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Thesis 50v ”19 Degree of M. S.
l‘iiCEiEi-i‘zéz‘é S‘E‘ATE UHEVEESETY
Jose Britto Figueiredo

1957

j not-«'E

(1-21

 

LIBRARY

Michigan State
University

 

A COMPARISON OF THE CALIFORNIA MASTITIS TEST
WITH THE OTHER COMMONLY EMPLOYED

DIAGNOSTIC TESTS

by

Jose Britto Figueiredo

AN ABSTRACT

Submitted to the College of Veterinary Medicine
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health
1957

Approved WM:
_ c)

*’<ZC%TQZQ:2£:"—“

" 0

2

JOSE BRITTO FIGUEIREDO ABSTRACT

The California Mastitis Test (CMT), a new indirect
test for detection of bovine mastitis, was applied in 70
milk samples and its value was compared with seven commonly
employed diagnostic tests.

All samples were from mid-lactation phase and from
chronic or sub-clinical mastitis.

The CMT is closely correlated with the Whiteside test,
but instead of sodium hydroxide, a surface active agent and
bromcresol purple are used as reagent.

The per cent agreementsamong the methods of diagnosis
used and bacteriological isolation and identification (con-
sidered standard method) were: pH test 19.6; leucocyte counts
35.1; catalase test 41.2; Hotis test 46.6; Whiteside test
47.5; CMT 48.8 and bacterioscopic examination 54.7.

Bacteriological isolation and identification were
made on Edwards' medium and on the Tellurite-Glycine Agar
medium described by Zebovitz, Evans and Niven (1955), which
is a modification of Ludlam's medium. This medium is very
efficient to isolate coagulase producing strains of micro-
cocci and showed a per cent agreement of 83.0 as compared
with the test of coagulation of blood plasma.

The literature of the micrococci pathogenicity,
evaluated by their ability in producing cogulase and hemo-
lysins, was reviewed and discussed. The total agreement
between coagulase and hemolysins production occurred only

with the alpha-beta hemolytic Micrococcus pyogenes.

 

 

A COMPARISON OF THE CALIFORNIA MASTITIS TEST
WITH THE OTHER COMMONLY EMPLOYED

DIAGNOSTIC TESTS

by
Jose Britto Figueiredo

A THESIS

Submitted to the College of Veterinary Medicine
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1957

This thesis is dedicated
to my PARENTS and to my WIFE
who have been the inspiration of

my search for greater knowledge.

3U

 

 

.5. ‘”

vim-i. . J

ACKNOWLEDGMENTS

The author wishes to thank the following individuals
for the part they played in the preparation of this thesis.

To Dr. Lloyd C. Ferguson for his suggestions and the
stimulating conversations we had.

To Dr. Albert R. Drury who gave much of his time to
guide me in the research which has been presented in this
paper.

Besides his technical assistance in all the phases
of this work, Dr. Drury also made this stay at Michigan
State University a pleasant experience which I shall never
forget.

I should also like to thank Mrs. Betty R. Leiby for
her help in the course of this study.

The author is indebted to the staff of the Department
of Surgery and Medicine and particularly the Bovine Mastitis
Research Laboratory where this research was conducted.

The writer deeply appreciates the financial support

of the Universidade Rural do Estado de_Minas Gerais (Brazil)

 

 

and of the Rockefeller Foundation, which made it possible

for him to complete this work.

.-
n

a“.
‘1-4
L..

 

 

TABLE OF CONTENTS

CHAPTER PAGE

I. INTRODUCTION. . . . . . . . . . 1
II. MATERIAL 3
III. METHODS 4
Leucocytes counting. 4
Modified Whiteside test 5
Catalase 5
California Mastitis test (CMT) 5
Hydrogen-ion concentration 6
Bacterioscopy. 6

Hotis test. 6
Bacteriological isolation and identification 7

IV. RESULTS . . 9
Isolation and identification. 9
Leucocyte counts. ll
Hydrogen-ion concentration . . . . . . l2
'Whiteside test . . . . . . . . . . 12
Catalase . . . . . . . . . . . . l2
Bacterioscopy. . . . . . . . . . . 12

Hotis test. . . . . . . . . . . . 13
California Mastitis test (CMT) . . . . . 14

Per cent agreement . . . . . . . . . l4

CHAPTER
V. DISCUSSION.
Leucocyte counts
Hydrogen-ion concentration.
Modified Whiteside test.
Catalase test
Bacterioscopy
Hotis test
California Mastitis test (CMT)
Isolation and identification
The micrococci.
Catalase production by cocci
The streptococci
VI. SUMMARY AND CONCLUSIONS

BIBLIOGRAPHY.

PAGE
15
16
19
20
22
23
25
28
BO
31
38
39
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LIST OF TABLES

PAGE

TABLE

I. Efficiency of the Tellurite-Glycine Agar

medium for the isolation of coagulase
producing micrococci. 10

II. Efficacy, in terms of per cent agreement, of

the bovine mastitis tests which were studied

in comparison to the bacteriological

findings. . . . . 13
III. Comparison of bacteriological and other
diagnostic methods for the detection of
17

bovine mastitis (per cent).

 

 

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CHAPTER I
INTRODUCTION

If there is a subject in the entire field of animal
pathology to which basic research is always needed it is
mastitis or, more specifically, bovine mastitis.

Thousands of papers have been written and still many
points remain obscure or not well defined with respect to
pathology, etiology, diagnosis, treatment and, of course,
control.

The subject is really complex and, unfortunately,
the foremost research workers in this field very often have
different concepts. This situation led Murphy (1956) to
write, an excellent review of the whole picture, fixing some
important points and suggesting a general understanding.

