables IV en V).
Our examination of the results has failed so far to reveal any major factor
influencing the new infection rate within the treatment groups and it is
quite possible that this is due to the relative infectivity of the different
strains of pathogens in the herds.

Percentage of cows with healthy teat skin, chapped teats, and
„infectious" teat sores.

% Healthy Teats
Date of examination

June/July Oct./Nov. Feb.
\'62 \'62 \'63

Staph. Strep. Strep. Strep. Others Total Suph. Strep. Strep. Strep. Others Total
aureus agal. dys. uberis aureus agal. dys. uberis

No. cows in
milk during
whole or part
of period

58
55
62
51

2
7
0
7
1
0

0
1
0
1
2
6
6

29
47
7
29
12

137

43
42

14
82

44
63

324

8
3
0
1
2
3

11
3
6
7
3

10
7

21
0
3
1
2

11
10
24
1

38
16

123

2
7
0
11
1
0

0
1
0
1
2
7
9

16
16

7
18

8
8

13
29
7
5

39
11
27

131

11
9
0
4

3
7

4
9

25
9

4
24
0
4
4
4

16
11
26
3
54
29
35

174

26
55
8
43
19
18

169

19
43
9
26
79
14
41

231

Hygiene herds 1
2

Total

Control herds 8
9
10
11
12
13

_14

Total

327

43
42

51
32

52
58
65

343

2
0
10
0
0
1
0

De spreker, Mr. R. G. Kingwill, houdt zich in deze voordracht vooral bezig met
de invloed van hygiënische maatregelen van de melker, de uier en de machine
op het voorkómen en ontstaan van uierontstekingen bij runderen.
De desinfectie van handen en uier geschiedt met chloorhexidine digluconaat (100
d.p.m.); det tepels worden direkt na het melken gedompeld in chloorhexidine (5000
d.p.m.) en de tepelvoeringen worden gedurende 5—8 seconden in stromend water
van 85° C verwarmd.

De waarde van hygiënische maatregelen berust op de hypothese dat de infectie —
veroorzaakt door de uier-pathogene microörganismen — gedurende het melken
ontstaat via de handen, uierdoek en tepelvoeringen. Met behulp van hygiënische
maatregelen kan men de overdracht van micrdSrganismen grotendeels beperken,
vooral indien de huid van de runderen in een goede conditie is.

Het doel van dit onderzoek is het vergelijken van het ontstaan van het aantal nieuwe
gevallen van uierontsteking op bedrijven waar onder extra goede hygiënische om-
standigheden wordt gemolken en op bedrijven waar deze maatregelen niet worden
genomen, gedurende een tijdvak van een jaar.

Een pasteuriseerapparaat werd geconstrueerd waarmee het mogelijk is ongeveer
2 liter water van 85—90° C gedurende 5—8 seconden te doen circuleren door de
tepelvoeringen en de melkslang. Deze desinfectie wordt gedurende het melken en
na afloop ervan voor elke te melken koe toegepast.

De hygiënische maatregelen, welke op de z.g. hygiënische bedrijven werden toe-
gepast, waren de volgende:

2. beide handen van de melker worden gedompeld in een hibitane oplossing
(100 d.p.m.);

3. de uier wordt met hibitane (100 d.p.m.) door middel van een steriele doek
(papier) gewassen en gedroogd;

5. voordat het apparaat afgenomen wordt, worden beide handen in een hibitane
oplossing (100 d.p.m.) gedompeld;

6. de tepelbekers worden in het pasteurisatieapparaat geplaatst dat half auto-
matisch werkt;

7. de tepels worden gebracht in een hibitane oplossing (5000 d.p.m.).
Op de controlebedrijven werden de volgende maatregelen getroffen:

1. De uier wordt met warm water gewassen en met behulp van een doek afge-
droogd ;

3. de koe wordt normaal gemolken, zonder enige bijzondere hygiënische maat-
regelen.

De diagnose van uierontsteking wordt gesteld aan de hand van het bacteriologisch
melkonderzoek, het uitwendige aspect van de melk en de Whiteside-rsactie, even-
tueel ook door celtelling.

Om het effect van de hygiënische maatregelen te controleren werden met behulp
van steriele „swabs" de tepels van elk rund bacteriologisch onderzocht, evenals de
handen van de melkers en de tepelvoeringen. Dit onderzoek gebeurde elk kwartaal.
Alleen de gevallen van klinische mastitis werden met antibiotica behandeld.
Met behulp van een ponskaartensysteem werden de gegevens genoteerd.
Tabel I geeft een overzicht van de 14 bedrijven die deelnamen aan de praktijkproef.
Tabel II geeft aan welk percentage „swabs" van de tepels der runderen op de
hygiënische bedrijven, 4 maanden na het begin van het experiment. Staphylococcus
aureus bevat vergeleken met de „swabs", genomen bij het begin van het experiment.
Tabel III geeft aan het aantal swabs met Staph, aureus die genomen zijn na de
pasteurisatie van de tepelvoeringen uit het inwendige en uit het uitwendige van de
tepelvoeringen. Hieruit blijkt dat de isolatie van Staph, aureus veel frequenter

plaats vindt aan dc buitenkant van de tepelvoering dan aan de binnenkant van dc
tepelvoering. Tabel IV toont dat het aantal runderen met kloven op de spenen en
met tepellaesies op de hygiënische bedrijven aanmerkelijk hoger in aantal zijn dan
op de controle-bedrijven.

Blijkbaar heeft de desinfectie van de tepel een nadelige invloed op de gaafheid van
de tepelhuid.

De laatste tabel, een voorlopige mededeling, geeft aan dat het aantal positieve bacte-
riologische onderzoekingen op de z.g. hygiënische bedrijven aanmerkelijk lager is
dan die op dc controle-bedrijven Dit geldt speciaal t.o.v. de isolatie van het aantal
stafylokokken, Str. agalactiae, Str. dysgalactiae en Str. uberis.

A preliminary report of a controlled field experiment with 700 cows in 14 herds to
measure the effect on the incidence of new udder infection of a hygienic milking
routine. The routine includes disinfection of hands and udders with chlorhexidine
digluconate solution (100 p.p.m.), pasteurising teat cup liners between each cow
(85° C. for 5—8 seconds) and teat dipping in chlorhexidine digluconate (5000
p.p.m.) immediately after milking.

The results of examination of swabs from teats, and teat cup liners are discussed in
relation to new udder infections found by examination of milk samples for bacteria,
Whiteside test reaction and cell count.

1. If you use heat to disinfect the teat-cups without use of rinsing
water, would you not get milkstone in your cup-liners?

2. When you use a disinfectant after milking without drying the teats,
don\'t you get some irritation at the teats of the cows?

We have not found that milkstone formation has occurred. I cannot
explain this, perhaps it is, because there is always rinsing between two
cows. The liners of the milking-machinc have lasted only about half their
normal length of life. Further, I think that synthetic rubber will be better
for this than natural rubber but we have done little work yet. It is not
necessary to cool these cups. We have seen no harm of the cow and no
irritation.

As to your question of wet teats. We have not found a direct relation
between the wet teats and irritation.

Mr. Kingwill, I should like to ask you about the temperature-co-
efficiënt of the disinfectant. There is a great difference between the
different disinfectants. Fenol acid has a low temperature-coëfficiënt and
chlorine has a very high one. Also the chlorhexidine has a high tempera-
ture-coëfficiënt.

When I saw the dipping of the hands of the people we have seen in your
film, I asked myself, what is the temperature-coëfficiënt of the disin-
fectant? Is it perhaps five degrees, ten degrees, twenty-five or forty-seven

degrees? For the disinfection of the hands, it was best the temperature
of the disinfectant is thirty-seven degrees.

Also I\'m interested in the temperature of disinfectants for disinfection
of the teatcups. According to the experience of Dr. Jaartsveld and
me, in which we used 0,4\'% chloramine at about 20 degrees, we got a
total disinfection of germs of the skin of the hand within three minutes.
You didn\'t get a whole sterilization of the cloths and I ask how is that
possible ?

About your first question. The disinfectant for the first cow was prepared
at a high temperature, about 40 to 45 degrees C°. The temperature
declined, perhaps at the end of milking to 20 degrees. When we made
the comparison for hand disinfection the temperature of the disinfectant
was about 40 degrees C°.

For disinfection of the teat-cups a fresh solution of disinfectant was
always used. In this way the disinfectant was not contaminated except
from the milk of the clusters.

We have seen this disinfection-machine in a milking-parlour, but I should
like to ask you, if it\'s possible to use this machine in a milking cow
shed. The machine would have to be moved over some distance.
In these results we have seen, it is perhaps a disappointment to many of
us who have worked in mastitis control to find, that the staphylococci
; infection is the most difficult infection to control by disinfection.

It\'s possible to get a moving disinfection-machine. In a rescarchherd we
j started this work in the cow shed, and developed a machine on wheels.

.About the second question; I agree with you about the disappointment in
not reducing the staphylococci infection with these methods of di.sinfection.
One point is that the disinfection of teats is always incomplete. This is
one important thing. You will be familiar too with the paper of D a v i d-
s o n. I think from his work is does seem likely that if one can reduce the
level of udder infections, the risk of survival of staphylococci will be less.
We will hope that as we make a little more progress with the disinfection
of the teats, we will get better results.

There is another aspect too. Wc have made no attempt to segregate or
I reduce the chronic infected cows nor to use antibiotics in the infected

common herds clearly one should use all the possibilities for controlling
mastitis. One would be wise, one would eliminate the cows with chronic
j mastitis and one would disinfect as well as possible.

On the first question the answer is „no"; we have no evidence that
hibitane gives a better protection of the teats. In a survey we have seen
that by hibitane we got more teat lesions than by hypochlorite. Maybe
this was caused by the high concentration of a detergent that was added

to hibitane. Now the hibitane is diluted in less detergent and I hope
in the future we get better results.

The second question was about the skin contact between the different
disinfectants. Chlorine appeared to be shghtly inferior to hibitane.
Possibly, but I have no evidence, because there is an accumulation of the
bactericide chlorhexidine on the skin. This is one possibility.

I should like to consider further the matter of disinfection after milking.
There is a very good work of Dr. T h i p a t h y of the Cornell University,
a thesis of the former year. Systematically disinfectants — also G-11 —
were tested in disinfection the teats after milking.

The result was, that disinfection after milking had no results at all on the
number of bacteria, found at the moment for the following milktime. So
the contamination by the bacteria and other agents is so heavy that
disinfection after milking has no worth.

But there is a second thing and that is that disinfection after milking gives
more cracks and lesions of the teats and that should be avoided. Keep
the skin of the teats in good health. That is the first thing we have to do
and therefore we should avoid every disinfection.

I agree that the health of the skin is very important and we have been
aware of the danger of disinfecting the teats after milking. We have to do
more work, but we have found that the aqua solution of hibitane is not
harmful.

Concerning the report you have spoken of, I must say, we have found the
reverse. We have made ifomparisons of disinfectants for washing the
udder and teats of the cow, and we have dipped teats after milking. We
have taken swabs of the udder pjid teats, firstly before the cow is milked
and after the cow has been washed.

It does appear that teat dipping has a distinctly beneficial effect, but
maybe it is better we will discuss this problem later on.

In The Netherlands every province has its
own Animal Health Service. There are eleven
Provincial Animal Health Services; they are
real farmers\' organisations.
After the second world war the activities
started with the eradication of tuberculosis,
soon followed by the control of brucellosis.
In the meantime the Animal Health Service
got the supervision of the artificial insemina-
tion of cattle and swine, and began an orga-
nization to eradicate swine- and poultry di-
seases. Veterinary surgeons and farmers can
be assisted in individual disease problems.

For the control of brucellosis of cattle, blood and milk-samples are exa-
mined on brucellosis antibodies. At least every quarter of a year milk-
samples are taken at the dairy-factories out of all cans for the examination
on brucellosis-antibodies, with the Abortus Bang Ring-reaction (A.B.R.).
In order to transport these milk-samples easily, we make use of milk-
chests, in which several tube-containers are placed, each containing 100
tubes.

By means of an instrument milk-samples are taken out of every can deli-
vered at the dairy-factory. A milk-sample of 1 ml is transferred to a tube.
When the tube-containers arrive at the laboratory one drop A.B.R.-antigen
is added to each of the 100 tubes with a special drop-apparatus. The anti-
gen and the milk are mixed, placed at 37° C. for one hour, after which
the result of the A.B.R. of the milk-samples is noted.
It is also possible to use the same milk-samples for the mastitis-reaction. The
appearance of a great number of cells in the milk is the most important
criterion for mastids. Therefore the milk is mixed with T-pol or sodium
laurylsulfate. Those reagents are capable to break open the nuclei of the
cells (leucocytes). The desoxyribosenucleic acid (DNA), originating from
the nuclei of the cells in the milk, causes a sharp rise in viscosity and the
more cells the milk contains, the more ropy the mixture is. The ropiness is
measured by means of the Brabant Mastitis Reaction in determining the
rate of flow through capillary tubes.

0.6 ml of milk and 0.4 ml of T-pol 414 or sodium laurylsulfate 2% are
mixed and put in the funnel. There is a relation between the dme of
flowing through the capillaries and the number of cells in the milk. The
dme of flowing through is measured after 5, 10, 20 and 60 seconds. The
samples sdll remaining in the funnels after 5 seconds are marked with one
dot ( • ), the samples sdll in the funnels after 10 seconds are marked with
a second dot ( ••). Those that are still in the funnels after 20 seconds get

1 F. H. J. Jaartsveld D.V.Sc.; veterinary surgeon to the Animal Flealth Service in
North-Brabant; Rechterstraat 80, Boxtel; The Netherlands.

three dots ( • ¹ • ) and those that do not flow out after 60 seconds get four
dots (••••).

In order to apply the B.M.R. in a uniform way the milk-samples with
which the A.B.R. is done, are shaken very well afterwards. In this way the
cream is mixed with the milk and the leucocytes are homogeneously mixed
in the whole milk-sample.

By means of an sucking-apparatus, the total volume of the milk-samples in
the tubes is reduced to 0.6 ml milk, so every tube contains 0.6 ml milk.
Afterwards with a pipetting-apparatus, 0,4 ml of sodium laurylsulfate 2%
is added to the 0.6 ml of milk.

The liquids are mixed and decanted by means of a perforated interplate
in 100 flow-through capillaries, which are fixed on a plate covered with
a sheet of foam-rubber. In this way the flow of the capillaries is blocked.
In order to register the B.M.R. the apparatus with 100 flow-through ca-
pillaris is lifted up and placed below a film camera and after 5, 10, 20 and
60 seconds a picture is taken.

The milk-samples which flow through the capillaries within 5 seconds, have
a negative B.M.R. The milk-samples which do not flow through the capil-
laries after 5 seconds get one dot, after 10 seconds two dots and so on.
There exists a very close relation between the B.M.R. and the mean total
of cells in the milk-samples. The B.M.R. is called positive if 3 or 4 dots are
recorded, doubtful if 1 or 2 dots are recorded and negative if no dots are
recorded. This reaction is done with milk-samples out of cans, pails or
quarters of the udder. Six laboratory workers are capable of examining
10.000 milk-samples in one hour by means of the B.M.R. in a simple, accu-
rate and cheap way.

A regular examination by means of the B.M.R. of all cans delivered at the
dairy-factors gives a clear impression of the farms with serious or no com-
plaints of mastitis. The B.M.R. is a very simple reaction, therefore the
different laboratories may gain the same results with the same milk-samples.
By means of the B.M.R. of the milk out of the cans delivered at the dairy-
factories it is possible to indicate the farms with mastitis problems.
By means of the B.M.R. of the pail- or quartermilk-samples one is capable
to indicate the cows with mastitis at these farms. The primary cause of
mastitis in most cases is a traumatic one, for example hurts and contusions.
Bacteria play a secondary role. If it is possible to find and to take away
these causes the number of cases of mastitis will sharply decrease.
The B.M.R. is suited also to the determination of the leucocytes of blood-
samples. At first the blood-samples with heparin were diluted 1:25 with
water and then the B.M.R. was carried out. This method gave bad results,
especially if the blood-samples were diluted some time before the B.M.R.
was performed.

We got better results if the blood-samples were diluted 1:20 with saline
(0,9%), before the B.M.R. was performed.

The graph shows the relationship between the B.M.R. on the diluted
blood-samples and the total leucocyte-count per mm^.

In addition to the examination for brucellosis and mastitis it is also pos-
sible to examine the milk-samples for the presence of antibiotics. There-
fore rectangular plates are made with the same surface as the tube-con-
tainers. In the future we will use plastic rectangular plates for this purpose,

Relationship of the B.M.R. on diluted blood samples and the total
leucocyte-count.

Het te onderzoeken bloed wordt met behulp van heparine onstolbaar gemaakt.
Daarna wordt het met fysiologische NaCl-oplossing verdund tot b.v. 1 : 20, waarna
met deze oplossing de B.M.R. wordt uitgevoerd (zie grafiek).
De melkmonsters die onderzocht worden met behulp van A.B.R. kunnen door een
eenvoudige techniek eerst op het voorkomen van groeiremmende stoffen worder.
onderzocht, vóórdat de B.M.R. wordt uitgevoerd.

Voor de uitvoering van het onderzoek kan verwezen worden naar de laboratorium-
demonstratie, blz. 95 e.v.

het kader van een georganiseerde bestrijding. Thesis. Utrecht 1961.
Beekers-Heitkamp und Jaartsveld F. H. J.: Massenblutuntersuchung
zur Orientier»ng hinsichtlich der Anzahl kernhaltiger Zeilen im Blut. Arch. Le-
bensm. hyg., 15, 79, (1964).

First I like to consider the presence o.f leucocytes in the milk. It is
dangerous, as Prof. Schalm has told us, to decrease the leucocytes
below a minimal level in the milk.

I have had some experience about leucocytes, but this was a quite dif-
ferent problem. It was the problem of bacteria in vaccinia. Formerly
vaccinia contained some millions of staphylococci pro gramme of vaccinia.
After the first world war the vaccinia could be made better as it was told.
Therefore vaccinia with a very small number of staphylococci was pro-
duced. In 1927 some cases of encephalitis have been seen in Holland
and I had the opportunity to study this problem.

The post vaccinal encephalitis was supposed to be caused by another
virus. It was also possible that the virus had become more virulent than
before, but the real cause was the very low content of staphylococci in the
vaccine. The strain of vaccinia, which was very mild and which had
never given an encephalitis contained many staphylococci.
I tried to make the vaccinia more mild by mixing it with staphylococci.
The staphylococci give a zóne of leucocytes round the place of injection.
This is the reason why the old vaccinia virus was much milder, than
the new vaccinia virus which had a very low content of staphylococci.
The same results I got when I Injected vaccinia with a few staphylococci
mixed with turpentine. In this way it was possible to render the neuro-
lapine milder.

The leucocytes prevent the generalization of the virus and this is the
same problem Prof. Schalm has found with coliform mastitis.
For the mastitis campaign it is important to know how far one can
reduce the total number of leucocytes in milk without permitting every
cow to get mastitis of bactcrial origin.

We have also some experience with the coliform mastitis and we found also
that the total bacteria used for an infection is very important to get an
inflammation of the udder. We had to use more bacteria to get an
infection than Prof. S c h a 1 m. All test objects were cows of the slaughter-
house. Most of them had a severe or chronical mastitis and therefore they

had many leucocytes in the milk. That was the reason that we had to
take more bacteria to get an inHammadon of the udder. We used about
20.000 micro-organisms.

I should like to ask Dr. Schalm which kind of mastitis you found in
U.S.A. We found acute cases of mastitis and more chronical cases of
mastitis, especially in case of wounds on the teats. Is it possible that the
Aerobacter aerogenes also causes a chronic form of mastitis?

We did not find chronic mastitis caused by E.coli, but with Aerobacter
aerogenes — even in our experimental cows — we had one animal
that failed to recover completely from Aerobacter aerogenes-infection. This
cow has been infected several times. She carried the infection for months.
We had to incubate the milk to prove that the Aerobacter aerogenes was
still in the udder.