Outbreaks of milk-borne human diseases have declined
due to extensive milk pasteurization and several types of
bacteriological and serological diagnostic methods, but
bovine mastitis continues as a very important dairy herd
infection.

The significance of carriers is great in almost all
animal infections, particularly in diseases like bovine
mastitis where no conclusive evidence has been found for an

artificial or even natural lasting immunity. Thus a rapid

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and precise diagnostic method is desired for the efficient
treatment and establishment of a practical control program.

With this objective in mind research workers at the
University of California recently developed a new test for
the detection of abnormal milk, which is called California
Mastitis Test (CMT).

The purpose of this study, which has no pretension
to fill deficiencies or vacuums, is to compare the new CMT
with 7 currently employed laboratory methods of diagnosis
for mastitis along with the medium described by Zebovitz,
Evans, and Niven (1955) for the isolation of coagulase

positive micrococci.

CHAPTER II

MATERIAL

Samples were collected from five different farms
located in four counties in Michigan.

The udders were washed with warm aqueous chlorine
solution containing 200—4OO parts per million of available
chlorine. Special care was taken on the end of the teat
which was re-washed in order to get the teat meatus as
bacterial free as possible.

The foreqnilk was used to perform the CMT, and sub-
sequent streams were used to carry out other tests.

Samples were always collected from each quarter Just
before milking, however, composite samples were also taken
from known negative cows.

All milking cows from which samples were collected
were in the mid-lactaction phase in order to avoid expected
alterations in the milk, since leucocyte counts are known
to be higher in the early and the late lactation.

In this thesis were included only chronic mastitis
Or'sub—clinical infections, which are latent and hence
anaxiliary methods of diagnosis must be applied.

Seventy milk samples were obtained from normal and

abnormaltmders.

CHAPTER III

METHODS

Generally standard or well accepted techniques were

utilized whenever available.

Leucocytes Counting

 

The technique of Prescott and Breed (1910) was used
with the following modification. Instead of measuring the
milk by means of a graduated pipette,a.closed platinum loop
which delivered a known amount was used for spreading the
milk on the guide area. This procedure is authorized by

the Standard Methods for the Examinations of Dairy Products

 

 

 

(1948). According to Malcolm and Smillie, cited by Black-
burn, Laing, and Malcolm (1955), there is no significant
variation between the two procedures.

Samples were examined after incubation. No signifi-
cant variations occur in cell counts if the film is made
before or after incubation. This was suggested by Bryan
(1935), and established by Slanetz and Naghski (1939).

The staining method employed was that suggested by
Charlett (1954) which is similar to that of Broadhorst-Paley
but does not require previous fixation and is performed in
10-12 seconds. The differential coloration makes leucocyte
counting easier and more accurate. Cells in 25 fields were

counted to determine the number per ml. of milk.

Modified Whiteside Test

 

This test described by Whiteside (1939) was performed
in accordance with the modification of Murphy and Hanson
(1941) and Murphy (1942). Two drops of normal sodium
hydroxide and 5 drops of fresh milk, or 1 drop of normal
sodium hydroxide h35 drops of refrigerated milk were mixed
on the surface of a plate commonly employed for the Brucella
agglutination test.

Readings were taken 20 seconds after mixing with a
toothpick. The reactions were scored according to the

directions given in that paper.

Catalase

The method used was suggested by Merchant and Packer
(1945) in which 5 m1. of 1 per cent hydrogen peroxide were
added to 15 ml. of fresh milk in a Smith fermentation tube.
Positive reactions were those in which over 1.5 m1. of gas
was accumulated in the blind arm of the tube after incubation

at 37Oc. for 3 hours.

California Mastitis Test (CMT)

 

The technique of Schalm and Noorlander (1957) was
employed. Two or 3 ml. of milk were drawn into a special
plastic paddle from each quarter of udder. A similar quantity
Of reagent was added by using a polyethylene wash bottle.
Positive reactions appeared immediately after the mixing of

reagent and the milk. During the period of observation,

the paddle was maintained in a gentel rotating motion.
Reactions were scored in the prescribed manner, that is,

based on the presence and intensity of precipitate or gel
formation. However, a classification designated as 4 was
utilized when an udder secretion was abnormal to the eye which
is commonly designated as garget. Also, pH was indicated by
changes in the color of the indicator.

Schalm (1957) explained the chemical basis of the CMT
in the following statement: "Chemical compounds belonging
to the group of surface active agents containing long-chain
hydrocarbon salts have been found to become visibly altered
in the presence of native proteins of cellular origin." The
surface-active agent used is anionic and bromcresol purple

is added as an pH indicator.

Hydrogen-ion Concentration

 

*The electrometric method employing a Beckman Glass

Electrode pH Meter, model H 2, was used.

Bacterioscopy

 

A thin smear was prepared and stained with the
Charlett (1954) improved staining method after an incubation
period of 12-20 hours at 370C., as suggested by Bryan (1935,
1941) and Little and Plastridge (1946).

Hotis Test

 

This method was proposed by Hotis and Miller (1936)

for the detection of mastitis due to streptococci. Further

7

observations permitted it to be used for the other mastitis
organisms. To carry out this test, 9.5 ml. of fresh or
refrigerated milk were added to 0.5 ml. of a sterile aqueous
bromcresol purple solution.

Two readings were made: one at 24 hours and another

at 48 hours of incubation at 370C.

Bacteriological Isolation and Identification

 

Two loops (4 mm. diameter) of fresh or refrigerated
milk were plated on Edwards' medium (1933) and on the

Tellurite-Glycine Agar mediuml

described by Zebovitz, Evans
and Niven (1955), and then incubated at 370C. This medium
is a modification of Ludlam's medium and as such, has

potassium tellurite and lithium chloride as selective agents.

Glycine is added to increase selection. The final pH of the

medium is adjusted to 7.2. By the use of these two media,

 

cocci and bacillar organisms (Pseudomonas sp., E. coli and
Proteus s2.) can be easily isolated, with the advantage that

coagulase positive Micrococcus pyogenes is identified in

 

24 hours by its black colonies on the Tellurite-Glycine Agar
medium. Very few other micro-organisms can grow on this
medium. Micrococci which are coagulase negative are recog-

nized because they grow in grey colonies.

 

1Supplied by Case Laboratory, Chicago, Illinois.

To confirm the ability of the black colony in
coagulating the plasma, it was transferred to a Brain Heart
Infusion and, after 16-24 hours of incubation at 370C., 2
drops were used as inoculum to 0.5 ml. of plasma solution.

Bacto-coagulase plasma,2

which is desiccated rabbit plasma,
was used. Boyd (1956) compares the use of‘desiccated plasma
with fresh citrated plasma and reported identical results.
The presence of catalase, gelatinase and hemolysin
production on 5 per cent bovine blood agar were also checked
in all cultures of cocci. Also, the capacity to hydrolize
sodium hippurate and metabolize some carbohydrates like
mannitol, esculin and insulin were tested.
Brilliant-Green-Bile 2 per cent broth, gelatin,

litmus milk, pigment production and a few sugars were used

to isolate and identify Pseudomonas sp. and Escherichia coli.

 

 

The Gram's staining characteristics were checked for

all organisms isolated.

 

2Supplied by Difco Laboratories, Inc., Detroit,
Michigan.

CHAPTER IV
RESULTS

A criterion to compare efficiency of the CMT and
other currently used methods of diagnosis of mastitis was
formulated based primarily on the isolation and identifi-
cation of the etiological agent.

A complete identification of each etiological agent
was not made, because it was judged unnecessary for the
objective of this work. The tests carried out were con-
sidered sufficient to permit grouping them in the following

categories of infections: (1) due to Streptococcus

 

agalactiae, (2) due to other streptococci, (3) due to

 

Micrococcus pyogenes, and (4) infections due to Pseudomonas

 

 

s2. or Escherichia coli. These categories were stated by

 

Murphy (1956) as accounting "for at least 99 per cent of

all mastitis."

Isolation and Indentification

 

In this study, a total of 54 micrococci were isolated
from 70 milk samples. Of these, 29 were coagulase positive
and 25 were coagulase negative strains.

The selective medium employed to isolate coagulase
positive strains, gave typical black colonies (coagulase

producers) in 32 instances, and grey colonies (coagulase

 

 

10
negative) in 29 instances. Twenty-nine of the 32 black
colonies were confirmed as coagulase positive and all the
grey colonies were coagulase negative. Thus, false positive
results were found 3 times. The per cent agreement of these

results are given in Table I.

TABLE I

EFFICIENCY OF THE TELLURITE-GLYCINE AGAR MEDIUM FOR
THE ISOLATION OF COAGULASE PRODUCING MICROCOCCI

 

 

Coagulase Producers

 

 

 

Compared Positive Findings Negative Findings P:r Cent
ree-
Medium Positive Negative Positive Negative gent
No. % No. % No. % No. %
Tellurite-
Glycine
Agar 29 100. 0 0.0 3 12.0 22 88.0 83.0

 

Gelatinase production-~Of the coagulase positive
micro-organisms, 81.4 per cent were gelatinase negative,
whereas only 18.6 per cent were gelatinase positive.

Hemotoxins--Of the total micrococci identified, 36

strains were hemotoxigenic as follows: 16 alpha-beta;

 

17 beta and 3 alpha. The 29 coagulase producing strains

were: 13 beta hemolytic and 16 alpha-beta hemolytic. The

 

25 coagulase negative were 4 beta hemolytic, 3 alpha hemolytic
and 18 non—hemolytic. The total agreement between coagulase
positive and hemolysin production occurred only with the

alpha-beta hemolytic Micrococcus pyogenes.

 

 

ll
Catalase--All isolated micrococci, coagulase negative
or coagulase positive, were datalase positive whereas the
streptococci were calalase negative.
From 70 milk samples, 21 streptococci were isolated.
Based on the results obtained from the tests used, the micro—

organisms were thus grouped: 4 Streptococcus agalactiae;

 

17 other streptococci (3 were Streptococcus uberis and 4

 

Streptococcus dysgalactiae). Mixed infections were found in

 

7 instances with Micrococcus pyogenes, and in 3 samples with

 

Pseudomonas EB' or E; coli.

 

 

With hemolysin production, the findings were: 9 alpha
(virescent), 4 beta and 8 gamma (non-hemolytic).
Bacillary infections were found 11 times due to

Pseudomonas sp. or E; coli. The latter was isolated in 10

 

instances and the first in one instance. Mixed infections
were found 3 times with streptococci and 5 times with micro-
cocci.

Of the 70 milk samples collected, 51 were bacteriolo-

gically classified as positive and 19 as negative.

Leucocyte Counts

 

In this study, 29 samples were found to be positive
(leucocyte counts above 500,000 per m1.) and 41 negative.
False positive were found 3 times. The 41 leucocyte counts
(58.5 per cent) that were found negative, contained less

than 500,000 pertml.; 11 samples (15.7 per cent) contained

12
500,00 to 1,000,000 per ml., and finally 18 samples (25.7
per cent) had 1,000,000 or more leucocytes perinl.
The results refer to the total cells. Differential
and total counts have no market advantage, except in milk

from late lactation. (Blackburn, Laing and Malcolm--l955).