It must be possible that if a cow is infected with Aerobacter aerogenes
she fails to recover completely and carries this infection for months. We
must incubate the milk to prove that the Aerobacter aerogenes is still
there, but periodically about every three weeks we get a clinical flare-up
in this udder

For two or three days there is a very high leucocyte-count. Then the
inflammation declines, but the organisms arc still there and then the
leucocyte-count comes almost to normal.

And so with Aerobacter aerogenes infection we may have a chronic
mastitis. In the diagnosis of these coliforms one should culture the milk
fresh, but also incubate the milk if the fresh milk-samples show no growth.

In the experimental cases we have learned that we must make cultures of
at least one millilitre of milk.

What we do, we have a number of plates with one millilitre, half a
millilitre, a tenth of a millilitre and a hundredth of a millilitre. Then
we usually isolate a small number of micro-organisms in these high
quantities of milk. One must use higher quantities of milk to isolate the
micro-organisms.

I feel that there are quite more problems about the resistance to masitis
besides the low cell-count of the milk.

I have carried out some experimental work of staphylococci-infections of
the udder. We infected cows artificially with very small numbers of
staphylococci, between 40 and zero; mostly between zero and 5 staphy-
lococci.

The heifers, which we tested before they were infected, were examined on
chemical substances of the milk. The milk was also examined on the total
cell-counts as well and before infection of these heifers; we were sure
that the milk had a normal cell-count and a normal chlorine and glucose-
content.

At the end of this experiment the difference of infections could be con-
nected to the pathogenicity of the staphylococci-strains more than to the
individual cows. I think that the general condition of the cow is quite
important.

We must be careful not to consider only the leucocyte-count of the milk.
In my opinion there are many more problems.

It would be wrong if the participants of this congress went home with the
impression that we must have necessarily leucocytes in the milk, or we are
lost if this is not the case.

I am always speaking about the pathogenicity of coliform-strain;. At least
with our experimental coliform-strains clinical mastitis is partially or
completely prevented when we have the leucocytes on the upper normal
cell-count, that is to say about 300.000 till 500.000 cells per ml. of milk.
This count is not normal, it indicates some irritation of the udder.
In Germany there is some feeling that feeding conditions will lead to coli-
mastitis, coming by way of the blood into the udder. I do not know if
this is a possibility, but I would say that if the coli comes by way of the
blood, it would enter in the basis of the gland rather than down in the
teats.

In chronic mastitis my experience indicates that the inflammatory reaction
begins in the lower ends of the gland. If we dissect the mammary gland we
will find that the inflammatory zone is in the cystem of just about the
cystern. I think the coliform-mastitis comes by teat-road and especially
those cows with a very low cell-count will get the coliform mastitis.

I should like to ask Prof. Schalm: which are predisposing conditions
for the Escherichia co/i-infections in cows?

1 remember a visit to the herds of Prof. Schalm and we talked about
the same problem and I can remember that he told 13 years ago that the
Gram-negative organisms were of considerable importance. Up to that
time the Gram-negative germs were not important in our country.
I would like to ask him more especially what factors of hygiene and
management may be responsible for the occasional infections with
Escherichia coli. Important was, I think, not the buildings but the floors.
In other words do these cows lie on straw or another bedding that may
be responsible for the infection of Escherichia coli?

It is important in experiments to know if there arc some typical serological
types. As in other diseases it is important to know with which kind of
Escherichia coli we are dealing. We know that there are very important
serological types of Escherichia coli in pig diseases.

I should like to ask Prof. Schalm if he was able to find a typical
serological type of Escherichia coli, which is connected with an infection
of the udder.

These particular herds Dr. Edwards mentioned, have the most ideal,
sanitary and hygienic condition I ever worked with.

The cows are never out on the ground. Thirty cows are within an
enclosure, covered with cement that is well bedded with straw and a roof
is present to protect them from rain. After each milking the cup-liners are
sterilized and between milking of cows the teat-cup are dipped in chlorine.
Chlorine we used at the time before the discovery of hibitane. Everything
was ideal. Even the control on streptococci and staphylococci mastitis was
ideal. In our institute we don\'t type the different colis.
You cannot type the aerobacter serologically. In U.S.A. there are more
herds with considerable problems with a coliform mastitis. I may be
wrong in this, but I feel that the coliform-mastitis will become much
more important in future.

Prof. Schalm talked about coli-mastius in Germany. We found it in
single quarters. Geneially the coli-mastitis occurs in one quarter.

I should like to tell something about the cohform-mastitis in Holland.
It is now about ten years ago, I think in the early fifties, that my colleague
B a u d e t came to Leeuwarden one day with ten cultures ot coliforms.
I have typed them and it was shown that it was all Aerohacter aerogenes.
Now we call them Klebsiella. That means that in that time Klebsiella
pneumoniae was present in the province of Gelderland.
In the Clinic for Internal Diseases of the Veterinary Faculty at Utrecht
we sometimes find acute coh-mastitis. In many cases the animal will
die. And in most cases we found coli together with Clostridium
perfringens. If we would not have carried out anaerobic culturing of the
sample of the milk, we would not have found Clostridium perfringens, for
this only grows anaerobically.

If there is gas in the udder, if the animal is severely ill, you should always
use both culture-methods aërobical and anaërobical, to find out which
germs are present in the udder.

We never found merely Escherichia coli in the udder from those animals,
which were severely ill.

Dr, Brus: Mr. Cazemier, when we started al-
most two years ago to investigate in cooperation
with you and Mr. de Rooy the correlations
in management of the dairy farm and mastitis,
we were very glad.

It will always remain interesting to discuss the
importance of bacteria on one side and the
situation of the udder on the other side.
I think the answer will not be black or white,
but grey. It is the task for the investigators
in the field to find out how grey it is. It\'s
not possible to do this in the laboratory.
We are interested to learn of your first results.

For many years the technique of milking has been looked at as a factor
of importance in the prevention and eradication of mastitis. When antibio-
tics were discovered and these — how valuable as an aid in mastitis
control they may be — had not brought the originally expected progress,
even more attention was paid to milking.

The research-work carried out in this field (mechanical milking) up to
now, has by no means led to agreement on all points. The main factors
which have been put forward can be mentioned as follows:
with respect to milking:

insufficient preparation of the udder before the machine is put on;
too much time between preparation and putting the machine on;
letting the machine work on the udder, after milkflow has already
ceased;

taking off in a rough way (under vacuum);
rough and prolonged stripping;
with respect to machine:

vacuurnpump with insufficient overcapacity, resulting into an irregular
and occasionally too low vacuum;
too high vacuum;

irregularly working pulsator with an insufficient sharp transition from
vacuum to atmospheric pressure and vice versa between teat-cup and
liner;

teat-cup liners: worn out, not soft enough and elastic enough, hard
mouth piece and a too wide bore.

Some of the faults mentioned above are due to the facts that one man has
too many milking units at his disposal and to imperfect organization of the
work. Practical research and experience especially have drawn attention
to these factors.

1 Ir. C. H. Cazemier; member of the Scientific Staff of the Research Institute for
Animal Husbandry „Schoonoord"; Driebergsewcg lOd, Zeist; The Netherlands.

Investigations made into the influence of special factors, have often given
somewhat contradictory results and have to a large extent not been able to
confirm the results of this research work in praxis. In 1961 Neave and
D o d d declare that there may be a relation between machine milking and
mastitis and that the type of teat-cup liner is certainly of importance.
They however state that it has not been proved that masdtis outbreaks
can be prevented by the single careful use of the milking machine under
the application of a certain number of rules, as is maintained by some
authors. They consider a well-designed practical investigation to be es-
sential, although this would be difficult to arrange.

As far as the influence of the teat-cup liner mentioned above is concerned
it may be said that there is rather a lot of evidence, that the so called
moulded liner in general has a less favourable effect on udder health as
compared with the type of liner described as extruded liner.
That there is inconsistency between the results based on investigations in
praxis, and those reported from the more fundamental research work, is
not in our opinion very surprising.

Milking is influenced by the personal character of the milker, a complete
standardization of it is actually impossible, as is a real imitation of the
great number of types of milking which can be found in praxis. Besides
milking, hygiene both during milking and in the stable, is of importance
and in praxis can not be dissociated from milking as such. On the other
hand most scientific research work is based on a relatively small number
of animals, often heifers. Differences in susceptibility between the animals
and the average susceptibility in the entire group can under similar con-
didons, considerably influence the results. We have fully encountered the
difficulties of research work in praxis, in a research project on this subject
now being carried out in The Netherlands.

Up dll now little systematic research in this field has been done in our
country. In praxis, by far the most important factor in this respect has
always been considered the fact that the cows should be stripped well.
The investigation mentioned above was started in 1962, being carried out
in cooperation between the Provincial Animal Health Service in North-
Brabant and the Research Institute for Animal Husbandry „Schoonoord"
at Zeist. The investigation first included 38 herds, which number was
extended later to 57 (approx. 750 cows), nearly all mixed farms situated
in the area of the Co-operative Dairy Factories at Dongen and Oosterhout.
The herds were classified into two groups on the basis of 13 3-weekly
investigations (in canmilk samples) on B.M.R. (Brabantic Mastitis
Reaction) from February till September 1962. A B.M.R. of 2 points in
a can sample was considered as being a positive reaction. The average
percentage of B.M.R.-positive can samples in the entire group during the
y>eriod mentioned above was 11.65 Also for the endre group the average
number of times that one or more cans were positive was 4.28.
Herds in which during that period at least 11% of the cans delivered
was positive, were considered to be test-herds. This group included 27
herds with an average percentage of positive can samples of 18.4. The
remaining herds were called control-herds. This group consisted of 30
herds, their average percentage of positive can samples was 3.4.
Based on what we know about the physiology of milk ejection and on
what is common pracdce f.i. with respect to pulsation rate and height of

vacuum, one can imagine a method of milking which as much as possible
promotes a good and rapid milking of the cow without irritation of the
udder tissue. Descriptions of such a method of milking can be found in
many publications e.g. in „Machine Milking" (London 1959).
This method of milking has served as a standard for judging the milking
technique in the herds included in this investigation. To get an evaluation
as objective as possible, time studies were made on all herds. More atten-
tion was paid to the degree of having a good idea of the job, than to beauty
faults and perfection.

In table I a survey is given of the evaluation of the various factors taken
into account, for both groups of herds.

A strict definition of what is exactly meant by „good", „moderate", „bad"
etc. is difficult to give. As important points with regard to preparation may
be mentioned: cleaning, if necessary, a clear but not rough massage, any
pre-milking of a few jets of milk.

With regard to the application of the teat-cups: is the teat clearly led into
the chamber, is there a visable turning around of the teat-cup after the
milker has moved his hand away; with regard to the removing of the teat-
cups: is this or is it not done in a rough manner and/or under vacuum;
hand stripping: is this done quickly up to approx. one min., with the whole
hand and clearly directed towards removal of the last milk that can be
got, from the places where it may be found. Or does it take a long time
(more than 3 min.), is it done perhaps with wet hands, more or less
rubbing of the teat between thumb and forefinger, without there being
clearly looked for the last milk that eventually could be obtained.
Also the evaluation of the organization of the work cannot be described
clearly. A logical sequence of the activities is of importance here. If this
is lacking, the result is often that the times used and needed for the
various activities becomes either too short or mostly too long (waiting time
between preparation and putting the machine on, machine milking time,
waiting time between taking the teat-cups off and starting hand stripping).
,\\s regards the point: „state of repair of the machine", the quality of the
teat-cup liners and the action of the pulsator have especially been taken
into consideration.

From the survey given in Table I it can be seen that the factors „change
of milkers", „preparation of the cows", „putting the teat-cups on and
taking them off", „hand stripping" and the organization of the work
(closely connected with the total treatment and machine ovcrmilking
time), must be considered as relatively unfavourable for the testherds.
When the total care given to milking and to the milking machine is
classified according to the classes „sufficient" and „insufficient" the figures
in Table II are obtained. When the same is done for herds which never
had a positive sample (10) and for those which for at least 8 of the 13
times had one or more cans positive (12), we obtain the figures given in
Table III.

There need be no doubt on the general conclusion that can be drawn from
these figures, namely that surely milking on the test-herds is generally
done much more carelessly and with less interest.

Vacuum in cmJHg
(Vacuum in cm\\Hg)
Aantal pulsaties/min.
(Pulsation ratelmin.)

Aanbrengen van de tepelhouders
(Applying of the teat-cup liners)
Stand van het apparaat
(Position of the appartus)
Afnemen van de tepelhouders
(Removing of the teat-cup liners)
Handnamelken
(Hand stripping)
Machinaal namelken
(Machine stripping)
Organisatie verdeling v/h werk
(Organisation & division of work)
Staat van onderhoud v/d machine
(State of repair of the machine)

6
1
5
1

15
1
18
9

9
12

5
9
7
2

6
10

This conclusion is confirmed by the figures in Table IV, where a survey is
given of the results of milk quality analysis in both groups of herds from
January to September 1962.

Overzicht van de percentages bedrijven met afwijkingen bij het kwaliteits-
onderzoek van de melk (gemiddelde over 21 onderzoekingen).

Survey of the percentages of herds with deviating quality of the mi k
(average of 21 investigations).

At the same time however these figures underline the question whether the
less care given to milking directly and as such on the test-herds or the

less hygienic way of working closely connected to this, will be primarily
responsible for the higher degree of mastitis found in these herds.
Some indication in this direction might be obtained by considering the
results of quality analysis for the classes „sufficient" and „insufficient
milking", separately, as is done in Table V.

Overzicht van de percentages bedrijven met afwijkingen bij het kwaliteits-
onderzoek van de melk (gemiddeld over 21 onderzoekingen).

Survey of the percentages of herds with deviating quality of the milk
(average of 21 investigations).

From these figures it can be seen that in the two groups where milking was
considered to be sufficient, the average quality and especially the purity
of the milk is less favourable in herds with more mastitis. The same dif-
ference is observed on the herds on which milking was characterized as
insufficient. Within the two groups of herds as a whole (test-and control-
group resp.), there is, between the classes „milking sufficient" and „in-
sufficient" no great difference in the quality of the milk, although in both
cases the quality is somewhat inferior in the class „milking insufficient".
It should be recognized especially that on the testherds classified in the
group „milking sufficient", the quality of the milk seems to be inferior
than on the control-herds classified as „milking insufficient".

Uitkomsten kwartiermonster onderzoek, bacteriologisch en op B.M.R. naar
proef- en controlebedrijven, over 3 onderzoekingen in de periode jidi t.m.

Results of bacteriological and B.M.R. research of cjuarter samples. Data
of 3 investigations from test- and controlherds during the period
July - September 1962.

De mate van optreden van nieuwe injecties. Aantal koedagen per nieuwe
infectie (in kwartieren) of positieve reactie op B.M.R.

Rate of new infections. Number of cow-days per new infection (in quar-
ters) or positive reaction on B.M.R.

Reactie percentage in kwartieren, waarin bij een voorgaand onderzoek geen
enkele reactie of besmetting werd gevonden, resp. reactie percentages in
kwartieren, waarin bij een voorgaand onderzoek uitsluitend een positieve
B.M.R. werd gevonden.

Percentages of reaction in quarters, which were negative in the foregoing
sampling, respectively percentages of reaction in quarters, which in the
foregoing sampling were only positive on B.M.R.

Reactie percentages in kwartieren waarin bij een voorgaand onderzoek uit-
sluitend Strept. agalactiae werd gevonden.

Percentages of reaction in quarters which in foregoing sampling were only
positive on Strept. agalactiae.

These observations in our opinion give some support to the idea that at
least in this case, if more mastitis results from bad milking techniques, it
are in the first place the bad hygienic conditions closely connected with
these bad techniques, that must be regarded for as being responsible.

Interesting also in this connection are some of the figures than can be
obtained from the investigations into the bacteriological picture and the
B.M.R. of quarters.

About once every 4 or 5 weeks the quarter samples of all cows are
examined bacteriologically and on B.M.R. In Table VI a survey is given
of the first three samplings (July to September 1962). As the figures reveal,
during this period this part of the investigation was restricted to a smaller
number of herds. Especially the degree of Streptococcus agalactiae infection
appears to be much higher in the test-herds than in the control-herds.
During the three tests, 50—60% of the cows on the test-herds showed one
or more infected quarters. In the control-herds this percentage was 30—
40%. In Table VII the rate of new infections in quarters is given based
on the number of cow days in both groups. Also the rate of new positive
reactions on B.M.R. (3-f) in quarters is given.

In the test-herds there is a definitely higher frequency of new infections
and of newly B.M.R. positive quarters than in the control-herds. Some
doubt may be put to the significance of a Staphylococci-infection, not ac-
companied by a positive reaction on B.M.R. Based on what is said before
the quesdon arises as to what may be the reason for the foregoing higher
number of new B.M.R.-positive quarters in the test-herds. The inferior
milking technique as such with consequently a higher degree of irritation
of udder tissues, or the less hygienic way of milking resulting from it and
hence a higher degree of contamination.

Table VIII summarizes the reactions found in quarters in which nothing
whatever resp. only a positive B.M.R. was found in the foregoing sampling.
In few words: what happens to quarters now found to be negative in all
respects, between the present and the following sampling.
The figures given show that starting from these negative quarters, the rate
of newly B.M.R.-positive quarters is practically the same in test-herds as
in control-herds. Indeed the total number of newly B.M.R.-positive
quarters is relatively higher in the test-herds, but this difference is caused
by quarters in which at the same time one of another infection has been
established. This difference has been proved to be statistically highly
significant. Thus in our opinion these data once again lend support to the
idea that at least in our group of herds, as far as bad milking techniques
have an unfavourable influence on udder health, this is mainly caused by
the higher degree of contamination that in practice, will nearly always be
closely connected with bad milking techniques.

As an interesting feature as such it can be seen from these figures that
as well in test- as in control-herds, B.M.R.-positive quarters are more
likely to get infected later on, than are negative quarters.

In Table IX it is shown that in the test-herds quarters positive on Strept.
agalactiae are somewhat (not significant) more likely to get a positive
B.M.R., than in the control-herds. Here one could think of an influence
of milking techniques, but also on the fact that contaminations will be
more frequently and heavier in the test-herds.

In summary, according the literature, under good milking conditions there
still may be an influence from the type of teat-cup liner on udder health.
Also according to the literature, where inferior milking techniques are
employed in pracdce, an unstable udder health may in general be one of
the results. This conclusion is supported by the data presented in this
paper.

At the same time some evidence is given that it may be not the bad milking
technique as such that causes the higher degree of mastitis, but more the
enlarged chances of contamination, by which in practice bad milking tech-
niques are very often accompanied.

Sedert lang wordt de melk-techniek, .speciaal van het machinale melken, als een
belangrijke factor gezien bij de preventie en bestrijding van mastitis.
In praktijk-onderzoek werd hoofdzakelijk aandacht besteed aan de volgende punten:

1. vacuum-pomp: eventueel onvoldoende overcapaciteit, waardoor een regelmatig
en soms een te laag vacuum ontstaat;

4. pulsator: eventueel onregelmatig of een onvoldoende scherpe overgang tussen
zuig en pers onderbrekingsslag;

5. tcpelvooringen-dikbuikig, te hard, te weinig elastisch, stugge kop, te wijde
boring etc.

De kwartier melkmonsters werden bacteriologisch en met behulp van de Brabantse
Mastitis Reactie (B.M.R.) onderzocht door de Provinciale Gezondheidsdienst voor
Dieren te Boxtel.

Op grond van het B.M.R.-onderzoek van de busmelkmonsters van de zuivelfabrieken
Dongen en Oosterhout, dat elke 4—5 weken plaats vond, werden twee groepen be-
drijven uitgezocht nl. de groep van de positieve-bedrijven en die van de negatieve
bedrijven, in de tabellen resp. proefbedrijven en controlebedrijven genoemd. De
positieve bedrijven hadden in de eerste periode van de proef steeds een positief
B.M.R.-onderzoek van de busmelkmonsters, terwijl de negatieve bedrijven in de eerste
periode van de proef steeds of vrijwel steeds een negatief B.M.R.-onderzoek van dc
busmelkmonsters haalden.