Hydrogen-ion Concentration

 

The characteristics of milk used in this study made
it possible to regard those which had a pH value of 6.9 or
more as positive samples.

Ten samples were positive (14.3 per cent) and 60 were
negative (85.7 per cent). Forty-one negative samples came
from mastitic milk recognized by bacteriological findings.

This means that 80.4 per cent were false negative results.

Whiteside Test

 

The results were: 50 positive samples (71.5 per
cent) and 20 negative samples (28.5 per cent). False posi-
tives were obtained 7 times (14.00 per cent) and false

negative 8 times (40.0 per cent).

Catalase
Thirty samples (42.8 per cent) were classed as
positive and 40 as negative (57.2 per cent). False negative

results were found in 23 samples (57.2 per cent).

Bacterioscopy

 

The results refer only to cool-streptococci or

micrococci. Bacteriologically, 43 samples (61.4 per cent)

13
were found to contain pathogenic cocci, in pure or mixed
infections, and 27 non-pathogenic cocci. Size and morpho-
logic characteristics were the criteria for classification.
The bacterioscopic findings were positive in 52 instances
and negative in 18 instances. The distribution of false

results are given on Table II.

TABLE II

EFFICACY, IN TERMS OF PER CENT AGREEMENT, OF THE
BOVINE MASTITIS TESTS WHICH WERE STUDIED IN
COMPARISON TO THE BACTERIOLOGICAL FINDINGS

 

 

 

 

Bacteriological Per
Compared Positive Findings ‘Negative Findings Cent
Tests Positive Negative Positive Negative Agree-
No. % No. % No. % No. % ment
Leucocytes
counts 26 50.9 25 49.1 3 15.8 16 84.2 35.1
pH 10 19.6 41 80.4 0 0.0 19 100.0 19.6
Whiteside 43 84.3 8 15.7 7 36.8 12 63.2 47.5
Catalase 28 54.9 23 45.1 2 10.6 17 89.4 41.2
Bacterio-
scopic* 41 95.4 2 4.6 11 40.7 16. 59.3 54.7
Hotis* 36 83.7 7 16.3 10 37.1 17 62.9 46.6
CMT '41 80.4 10 19.6 6 31.6 13 68.4 48.8

 

 

*Examination for both cocci groups, only.

Hotis Test

 

The criterion used to score Hotis reactions due to
streptococci was that described by Hotis and Miller (1936)
and Miller (1943). To read the reactions supposedly due to

Micrococcus, the characteristics described by Miller (1943)

 

and Schalm (1948) were used. This is based mainly on the

digestion of the tested milk by the pathogenic micrococci.

14
In the 70 milk samples the Hotis test was classified
as positive 46 times and as negative 24 times. Comparisons

with the bacteriological findings are presented in Table II.

California Mastitis Test (CMT)

 

This test gave 47 positive results and 23 negative
results. Some positive and negative results were classified

as false and their distributions are found in Table II.

Per Cent Agreement

 

The comparison of the results from each type of test
with the bacteriological findings was made in the form of
per cent agreement. This was calculated as follows: the
sum of the true results in per cent minus the sum of false
results in per cent, all divided by two. Per cent agreement
was used to express the relative efficacy of the tests
employed. This method gives an idea of the real value of
the figures obtained.

There were cases in which the test gave 100 per cent
agreement in judging negative samples, such as pH, but
showed very low relative efficacy in respect to per cent
agreement. A low per cent agreement means, in this case,

a high number of false results.

CHAPTER V
DISCUSSION

The standard of comparison used in this thesis was
the bacteriological findings. This criterion can be criti—
cized because it is possible to find pathogenic bacteria in
the milk but no clinical disease. 0n the other hand,
inability to isolate pathogenic micro—organisms does not
mean, necessarily, that the udder is free of infection.

Also, some physical-chemical changes in the milk can
13ersist after the udder is made free of infection. Irritating
eéffects due to injection of some drugs into the udder may be

Iwesponsible for the increase in the abnormality of resulting
Seecretion. Drury (1952) has demonstrated this in the case
le Neomycin sulfate. He states that, "the irritation from
tide treatment was not as severee as the infection when
.Tudged on the basis of the leucocyte counts, choloride con-
‘tent, pH and resazurin class." Since samples were taken
Lfrem.untreated cows or from those treated sometime ago and
Thaw with chronic or sub—clinical infection, some of the
O‘bstacles were eliminated.

The choice of the methods used for comparison was
lGased on their relationship to the new CMT. Others were

em1ployed because they are commonly applied in the routine

16

control of bovine mastitis. A comparison of some of these

tests to cultural examination as reported in the literature

is summarized in Table III.

Letmocyte Counts

In normal milk cows, there is variation of cell number
from early, to mid and late lactation, also during the season
caf the year, and even between morning and evening milkings.
The borderline of normality is not sharp and has received
several interpretations from different workers.

Plastridge, Anderson and Williams (1939)found that
80.23per cent of 2,125 milk samples free of mastitis
(Jrgarusms, had under 100,000 cells per1m1.; 17.7 per cent
turd between 100,000 to 500,000 cells per1m1,; 2.0 per cent
hari 500,000 to 1,000,000 cells per ml. and only 0.1 per cent
Ccnltained over 1,000,000 cells per ml.

Recently, Schalm (1957) reported similar results on
69%) normal milk samples from the second week to the seventh
mcnlth of lactation. He found that 76.52 per cent had cell
Ccflants under 100,000 per m1.; 19.99 per cent between 100,000
to 500,000 cells per m1.;. 2.31 had 500,000 to 1,000,000
1991? m1. and finally, 1.15 per cent of the samples were
Classified as over 1,000,000 cells per m1.