In tabel I wordt een overzicht gegeven van een aantal waarnemingen. De waardering
„goed", „matig" of „slecht" zijn moeilijk exact te omschrijven, evenals een aantal
andere begrippen in tabel I genoemd. Uit tabel I blijkt, dat op de positieve be-
drijven in verschillende opzichten zowel wat de melktechniek, de staat van onderhoud
van de machine, als de organisatie van het werk betreft, minder goed zijn dan de
negetieve-bedrijven.

Dit komt nog duidelijker tot uitdrukking in de tabellen II en III.
Dat ten gevolge van het minder accuraat melken op de positieve bedrijven de
kwaliteit van de melk zoals deze nu wordt bepaald, minder is dan die van de
negatieve bedrijven, is duidelijk te lezen uit de tabellen IV en V.
In tabel VI wordt een overzicht gegeven van de eerste 3 melk-onderzockingen. Op-
merkelijk is het verschil in percentage Str. agalactiae-inkcties op de positieve en de
negatieve bedrijven. Het perecentage kwartieren met stafylokokkeninfecties is onge-
veer gelijk.

Tabel VII geeft aan dat er op de positieve bedrijven een hogere frequentie is
betreffende het ontstaan van nieuwe infecties of nieuwe positieve B.M.R.-uitslagen
dan op de negatieve-bedrijven. Deze grootheden zijn uitgedrukt in koe-dagen per
infectie of per positieve B.M.R.

Samenvattend kan worden gesteld dat de melktechniek in het algemeen van groot
belang is bij het ontstaan of voorkomen van uierontsteking bij het rund. Daar,
waar slecht wordt gemolken, ontstaan meer uierontstekingen.

Over het algemeen is de kwaliteit van de melk op bedrijven waar slecht wordt
gemolken, minder dan op bedrijven waar goed wordt gemolken.

The overmilking-time is commoner in the test-herds than in the control-
herds, however this difference is not very great.

1. It is very difficult to understand one thing, a very important thing.
What is the meaning of control- and test herds here?
Test-herds; that means you have done something and the control-
herds you have done nothing? That is the first question.

2. And next, what is the significant difference between the control-herds
and the test-herds, applied to your difference here? Formerly — I

think — you said the machine wat not so important as hygiene.
I think that is a very good summary.
3. And the matter of ovcrmilking I think is emphasized by you. I think
I agree with many workers who are here, that overmilking is the
most dangerous of machine milking.

As to the question of test-herds and control-herds, we must have two
names for the two groups. The test-herds are the herds which have more
mastitis than the control-herds according to the B.M.R. The control herds
have less mastitis according to the B.M.R. It was only a matter of name.
An especial test has not be done with the test-herds in relation to the
control-herds, up to now at least. The test-herds have a high percentage
of positive B.M.R..

This division in two groups is not a very sharp one, but based on the data
we had, it was the best one to be reached.

.\'Vnd now the question of overmilking time. Of course we cannot judge the
influence of special factors in such a research. There are a number of
factors which influence the coming into existence of mastitis. In literature
some experiments are described which give a clear difference between
normal milking and overtime milking, but there are other experiments in
which the difference between normal time milking and overmilking-timc
concerning mastitis is not clear. I remember the reports of Neave and
D o d d in Kopcnhagen last year. There was a combination of high vacuum
and overmilking time.

So two factors, namely a vacuum of about 50 centimeters and an over-
milking time of 5 minutes. Neave and Dodd could not find a great
difference between these two groups of herds, although in the groups of
ovcrmilking time and high vacuum there were more lesions on the teats.

I agree with the speaker that bad-milking is mostly combined with a bad
milker and so quite a lot of different troubles fall together.
It is quite interesting to watch what happens if by a good milker and good
hygienic conditions some or more changes are made in the milking tech-
nique. For instance we have had a good milker and we had good hygienic
conditions, but the machine milking was changed by a pipeline-condition
with too high a vacuum and prolonged milking time. The result in fact
was mastitis.

So I think it is very clear from this example that a bad milking technique
can be the reason of many troubles.

In both groups, the test-group and control-group, there are different herds
with a vacuum which was too high. I wonder you did not compare the
incidence of infections from especially staphylococci or the incidence
of mastitis in general or the incidence of B.M.R. between the herds with
a high vacuum and the herds with a normal vacuum.

Up till now we have not done so. The vacuum which was too high was
more than 40 c.m. Hg. and the vacuum which was too low was less
than 30 cm. Hg. The vacuum should be normally between 35 and 37 cm.
Hg. Maybe there will be some difference between the different groups as
you suppose. We will try to find it out.
I thank you for the suggestion.

Dr. Brus; Unserer Dienst hat mit dem Gesund-
heitsamt in Hannover schon sehr viel über Mastitis
und Mastitis-Bekämpfung diskutiert. Wir wissen
dass Sie speziell in der Diagnostik viel Erfahrung
haben. Es ist mir darum eine ganz besondere
Freude Sie, Dr. Scheiner, jetzt zu Ihrem Vortrag
bitten zu dürfen.

Vor einigen Tagen hat das Tiergesundheitsamt Hannover sein 50-jähriges
Jubiläum gefeiert und rückblickend allen in der Land- und Milchwirtschaft
tätigen Personen aufgezeigt, was in diesen 50 Jahren an wissenschaftlichen
Arbeiten und tierärztlichen Untersuchungen geleistet worden ist. In den
weit umfassenden Rahmen dieser wissenschaftlichen Arbeiten und tier-
ärztlichen Untersuchungen fallen zu einem sehr gewichtigen Teil die
Euter- und Milchuntersuchungen.

Mit diesen Euter- und Milchuntersuchungen hat sich das Tiergesund-
heitsamt sehr eingehend befasst. Dabei kam ihm sehr zustatten, dass es
stets einen engen Kontakt zu den praktizierenden Tierärzten und zu den
Bauern hatte. Es konnte die Untersuchungen nicht nur im Institut, sondern
in der Hauptsache draussen im Stall an der Kuh vornehmen und über-
prüfen.

Bedeutende Arbeiten über die Feststellung der Rinderbrucellose mit Hilfe
der Milchuntersuchungen sind von Prof. Karsten im Tiergesundheits-
amt durchgeführt worden. Aber auch an der Erforschung, Ermittlung und
Bekämpfung des gelben Galtes ist vor allem von Dr. Ehrlich, dem
späteren Direktor der Tiergesundheitsämter Stettin und Münster, gear-
beitet worden. Das schwarze Seihtuch nach Ehrlich für die Vormelkprobe
und die nach Ehrlich modifizierte Schnellkatalaseprobe sind feste Begriffe
in der Geschichte der Milchhygiene geworden.

Ehrlich hat seine Erfahrungen und Kenntnisse bei den Milchunter-
suchungen und in der Bekämpfung der Euterkrankheiten vor allem in
Vorzugsmilchbeständen erworben, die in den 20-iger Jahren hauptsächlich
mit seiner Hilfe und Beratung aufgebaut und überwacht wurden. Bis auf
den heutigen Tag bestehen diese Vorzugsmilchbetriebe in Deutschland, die
eine rohe Milch bester Qualität in den Handel bringen.
Für diese Vorzugsmilchbetriebe führte Ehrlich eine freiwiUige, tierärztliche
Milch- und Stallkontrolle ein und arbeitete Überwachungs- und Unter-
suchungsverfahren aus, die von den heute gültigen, gesetzlichen Vor-
schriften für die tierärztlichen Untersuchungen in Vorzugsmilchbeständen
kaum abweichen.

1 Dr. A. Scheiner; Direktor des Tiergesundheitsamts Hannover; Vahrenwalder-
strasse 133, Hannover.

zugsmilchbestände monatlich, also 12 x im Jahr, auf Euterkrankheiten
durch den beamteten Kreistierarzt (Veterinärrat) kontrolliert. Er lässt,
nachdem er die Euter untersucht hat, unter seiner Aufsicht Milchproben
von jeder einzelnen Kuh entnehmen und schickt diese an das Tiergesund-
heitsamt. Die eingesandten Milchproben werden auf alle milchhygienisch
wichtigen Krankheiten zytologisch, bakteriologisch, serologisch und durch
den Tierversuch geprüft.

Da diese Vorzugsmilchbetriebe von Anfang an tierärztlich untersucht
worden sind, und sie auch bis auf den heutigen Tag eine Trinkmilch
bester Qualität in den Verkehr bringen, sind sie immer wieder von nam-
haften Milchhygienikem als ein Musterbeispiel hingestellt worden, wie
Trinkmilch bester Quaütät erzeugt werden kann. Ähnlich wie in diesen
wenigen Vorzugsmilchbetrieben — im Bereich des Tiergesundheitsamtes
sind es etwa 50 Betriebe mit etwa 1.000 Kühen — wollte man eine tier-
ärzdiche Milch- und Stallkontrolle auch in den übrigen milcherzeugenden
Betrieben einrichten.

Für diese Betriebe, die ihre Milch zur Pasteurisierung an Molkereien
lieferten, war aber im Deutschen Milchgesetz eine regelmässige tierärztliche
Überwachung der Milchkühe nicht vorgesehen. Schon 1925 hatte
Ehrlich für diese Betriebe ein freiwilliges Streptokokken-Bekämpfungs-
verfahren eingeführt. Regelmässig konnten vierteljährlich Einzehnilchpro-
ben aller Kühe eines Bestandes mikroskopisch auf Milcheiter und Strepto-
kokken im Tiergesundheitsamt untersucht werden. Der Besitzer und der
Tierarzt erhielten eine Abschrift des Untersuchungsbefundes. Bei festge-
stellter Erkrankung sollte der Besitzer die Tiere absondern und nach
tierärztlicher Anweisung behandeln.

Diesem Verfahren konnten sich die Besitzer einzeln oder über ihre Mol-
kerei anschlicssen. Ausser einer Reihe von Einzelanschlüssen, meist grössere
Betriebe, die die Arbeit ihrer Melker kontrollieren wollten, schlössen sich
3 grosse Molkereien mit allen milchliefernden Betrieben diesem Über-
wachungsverfahren an. Jedoch führte nur eine Molkerei die Unter-
suchungen 4 x im Jahr durch, während die beiden anderen Molkereien sie
2 x im Jahr durchführten.

Die praktizierenden Tierärzte oder Vertrauenstierärzte des Tiergesund-
heitsamtes suchten die Betriebe auf und prüften mit einer schwarzen
Milchprüfschale die Milch aus jedem einzelnen Viertel. Nur beim Ver-
dacht einer Eutererkrankung wurde eine Milchprobe aus dem betreffenden
Viertel und eine Milchprobe aus den anderen 3 Vierteln dem Tierge-
sundheitamt zur mikroskopischen Untersuchung eingeschickt. Dieses frei-
willige Untersuchungsverfahren zeigte aber gleich zu Beginn Mängel. Von
den Tierärzten wurden die Untersuchungstermine in den seltensten Fällen
innegehalten. Vor allem nicht in den Sommermonaten, wenn die Kühe auf
der Weide waren. Ausserdem wurde von ihnen regelmässig eine grosse
Anzahl kleiner und mittlerer Betriebe kontrolliert, in denen sie nie Euterer-
krankungen feststellten. Daher empfanden sie und die betreffenden Bauern
diese durchgeführten Kontrollen als überflüssig und lästig.
Durch den Krieg wurde dieses freiwillige Streptokokken-Bekämpfungs-
verfahren zwangsläufig wieder eingestellt. Immerhin sind vor dem Kriege
jährlich etwa 100.000 Milchproben im Tiergesundheitsamt untersucht
worden.

1950 wurde erneut im Tiergesundheitsamt Hannover mit der Einrichtung
eines Euterüberwachungsverfahrens begonnen. Dieser neue Eutergesund-
heitsdienst wurde so aufgebaut, wie ihn das Tiergesundheitsamt in Kiel
eingerichtet hatte.

Auf freiwilliger Basis konnten sich ihm wieder die Besitzer einzeln oder
über ihren Milchkon trollverein oder ihre Molkerei anschliessen. Von einer
generellen tierärzthchen Euteruntersuchung wurde aus den oben ange-
führten Mängeln und aus personellen und materiellen Gründen Abstand
.genommen. Die Tierärzte sollten erst nach erfolgter Feststellung von Eu-
terkrankheiten hinzugezogen werden. Daher wurde mit der Entnahme und
Einsendung der Einzelmilchproben das Kontrollpersonal der Milchkon-
trollvereine und Molkereien beauftragt. Sie schickten 2 x im Jahr Ein-
zelmilchproben von jeder Kuh des Bestandes zur Untersuchung auf Se-
kretionsstörungen ein.

Die Milchproben wurden im Tiergesundheitsamt nach dem Sedimentier-
verfahren beurteilt und nur die verdächtigen Milchproben auf Erreger von
Euterentzündungen untersucht.

Beim Sedimentierverfahrcn werden 10 ccm Milch 15 Minuten bei 3.500 Umdre-
hungen zentrifugiert. Anschliessend wird das Sediment beurteilt und nur bei
verdächtigem Sediment eine mikroskopische und kulturelle Untersuchung (TKT-
Agar, Blutagar, Traubenzuckerbouillon) auf Euterkrankheiten und ihre Erreger
durchgeführt.

Mit Hilfe der Abortus-Bang-Ringprobe wurden die Milchproben ausserdem auch
auf Brucellose und anschliessend ebenfalls nur die verdächtigen und positiven
Proben mit der Milchserum-Langsamagglutination nachuntersucht. Sämtliche
Milchproben wurden in Gruppen von 10—20 Milchproben auf Meerschweinchen
verimpft, um auch Kühe mit Eutertuberkulose zu ermitteln.
Es wurden also mit diesem neuen Eutergesundheitsdienst die Tierbesitzer
über 3 verschiedene Krankheiten informiert, die in ihren Beständen vor-
kommen konnten:

Da das Tiergesundheitsamt in den ersten Jahren nach der Währungs-
reform noch nicht über genügend Arbeitsrainn verfügte, konnte sich der
Eutergesundheitsdienst trotz regen Interesses der Molkereien nur ganz
allmählich erweitern. Erst 1957 war es möglich, im Hinblick auf die Trink-
milchverordnung Niedersachsens sämtliche Trinkmilch-liefernden Molke-
reien anzuschliessen. Da aber das Tiergesundheitsamt gleichzeitig auch mit
der Untersuchung der Milch- tmd Blutproben im Rahmen des freiwilligen
staatlich geförderten Brucellose-Bekämpfungsverfahren betraut worden
war, konnte der grösste Teil dieser Molkereien nur über die Untersuchung
von Kannenmilchproben dem Tiergesundheitsamt angeschlossen werden.
Sowohl die Kannenmilchuntersuchungen als die Einzelmilchuntersuchun-
gen des Eutergesundheitsdienstes wurden so durchgeführt, dass sie jederzeit
auch für die vorgeschriebenen Brucelloseuntersuchungen im Rahmen des
Brucellose-Bekämpfimgsverfahrens mit ausgewertet werden konnten.
Die Untersuchung von Kannenmilchproben im Rahmen des Eutergesund-
heitsdienstes sollte allerdings nur eine Notlösung sein. Jedoch wurden wir
in der folgenden Zeit davon überrascht, dass vor allem auch die Molke-
reien und die Oberkontrollassistenten Gefallen an diesem leicht durch-

zuführenden Verfahren bekamen und auch Molkereien, die vorher die
Einzelmilchuntersuchungen in ihren Einzugsgebieten durchgeführt hatten,
nun ebenfalls zur Kannenmilchuntersuchung übergingen. So waren z.B.
1955 119 Molkereien durch Einzehnilchuntersuchungen dem Tiergesund-
beitsamt angeschlossen. 1963 sind es nur noch 77 Molkereien, während
117 Molkereien über die Untersuchung von Kannenmilchproben dem
Eutergesundheitsdienst angeschlossen sind.

Von Anfang an waren wir im Tiergesundheitsamt mit dieser Entwicklung
nicht einverstanden, da die Untersuchung der Einzelmilchproben viel ge-
nauere Ergebnisse lieferte als die Untersuchung der Kannenmilchproben
mit Hilfe des Sedimentierverfahrens.

Auf der anderen Seite waren aber die Entnahme und Einsendungen von
Einzelmilchproben aus sämtlichen Beständen und ihre Untersuchung aus
Personalmangel in den Molkereien und in den Instituten nicht durch-
führbar. Wir hatten auch statistisch festgestellt, dass die Probenehmer
in jedem Jahr etwa 90% der Bestände umsonst aufgesucht und hier die
Proben entnommen hatten, da Jahr für Jahr bei nur etwa 5—10% der
Bestände Sekretionsstörungen festgestellt wurden. Dieser Leerlauf bei der
Probe-entnahme und bei der Untersuchung im Institut hat uns viel be-
schäftigt.

Nimmehr stehen wir mit unserem Eutergesundheitsdienst an einer Wende.
Personalmangel in den Molkereien und in den Instituten zwingen zu einer
neuen Lösung; ebenso die z. T. unbefriedigenden Ergebnisse der Kannen-
milchuntersuchungen mit Hilfe der Sedimentier- und Mikroskopierme-
thode. Die Vorwürfe, die verschiedentlich geäussert worden sind, dass
dieser Eutergesundheitsdienst nur ein Feststellungsverfahren sei, zwingen
ebenfalls dazu, uns auch mit dem Einbau einer gezielten tierärztlichen
Überwachung und Sanierung der an Mastitis erkrankten Bestände zu be-
fassen.

Ausschauhaltend nach einer anderen Möglichkeit der Durchführung eines
Eutergesundheitsdienstes haben wir festgestellt, dass es in Deutschland sehr
unterschiedliche Eutergesundheitsdienste gibt:

1. Die monadiche tierärztliche Untersuchung der Euter in Vorzugs-
milchbeständen. Einsendung von Einzelmilch- oder Gruppenmilch-
proben.

2. Eine vierteljährliche tierärztliche Untersuchung der Euter in Mar-
kenmilchbeständen. Einsendung von Einzelmilchproben.

3. 2 mal im Jahr durchgeführte tierärztliche Untersuchung der Euter
in Markenmilchbeständen und versuchsweise in einem politischen
Kreis. Einsendung von Einzelmilchproben aller milchgebenden
Kühe oder nur der klinisch verdächtig erscheinenden Kühe.

Alle bis jetzt genannten derärzdichen Eutergesundheitsdienste kamen nur
bei einem geringen Teil der milchliefernden Bestände zur Durchführung.
Bei sämdichen milchlieferden Beständen oder nur bei den Trinkmilch-
liefernden Beständen der Molkereien oder bei Mitgliedern von Herdbuch-
gesellschaften werden durchgeführt:

4. Einsendung von Einzelmilchproben 2mal im Jahr durch Probe-
nehmer, Hinzuziehung des Tierarztes nur zur Behandlung erkrank-
ter Kühe.

5. Einsendung von Einzeliniichproben Imal im Jahr, Einsendung von
kannenmilchproben einmal im Jahr. Die Einsendung erfolgt durch
Probenehmer.

7. Untersuchung der Milch an der Kuh durch Kontrollassistenten mit
Hilfe der schwarzen Schale, der Katalase-Thybromolprobe oder des
Indikatorpapiers. Einsendung von Milchproben nur in Verdachts-
fällen. Hinzuziehung des Tierarztes auch in diesen letzt genannten
Eutergesundheidtsdiensten nur zur Behandlung der erkrankten
Kühe.

Daraus ist zu ersehen, das es keine Einheitlichkeit in der Durchführimg
des Eutergesundheitsdienstes und auch keine Einheitlichkeit in der Er-
kennung und Bekämpfung der Euterkrankheiten gibt.

Jedem dieser Eutergesundheitsdienste sind aber Grenzen gesetzt.
Bei den zu untersuchenden Vorzugsmilchbeständen, die monatlich unter-
sucht werden müssen, ist es die begrenzte Anzahl der Bestände.
Bei den zu untersuchenden Markenmilchbeständen ist es ebenfalls die be-
grenzte Anzahl der Bestände, da für eine viertel- bzw. halbjährliche Über-
prüfung der gesamten milchliefernden Bestände die vorhandenen Tier-
ärzte nicht ausreichen würden, um diese Untersuchungen durchführen zu
können.

Einer solchen allgemeinen tierärztlichen Euterüberwachung Vv\'ären aber
auch im Hinblick auf den Milchpreis und die Honorierung der Tierärzte
Grenzen gesetzt.

Grenzen sind aber auch einem allgemeinen Eutergesundheitsdienst ge-
setzt, der sich auf die Einsendung von Einzelmilchproben durch Probe-
nehmer und deren Untersuchung in einem Institut beschränkt und nur in
den Fällen, wo Sekretionsstörungen in Beständen gefunden werden, den
Tierarzt einsetzt; denn leider stehen uns z. Zt. die erforderlichen Probe-
nehmer auch nicht mehr so ohne weiteres zur Verfügung. Personalmangel
und erhöhte geldliche Forderungen erschweren auch diesen eingeengten
Eutergesundheitsdienst.

Grenzen sind aber auch einem allgemeinen Eutergesundheitsdienst gesetzt,
wenn man ganz klar und nüchtern überrechnet, wieviel Milchproben ein
Institut täglich untersuchen kann. So müssten z.B. bei einer 2mal im Jahr
erfolgenden Einsendung von Einzelmilchproben aller milchgebenden Kühe
vom Tiergesundheitsamt Hannover 1.200.000 Proben untersucht werden.
Das wären rund gerechnet 5.000 Proben pro Tag, wobei man bedenken
muss, dass aber die Einsendungen fast ausschliesslich in den Wintermona-
ten erfolgen, somit also in diesen Monaten etwa 8. - 10.000 Proben pro Tag
imtersucht werden müssten. Hier taucht dann immer wieder der Gedanke
auf, ob dieser Riesenaufwand wirklich erforderlich ist, wenn bei den Unter-
suchungen Jahr für Jahr nur in etwa 10% der Bestände Sekretionsstörun-
gen festgestellt werden.

Idealzustand wäre selbstverständlich eine monatliche Untersuchung von
Einzelmilchproben aus allen milchliefernden Betrieben, wie das in Vorzugs-
milchbeständen der Fall ist. Das wäre ohne weiteres möglich, wenn es sich
dabei um einige 100 oder 1.000 Bestände handelte. Es geht aber beim
Eutergesundheitsdienst des Tiergesundheitsamtes um etwa 660.000 Kühe in

nahezu 115.000 Beständen. Diese Bestände müssten alle wegen des verbor-
genen Charakters der Sekretionsstörungen häufig aufgesucht und die Milch
der Kühe untersucht werden, da sie ja von heute auf morgen Sekretions-
störungen aufweisen können, die kürzere oder längere Zeit anhalten und
auch wieder verschwinden können.

Müsste denn nicht ein allgemeiner tierärztlicher Eutergesundheitsdienst ge-
rade daran scheitern, dass ein Tierarzt höchstens 3 Betriebe am Tage auf-
suchen könnte, wenn er vorbildlich und einwandfrei arbeiten möchte; denn
er braucht das steril entnommene Anfangsgemelk von jeder einzelnen Kuh
für die Untersuchung auf Sekretionsstörungen und muss doch warten, bis
die Kühe ausgemolken sind, um auch die Euter gut durchtasten zu können.
Wie soll er aber in den Sommermonaten diese Untersuchungen durch-
führen?

Es stünden ihm also nur die Wintermonate mit der ohnehin sehr grossen
Arbeitsüberlastung zur Verfügung. Mehr als 1 mal könnte er die Bestände
in dieser .Zeit nicht untersuchen.

Ähnlich ist es auch mit der Durchführung eines Eutergesundheitsdienstes,
bei dem 2mal im Jahr — weil auch hier die Weidemonate ein häufigere
Probeentnahme nicht zulassen — die Probenehmer der Milchkontrollver-
eine oder Molkereien in jedem Bestand die Proben entnehmen, und bei
denen das Ergebnis in 90% der Bestände und mehr negativ lautet. Dabei
stellt man sich immer wieder die Frage, ob eine zweimalige Untersuchung
ausreicht, um auch wirklich sämtliche Sekretionsstörungen erfassen zu kön-
nen und ob dieser starke Leerlauf in 90% der Bestände nicht doch ver-
mieden werden kann.

Dieser letztere Gedanke hat uns schon seit 1951 bewegt. Gerade in dieser
Zeit wurde die ABR-Probe nach Fleischhauer mit in den Euter-
gesundheitsdienst eingebaut und hat sich hierbei und später bei der Durch-
führung des Brucellose-Bekämpfungsverfahrens vorbildlich bewährt. War
es da verwunderlich, dass uns der Gedanke nach einem ähnlichen Ver-
fahren zur Feststellung der Euterentzündungen kam? Immer wieder wurde
dieser Gedanke von Karsten vorgetragen rmd hat vms auch unablässig
beschäftigt, bis wir dann durch den Whiteside- imd den Galifornia-Mastitis-
Test zu Versuchen angeregt wurden, die aber scheitern mussten, da wir
diese neue Untersuchungsmethoden anfangs falsch bewerteten.
Erst durch Dedié, Kielwein, Christ, Leidl und S c h a 1 m wur-
den wir erneut zu Versuchen angeregt.

Wir waren aber erst auf dem richtigen Wege, als wir die Dissertation von
Herrn Kollegen Jaartsveld erhielten. Seine Brabanter Mastitis-
Reaktion fanden wir geradzu ideal für unseren Eutergesundheitsdienst, da
sie in so vorbildlicher Weise auch mit der Untersuchung der Milchproben
auf Brucellose mit Hilfe der .A.BR gekoppelt ist. Zudem ermöglichen die kon-
struierten Apparate eine schnelle Entnahme und Untersuchung der Milch-
proben. Im Vergleich zu den bis jetzt von uns durchgeführten Eutergesund-
heitsdienstes wird die BMR auch kostenmässig am besten abschneiden.
In einem Grossversuch, der sich über ein halbes Jahr hinzog, hat Kollege
Matschullat 12.000 Kannenmilchproben und Einzelmilchproben ver-
gleichend untersucht.

Beurteiling Anzahl der Anzahl suchung Mastids-
B.M.R. Betriebe der Kühe o.B. Sekretions- problem

Abschliessend stellt er aufgrund seiner Untersuchungen mit der Kannen-
milch-BMR fest:

1. Dieses zwar grobe doch in der Praxis für Massenuntersuchungen ge-
eignet erscheinende Verfahren ermöglicht es, in kurzer Zeit auf ein-
fache Weise einen Überblick über die Masdtishäufigkeit in einem grös-
seren Gebiet zu gewinnen.

2. Die Einfachkeit der Technik der BMR erlaubt ohne grösseren Aufwand
kurzfristige Wiederholungsuntersuchungen, so dass man ständig einen
Überblick über alle milchliefernden Betriebe hat.

Gleichzeitig kann man durch Zwischenschaltung der ABR-Probe nach
Fleischhauer die heute üblichen Intervalle zwischen den Brucel-
lose-untersuchungen wesentlich abkürzen.

3. Die BMR ermöglicht mit relativ grosser Sicherheit die Ermittlung be-
sonders der Betriebe, in denen die Masdtis ein Herdenproblem dar-
stellt.

In einem Eutergesundheitsdienst, dem als Basis die Brabanter Mastitis-
Reaktion gewissermassen als Sortiermethode zugrundeliegt, wird es so-
mit möglich sein, den eigendichen Mastidsbetrieben erhöhte Aufmerk-
samkeit zu schenken.

Dort kann der zuständige Tierarzt mit Hilfe klinischer Euteruntersu-
chungen und bakteriologischer Prüfung von Viertel- und Einzelgemelks-
proben gezielt nach den Ursachen forschen und mit entsprechenden
Massnahmen die Sanierung vorantreiben.

Wir sind daher der festen Überzeugung, dass wir mit Hilfe der BMR die
Bestände mit einem Mastitisproblem mit allergrösster Sicherheit ermitteln
können.

In Duitsland worden reeds jaren bedrijven op mastitis onderzocht. De z.g. „Vor-
zugsmilchbetriebe", die rauwe melk voor consumptie mogen afleveren, worden elke
maand door een inspecteur van de Veeartsenijkundige Dienst (Kreistierarzt) be-
zocht.

Naast khnisch onderzoek van dc uiers worden koe-mclkmonsters onderzocht. Dit
betreft echter maar 50 bedrijven met 1000 runderen in een gebied met 115.000
veehouders.

Daarnaast is ook wel, op vrijwillige basis, door veehouders direct of via een zuivel-
fabriek aan een dergelijke mastitiscontrole deelgenomen. Halfjaarlijkse tot kwartaals-
gewijze bezoeken met klinische controle en onderzoek van koemonsters geeft bij ±
10% een positieve bevinding.

De monsters worden op het laboratorium bacterioscopisch en zonodig ook bacterio-
logisch onderzocht.

Terwijl in het gebied van de Gezondheidsdienst Hannover deze bezoeken door een
praktizerende dierenarts of een dierenarts van de Gezondheidsdienst worden gebracht,
komt het in Duitsland ook voor dat hulpkrachten zijn ingeschakeld. Het klinisch
onderzoek vervalt dan.

Afgezien van het feit dat deze methodiek duur is, wordt bij gebrek aan arbeids-
krachten de uitvoering ervan zowel op het bedrijf als in het laboratorium onmogelijk.
Dit zou nog sterker gelden indien het onderzoek algemeen zou worden.
In Hannover was men reeds in 1957 begonnen met een sedimentonderzock van bus-
melkmonsters. Hoewel deze dienst zelf het koemonster-onderzoek prefereert, waren
de zuivelfabrieken enthousiast over dit busmonsteronderzoek. Alleen de positieve be-
drijven werden bezocht en nader onderzocht; de hoeveelheid werk verminderde sterk
en het aantal positieve bevindingen nam daardoor sterk toe. Naast het sediment-
onderzoek van busmonsters werd nadien ook de C.M.T. beproefd voor onderzoek
van de busmonsters.

De laatste jaren werd de B.M.R. toegepast. Een vergelijking van de B.M.R. op bus-
monsters met onderzoekingen op de desbetreffende bedrijven gaf een goede over-
eenstemming.

1, de B.M.R, een grove doch praktische methode voor massale onderzoekingen is;
op een eenvoudige manier kan een overzicht, betreffende het voorkomen van
mastitis bij runderen op de bedrijven in een bepaald gebied, verkregen worden,

2, de eenvoud en geringe kosten van het onderzoek een frequent busmonsteronder-
zoek rechtvaardigen.

Het B,M,R, onderzoek kan, indien nodig, geschieden met melkmonsters waarop
de A,B,R. reeds werd uitgevoerd,

3, men met de B.M.R., met relatief grote zekerheid de bedrijven die met een
mastitisprobleem kampen, kan opsporen. Aan deze bedrijven kan men dan
speciale aandacht gaan besteden.

Dr. Brus: May I now introduce to you Dr.
Richter from Miinich, Germany.
He is director of the Animal Health Service
of that country and has the service of a mo-
dern laboratory where work is done in blood-
group typing of cows and pigs.
He also has carried out investigations for some
years in mastitis.

It is a well known fact that mastitis is a primary cause of cattle disease in
all dairying countries. Every 3rd or 4th cow is assumed to have suffered
from it one or another time of her life. The fact of mastitis being on the
increase instead on the decrease makes it even more serious.
Eradication of tuberculosis and brucellosis and coital infections being
finished in the herds, there was a demand for systematic mastitis control
in Bavaria.

Only if the cause and the nature of a disease is known, a control can be
planned. There are a series of mastitis definitions. The definition of mastitis
being a wearing off disease of cows seems to me the best one.
Thus the most effective method for preventing this wearing off disease
would be to make milk production cease altogether. This being impossible
there is no other way but to look for any factors causing the disease and
consequently eliminating them. This is the situation we shall have to face
in mastitis control.

High milk production being the real cause of the disease cannot be chang-
ed. Our only possibility consists in establishing conditions under which the
high productivity can be kept without any suffering of the organism or the
udder.

This general explanation already marks the limits and possibilities of mas-
titis control.

parasitic diseases, traumata or hormonal influences);
genetic factors and
bacteria.

In Bavaria we have established a mastitis control program since 1959. We
think that this program should consist of:

1 Dr. O. Richter; Direktor des Rindergesundheitsamts; Haydnstrasse II, München
15, Deutschland.

1. an efficient milk control service. This examination should make pos-
sible a perpetual and general supervision of the udder;

2. the employment of trained technicians being capable of diagnosing
the predisposing factors and of preventing the causes;

3. a systematic and planned medicamental therapy with the aim of eli-
mination of the mastitis germs out of the udder.

A current and general milk control is only possible with the help of a rapid
and cheap information test. For this purpose we use the California Mas-
titis Test (C.M.T.) for years and test with this method the churn milk 3 oi
4 times a year.

Up to date our experience shows that it is possible to detect mastitis herds
by means of a churn-milk-test. Since one year we use in addition for a com-
parison the Brabant Mastitis Reaction (B.M.R.), developed by Dr. Jaarts-
veld. We think to be able to detect more herds affected with mastitis by
means of the C.M.T. than by means of B.M.R.

Of course, the churn-milk-test must be considered only as an information
test. For a systematic mastitis control a cytological and bacteriological exa-
mination of milk-samples of each cow is necessary. This work is conducted
by trained technicians, who collect milk-samples from each cow in herds
suspicious of mastitis and deliver it at the laboratory of the animal health
service.

We have tried to collect simultaneously informations on the housing con-
ditions. This proved to be very useful, for systematic mastitis control should
be initiated only in those herds, where the milking and housing conditions
of the animals and the interest of the owner justifies the therapy. This is
very important because we must be aware that every failure in therapy will
affect the reputation of the udder health service.

Cows with severe pathological alterations of the udder don\'t pay for any
therapy and should be culled.

For bacteriological examination we take a sample of each quarter of the
udder under conditions as sterile as possible. Quarter samples have the fol-
lowing advantages as compared with milk-samples from all quarters mixed
in one tube:

1. they allow a comparison among the udder quarters and a better dis-
dnction between physiological and pathological affects in the udder;

3. a further advantage of examination milk from the single quarters
is a rapid, simple and exact diagnosis, because there is no conta-
mination by unspecific germs.

As soon as the result of cytological and bacteriological examination and
the result of herd inspection is available, we are able to decide what to do
further. According to the conditions encountered either veterinary therapy
or an improvement of milking technique, feeding and housing is recommen-
ded. In most cases both is necessary. The succes of therapy is controlled
at least twice by means of a single milk test. Animals resistant to therapy
have to be culled.

It is also of great importance to give adequate and systematic information
to the owners of herds on the influence of correct milking, feeding and

keeping conditions on the udder function by means of lectures and publi-
cations in agricultural journals.

It appeared that among all factors causing mastitis bad milking technic,
especially hygienic and technical defects of milking machines take the first
place. I am sorry to say that in my country though many milking machines
are sold, there is no well organized customers service. If udder health ser-
vice is to be successful we have to take care of this problem.
Veterinary therapy of the udder by means of antibiotics is only reasonable,
when the predisposing factors can be abolished simultaneously. Mastitis
eradication proved to be most successful with Str. agalactiae infections,
provided the eradication program was continuously and thoroughly super-
vised. With staphylococcus infections a complete elimination of the germs
was not obtainable, but in many herds a distinct decline in the incidence of
the mastitis was obtained.

In my opinion with our present knowledge an eradication of mastitis is not
to be expected. But if we are successful in curing herds and in controlling
the total incidence of mastitis we shall be able to assume that the mastitis
control program has been justified.

In alle landen waar melkvee wordt gehouden, is mastitis een belangrijke ziekte.
Men neemt aan dat 25 a 35% van alle koeien eens in hun leven mastitis hebben.
Spreker vindt dit probleem zo belangrijk omdat bovengenoemd percentage toeneemt.
In Beieren, waar tuberculose, abortus-Bang en de z.g. dekinfecties zijn uitgeroeid,
vroeg de veehouderij om een systematische aanpak van het mastitisprobleem.
Om een ziekte te kunnen bestrijden is het noodzakelijk dat men de aetiologie van die
ziekte kent. In dit kader zou mastitis een slijtageziekte genoemd kunnen worden. De
meest „effectieve methode" zou dan zijn om met het melken te stoppen. Men za!
de koeien moeten gaan houden op een manier waarbij het mogelijk is, hoge produktie
met goede gezondheid te combineren.

De factoren die het ontstaan van mastitis bevorderen, zijn slecht melken, slechte
hygiënische- en stalomstandigheden, biologische omstandigheden als trauma, hor-
monale invloeden, parasitaire en infectieuze aandoeningen en verder genetische
factoren.

In Beieren is men in 1959 begonnen met opbouw van een programma ter bestrijding
van mastitis. Hierbij denkt men aan: controle op de uiers en het melken, getrainde
hulpkrachten die de predisponerende factoren kunnen onderkennen en dienaan-
gaande adviezen kunnen geven, een sytematische medicamenteuze behandeling om de
uicrbacteriën te elimineren. Dc Gezondheidsdienst in Beieren gebruikt voor het mas-
sale nu-lkonderzoek reeds jaren dc C.M.T. en sinds een jaar daarnaast de B.M.R.
Met de C.M.T. zijn volgens spreker meer positieve bedrijven te vinden dan met
de B.M.R. Het massaal busmelkonderzock kan alleen als eerste informatie dienen.
Voor een systematische mastitisbestrijding acht spreker cytologisch en bacteriologisch
onderzoek van kwartiermelkmonsters noodzakelijk. Bovengenoemde hulpkrachten
zouden bij de monstrmame kunnen worden ingeschakeld.

Medicamenteuze behandeling acht spreker alleen verantwoord, indien de veehouder
en de bedrijfsvoering aan bepaalde kwaliteiten voldoen, anders heeft de therapie geen
resultaat en loopt de „uiergezondheidsdienst" gevaar haar naam te verliezen.
Koeien met ernstige afwijkingen dienen niet behandeld, doch geslacht te worden.
Spreker geeft de voorkeur aan het onderzoek van kwartiermelkmonsters boven koe-
monsters.

Na het onderzoek van een bedrijf wordt beslist wat te doen. Veelal komt dit neer
op verbetering van het melken en een medicamenteuze behandeling. Nadien vinden
controles plaats om het resultaat te beoordelen.

Voorlichting via lezingen en publikaties vindt spreker zeer belangrijk. Naar zijn me-
ning zijn slecht melken en defecten aan de melkmachines dc voornaamste oorzaken
van mastitis, In West-Duitsland is volgens spreker wel een goed verkoopapparaat
voor melkmachines, maar geen goede service in de ruime zin van het woord.
Medicamenteuze behandeling heeft alleen nut als de predisponerende factoren ook
weggenomen worden. De Str. agalactiae kan mogelijk uitgeroeid worden, de stafylo-
kokken echter niet. Uitroeiing van mastitis achtte spreker niet mogelijk, wèl het
terugbrengen van het aantal mastitis-gevallen tot een redelijk minimum.

following the lectures by Dr. Scheiner and Dr. Richter
Question: Prof. Van der Schaaf (The Netherlands) :

Did you use sodium lauryl sulphate with an alkaline reaction of pH 12
or did you use sodium lauryl sulphate of pPI 6?

I cannot understand how you found a difference between the results of
both tests and I should like it very much that you explain the difference
in these results.

In our district we have the so called „Fleckvieh" and we have many
troubles with staphylococci- and streptococci-mastitis. These two kinds
of mastitis occur both in about 50 percent. The total cell-count of the
staphylococci-mastitis in our „Fleckvieh" is very low in comparison with
the other breeds of cattle.