McFarlane and Blackburn (1949) in comparing the cell
CCHMnts of the milk with "post mortem" histological findings
irl the udder, reported an agreement in 92 per cent of cases.

Er“phasis must be placed on the fact that those authors have

l7

 

 

 

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19
considered as positive "an animal in mid lactation if the
average count of the last two was over 100,000 per ml."

The level of leucocytes alone cannot be accepted as
satisfactory for diagnosis. However, in combination with
(abservation of bacteria in the smears a probable diagnosis
inay'be made. When the samples come from a mid-lactation
(HDW, as in this study, it is safe to consider the finding
(3f 500,000 or more leucocyte per m1. as a good indication
(of infection.

The results obtained were very low, as compared to
the ratios of efficiency generally found in this method.

It: can be explained due to the following factors: high
runnber of Micrococcus pyogenes isolated because of the use
017 a selective medium; in sub-clinical and latent infections,
firms leucocyte increase is not quite as high, and micrococcal
”Mastitis produces a moderately high or not so constant

l€nacocyte increase.

Eiyglrogen-ion Concentration Test
The pH of the normal milk can vary from 6.4 to 6.8,

‘bLIt is usually 6.5 to 6.6. Physiological variations are
fcnand in the colostrum. McIntyre, Parrish, and Fountain
(3&952) give the average of 6.28 and a range of 6.00 to 6.61.
'TYKe pH in the milk from cows which are drying off shows a
"Klre alkaline reaction.

Mastitic milk generally shows an increase in alkalinity

but when a high amount of lactose is metabolized by the

fl .flil. HUN drill—load”... i

 

20

infecting bacteria, the milk may show an acid reaction. The
variations of the milk in physico-chemical characteristics
are a result of injury and inflammation of the mammary gland,
(hie to the presence of the pathogenic agent.

It seems logical, in this sequence of thinking, that
the change toward the alkalinity point can be explained as
a result of the abnormal passage of blood elements into the
rnilk. The findings of high cell counts and the presence of
abnormal amounts of fibrin could be sufficient basis for
this hypothesis. However, Stableforth (1930) studying this
jpossibility, stated: "No evidence has been found to support
the view that the abnormal reactions is due to an influx of
kilood serum; the reaction of the milk shows no constant
IYEIation to its antigenic titre in serum-precipitin tests."

Espe and Smith (1952) suggested that the increase in
D}I values is "probably" due to the presence of carbonates.
NE) evidence was given.

It is known that fore-milk is more alkaline than the
Subsequent streams. In this study, the fore-milk was used

tC) perform the CMT, thus a low efficiency should be expected.

Minded Whiteside Test
In 1939, Whiteside described a reaction of abnormal

n111kfollowing the addition of a normal sodium hydroxide
SOilution, as the appearance of a "viscid mass."
Murphy and Hanson (1941) found a close parallelism

between the positive Whiteside test and the leucocyte content

 

11.14%??11
. V
flint]:

 

21

of the milk. When samples were scored as 2 +, 100 per cent

contained 500,000 or more leucocytes per ml., while samples

scored as 1 +, 73.1 per cent had 500,000 or more leucocytes

per'ml.

Schalm and Gray (1954) applied the test to about
5,000 milk samples and found that 85.14 per cent scored as
1 + with a leucocyte count of over 500,000 per m1. High
agreement (99.70 per cent) was found between reactions
scored as 2 + and leucocyte counts of over 500,000 per m1.

Dunn, Murphy, and Garrett (1943) concluded that the
reaction is mainly dependent on the leucocyte content in the
Inilk, directly or indirectly.

Petersen, Grimmel, and Schipper (1950) postulated
ttuat “the reaction is caused by absorption of fibrin on the
Vfllite cells. . . ." They observed the whiteside phenomenon
Wixth.charcoal in very fine powder added to blood plasma.

Schalm and Noorlander (1957) analysed the gel clumps
Seen in the Whiteside reaction and found a fat content of
£431jper-cent. They were also able to verify with microscopic
exEunination the leucocyte desintegration due to the addition
Of‘ sodium hydroxide. They concluded that fat has a secon-
(iaJ?y role in the Whiteside reaction.

Thus, since a high leucocyte count is often regarded
3&5 a result of an injury to the epithelium of the mammary
glimnd, the Whiteside test should be an accurate indication

of abnormal milk, at least in mid lactation.

22
The figures found for this test may be considered
satisfactory and the considerations made in regards to

letwocyte counts, may be applied to this test.

Catalase Test

Koning in 1908, cited by Monlux (1948), was the first
to apply the catalase test for the diagnosis of bovine
inastitis. The enzyme catalase is found in several kinds of
living cells both vegetable and animal. In animal cells,
the leucocytes are those in which it is found in larger
announts.

Prouty (1934) found that samples having leucocyte
ccnmnts of 0 to 100,000; 500,000 to 750,000; 750,000 to
l,cm00,000 and over 2,000,000 per m1. contained 11.5, 69.0,
923.0, and 96.9 per cent catalase respectively.

Monlux (1948) explained various factors which inter-
fttre with bovine mastitis diagnosis using the catalase test.

The catalase test showed an agreement which is about

ttus same as that reported by other workers.

gaiterioscopy

Direct microscopic examination means, of course, the
‘ViAsualization of the milk abnormality in a stained film.
3411s test, which is simple and accurate, shows a general
Iricture of the degree of udder inflammation through the

IIPesence of the etiological agent and a significant increase

of cells.