Maybe the reaction of the udder to a staphylococci-infection is much lower
than the reaction in other catde. With staphylococci-mastitis the total
cell-count according to P r e s c o 11 and Breed is about 400.000 till
500.000 cells. This kind of mastitis we cannot find when we examine the
can milk samples with the B.M.R. When we examine the quarter milk
samples, we get good results with the B.M.R.

Your next question was how we compared the C.M.T. and the B,M,R,
and I must say we have not done that at the same time. The difference
in time v/as about 40 days. With the C.M.T. reaction we got about 10
to 12% positive herds by examination of can-samples and with the B.M.R.
test we got about 5 to 6% positive herds.

Some time ago colleague Jaartsveld was in Munich and it was
noticed that the pH was low, about 5 to 6. Afterwards we brought the
pFI to 12. With this reagent we examined 2 or 3 dairy factories, but we
now found also more positive reactions with the Schalm-test than with
the B.M.R.

It was remarkable that in Munich the results of the B.M.R, of quarter
milk samples were very good, but the results of can samples were not
right. It is important to get a good correlation between the total cell-
counts of milk and the B.M.R. I cannot explain why the B.M.R. of the
quarter milk samples gave good results and the B.M.R. of the can milk
samples gave bad ones. And what about milk samples with a total cell-
count of 400.000 per ml. out of which staphylococci are isolated?
Should one consider this milk as mastitismilk, or should one consider these
staphylococci as contaminants?

I will emphasize the necessity to use in all German laboratories the same
methods and the same reagent.

During the demonstration at the farm we have seen the following in-
teresting facts. We have seen a cow with a staphylococci mastitis, one
quarter was atrophied.

The C.M.T. reaction was made from all the quarter milk samples. Wc
have seen that the C.M.T.-reaction of the atrophied quarter was less
than the reaction of the other quarters. The bad quarter with the chronic
disease was less positive than the other three quarters, with more acute
mastitis. Maybe from the atrophied quarter there are less leucocytes in
the milk.

It would be very interesting for us to know. Dr. Jaartsveld, in which
cases this method does not give good results.

Last Monday we did the B.M.R.-tcst of the 4 quarters of this cow and the
B.M.R. test of all quarters was three or four points.

During the demonstration we should have done the C.M.T.-rcaction and
B.M.R.-test at the same time. In that way wc could have tested the

B.M.R. better. I agree with you that the C.M.T.-reaction of this atrophied
quarter was less positive than the C.M.T.-reaction of the other quarters.
It is possible that the total cell-count this morning at the moment wc
examined the atrophied quarter of the ccw was less than some days ago.

We have had the following experience with staphylococci mastitis in cows
in our district, in which we have „Fleckvieh" and „Braunvieh". After the
tuberculosis-campaign we also have black-white and red-white cows.
We have compared a great number of results of milk-examinations. The
„Fleckvieh" has about 0,4 percent Streptococcus agalactiae-iTilecXian?,,
The black-white about 3,5 percent and the red-white cows have about 9
pcrcent of Streptococcus agalactiae-iniections. The cases of staphylococci-
mastitis wc have divided into two groups.

The first group are those milksamples out of which we have isolated
Staphylococci and which milksamples have no more than 150.000 cells
pro ml., the C.M.T.-reaction being negative.

The second group arc those milksamples out of which wc have isolated
Staphylococci, the milksamples have high cell-counts and a positive

The „Fleckvieh" cows, 11 animals, have a staphylococci-mastitis with
a high cell-count, while 7 animals show a staphylococci-mastitis with a
low cell-count.

In the red-white breed this figures are 8 : 1. I agree with Dr. Richter
that the „Fleckvieh" has a less inflammatory reaction with a staphylococci-
infcction than the other breeds of catde.

Dr. Bra tlie said that the C.M.T.-rcaction in the milksample of the
atrophied quarter was less positive than the C.M.T.-rcaction of the other
quarters. I think it\'s quite nonrial, during the period the udder has
mastitis, the cell-count will differ from time to time as well.
Question: Dr, K r a u s (Germany) :

In order to compare the mastitis reaction it should be desirable to make
the method uniform in order to detect the same cell-count of the milk-
samples. The question is, what test we have to use; Schalm-reaction,
catalase-rcaction, B.M.R.-test or some other kinds of reactions.

This problem is very important and therefore I ask this meeting: how
to solve ihis problem?

We have seen the very good standardized method to detect the total cells
in the milk with the help of the B.M.R.-test at Boxtel. Professor K a s 11 i
in Switserland used the modified Whiteside-reaction,
At Aulendorf we used the Schalm-test, but we take this reaction in tubes.
I think it would be more important that each of us continues, to sec,
what is the most suitable method for detecting abnormal milk.
I agree with Dr, K r a u s it would be excellent to use the same methods
in all countries. I should prefer to wait one year or some more years.
The time will come quick enough that wc can judge the best method.

In order to compare the different methods to detect bad milk I should
like to tell you which method we use for this purpose.
About every two months about 35 milksamples arc sent from one of the
official laboratories to all laboratories of the Provincial Health Services,
the University of Utrecht and the Central Veterinary Institute at Rot-
terdam, .All laboratories perform the bacteriological examination, deter-
mine the total cell-count and perform the B,M,R,- test.
In this way it is possible to compare the results of the different mediods
from the different laboratories. Maybe it is also possible in Germany or
other countries to do likewise. In this way you can proceed in standardizing
laboratory methods.

I think the problem is not very simple, bccause if we make comparisons
between the different methods, we compare only the cell-count.
As I said before, in our country wc have cases of clinical staphylococci
mastitis in „Flcckvieh" with a lower content of cells than streptococci
mastitis in for example the black-white cattle.

We have found clinical staphylococci mastitis with a cell-count of 500.000
till 600,000 pro m,l. If we compare these methods, we compare the cell-
counts and that is not the problem.

I should like to ask Dr, Richter what he means by the word cell-
count. Do you mean a total cell-count or the number of leucocytes
pro millilitrc of milk?

So we should better use the term leucocytc-count, bccause I feel that much
of the trouble, arising in diagnostic methods, comes from the misunder-
standing of the terms: total cell-counts and Icucocyte-counts, I should
like to hear the opinion of this congress.

Mr, Chainnan, I should like to ask colleague Jaartsveld: what does
he understand by the word cell-count?

Wc have the opinion, that in many cases it is very difficult to differentiate
between different kinds of cells. Therefore, for standardization of the
B,M,R,-test we have used the total crll-count.

If one starts an eradication programme for a
disease, you first need a method of diagnosis
which is as simple, cheap and reliable as pos-
sible. In the case of mastitis this method must
give the opportunity to divide herds into po-
sitive and negative ones. As regards the posi-
tive herds you have to investigate the indivi-
dual animals, sometimes according to the
5ame method of investigation, but mostly with
other methods or combinations.
In the fight against Brucellosis in cattle the
Abortus-Bang-Ringtest is such a method.

Periodical investigations of can-milk-samples can point out the positive
herds. In these herds we use the serum agglutination and the Complement
Fixation Test to detect the infected animals.

It must not be necessary to talk long about the borders between "negative,
suspicious or positive" results of mass tests. All reactions near borders give
too big a chance to make a false conclusion and to take a wrong decision.
Conclusions may therefore only be drawn from real positive and real nega-
tive reactions.

If one declares a herd infected after one investigation, one has to be sure.
If one likes to declare a herd free one can only do so after several perio-
dical investigations. These investigations have to be done frequently. If
there are suspicious reactions, decisions must be made in the following in-
vestigations.

Can we find such a simple method and can we use it as a „police-dog"
in the fight against mastitis?

Cultural investigations of can- or tank-samples on the dairy-factories will
give too many false positives, it will sometimes reach a 100% in tank-
samples on the milk-delivery. Cultural investigations of cow- or quarter-
samples will give purer results. But this sample-taking requires much work
and labour is very expensive and in our country it will even become more
expensive.

If we started a follow-up programme i.e. of cow- and quarter-samples cul-
tural investigations every two months, it would be too expensive.
If you investigate once or twice a year, you miss a lot of positives. You
will come much too late and it is still too expensive.

Bacterioscopic investigation of sediments of tank- or can-samples in the
dairy-factories do not require so much work but yet it is too expensive to
do it frequently.

We hope and think that the method of colleague Jaartsveld which is
the Brabantic Mastitis Reaction, will help us in mass diagnosing of mas-
titis, in dividing herds in groups with and without mastitis problems. In

1 D. H. J. Brus D.V.Sc. ; director of the Animal Health Service of the Province of
North-Brabant; Rechterstraat 80, Boxtel; The Netherlands.

1. are there seasonal differences in the percentage of herds with one or
more positive can-samples?

The tables I and II show that there are more herds with positive reactions
in spring and summer than in autumn and winter.

We see that these seasonal differences are more pronounced in the western
part (Frisian cattle) than in the eastern part (Meuse-Rhine-Yssel cattle).
We don\'t think that this is a difference in breed, but in the eastern part cal-
ving is more spread (1962: in the western part 25,4% of the first insemi-
nations in July till December, in the eastern part 41,3%). May be you find
more mastitis in cows in the first months of lactation. This can be the
reason, but we have to do more investigations about this question.
There are also local differences as the maps show. These differences are
not constant, but we find a trend in it. There are dairy-factories which are
always black (as high as 20% herds with positive reactions) and there are
some which are always white (less than 10%).

We can ask ourselves, whether it are always the same herds in which we
find positive reactions?

We looked for them in three dairy-factories for the year 1962.,We have
taken three dairy-factories with successively a few, moderate and many po-
sitive reactions.

I considered these questions with Mr. K e r k h o f and Mr. P e ij n e n-
b u r g, directors of the "Regionale Organen" which means organisations of
dairy-factories, which look for the quality of the milk.
As you know, there is a difference in the price of good and bad milk; the
difference is now about 10%, but this will grow in future.
The two gentlemen told me, „we see some agreement between your "mas-
titis-maps" and our "milk-quality-map" especially in the extremes".
Up till now, only in the western part of Holland are mastitis-tests incor-
porated with these payments orders (measures) and in this area the milk-
quality investigations are:

For every test, the milk can get one to three points from good to bad. The
final classification is the highest number of one of the three tests.

The methylene blue reduction test is done as follows: to 20 ml of milk is added
1 ml. of a methylene blue solution (1 iablet of 14 mgr. methylene blue chloride
to 400 ml. of water). The tubes are placed in a waterbath of 37° C.
Interpretation: Decolorization within I/2 hour class 3, not decolorated in 3 hours
class 1, dependent on the outside temperature.

Most of the bacteria present in an inflamed quarter don\'t influence this
methylene blue test.

The sediment test is done to detect dirt in the milk. But a large amount of
cells (leucocytes) can also give a positive test.

The organoleptic test of can-samples also v^\'ill be influenced in very few
cases only by mastitis-milk.

There will not be a direct correlation between the quality tests we use now
in this country and the occurrence of mastitis at a farm. If we find a cor-
relation between the quality of the milk delivered by farmers and the occur-
rence of mastitis in their herds, this correlation must be indirect. We hope
to test this in the coming year for the whole province.
Up to now we have done this at four dairy-factories. These four factories
have 1629 farmers.

In order to compare the milkquality and the occurrence of mastitis, we
have given every herd a figure for milkquality and a figure for the B.M.R.
test. For the milkquality there are 26 investigations of can-samples in a
year. For this comparison we have taken the investigation in the days
nearest to the day of B.M.R. testing. For the quality, the farmer gets every
time a figure 1, 2 or 3. We have taken the average of the 4 investigations
(also a farmer with successively 1, 2, 1 and 2 class of milk gets the fieure
6:4 1,5).

We have done the same for the mastitis (B.M.R. test) as follows: all can-
samples with a negative B.M.R. get 1 point, one or more dubious ones
get 2 points, one or more positive cantests get 3 points. The sum-total of
points of the mastitis investigations divided by the number of investigations
is called the Total Mastitis Investigation:

Table III shows that from 1629 herds there are 974 which are negative
in B.M.R., 422 which are dubious and 233 which are positive. The per-
centage of first class milk decreases from 44,8 to 39,1 till 30,9.
The difference between the first- and second group is significant on a 5%
level, the same holds good for the difference between the second and third
group. When we look for the difference between the first and third group,
it is highly significant (0,1% level).

The percentages of second class milk in all the three groups is almost the
same. The third class milk increases from the negative to the positive mas-
titis group.

Here the difference between the negative and dubious group is significant
on a 5% level and the difference between the dubious and positive group is
highly significant (0,1% level), the same holds good for the difference be-
tween the negative and positive group.

b. The fight against mastitis should be incorperated in the fight for better
milk-quality. Not only in an organized way (payment of milk) but also
in a technical way.

c. Penicillin and other antibiotics don\'t give us a solution for the mastitis
problem. They can only help to diminish the direct results of the in-
flammations. I will call it "Tranquillizer for the farmer".

Om een ziekte massaal te kunnen bestrijden, moet men vooreerst over een diagnostiek
beschikken, die zo betrouwbaar, eenvoudig en goedkoop mogelijk is.
De Abortus-Bang Ring-reactie — A.B.R. — was een reactie die aan bovengenoemde
voorwaarden voldeed in het kader van de Abortus-bestrijding. Met behulp van deze
methodiek is men in staat om door een onderzoek van busmelkmonsters bedrijven
op te sporen die verdacht of positief zijn t.o.v. Br. abortus.

De runderen van deze bedrijven kunnen nader onderzocht worden met behulp van de
serum-agglutinatie, complement bindingsreactie, A.B.R., enz. Bij elke massa-diag-
nostiek kunnen de uitslagen uitgesproken negadef, dubieus en uitgesproken positief
uitvallen. Alle reactie-uitslagen die dubieus uitvallen, kunnen leiden tot foutieve
conclusies. Het is daarom aan te raden in dergelijke gevallen het onderzoek te her-
halen. Men moet zijn kracht niet zoeken in de verfijning van de reactie doch meer
in de veelvuldige herhaling.

Het is juist een bedrijf na één positieve uitslag als „positief" aan te merken. Wil
men echter een bedrijf „negatief" verklaren, dan moet het onderzoek vaker uitge-
voerd zijn met bepaalde tussentijden. Voor de massa-diagnostiek op mastitis is het
cultureel onderzoek van melkmonsters te omslachtig en te duur. Bovendien zijn er
veel mastitisgevallen (± 35%), waarbij het bacteriologisch onderzoek negatief
verloopt.

Wij menen dat de Brabantse Mastitis Reactie (B.M.R.), zoals deze door collega
Jaartsveld werd beschreven, als massaal diagnostisch onderzoek van mastitis
bij runderen aan de bovenbeschreven voorwaarden voldoet.

Tenminste viermaal per jaar worden aan de Provinciale Gezondheidsdienst voor
Dieren alle busmonsters die op de zuivelfabrieken geleverd worden (± 100.000)
met behulp van de A.B.R. op brucellosis onderzocht. Sedert 1962 worden deze
monsters ook met de B.M.R. op mastitis onderzocht. In totaal werden meer dan
een half miljoen busmelkmonsters onderzocht met behulp van de B.M.R.
Uit dit onderzoek blijkt dat het aantal positieve B.M.R.\'s in het voorjaar en de
zomer hoger is dan in de herfst en de winter. Ook zijn er grote regionale verschillen,
(zuivelfabrieken). Deze seizoensinvloeden zijn duidelijker waarneembaar in West-
Brabant (zwart-bont vee) dan in Oost-Brabant (M.R.IJ.-vee). Waarschijnlijk speelt
hierbij het feit een lol dat in West-Brabant het afkalven van de runderen meer in
het voorjaar is geconcentreerd.

In het gebied van drie zuivelfabrieken nl. Asten, Riel en Raamsdonk met resp.
weinig, matig en veel positieve B.M.R.\'s werd nagegaan hoe de kwaliteitsbeoordeling
van de melk, zoals deze nu wordt uitgevoerd, correleert met het voorkomen van
mastitis op deze bedrijven.

De kwaliteitsbeoordeling heeft plaats op grond van: 1. reductaseproef; 2. watten-
proef; 3. geur- en smaakproef.

De kwaliteitsbeoordeling wordt uitgedrukt in klassen, nl. Ie, 2e en 3e klasse, hier
uitgedrukt in cijfers 1, 2 en 3.

De B.M.R. wordt uitgedrukt in punten. Aan een negatieve B.M.R. wordt het cijfer
1 toegekend, aan de dubieuze B.M.R. het cijfer 2 en aan de positieve B.M.R. het
cijfer 3.

Op grond van deze cijfcrwaardering, zowel voor de kwaliteitsbepaling als voor de

B.M.R. worden de kwaliteit van de melk van de drie bovengenoemde zuivelfabrieken

Bezien we de drie zuivelfabrieken als geheel, dan is er in de eerste en derde groep
een sterk significant verschil.

Dat wil zeggen dat op de bedrijven waar de kwaliteit van de melk goed is, significant
minder mastitis voorkomt en omgekeerd. Het percentage van de eerste klas melk
daalt resp. van 44,8 naar 39,1 tot 30,9 (zie tabel Hl) van de „mastitis" — via de
dubieuze naar de vrije bedrijven.

De verschillen tussen deze grootheden van de Ie en 2e groep of de 2e en 3e groep
zijn eveneens significant (5% level). Deze significantie gaat niet voor elke zuivel-
fabriek apart geheel op, omdat hier de aantallen te klein zijn.

Zoals Ir, C a z e m i e r naar voren bracht, is het moeilijk vast te stellen welk punt
betreffende de bedrijfsvoering of welk punt bij hot machinaal melken extra belang-
rijk is voor het ontstaan van mastitis.

Er bestaat volgens de spreker een correlatie tussen de algehele bedrijfsvoering van
een veehouder enerzijds en het voorkomen van mastitis bij de runderen en het
resultaat van de kwaliteitsbepaling van melk andei-zijds. Vandaar dat het logisch is
de mastitis-bestrijding samen op te bouwen met die instanties, die zich bewegen
op het terrein van de kwaliteitsbepaling van de melk en de daaraan gekoppelde
uitbetaling.

Omdat het onmogelijk is de mastitis-verwekkende of mastitis-onderhoudende bac-
teriën uit te roeien, moet het doel van een mastitis-bestrijding voornamelijk gericht
zijn op het zo hoog mogelijk houden van de weerstand van de uier. Antibiotica als
zodanig vormen over het geheel genomen geen oplossing voor het mastitis probleem.
Tenslotte stipuleert de schrijver het heel duidelijk als volgt:

1. Mastitis bij runderen is in de eerste instantie meer „een ziekte" van de
veehouder dan van de runderen,

2. Zowel organisatorisch als technisch moet de mastitis-bestrijding worden op-
gezet samen met de instanUes die zich bezig houden met de kwaliteitsbc-
paling van de melk.

3. Antibiotica vormen als zodanig geen oplossing voor het mastitisprobleem. Het
zijn hierbij meer verdovingsmiddelen voor de veehouders, dan middelen om
de mastitis terug te dringen.

Brus, D. H. J. en Jaartsveld, F. H. J.: Bijdrage tot de diagnostiek van
brucellosis bij het rundvee. Tijdschr. Diergeneeskunde., 88, 547, (1963).

on September 25th, 1963, at the end of the second day of the Congress.
Remark: Dr. Klastrup (Denmark):

If you make the bacteriological examination of the can-samples and at the
same time you make a bacteriological examination of the quarter-milk-
samples you can get a correlation of about 100%, So we feel in Scandina-
via — I don\'t know if I can speak on behalf of my colleagues from Norway
and Sweden — that the bacteriological examinations arc reliable in the
control of mastitis.

seasons of the year, I don\'t know how the calving season is in your country.
In one dairy-district of about 220 herds, every week we made a total
cell-count, at the end of the year we compared the result with the calving
season and there was a correlation between the level of the total cell-
count and the calving season.

I should like to put forward that I also agree with you that the bacterio-
logical examination can be useful in controlling mastitis, but first I must
have a good and cheap method by means of which you can find out
the herds and the cows which have problems with mastitis. And when
I have the infected quarter, then I like to take a bacteriological
examination.

If you take the bactcriological way to detect the farm and the cows, it
costs a lot of money and a lot of labour. Labour is already very expensive
and is becoming more expensive. It is already very difficult to get people,
who take milk-samples in controlling the fat-content of the milk. And
therefore, when we want to start an organized campaign against mastitis
by taking samples from the quarters for bacteriological examination, it is
impossible for us to do so. There is a proverb in the Dutch language
that says „The calf will be bigger than the cow". Do you understand that?
It is not to be paid, it is too expensive.

And as to the other matter, I told you there is a difference of B.M.R.-
test during the different seasons of the year. The difference between the
seasons is more pronounced in the western-part of Brabant than in the
eastern-part.

In the western-part calving more or less takes place in the beginning of
the year. It is possible, but I am not quite sure, you have the greatest
frequency of mastitis in cows which have calved about five months ago.
We have to prove it.

It will be dangerous to conclude from these investigations that there will
be a narrow correlation between masdtis and the hygiene of the milk. It
can be a coincidence without a real relation.

For example: your percentage of positive herds is very closely related
with the number of cows in the herds. If you have a herd of 20 cows,
the chance of having a cow with masdtis is at least twice as great as in
case of a herd of ten cows.

The same holds good for the quality of the milk. With bigger herds you
have more people to handle milk than in smaller herds. On bigger farms
the quality of milk can go down without trouble of mastitis, so in fact
there seems to be a relation, but I do not think that there is a real
correlation.

I agree with you, you have a greater possibility to find one cow suffering
from mastitis on bigger farms than on smaller farms. But as far as we know
there is no correlation between the number of cows and the quality of the
milk delivered and the number of cells pro millilitre in this milk.

I am not the same opinion. If you compare herds of ten cows to herds
of 20 cows you don\'t find more than twice the number of cases of mastitis.
My conclusion is not that mastitis-milk always will be classified as of

third class quality. If you have a farmer, who manages his herd in a
specially bad way, you have more chance to get mastitis and to get third
class milk.

These two phenomerions: mastitis and a bad quality of milk have the
same cause, namely bad management.

Mr. Chairman, it will be good to prevent some misunderstanding.
Dr. Brus wants to state that farmers having poor management practices
also produce unclean milk. That is what Dr. Brus means.
There should be a relation between the hygienic quality, detei mined by
the sediment-test, methylen-bleu-test and the smell of the milk and of
the incidence of mastitis in cows. Poor practices of management of the
cows coincide with a bad quality of the milk and bovine mastitis.

Dr. Brus, I would like to ask you some questions.
You said that the penicillin is a tranquillizer for the farmer. That is a
very interesting remark. I cannot agree with you as regards that statement.
Penicillin will not remove mastitis, but we do know certain forms of
mastitis which can be eradicated by using penicillin, if applied properly.
For example the mastitis caused by Streptococcus agalactiae. I don\'t think
that you can generalize that penicillin is a tranquillizer.

I told in the beginning that I would tell you things very black and white.
I also exaggerate.

I agree with you that good results may be obtained writh penicillin in
a herd writh Str. agalactiae, in that you can cure some cows and also help
that farmer for a while. But when you like to help your country, to
decrease the number of problem herds, penicillin will not be the solution.
You know what L i v o n i found, when the Streptococcus agalactiae was
eradicated, another streptococci was coming. And therefore when we speak
about an organized fight against mastitis — not only against agalactiae-
mastitis — you do not come to the end with penicillin. If we find a
farmer, who is in trouble, we have to help him. But when I speak about
an organized fight against mastitis, I am thinking of preventing. And
when I come at a farm vnth a lot of mastitis,I come too late in the
sense of an organized mastitis control.

We also have to investigate what is the best way of management to prevent
mastitis on these farms.

In how many cases of masutis did you fail to have good results and what
have you done in such cases?

I only told you about the results of our mass investigation about mastitis
diagnosing and the correlation between mastitis and quality of the milk.
Therefore I can\'t answer your question.

I agree with Dr. Brus that the prevention of mastitis is very important
and I think all of us can subscribe that.

But another question is this: Dr. Brus said that L i v o n i has found that
when you eradicate Str. agalactiae, other infections come in. In my
opinion L i v i o n i didn\'t prove that.

If you have a herd, infected v/ith Str. agalactiae and no othei infection
can be seen, no other infection comes in when you eradicate Streptococcus
agalactiae. That is why we in Scandinavia prefer to eradicate this specific
diseases of Streptococcus agalactiae. But at the same time we carry out
cell-count or C.M.T. in order to find herds with mastitis problems caused
by other reasons.

I thank you very much. So you have in Scandinavia herds with mastitis
problems, in which you only find Streptococcus agalactiae. We seldom
find only one bacterium in our problem herds.

We have the impression that in many of our herds different micro-orga-
nisms play a part in causing mastitis. From one quarter one isolates Str.
agalactiae, out of the other Str. dysgalactiae, Str. uberis, staphylococci and
so on.

Therefore we have the impression that mastitis is primarily not an
infection-disease. Mastitis is caused by a general lowering of resistance of
the udder. And when the udder has a low resistance, that bacterium comes
in which is nearest to the udder.

I think you have two starting points in combating mastitis;
Firstly: by bacteriological examination of the can-samples.
Secondly: by estimation of the cell-count of the can-samples.
You can have a starting point of finding bacteria in can-samples and
then you find many herds with streptococci. We have found the same
thing, but when you come at the farms where Streptococcus agalactiae
is present, you don\'t always find a herd with a mastitis problem.
If you face mastitis in that way, you use can-samples for bacteriological
examination and if you have a good penicillin regulation, you get results
on these farms and you have a lowering of the cell-counts.
Next to this you have the other farms and I think that is the other point
of view. When you start the mastitis- campaign to find out the problem-
herds by estimating the total cell-counts of the can-samples, you can start
with these problem-herds, because you cannot go to all the farms. It is
impossible to do so. You have to go to the farms on which they struggle
with the problems.

In some herds Streptococcus agalactiae is very infectious and in other
ones Streptococcus agalactiae is not infectious.

If we will facc the mastitis in all cases for an esthetic aim as well we try
to get a low cell-count in the milk of all farms.

The efficiency of antibiotics and of penicillin especially, is very different
on streptococci and against staphylococci. I think we will all agree in this.
I think in England it is the opinion that sincc the eradication of Strepto-
coccus agalactiae-infectiom in many herds the incidence of three quarter
cows is less common. In the last years\'work we have obscr\\\'ed that in many
cases the serious clinical cases of mastitis have been caused by streptococci
namely Streptococcus agalactiae-, dysgalactiae- or uberis.
Although the staphylococci infection is a great problem, and in some
herds it is the most common pathogen to be found, it is relatively not so
important in chnical disease. In our experimental work the new infections
with staphylococci remained high. It is more encouraging that the strep-
tococci infection can be reduced to a greater extent.

In Middle-Sweden we have a higher occurrence of mastitis in small herds
than in big herds. Maybe the reason is, that in big herds they have more
young cows than in small herds. In middle Sweden wc have very few
infections with Streptococcus agalactiae. Streptococcus agalactiae is no
problem in Middle Sweden. Maybe this is another reason why we have
more infections in small herds. We possess a health control that agrees
with the method Dr. Richter reported to a large extent.

I should like to make some remarks about the difference in pathogenicity
of Streptococcus agalactiae and Staphylococci.

In 1930 Stableforth, Edwards and M i n e 11, were perhaps
the first people to show that Streptococcus agalactiae can f>e eradicated.
They were also the first to show that one can live with Streptococcus
agalactiae in case of good management.

Unfortunately the moment comes in the bovine lactation cycle, that she
must be dried off and you can\'t milk her completely and regularly. And
it is my observation that if Streptococcus agalactiae will be left in the
udder of the cow during the dry-period, more scar tissue will penetrate in
the udder.

With staphylococci, I believe there is less scar tissue and personally, if I
were a farmer and I know about mastitis what I know now, I should elect
stapylococci instead of Streptococcus agalactiae in my herd, during the
dry-period, and if possible I should do my utmost best to eradicate Strep-
tococcus agalactiae.

I should like to put to Dr. Richter the following question.
He said that after some bacteria were isolated out of cases of mastitis he
checks these cases after three weeks and later on after three months.
I should Hke to ask him: what results did you have by this checking?

It is not possible for me to answer this question exactly. I agree with
Dr. K i n g w i 11 that if it is possible to eradicate the chronic mastitis
cases and to use penicillin with the non-chronic cases, we may have a
good result in about 85%.

Question: Prof. Van der Schaaf (The Netherlands) :
There is one question I didn\'t hear up till now.

Are there some figures about decrease of the production of the cgw in
kilograms of milk, when there is an infection by Streptococcus agalactiae
or a staphylococci infection?

I think we have two groups of mastitis; there is one group caused by
Streptococcus agalactiae and one group caused by the other udder
streptococci and staphylococci.

Is there anybody who can tell something about the diminishing production
in case of mastitis?

I can tell about the results of Wilson about 1947. He found a lower
yield of milk of the cows infected with Streptococcus agalactiae of 10
till 15% and in addition I can mention the examinations of Dr. L i v o n i.
He compared the successive lactations of a group of cows suffering from
S.agalactiae-\'mfections to those of a group of cows which were not infected

with this germ. He found that the diminishing of milkproduction of the
S. agalactiae was about 8%.

During this discussion the following statement of Dr. W i s n i o w s k i was read
out:

The Statement of the Lecturer Dr. George Wisniowski (Poland) during
the Congress at Boxtel, North Brabant, The Netherlands) in September 1963.

Feeling honoured by your invitaUon to the Mastitis Congress I must apologize for
my absence caused — to my regret — by having got the invitation too late. I want
to emphasize that I am fully aware of the importance of such a meeting of the
researchers interested in the problems of mastitis. I hope that such meetings will take;
place in future, too.

Being the only representative of my country I wish to assure you that — as Poland
is the fifth of the producers of milk in the world — (12,5 milliard litres yearly) we
are gready interested in the progress of science and in the organization of mastitis
control in cows. Our economy and agriculture fully appreciate the negative influence
of mastitis upon the efficiency of milk in cows. The result of this opinion is the
concentration of the investigadons of mastitis in the Department of Animal Hygiene
of the State Veterinary Institute. In our Department of Animal Hygiene we make
immunological investigations planned for a long time, and besides this, since two
years, we make observations as to the usefulness of two screening tests in mass
diagnosis such as the H o t i s test and the cytological test according Schalm.
Both these tests seem to be very valuable in comparative investigations (based on the
classical bacteriological test and on the cytological analysis and cell count de-
signation). Here I want to say, that we met with the kind understanding of Professor
Schalm at the adaptation of the Californian Mastitis Test and got concrete help
from our colleague Doctor Jaartsveld whom I want to say here my best thanks.
It is our opinion that — at least in the conditions of our country — the introductory
mass diagnosis should be based upon both of the mentioned tests. They supplement
very well, one describing the ethiology of infections for orientation, the other one
describing the irritation of udder, and it is of greatest importance that they may
be applied by practical veterinarians in the field (terrain). The results of the investiga-
tion of individual cows of great herds when both the tests were applied, allow to make
a rather detailed appreciation of the herd and to get a suggestion as to the neces-
sity of the ehmination, the treatment and the sanitary plan of milking. This may
be done by the owner, whereas the detailed investigations in particularly justified
cases made with clas.sical methods would be — such is our opinion — the task of the
specialistic laboratories.

However, as we have not such a large experience as, for instance, our Hosts, therefore
this Congress is of a still greater value for us. I am sure, that it is a rather actual
and necessary meeting which ought to initiate further congresses of this kind.
I also beg to notice, that in case of the standardization of methods and the working
out of some organizing scheme for the uniformity of mastitis control, valuable
comparable results could be .got in the particular countries taking part at this Con-
gress, and those results could be the subject of further mutual discussions.
At the end I want to thank my Colleagues and Hosts for giving me the possibility
to take the word at least in this way.

Deense onderzoekers hebben aangetoond, — en in vele
andere landen hebben wetenschapsnfiensen dit bevestigd -
dat door sprayen nnet MAST-O-FINI (op basis van hexachloro-
feen) mastitis kan worden voorkomen en bestreden.

Indien antibiotica niet nodig of zelfs ongewenst zijn, dan is
MAST-O-FINI het preparaat Uwer keuze.

277 LAAN VAN MEERDERVOORT - \'s-GRAVENHAGE - TEL. 070 - 394481
Gaarne zenden wij U een brochure toe.

Volledige voorlichting en assistentie bij
vestiging, prakfijkovername of associatie

Every three months milk-samples have to be taken from 64 dairy-factories
with a total of 70-90.000 cans. Therefore the province "Noord-Brabant"
is divided into several sections each of 3-5 dairy factories and in each
section a man is employed to take the samples.

The laboratory at Boxtel has a capacity for the examination of 10-15.000
samples in the afternoon, so execution of this test takes 9 days. The deli-
very and collection of the sampling outfit and the samples is carried out
by car.

3. Penspex tube-containers: for a hundred milk-tubes. Every container
has a serial number. The places for milk-tubes are numbered from 1
to 100.

4. Milklist: A list in duplicate. The numbers of the milk-cans and the
serial number of the tube-container are noted on this list, so it is pos-
sible to trace the cans back to their origin.

5. Milkchest: There are two kinds of chests; one contains 600 tubes, the
other one 1000 tubes. They serve for the transport of the samples. Two
foamrubber pads cover the tubes in the containers.

The containers and milklists are placed into the chests, ready for trans-
port to the laboratory.

Tenminste eenmaal per kvvartaal moeten 70—90.000 melkmonsters genomen worden
op 64 zuivelfabrieken. Cm dit te organiseren is Noord-Brabant in 2 delen verdeeld.

1 Mr. M. Galema; Animal Health Service of North-Brabant; Rechterstraat 80,
Boxtel, The Netherlands.

Een deel omvat alle zuivelfabrieken ten Oosten van Boxtel, het andere deel de fa-
brieken ten Westen van Boxtel. Ieder deel is onderverdeeld in rayons van 4—5 zuivel-
fabrieken, waarbij voor elk een monsternemer is aangetrokken.
Door op een onderzoekdag uit ieder rayon van resp. Oost- of West-Brabant een
zuivelfabriek te bemonsteren kunnen in 4 dagen alle zuivelfabrieken in het Oostelijk
deel en in 5 dagen alle fabrieken in het Westelijk deel van Brabant bemonsterd
worden. Per dag worden zo 10—15.000 monsters genomen en op het laboratorium
onderzocht. Het bezorgen en ophalen van de v5or het monsternemen benodigde
materialen wordt door de Gezondheidsdienst verzorgd.

2. Plastic schermkap: wordt na iedere 20 monsters over de genomen monsters ge-
schoven en voorkomt, dat melk gemorst wordt in de andere buisjes.

3. Meikrek: Een uit kunststof vervaardigd rek met plaats voor 100 melkbuisjes.
Ieder rek heeft een reknummer. De plaatsen van de 100 buisjes zijn genummerd
van 1 t/m 100.

4. Melklijst: Een lijst in duplo waarop de busnummers van de genomen monsters
genoteerd worden. De no\'s 1 t/m 100 corresponderen met de nummers van de
melkbuisje. Het genoteerde reknummer komt overeen met het rekno. waarin de
monsters geplaatst zijn.

5. Melkkist: Een houten kist met plaats voor 6 of 10 melkrekken. Een met plastic
folie overtrokken plaat schuimplastic voorkomt, dat tijdens het transport melk
gemorst wordt.

Uit iedere aangevoerde bus wordt een melkmonster van ± 1 ml. genomen. De melk-
monsterlepel wordt geopend in de melkbus gestoken en na enkele malen goed roeren
er gesloten uitgehaald. Op deze manier is het niet nodig, dat de lepel na ieder
monster wordt schoongemaakt. Praktijkproeven hebben uitgewezen, dat door de grote
verdunning van de achtergebleven melk bij A.B.R. en B.M.R. geen invloed wordt
uitgeoefend op de uitslag van de reactie. Nadat de monsters genomen zijn, worden de
rekken in de kisten geplaatst en zijn dan klaar voor transport naar het Laboratorium
te Boxtel.

When we started with the investigations of mastitis, the Whiteside-
reaction was used. 3 Drops of milk were transferred on a glassplate and
mixed with 1 drop of NaOH 1 n (4%).

This method was followed by a plate-reaction between milk and a sur-
face-active agent, Na-T-pol (Shell). Therefore 3 drops of milk were mixed
with 3 drops of a 10% T-pol solution, with the aid of a sdrring rod.
A very great number of milk-samples^ which we received in plastic tubes
at our laboratory, had to be examined. The plate-method was modified
into the tube-method. The California Mastitis Tube Test (C.M.T.T.) was
born.

The amount of inflammatory cells in the milksamples was also counted.
It seemed that there was a very good correlation between the number of
inflammatory cells in the milk and the viscosity, which arised by mixing
in a tube 0,6 ml. of milk and 0,4 ml. of a 10% Na-T-pol solution, with
the aid of a stirring rod. Later on a 2% sodiumlaurylsulfate solution was
used.

When a dilution of the milksamples was made with negative milk, an
impression was obtained regarding the amount of inflammatory cells,
which was present in the milk.

In order to estimate the number of cells without dilution, the viscosity
of the mixture of milk and Na-T-pol can be determined using a glass
funnel with a capillary of 20 mm. length and a diameter of 1,3 mm.
In this way the Brabant Mastitis Reaction (B.M.R.) is performed.

The milksamples, used for the investigations on mastitis, are first used for
the brucellosis investigation by applicating the Abortus Bang Ring-reaction
(A.B.R.).

Therefore to each tube containing 1,0 ml. milk is added 1 drop of A.B.R.-
antigen. The samples are shaken well, placed in an incubator and after
1 hour they are judged. A positive A.B.R. reaction shows a blue coloured
cream layer and decoloured milk, and a negative reaction shows blue
coloured milk and a white cream layer.

After this, the samples are used again for the investigations as to mas-
dtis. As most cells are to be found in the cream layer, the samples are
shaken well to mix these cells homogeneou.sly through the milk.
We use 0.6 ml. of this milk and therefore the supernatant layer is re-
moved by means of a sucking apparatus. In order to prevent inflammatory
cells from remaining on the needles of the sucking apparatus, the latter
are rinsed with running water, every time after use.

1 Mr. E. van Werven; Animal Health Service of North-Brabant; Rechterstraat
80, Boxtel, The Netherlands.

To each tube is added 0,4 mb of a 2% Sodium Laurylsulfate solution.
The pH of this solution is corrected by NaOH to pH 12. In this way we
can examine the milksamples after a longer time (± 48 hours).
The samples are shaken well. By the presence of inflamn^atory cells the
nucleus breaks open and the desoxyrubosenucleic acid (D.N.A.) comes
free.

This D.N.A. lends a rise in viscosity to the samples. This viscosity is ac-
cording to the amount of inflammatory cells, which is present in the
milk. The grade of viscosity is determined by means of the time of flowing
through.

For this, the samples are put in capillaries. This takes place by means of
tumbling 100 samples at a time in 100 corresponding capillary tubes.
These tubes are blocked by a block-plate. Now the time of flow through
is determined. As soon as the tubes are lifted from the block-plate, the
time of flow through starts, and after 5 seconds a picture is taken. This
is repeated after 10, 20 and 60 seconds. Samples, which have flown
through the capillaries within 5 seconds have a negative B.M.R. The
amount of cells is not greater then 200.000 per ml.

Samples which are in the tubes after 5 seconds get 1 dot; it is the milk
which contains about 400.000 cells per ml. Samples which are still in
the tubes after 10 seconds get 2 dots, corresponding with about 800.000
cells per ml. 3 dots are noted for those samples, which contain about
2.000.000 cells per ml. Samples, which are in the tubes after 60 seconds,
are marked with 4 dots. The amount of inflammatory cells is about
5.000.000 per ml.

When time is up the sets with capillary tubes are removed quickly to
make place for the following sets. In this way it is possible to investigate
about 10.000 samples an hour with the aid of 6 laboratory assistants.
The following day, the results are noted on the corresponding lists.