. 23
Baker and Breed (1920) suggested this procedure for

the detection of streptococcal mastitis. Bryan (1935)
classified infected milk by the detection of chain of 6 or
HKDPe cocci, in sample which were enriched by incubation at
370C for 12-15 hours.

Brown and Bryan (1950) reported on the daily obser-
vation for 6 months of one cow infected with Streptococcus

:agalactiae and another cow with no mastitis, that the test

 

was negative at all times with the non-infected cow and
zalways positive with the infected cow.
The regularity of elimination of Streptococcus

agalactiae in the milk gives this test a high score of

 

efficiency.

Bryan and Devereux (1937) using the microscopic
e)mamination as a standard, found an agreement of 86.6 per
Ceflt with the blood agar plate method.

Fay (1938) reported 96.0 per cent positive results
‘Wd:th this test and 9.0 false positives by comparison with
bacteriological identification. Using the same type of
COmparison, Slanetz and Naghski (1939) found 92 per cent
aruj 2.8 per cent positive and false positive results,
I‘espectively.

Recently, Narayanan and Iya (1954) also using the
SEune method of evaluation, found a very high figure of
eff‘iciency for streptococal infection as 98 per cent posi-

iiive and only 0.5 per cent false results.

 

 

 

 

24

The bacterioscopic test requires some skill in order
to get an accurate interpretation. Some factors which most
commonly result in errors are appearance of micrococci ex-
hibiting chain formation; the presence of saprophytic strains
often found among the micrococci and less frequent with
streptococci; the irregular shedding of the causative agent
in the milk and the presence of contaminanting bacteria. By
combining the observations on bacteria and leucocytes with
repeated examination, such factors are controlled.

There are chronic mastitis cases, however, in which
infection is so mild that the leucocyte count is within the
llormal range or, on the other hand, the leucocyte counts
.found may be due to an expected or physiological increase
(luring early and late lactation. In some instances, the
Sinmfle presence of bacteria is not enough for an accurate
CLLagnosis and more tests must be carried out to determine
ttue pathogenicity of the strains.

Some morphologic characteristics of the cocci may
iruiicate the presence of saprophytic or pathogenic bacteria
blrt this process is very inefficient. Of course, it is not
DCHSSible to differentiate coagulase-producing micrococci in
Stkiined smears. Since a selective medium for the detection
Of' the coagulase-producing micrococci was employed, it is
LIINierstandable why poor results were found. If the blood
agmtr plate method was used (hemolysin patterns), the results

'WOLle be better because 7 coagulase negative micrococci

25
strains were hemolytic, diminishing in this way the number
of false positive scores. If the blood agar plate was the
standard method, the per cent agreement would be 76.0.

Also, if bacterial findings were combined with the leucocyte
counts, the per cent agreement would be better. But in this
study, because the mastitis cases examined were chronic or

sub—clinical infections and due in most cases to micrococcal
infection, this combination was not to be so conclusive and

the agreement of 78.1 per cent confirms this supposition.

HOtis Test
This test was described by Hotis and Miller (1936)

for-the detection of Streptococcus agalactiae infection.
These authors compared the test with blood agar plate
.isolation and found that of 753 samples tested 95 per cent
Eugreed.

Murphy (1939), also using blood agar plate isolation
as; a standard, found that 94.86 per cent of the quarter
Seunples contained Streptococcus agalactiae while 53.63, 19.61,
arni 10.53 per cent contained Streptococcus dysgalactiae,
§j¥§§ptococcus uberis and atypical streptococci infections,
FEHspectively. False negative results for the same bacteria
'Welfie 5.14, 47.37, 80.39, and 89.47 per cent, reSpectively.
Orfily'0.79 per cent micrococci agreed and 99.24 per cent were
false negatives. The false positive reactions for Strepto-

EfilEfizgg agalactiae were 1.05 per cent with quarter samples.

 

26

Miller (1943) using the same method in the comparison
of 10,000 to 15,000 milk samples tested at the U. S. D. A.
Animal Disease Station, Beltsville, Maryland reported a mean
agreement of 85 to 90 per cent positive results. In the
same paper reactions due to other pathogenic organisms found
parasitizing the cow's udder were described.

By using Edwards' medium for the isolation of strepto-
cocci in 4,132 milk samples, Schalm (1944) found the
following results: 84.3 per cent positive reactions and
19.3 per cent false positive reactions with respect to

Streptococcus agalactiae. In samples yielding Streptococcus

 

dysgalactiae, Streptococcus uberis and atypical streptococci,

 

the Hotis test was positive only 11.6 per cent, suspicious
111 46.5 per cent, and negative in 41.8 per cent. With Migro-
cxbccus infection, 1.8 per cent were positive, 36.8 per cent
vwere suspicious, and 61.3 per cent were negative.

Later, Schalm (1948) studying the correlation of
CCHagulase positive micrococci and Hotis reactions, found
ttuat 80 per cent showed positive reactions and 7.1 per cent
Stubwed false positive reactions.

Brown and Bryan (1950) carrying out the modified
HfYtis test daily for 6 months, on samples from an infected
COVV having Streptococcus agalactiae and in samples from a
non-—cliseased cow, reported positive results in 100 per cent
0f"the mastitic milk; 15 per cent of false positive reactions

‘WeIRe observed in the non-infected animal.