In een overzicht wordt de ontwikkeling van de B.M.R. gegeven. Via de Whiteside
reactie (melk loog), de C.M.T. (reactie tussen melk en T-pol op een glasplaat),
de C.M.T.T. (melk T-pol in een buisje) is de huidige B.M.R. ontwikkeld.
Deze reactie maakt het mogelijk een groot aantal melkmonsters op snelle en goed-
kope wijze te onderzoeken op aanwezigheid van ontstekingscellen.
Eerst worden de melkmonsters voor het abortus-onderzoek gebruikt. Dit geschiedt
met behulp van de Abortus Bang Ringrcactie (A.B.R.)

Direct nadat deze reactie is afgelezen, wordt met de B.M.R. begonnen. Deze directe
opeenvolging is gunstig voor het verloop van dc B.M.R., gezien de optimum tempe-
ratuur bij 20 k 25° C.

Daartoe wordt het volume van ieder buisje tot 0,6 ml teruggebracht en hieraan 0,4
ml T-pol oplossing toegevoegd. Bij aanwezigheid van ontstekingscellen worden door
een reactie tussen deze cellen en het toegevoegde reagens de ontstekingscellen stuk-
gemaakt, waarbij een slijmige kernvloeistof (D.N.A.) vrijkomt.

Naarmate het aantal ontstekingscellen groter is, neemt de slijmigheid toe. Deze
slijmigheid wordt met behulp van nauwe doorstroomcapillairen gemeten.
De uitslagen worden fotografisch vastgelegd.

In order to test many milksamples for the presence of antibiotics, we
have taken the view that the tube-containers form the basis of the me-
thod. Therefore rectangular moulds are made, with the same surface as
the tube-container.

About 160 ml. 2% agar medium is mixed with ± 25 ml. Sarcina lutea-
culture and poured out in this mould.

After that a punching-apparatus with 100 legs (photo 1) is placed on
the bottom of this mould. When the agar is coagulated the punching-
apparatus is removed from the filled mould and 100 little holes are left.
With an antibiotic-drop-apparatus (photo 2), three drops of milk are re-
moved out of every tube of the tube-container (photo 3) and transferred
in the corresponding hole of the mould (photo\'s 4 and 5).
The mould is incubated at 37° C for about 20 hours.
All milksamples examined for brucellosis antibodies with the Abortus-
Bang-Ring-reaction are as well suited for the method of penicillin de-
tection as the fresh milksamples are.

In this way it is possible to examine the milksamples as to brucellosis, as
to the presence of antibiotics and of inflammation cells with the aid of the
B.M.R. and in a simple, easy and cheap way.

Hierbij wordt uitgegaan van de bestaande melkrekken, die worden gebruikt voor het
A.B.R.- en B.M.R.-onderzoek.

De antibiotica in de melk worden aangetoond in rechthoekige platen, welke dezelfde
oppervlakte hebben als de bovengenoemde melkrekken. Deze platen worden gegoten
met ± 160 ml 2% agar en ± 25 ml Sarcina lutea-cuXiwxr. Daarna wordt in dc
vloeibare agar een ponsapparaat geplaatst met 100 pootjes die onderling dezelfde
afstand hebben als de buisjes in het meikrek.

Als de agar gestold is, wordt het ponsapparaat verwijderd zodat 100 gaatjes in de
agar overblijven.

Met behulp van een antibioticum-druppel apparaat wordt in één keer vanuit de 100
buisjes staande in het meikrek 3 druppels melk opgezogen en overgebracht in de over-
eenkomende holten van de agar-plaat. Deze agar-plaat wordt ± 20 uur bij 37° G
bebroed.

De melkmonsters die een remmingszóne vertonen kunnen dan direct onderkend
worden. Melkmonsters die meer dan 0,02 E penicilline per ml bevatten, kunnen met
deze methode worden aangetoond.

Op deze wijze is het mogelijk alle kwartaal-busmelhmonsters — elk van 1 ml melk
— komende van de zuivelfabrieken te onderzoeken met behulp van de A.B.R. op
abortus Bang, met behulp van de bovenbeschreven methode op het voorkomen van
antibiotica en met behulp van dc B.M.R. op het voorkomen van ontstekingscellen
in de melk.

1 F. H. J. Jaartsveld D.V.Sc.; veterinary surgeon to the Animal Health Service of
North-Brabant; Boxtel, Rechterstraat 80, The Netherlands.

Photo 3. Three drops of milk are sucked by the
antibiotic-drop apparatus out of the tubes placed
in the tube-container.

With the help of a platinum wire 1/50 ml. of milk is taken out of a ste-
rile, uncentrifuged milksample and transferred to a blood and H.E.T.-
medium, which now is incubated at 37° C for about 20 hours.
A rectangular collapsible plate was designed for examination by culture
of milksamples taken under sterile conditions. The rectangular plate will
be replaced in future by a plastic one, which will be used only one time.
This plate is suited also for the detection of antibiotics in milk. The plate
is divided into two unequal parts by a threshold running lengthwise.
Five to ten percent blood agar is poured into the left (greater) half and
H.E.T.-medium into the right (smaller) half of the plate. The samples of
milk to be examined are inoculated very simply and quickly on the two
media in the order of the numbers marked on the threshold. The following
day — after incubation — it is possible to determine whether any growth
has occurred and the species of bacterium concerned.
The cultures of bacteria obtained from the milksamples can then also
rapidly be examined for their sensitivity to penicillin and terramycin.
Long strips of filterpaper containing a standardized quantity of these anti-
biotics are used for this purpose. For that purpose filterpaper "What-
mann 3" is used. The length is 300 mm and the breadth 2 mm. For %
of an hour they are sterilized at 120° C. Afterwards 0,225 ml. of a stan-
dardized penicillin and terramycin solution is spread over the whole strip.
The first solution contains 1200 LU. pen/10 ml. aqua-dest, the second solu-
tion contains 6000 I.U. terramycin/10 ml. aqua-dest.

and 60 seconds, and respectively indicated with B.M.R. —, •, • •,
• • • • • • •

Before the culture media are examined, we first look with the aid of an
ultra violet lamp whether there is any reaction with aesculine on the
H.E.T.-media.

1 F. H. J. Jaartsveld D.V.Sc.; veterinary surgeon to the Animal Health Service
of North-Brabant; Rechterstraat 80, Boxtel; The Netherlands.

The determination of Streptococcus agalactiae does not give any trouble
with this method.

Streptococcus dysgalactiae is characterized by its flat bacterial colonies
on the H.E.T. medium. To check this determination the culture is trans-
ferred to a horse-serum-agar medium. After the bacterial colonies have
grown, a turbidity arises under the colonies.

The aesculine-decomposing streptococci, which were already marked on
the culture-disks, cannot be determined by this scheme only.
Streptococcus faecalis mostly gives a greenish discoloration on the blood
medium (alpha-hemolysis).

For further determination aesculine decomposing bacteria are transferred
to media named in table 2.

In extra-ordinary cases Listeria monocytogenes can be isolated. This bac-
terium also decomposes aesculine. However it is a Grampositive small rod,
it decolorizes the litmusmilk (a so called white foot). Further examination
is asked then.

Also the determination of Streptococcus zooepidemicus, — that appears
rarely — gives no problems.

Corynebacterium pyogenes has grown very badly after one day on the
blood medium. On the H.E.T. medium it does not give any growth. This
bacterium is mostly found in very abnormal milk. It is recommended to
transfer directly, very abnormal milk on a Loffler medium.
C. pyogenes liquefies the Loffler medium in places where the bacterial
colonies have grown; there a bit brown-coloured excavation arises. For
further determination we make a Gram staining of the bacterial film.
Then the bacterium is transferred to the determination sugars.
Staphylococci. These are growing luxuriantly on the blood medium, they
are catalase positive, in contrast with the streptococci.
Staphylococci are usually found on the skin. They are called pathogenic
in mastitis research when the B.M.R. of the milk is ••• or****.
Escherichia coli is also marked as a cause of mastitis, when the growth is
accompanied by a positive B.M.R. In that case the milk is very abnormal.

Aerobacter aerogenes re.sembles in proporties Klebsiella pneumoniae type B.
(animal pathogenic form). This forms ammonia from pepton, so that the „slant"
in the T.S.I, often becomes red after 2 x 24 hours, this is also the case with the
yellow bacterial colonies on the Kauffmann medium.

\'■\') C. pyogenes grows badly into the litmus-milk, it grows well after adding a
little bit of liver-broth.
Listeria is catalase positive.

In other cases, the growth must be seen as defilement. For further deter-
mination this bacterium is placed on the media mentioned in table 3.

This solution is boiled for 10 min. on a open flame, cooled and filtered. The pH is
adjusted to 7.3 and now sterilization for 10 minutes at 120° C. takes place. After
cooling, filter through double paper till it\'s clear, and dispense the broth in flasks.
These now arc sterilized for 20 minutes at 100° C. The broth has to be clear.
After cooling 10% sterile serum is added to the meat broth.

Dissolve in the Koch-apparatus. Adjust the pH to 7,3. Sterilize in autoclave for 15
minutes at 110° C. Cool down at about 40° C. Add 10% horse-serum, sterile.
Rotate carefully (no formation of bubbles), pour sterile in Petridiscs.

To dissolve the agar well, it is placed during one hour in the Koch apparatus. Then
it is sterilized for 15 minutes at 120° C. After coohng down at about 50° C., add
under swinging about 25 ml. cow-blood, (defibrinated or citrateblood) and then the
plates can be poured.

The citrateblood is prepared by adding ± 800 ml. cow-blood to 50 ml. of sterile 10%
sodium citrate solution.

Fresh 1% thalliumacetate solution 33 ml.
Add aqua-dest till a volume of 1000 ml.

For a good solution the agar is placed for about 1 hour in the Koch, and then
approximately 250 ml. are filled in bottles, during 20 min. sterilized at 120° C.
After cooling down at about 45° C. with good mixing add 35 ml. cow-blood with
crystal violet (C.D.I.)i) and 0,5 ml. B. toxine (C.D.I.) per 500 ml. agar.
Now the plates can be poured, after coagulation they are wrapped in brown paper,
to protect this medium against light, which produces hemolysis.

0,2 % phenolred solution in aqua-dest 50 ml.
For a good solution all the ingredients are boiled together for about 10 minutes
and after cooling down the pH is adjusted to 7,3. Filter now and sterilize for 10
minutes at 130° C. After cooling down filter the turbid solution up to clear.

Pour in culture tubes with a little Durham tube. The sugars are now placed in the
Koch, so that at the same time the air-bubbles in the Durham tubes, arc driven away.

Add to this milk 10% litmus-solution of Kubel and Tieman (BsS), filter. Pour
culture tubes about 7 cc. per tube. Sterilize twice for J/j hour in the Koch, with an
interim of 24 hours. (By sterihzing during a longer time the milk is discoloured).
Instead of skimmed milk one can also use the solution: Skim-milkpowder 20 gram
in 200 ml. aqua-dest).

After mixing well and dissolving without heating the pH is adjusted to 7,6. The
solution is poured into sterile culture tubes about 5-6 ml. These tubes are placed
during 3 hours on 2 successive days in the scrum-coagulation warmer at a tempe-
rature of 85° C.

Warm up for /a hour in the Koch at 100° C., then adjust the pH at 7,5—7,6.
Filter through a „faltcnfilter" till it\'s clear. Pour culture tubes, and sterilize on

2 successive days at 100° C. during yi hour. Then cool it down, so the tubes
coagulate in upright or sloping position.

Jaartsveld, F. H. J.: Massaal bacteriologisch onderzoek van melkmonsters voor
een georganiseerde mastitisbestrijding bij runderen. Tijdschr. Diergeneesk., 87,
1088, (1962).

Op deze demonstratie wordt de rechthoekige schaal vertoond waarmee het mogelijk
is het bacteriologisch onderzoek van een groot aantal melkmonsters in een korte tijd
uit te voeren.

Deze schaal wordt in de lengterichting door een drempel in twee ongelijke delen
verdeeld. In de grootste helft wordt 5-10% blocdagar en in de kleinste helft wordt

het H.E.T.-medium gegoten. Volgens de aangebrachte nummering wordt de plaat
met de te onderzoeken mclkmonsters op beide media geënt. De bactcric-culturcn,
afkomstig uit de melkmonsters, kunnen vervolgens op een snelle wijze onderzocht
worden op hun gevoeligheid voor antibiotica zoals penicilline en terramycine. Dit
gaat met behulp van lange strips van filtrecrpapier, die cen gestandariseerde hoe-
veelheid van het antibioticum bevatten.

Aan 0.6 ml te onderzoeken melk wordt 0,4 ml Na-laurylsulfaat 2% (pH 12) toege-
voegd. Dit wordt goed gemengd en overgebracht in z.g. doorstroomcapillairen. Dc
tijd die dit mengsel nodig heeft om vanuit de trechter via de capillair uit te stromen,
is cen maat voor de slijmigheid van het mengsel, die op haar beurt een maat is voor
het aantal cellen dat bij benadering in de melk aanwezig is.

Betreffende de determinatie van de bacteriën en de te gebruiken voedingsbodems
worden uitgebreide voorschriften gegeven.

Demonstration on „Campina-dairy" at Eindhoven and on
the laboratory of the Provincial Animal Health Service at
Boxtel.

In order to get an impression about the difference in quality of dairy-
products made from s.c. "mastids-milk" and normal milk, the following
investigations are performed.

By means of the BrabanUc Mastitis Reaction (B.M.R.) at the Campina-
dairy, the supphed milk is divided in normal milk and masdtis-milk.
Milksamples are taken out of all cans and examined by the B.M.R. The
milk out of the cans with a negative B.M.R. is collected in one tank,
the milk out of the cans with a positive B.M.R. (three or four points) is
collected in an other tank. In this way mastitis-milk means milk of which
15-20% is drawn from abnormal quarters.

.«After working up the daily milk, about 4000 kg is pumped separately
into two different buffertanks and is separated by two different and
clean cream separators into two kinds (mastitis and normal for control)
of cream (15 to 20% fat) and skimmed milk, leaving behind different
quantities of separator slime.

Both kinds of cream are pasteurized in the usual way, soured and churned
in a normal churn into butter and buttermilk.

Recombined pasteurized milk was made from separated cream and skim-
med milk for the yoghurt making in the usual manner.

Two kinds of milk collected in the same manner, were transported to the
cheese-factory "Dongen". Two batches of cheese were made without pas-
sing the milk through the cleaning-separator.

As the first experiment suggested that there was a difference in copper
content between mastitis-butter and normal or control-butter precautions
were taken to prevent any copper contamination in the "milk-way" and
routine samples of both kinds of milk and products were taken for exa-
mination of copper content by this institute and the laboratory of the
Association of co-operative Dairying in the south of The Netherlands
(G.Z.N.Z.) at Roermond.

Organoleptic and other tests were carried out by the laboratories of the
C.Z.N.Z. and the N.I.Z.O. (Institute for Research in Dairies in The Ne-
therlands) and the department of the Z.K.B. (Dairy Quality Control Bu-
reau) at this town (Boxtel).

a. B.M.R. and cell-counts of milk; quantity of se-
parator-slime.
Table 1 shows that the quantities of separator-slime vary with the kind
and quandties of milk, but the mastitis-milk contains more than double
the quantity of slime.

Both fresh and after 5 days keeping in the refrigerator no differences
were found in the organoleptic tests of the mastitis and controlproducts,
when these were made of recombined milk or separated and soured
cream.

There appeared to be some difference in taste (unfresh, unclean, salty),
when these products were made directly of pasteurized (not cream sepa-
rated) of naturally skimmed cow milk. You can try this in an organo-
leptic test afterwards. Up till now, no differences were found in copper
content of the routine samples.

The results of the organoleptic and other tests of the keeping quality of
mastitis and controlbutter of the first experiment dated 15/11 1962 are
summarized in table 2 and table 3.

Classification quality of mastitis and contro\'hutter in cold storage
according to Z.K.B. (three samples)

1. By means of the B.M.R. it is possible to separate at a dairy-factory
s.c. mastitis-milk from normal milk.

2. Mastitis-milk collected by this way has a high cell-count (about
1.500.000 cells per ml.); the quantity of separator slime is much more
than the quantity of separator slime from normal milk.

3. Yoghurt and buttermilk made from s.c. mastitis-milk and normal-milk,
both fresh and after 5 days keeping in the refiigerator, show no dif-
ferences in the organoleptic tests.

There appeared to be some difference in taste (unfresh, unclean, sal-
ty), when these products were made directly of pasteurized (not
cream-separated) or naturally skimmed cow-milk.

4. There seems to be a difference in the quality of butter made from
s.c. mastitis- and normal-milk, especially when the butter has been
stored for some months. The butter made from mastitis-milk deterio-
rates quicker.

5. There appeared to be a difference in taste, consistency and structure
of cheese made from the two kinds of milk. Cheese made of mastitis-
milk was of a lower quality.

6. It is very important to repeat investigations with more controls than
we have done here, in order to get a clear insight of these problems.

Op de zuivelfabriek „Campina" te Eindhoven werden enkele malen „mastitismelk"
(celrijke) en controle melk (celarme) verzameld.

Daartoe werd van elke bus een monster genomen en ter plaatse volgens de B.M.R.
onderzocht. De melk, afkomstig uit bussen met een positieve reactie, werd in een
aparte tank gestort, terwijl in een andere tank de „negatieve" bussen werden ge-
leegd. Beide groepen bevatten dus melk van dezelfde bedrijven.
Het gelukte zo 3000 a 4000 kg „mastitismelk" te verzamelen, waarbij kan worden
vermeld dat 15 a 20% van deze melk afkomstig is uit ontstoken kwartieren. Het
aantal cellen in de controlemelk bedraagt dan ± 200.000 per ml terwijl dc „mastitis-
melk" er ruim een miljoen per ml bevat.

Bij organoleptische keuring bleek yogurt en karnemelk, gemaakt van „mastitismelk"
van mindere kwaliteit (onfris-zoutig) dan dezelfde produkten gemaakt van controle-
melk. Dit verschil viel echter weg, indien de melk eerst gecentrifugeerd werd en dan
door menging van room en ondermelk opnieuw werd samengesteld. Bij centrifugeren
bleek, dat de mastitismelk belangrijk meer ccntrifugeslib gaf dan dc controlemelk.
Botcrpartijen, gemaakt van beide soorten melk, vertoonden in verse toestand geen
organoleptische verschillen. Bij bewaring in het koelhuis traden er na 2 a 3 maanden
kwaliteitsverschillen op. De boter gemaakt van mastitismelk werd dan vettig,
spekkig, tranig. Het viel op dat de „mastitisboter" meer koper bevatte dan dc controle-
boter. Dit was bij dc eerste proef (winter) duidelijker dan bij de tweede proef (zo-
mer), De keuringen vonden plaats door de CZNZ (Roermond), het NIZO (Ede) cn
ZKB (Boxtel),

Op de zuivelfabriek te Dongen werd kaas gem.aakt van resp, mastitismelk en con-
trolemelk, Deze melk was niet gecentrifugeerd. Ook deze beide partijen melk waren
op „Campina" te Eindhoven verzameld. Na enige weken bleek dc mastitiskaas wat
deegachtig en wat nestig, de korst was week. De kaas, gemaakt van controlemelk,
was normaal van kwaliteit. Gedacht wordt aan een vertraagde zuring als oorzaak
van deze verschillen.

Het is van groot belang dat deze proeven herhaald worden, opdat door een nadere
bestudering een beter inzicht verkregen wordt in deze problemen,

Darf ich mal einige Worte an Herrn Kollege Brus sprechen. Ich möchte,
auch in Ihren Namen, lieben Herren Kollegen, unseren Dank aussprechen
für die Einladung zu diesem Kongress über Mastitis-Frage.
Ich glaube, dass diese Tage sehr erfolgreich waren und dass sie uns allen viel
Nutzen gebracht haben.

Zugleich möchte ich Sie bitten, Dr. Brus, auch dem Vorstand Ihres Pro-
vinzialen Gesundheitsdienstes unseren Dank auszurichten.
Dass wäre dann, was ich zu sagen hatte.