27

Throop, Swanson and Mundt (1957) demonstrated the
efficiency of the Hotis test by comparing it with physical
examination of the udder, strip-cup, pH, leucocyte count,
microscopic examination and isolation of the etiological
agent. The criterion of mastitis diagnosis was shown by
direct test combined with l or more indirect test. Detection
of Streptococcus agalactiae infection by the Hotis test was
found in 82 per cent of the samples whereas Streptococcus

dysgalactiae and Streptococcus uberis were found only in 50

 

per cent of the samples. They concluded that the Hotis test

is an excellent method for detecting bovine mastitis due to
Streptococcus agalactiae but not for Streptococcus dysgalactiae
or'Streptococcus uberis because of the large number of nega-
‘tive and suspicious results.

The variations of results in scoring Hotis test may
b6? explained by several factors. Thus, the amount of
agmglutinins present is expected to be important because the
fOIfination of flakes or balls on the side or bottom of the
ttfloe represent "the alteration of the surface which causes
ttue organisms to clump. . . . " (McCulloch and Fuller--l939).
TTLis fact is not only observed with Streptococcus agalactiae
bud: also with Streptococcus uberis and Streptococcus dysga-
W- McCulloch and Fuller (1939) stated that it is
pcfissible to find typical Hotis reactions with any organism
Whixsh stimulates the production of agglutinins, grows in
pITESence of bromcresol purple, forms clump due to presence

‘Df zagglutinins and produces acid from lactose.

28

The number of bacteria present in the sample, the
presence of contaminants such as spore-forming bacilli, the
existence of more than one pathogen and the ability of the
bacteria to ferment lactose are other elements correlated
with the readings.

Pounden, et a1 (1957) stated that "some of the marked
variations encountered might well be explained by differ-
ences in the suitability of the milk as a medium for the
organism. . . ." Some limited evidence for this was
presented.

Perhaps these factors make it easier to understand
the statement of Plastridge and Hale (1939) when they called
the Hotis test "inadequate" for the diagnosis of bovine
Inastitis due to Streptococcus agalactiae.

The problem of contaminants, which can be diminished
by'rnicroscopic examination of the milk from the Hotis test
tlibe, is really important. This was found to be true mainly
‘W11dh micrococcal infections because spore-forming bacilli
WGIN? found 4 times giving a false positive reaction.

The data found in several papers refers mostly to
streptococcal infection (Table III). In this work, the
Hotxis test was used for cocci--both streptococci and micro-

C0C:<:i--which resulted in lowered efficacy.

.EéfigLfornia.Mastitis Test (CMT)
The California Mastitis Test is a new bovine mastitis

tefiit proposed by Schalm and Noorlander (1957). It is an

29

indirect test which is based on three known mastitic phe-

nomena: pH modification, Whiteside test and on the abnormal

number of leucocytes in mastitic milk.
Schalm and Noorlander (1957) presented data showing

a close relationship between CMT score and leucocyte counts

of the milk.

The characteristics of these tests were discussed

and almost all considerations, advantages and disadvantages,

can be applied to this test.
The characteristic readings, however, are principally
correlated with the Whiteside phenomenon which is based on

the abnormal leucocyte content in the milk which comes from

a diseased udder.
Whiteside described this phenomenon as a "viscid mass"
aund this description agrees with that reported by Puri and

Chapta.(l955) who stated: "Protein contributes by far more

tcnnards the viscosity of milk than any other major constitu-

erlt of milk." The increase of protein generally occurs in

rnaxstitic milk, as well as in early and late lactation.

The surface-active agent used eliminated the secondary

IYDle played by the fat content of the milk.

In this work, the CMT was found to be superior to
Otflier*test except the bacterioscopic examination.

These results were obtained from fore-milk and the
CkMiracteristics of the material employed eliminated the

pc”Ssibility of large number of false positive results, due

30
to the presence of physiological leucocyte increase in early

and late lactation.

Isolation and Identification
An increase of bovine mastitis due to micrococci has

taken place in recent years or, at least, more attention

has been paid to it, resulting in more reported cases.
Perhaps, because mastitis caused by streptococci is declining
as the result of increased use of antibiotic therapy, micro-
cocci infections due to antibiotic resistant strains are
often found. This problem has been brought into focus
recently. Dolman (1956) commenting on seven decades of
research on the micrococci, stated that: "Today, staphy-
lococcal infections appear more prevalent, more virulent,
euld more unavoidable than they were a quarter century ago,
irl the pre-penicillin era." This paper refers to human in—
ftections but, of course, the same statement can be applied
tC> bovine mastitis.

Schalm and Woods (1953) describing the mastitis
Ccnnplex concluded: "Cocci are the dominant organisms in
true bovine udder. Both streptococci and Micrococcus pyogenes
area highly invasive for that organ, but with gradual elimin-
aiiion of the former through the use of antibiotics, the
lEl‘tteris assuming even greater significance in the mastitis
COrYlplex."

These opinions are not generally accepted. Plastridge

arki Hale (1956) showed that Streptococcus agalactiae

31
infections have decreased but the micro-orgalism continues

to be considered as the principal agent of mastitis. This

evidence is basis on data collected from several years of

mastitis research in Connecticut.

These two adverse opinions may be the result of dif-

ferent methods of diagnosis, treatment, and control-programs

applied in those areas.

The micrococci. The criterion of judging micrococcal

 

pathogenicity, is based mainly on its ability to secret
toxins which have the capacity to lyse the red blood cells
43f certain animal species and its capacity to coagulate blood

plasma.of some species. Many papers are concerned with

rnicrococcal hemolysins and so far, four different ones have

'been.accepted.

The presence of hemotoxins elaborated from strains

Of‘lnicrococci, called "Hot-Cold" lysins have been described

Sixlce 1922 but a differentiation between these two was made
b3? Glenny and Stevens (1935) using specific neutralization.
Ttue terms alppa and papa were suggested by them.