1 Dr. K. Tilgner; Direktor des Instituts für Tiergesundheit, Güterbergstrasze 77,
23 Kiel, Deutschland.

At the close of this congress, I wish to speak a few more words.
I believe that I may say that we have had a successful congress. We
have listened to many speakers and we have heard various views, parti-
cularly during the last discussions, and I am glad that they differ.
I hope that these questions will induce everyone to start investigating.
An investigation will only be a good investigation if it supplies the answer
to a quesdon and at the same time raises two fresh questions which have
to be answered.

I hope that the various investigators will test one another\'s methods and
that they will write personally on the results obtained or, perhaps even
better, personally discuss these results.

May the personal contacts we have now established bear fruit in the
practical work of mastitis control.

I am glad that you have attended these congress days and I cordially
thank you for doing so.

All of you who have come from Finland, Sweden, Norway, Denmark,
Germany, Great Britain, Belgium, the United States of America and, last
but not least, The Netherlands.

A. special word of thanks is due to the speakers Dr. Schalm, Prof. Van
der Schaaf, Mr. Kingwill, Dr. Jaartsveld, Mr. Cazemier, Dr. Scheiner
and Dr. Richter.

Thanks also to Prof. Vandeplassche and Dr. Tilgner who conducted the
discussions.

I also wish to thank the management of the Co-operative Dairy Works
"Campina" as well as our own committee, who have enabled Dr. Jaarts-
veld and myself to organize this congress.

I hope that this congress will lead to many contacts in the future and
wish you all a pleasant journey home.

1 D. H. J. Brus, D. V. Sc.; Director of the Provincial Animal Health Service in
North Brabant; Rechterstraat 80, Boxtel, The Netherlands.

				Temperature of milk and reagent before mixing.
				35° C.	25° C.	15° C. 5°C.
Flowtime	in	seconds	1st	18	17	19 11
)i	»	a	2nd	17	29	13 11
»	>j	3J	3rd	14	19	13 5
		Milksample stored at ± 6°	C. during 20 hours¹)
				Temperature of milk and reagent before mixing.
				35° C.	25° C.	15° C. 5° C.
Flowtime	in	seconds	1st	78	46	52 31
»	>}		2nd	40	87	33 19
»		»	3rd	25	21	18 10

Zuig-persslag verhouding	50 — 50	70 — 30 of 80 — 20	50 — 50	70 — 30 of 80 — 20
(Pulsation ratio)	21	6	25	5
Wisseling van melkers	Normaal	Abnormaal	Normaal	Abnormaal
(Changing of milkers)	(normal)	( abnormal)	(normal)	(abnormal)
	18	9	26	4
Namelken	Met de band	Niet of machinaal	Met de hand	Niet of machinaal
(Stripping)	(by hand)	(not or by machine)	(by hand)	(not or by machine)
	24	3	25	5
Totale behandehngstijd	Binnen 9 min.	Langer dan 9 min.	Binnen 9 min.	Langer dan 9 min.
(Total time of treatment)	(within 9 min.)	(more than 9 min.)	(within 9 min.)	(more than 9 min.)
	11	16	21	9
Machine overmelktijden	Sporadisch	Veelvuldig	Sporadisch	Veelvuldig
(Machine over milking time)	(seldom)	(frequently)	(seldom )	(frequently)
	± 16	± 11	± 25	± 5
Werkmethode	PiAi P2A2 P1A2	P3A2 P2A1 PiAi P2A2 P1A2	P3A2 P2A1
(Work method)	18 2 5	2 0 17 6 6	0 1
Type melkmachine	Hangend	Staand	Hangend	Staand
(Type of milking machine)	(hanging)	(standing)	(hanging)	(standing)
	19	8	19	11
	Platte kop Bolkop	Platte kop Bolkop	Platte kop Bolkop	Platte kop Bolkop
Type tepelvoering	(flat cup) (spherical	(flat cup) (spherical	(flat cup) (spherical	(flat cup) (spherical
(Type of liner)	14 cup)	1 cup)	16 cup)	7 cup)
	5	7	3	4

	Proefbedrijven	Controlebedrijven
	(Testherds)	( Controleherds )
Melken voldoende	7	20
(Milking sufficient)
Melken onvoldoende	20	10
(Milking sufficient)
	Tabel III.
	Table III.
	10 bedrijven, nimmer bus-	12 bedrijven, minstens 8x
	monster positief B.M.R.	busmonsters positief
	(10 herds, never positive	B.M.R.
	B.M.R.-canmilk samples)	(12 herds, at least 8 times
		a positive B.M.R. canmilk
		sample )
Melken voldoende	7	1
(Milking sufficient)
Melken onvoldoende	3	11
(Milking insufficient)

Gem. % bedrijven met reductase 2	8.3	10.9
(Average % herds with reductase 2)
Gem. % bedrijven met reductase 3	4.3	8.9
(Average % herds with reducta.te 3)
Gem. % bedrijven met reinheid 2	8.4	17.7
(Average % herds with cleaness 2)
Gem. % bedrijven met reinheid 3	0.6	3.5
(Average % herds with cleaness 3)

	melken voldoende	melken onvoldoende	melken voldoende	melken onvoldoende
	( milking sufficient) (20)	(milking insufficient) (10)	( milking sufficient) (7)	( milking insufficient) (20)
Gem. % bedrijven met reductase 2 (Average % herds with reductase 2)	7.6	10.—	8.2	11.9
Gem. % bedrijven met reductase 3 (Average % herds with reductase 3)	4.3	4.3	8.8	9.3
Gem. % bedrijven met reinheid 2 (Average % herds with cleaness 2)	7.6	10.—	15.—	18.8
Gem. % bedrijven met reinheid 3 (Average % herds with cleaness 3)	0.7	0.5	2.7	3.8

				Proportion of cows	(%) infected	with		% cows
	No. of		Average						with	Milking	Machine
Herd	Breed	Staphylo- cocci	Str. agalactiae	Str. dysgalactiae	Str. uberis	All	Housing
	cows	Age (lact.)	types	chapped teats	Make	Type	in winter
1	30	Friesian	3.0	29	29	7	21	59	57	G	In-can	covered yard
2	46	Friesian	3.3	59	0	6	6	63	12	A-L	Jar-can	semi-covered yard
3	42	Guernsey	3.4	40	12	7	12	52	86	G	In-can	covered yard
4	42	Ayrshire	3.0	66	0	17	6	69	26	S	Pipeline	semi-covered yard
5	40	Friesian	2.6	55	0	8	4	60	33	A-L	In-can	semi-covered yard
6	51	Friesian	3.4	56	0	16	2	66	37	V	In-can	out-wintered
7	39	Shorthorn	3.7	59	38	10	0	74	54	A-L	In-can	out-wintered
8	32	Ayrshire	3.6	19	28	3	16	50	13	G	In-can	covered yard
9	34	Ayrshire	3.0	47	0	0	9	50	79	S	In-can	semi-covered yard
10	40	Friesian	3.0	58	28	3	3	68	59	A-L	In-can	semi-covered yard
11	26	Jersey	3.0	73	0	15	35	85	17	A-L	In-can	semi-covered yard
12	32	Friesian	2.3	84	0	3	3	84	19	A-L	Pipeline	covered yard
13	57	Friesian	3.7	32	0	9	44	60	11	A-L	In-can	semi-covered yard
14	45	Friesian	2.9	33	0	6	4	36	22	W	In-can	out-wintered

	Hygiene herds	Start of Expt.	After 4 months
No.	1	96	14
	2	95	41
	3	100	12
	4	100	40
	5	100	4
	6	100	60
	7	100	—
		Mean % 99	28

		Table III.
No. of swabs	with	Staph, aureus from	teat cup	clusters after
		pasteurisation.
Hygiene herds	Inside teat cup liner	Outside	of teat cups
No.	No. swabs No. -\|-vc	No. swabs	No. ve
1	17	0	17	1
2	22	2	23	6
3	24	0	24	0
4	28	0	27	0
5	22	1	22	6
6	18	0	18	1
Total	131	3	131	14

	Hygiene herds
1	Fr	13	47	15	57	34	70	43	3	26
2	Fr	61	46	4	12	41	89	4	0	27
3	G	0	14	3	86	68	94	10	0	0
4	Ayr	33	49	21	26	40	68	17	7	3
5	Fr	40	29	21	33	32	58	13	4	21
6	Fr	49	42	3	37	50	98	4	0	5
7	Sh	5		-	54	—		69
Mean %		33	38	11	43	44	79	23	2	14
	Control herds
8	Ayr	48	47	59	13	13	0	0	3	24
9	Ayr	3	17	3	79	43	71	18	13	42
10	Fr	10	14	17	59	44	78	27	19	8
11	J	4	57	29	17	24	46	0	0	0
12	Fr	25	47	35	19	40	58	9	5	16
13	Fr	67	0	0	11	9	88	0	100	47
14	Sh	40	11	0	22	51	83	5	27	72
Mean %		34	28	20	31	32	61	8	24	30

	Proef-	Controle-	Proef-	Controle-	Proef-	Controle-
	bedrijven	bedrijven	bedrijven	bedrijven	bedrijven	bedrijven
	(Test-	(Control-	(Test-	(Control-	(Test-	( Control-
	herds)	herds)	herds)	herds)	herds)	herds)
Aantal bed lijven	25	13	25	13	25	13
(Number of herds)
Aantal koeien	367	186	345	179	324	175
(Number of cows)
Aantal kwartieren	1460	743	1372	716	1288	699
(Number of quarters)
% kwartieren pos.
	16,8	8,1	15,2	9,1	16,1	7,2
(% of quarters positive
B.M.R. (>3^))
% kwartieren met
Strept. agalactiae	10,3	2,8	11,4	3,8	12,9	4,6
(% of quarters with
Strept. agalactiae)
% kwartieren met
Staphyloc. B.M.R.
^ 3	3,5	2,2	2,8	2,4	3,3	2,0
(% of quarters with
Staphyloc. li.M.R. >
3 )
% kwartieren met
Staphyloc. B.M.R.
<3	4,3	5,00	7,9	8,0	11,7	13,9
{% of quarters with
Staphyloc. B.M.R.
<3 )
% kwartieren
Strept. dysgalactiae	1,9	2,2	1,5	0,8	1,1	0,9
(% of quarters
Strept. dysgalactiae)
% kwartieren negatief	79,9	87,4	76,0	85,1	71,0	78,7
(% of quarters negative)
% koeien Strept.
agalactiae	24,8	8,6	22,0	8,9	30,8	13,7
(% of cowi Strept.
agalactiae)
% koeien Staphyloc.	16,6	16,1	19,1	20,1	24,1	22,9
(% of cows Staphyloc.)
% koeien Strept.
dysgalactiae	4,6	4,3	4,6	1,1	2,4	1,7
(% of cows Strept.
dysgalactiae)
% koeien positief
B.M.R. 3 )	41,4	22,6	37,7	25,1	38,3	20,6
(% of cows positive
B.M.R. (>3 ))
% koeien negatief
(bacteriologisch)	53,7	70,4	53,9	69,8	42,6	61,7
{% of cows negative
( bacteriological})

		Proefbedrijven (Testherds)	Controlebedrijven ( Controlherds)
Strept. agalactiae		173	424
Strept. dysgalactiae		1708
Staphyloe., tevens (together	with )
B.M.R. S 3		326	598
Staphyloc., tevens (together	with )
B.M.R. < 3		107	111
B.M.R. è 3		102	178

	1874 vrije	141 B.M.R.	1203 vrije	62 B.M.R.
	kwartieren	pos.	kwartieren	pos.
	( negative	kwartieren	( negative	kwartieren
	quarters)	(B.M.R.	quarters)	(B.M.R.
		positive		positive
		quarters)		quarters)
B.M.R. (^3 ) Strept.
agalactiae	1,8	12-	0,4	3,2
B.M.R. (^3 )	2,9	22,-	2,8	12,9
B.M.R. (§3 ) andere
besmetting (infection non
Strept. agalactiae)	1,8	11,3	0,8	3,2
Strept. agalactiae	1,8	5,8	1,2	4,9
Andere besmetting (in-
fection non Strept. agal-
actiae )	8,1	9,9	7,0	27,4
Vrij (Negative)	83,6	39,-	87,8	48,4

123	Proefbedrijven (testherds kwartieren (quarters)	Controle bedrijven ( Controlherds) 33 kwartieren (quarters)
B.M.R. (^3-t-) -(- Strept.
agalactiae	31,7	24,3
B.M.R. (^3-1-)	2,5	0,0
B.M.R. (^3-f) -1- andere
besmetting (infection non
Strept. agalactiae)	1,6	3,0
Strept agalactiae	31,7	21,2
Andere besmetting
(infection non Strept.	6,5	9,1
agalactiae)
Vrij (Negative)	26	42,4

iny opinion the value of this method is not so	1 much in the sensitivity, but
in the repeatability of this method. In the case of masdtis in my opinion
we have to test	it every month.
Four times a year we examine	in our laboratory can-samples of every herd
of our provmce,	for testing on brucellosis with the help of	the Abortus
Bang Ring-reaction. Since January 1962 we	made the B.M.R. on these
same samples as	well. Our farmers deliver about 100.000 cans daily. We
investigate also more than half	a million of milksamples with the B.M.R.
		Table I.
Percentage of mastitis farms and percentage	of cans with	B.M.R. •**
		or ••••
Period	% B.M.R.	Total of	% of cans	Total of
	farms	farms	B.M.R.	cans
			• ••or****
1st. quarter \'62	17,2	32.673	8.4	89.082
2nd	15.9	32.731	5.9	100.253
3rd	9.2	32.694	3.5	90.322
	8.3	32.844	-i.l	77.112
1st „ \'63	11.4	32.570	4.7	84.332
2nd	17.8	30.896	6.6	99.891
^rd „ „	11.9	31.552	5.7	92.336
		Table II.
Percentage of mastitis farms and percentage of cans with	B.M.R. •**
or	•••• in the western part (Frisian cattle).
Period	% B.M.R.	Total of	% of cans	Total of
	farms	farms	B.M.R.	cans
			****••• or
1st quarter \'62	21.8	9293	10.—	33131
2nd „ „	15.7	9423	5.1	39333
3rd „ „	8.1	6787	3.—	23923
4th „ „	8.6	9044	8.1	26931
1st „ \'63	14.2	8592	5.1	33368
Percentage of mastitis farms and percentage	of cans with	B.M.R. •••
or ••••	in the eastern part (Meuse-Rhine-Yssel ca	Utle).
Period	% B.M.R.	Total of	% of cans	Total of
	farms	farms	B.M.R.	cans
1st quarter \'62	14.7	17152	7.2	47884
2nd „ „	16.1	17228	6.4	61700
3rd	9.6	15222	3.7	51654
^th „ „	8.2	16740	3.7	48313
1st „ \'63	9.9	16430	4.3	49789
79

Dairy Factory	Asten	Riel	Raamsdonk
number of herds	308	118	196
always negative	62,9%	58,5%	40,3%
always positive	12,9%	24,1%	30,2%
varying	24,2%	17,4%	29,0%

Period:		Winter:	Summer;
Date of selection:		15-ll-\'62	18-4-\'63	27-8-\'63	28-8-\'63
Ohjccl of experiment:	buttcrmaking	checsemaking	buttermaking	clieesemaking
B.M.R. M-milk		• • •	• • •	• • •	• • •
N-milk		—	—	—	—
Cell-count M-milk		not detcrniined	I..\')00.000	1.300.000	1.275.000
N-milk		not determined	not delivered	90.000	120.000
Estimated tjuanfity	of both
milks		2.800 kg	5.500 kg	4.000 kg	4.000 kg
Quantity of ^	M-milk	1.120 gr	not separated	1.335 gr	not separated
scperator slime ƒ	N-milk	160 gr	not separated	533 gr	not separated

A,ge of the samples:	14 days	1 month 2 month	3 month	4 month	5	month
M-butter	I-	1 I —	11 fatty	III bacony	HI	train oily
I	I I	II fatty	111 bacony	HI	train oily
Mastitis	1 —	I I —	II fatty	iV bacony	HI	train oily
C-buttcr (control)	I — I 1 —	I I — I I 1 I	I — 1 —	I — I — I— a II		I — I — 1 —

Age of the samples:	14 days	1 month	2 month	3 month	4 month	5 month
Organol test	si. fatty	si, fatty	train-oily	very	very	very
M-Butter				train-oily	train-oily	train-oily
C-Butter	si sour	si, fatty	fatty	fatty	fatty	fatty
	si. fatty					>1. train-oily
r.B.A.-test
M-butter;	0,054	0,074	0,236	0,313	0,536	0,550
C-butter:	0,083	0,058	0,102	0,082	0,148	0,190
Peroxide-number
M-butter:	0,00	0,00	0,40	0,66	0,56	0,84
C-butter:	0,00	0,0>	0,32	0,19	0,24	0,32
Sour-number
M-butter:	0,59	0,59	0,56	0,60	0,64	0,58
C-bulter:	0,64	0,65	0,67	0,68	0,74	0,68
Copper-content
M-butter:	82 microgram/kg of buttc	:r
C-butter:	46 microgram/kg of butter.

from 1 to 1,5 the Total Mastitis Investigation is	called negative.
from 1,5 to 2 „ „	)> )> jj	„ dubious,
2 and more „ „	Ï» » J)	,, positive.
For instance:
B.M.R.		Total Mastitis Investigation
•----= 4 points	: 4 = 1	negative
± ----= 5 points	: 4 = 1,25	negative
± — ± — = 6 points	: 4 = 1,50	dubious
—• ± — =7 points	: 4 = 1,75	dubious
— — = 8 points	: 4 2	positive.

	Growth	Hemolysis	Growth	Hemolysis	Aesculine decom- position
Strept. agalactiae			-f		—
Strept. dysgalactiae			-1-		—
Strept. uteris	-1-		-t-	-(±)
Strept. lactis		-(±)	-h		-1-
Strept. faecalis			-1-		-f
Strept. zoöepidemicus	-1-		-f-1-		—
C. pyogenes	±		—
Staphylococci	-1- -1-	-I- —			—
E. coli	-1-	-1- —	-1- -f	-t—	—

	Str. lactis	Str. faecalis	Str. uberis	Str. bovis
InuHne	—		-1-	±
Raffinose	—	—	—	±
Sorbitol	—	-1-		±
Glycerol	—		—	—
Xylose		±	—	±
Litmus-milk	coagulation	reduction	no coagulation	usually
	acid, reduction	acid,	acid, reduction	coagulation
		coagulation		acid, reduction
Aesculine			-1-

	Escherichia coli	Aerobacter aerogenes^)	Pseudomonas aeruginosa	C. pyogenes^)	Listeria\'^)
Motility	motile	non motile	motile	non motile	motile (22° G)
Glucose	acid gas	acid -f- gas	no acid.	acid ±,	acid or neg.
			or acid	no gas
Sucrosc	neg. or acid	acid -f- gas	negative	acid ±,	no acid,
	gas	in 10 days		no gas	or acid.
					no gas
Lactose	acid gas	acid gas	negative	acid ±,	no acid,
				no gas	or acid.
					no gas
Indoi	positive	negative	njostly neg.	negative	negative
formation
Litnius-niilk	acid,	acid, no	coagulated.	coagulated	reduction
	coagulation	coagulation	alkaline,	at the bottom,	(white foot)
			reducted and	acid formation
			liquefacted	after
				48 hours; then
				peptonisation
Scrum broth	turbid	slimy ring	pellicle,	sediment	sediment 37° C.
			greenish		turbid (22° C.)
			fJiscolouration
Scrum agar	luxuriant	slimy bacterial	luxuriant	very small	small bact.
	growth,	colonies,	growth.	bacterial	colonics
	grey bactcrial	smell of	greenish	colonies
	colonies	trimethylamine	coloration
Gelatine	no liquefaction	no liquefaction	liquefaction	liquefaction	no liquefaction
Loffler	no liquefaction	no liquefaction	slow	liquefaction	no liquefaction