The aippa toxin causes total lysis of rabbit and
Skkeep erythrocytes while the papa toxin is a weak lytic
agant at 370C. but becomes rapidly hemolytic at lower tem-
Ipelfiatures. Hence, this phenomenon is termed "Hot-cold." It
Inabf be observed in species in which the red blood cells are
rHDt' lysed during incubation as occurs with man, rabbit as

'WGJJI as sheep and oxen. Sub—types of alpha and beta toxins

have been reported.

32
The gamma toxin was found by Smith and Price (1938)
and can lyse rabbit, sheep, bovine, rat, guinea pig, horse,
and human red blood cells.

The delta type hemolysins were described by Williams

 

and Harper (1947). These have the capacity to lyse sheep

red blood cells protected by alpha, beta or alpha-beta toxins.

 

This hemotoxin is active also against rabbit, human, horse,
rat, guinea pig, and mouse erythrocytes.

Elek and Levy (1950) described a new hemotoxin which
lyses rabbit and sheep erythrocytes which is called epsilon.
It was secreted by a coagulase negative micro-organism.

They concluded also thatialppa2,.aalpa and gapma are iden-
tical toxins. According to the results and suggestions

presented by them, Micrococcus pyogenes produces alpha, beta,

 

 

aalpa and epsilon hemotoxins. Only papa hemolysis is incom-
plete and more than one hemolytic toxins may be secreted by
‘Une same strains.

Minett (1936) stated that papa hemotoxin is a charac-
teristic of hemolytic micrococci from animal sources. He
later (1937) reported that bovine strains can secret aAppa
and papa toxins.

A similar statement was made by Elek and Levy (1950)
who also confirmed that papa is often elaborated by animal
Strains and is not common in micrococci parasitising man.

The ability of some Micrococcus strains to cause the

 

Coagulation of blood plasma has been known since 1903 through

33
original report of Loeb (cited by Smith and Hale--l944). It

was only after several years, however, that the presence of
this enzyme was proposed as the best single test to judge
micrococcal pathogenicity.

Human or rabbit plasma are generally used for this
test because of the consistent results obtained. Variations
in intensity of results take place with different plasma
sources. The explanation for the differences of reactions
was given by Smith and Hale (1944) who described micrococcal
coagulase as the percursor of a thrombin-like substance
formed in the presence of an activator normally found in
some plasmas. The failure of plasma to be coagulated is
due to the deficiency or absence of this activator. This
variation in activator content can be found also in the same
species as an individual characteristic.

Smith, Hale and Smith (1947) reported that coagulase
is closely related to the pathogenicity of certain strains
and described its role in development of infection. It is
important in the early stages when the micro-organisms are
establishing and starting to reproduce or, in other words,
it plays an important role in protecting the micrococci
against primary defenses of the host. This is made possible
through the fibrin clot formed around the cocci.

The association of hemolysin and coagulase production
with pathogenicity has been studied by a large number of

workers. Chapman, 23.21 (1934) found that of 690 Micrococcus

 

 

34

pyogenes var. aureus and 1,852 Micrococcus pyogenes var.

 

albus, 88 per cent and 11.9 per cent were coagulase positive,

respectively. With Micrococcus pyogenes var. aureus, they

 

reported: 78 per cent of non-hemolytic and coagulase nega-
tive strains did not kill rabbits in 10 days; 100 per cent
of coagulase and hemolysin positive strains killed rabbits

in less than 10 days. With Micrococcus pyogenes var. albus,

 

they found: 77 per cent of 17 hemolytic strains did not
kill rabbits in 10 days and 88 per cent of 16 coagulating
strains killed in less than 10 days.

Williams and Harper (1946) tested 100 coagulase
positive strains and reported that 93 were_alppa hemolytic
and 2 were papa hemolytic. The strains were, presumably,
from human sources.

In comparing laboratory methods accepted as evidence
of micrococcal mastitis, Plastridge, Weirether and Williams
(1938) found that the coagulase test was more nearly cor-
related with the micrococcal pathogenicity than the other
tests used. They reported hemolytic properties in 87 per
cent of the coagulase positive strains and 5 per cent from
the coagulase negative bacteria.

A high frequency of alpha-beta and beta hemotoxins to

 

bovine red blood cells was found by Schalm and Wood (1953a)
who tested 2,084 hemolytic strains, almost all from bovine
mastitis, with a coagulase positive score of over 99 per

Cent. Out of 209 micrococci non-hemolytic or weakly hemolytic

35

but showing no beta toxins, 17 were coagulase positive. They
stated that "for practical applications, manifestation of
beta toxins should be sufficient evidence that the Micro-

coccus is Micrococcus pyogenes." Coagulase tests should be

 

used as a decisive test in questionable cases. This can be
accepted because coagulase production is a special property

of Micrococcus pyogenes.

 

Slanetz and Bartley (1953) studied about 9,000 strains
of micrococci from mastitic milk and concluded that: (1)

They may be alpha-beta, beta or delta hemolytic to sheep,

 

 

cow, and rabbit red blood cells. In most cases they are

the alpha-beta type. (2) With the exception of weakly

 

toxigenic strains which are coagulase negative, these micro-
cocci are usually coagulase positive. (3) Leucocyte counts
are not significant for the diagnosis of chronic mastitis
infections due to micrococci.

Boake (1956) presented conclusive evidence on the
real importance of coagulase in micrococcal pathogenicity.
He found that rabbits actively immunized with coagulase were
more resistant to challenge with coagulase positive and
_a£pha hemolytic strains (mean survival time of 9.3 days)
than those immunized with aippa toxoid (mean survival time
of 1.3 days). Unprotected individuals had a mean survival
time of 1.5 days. On the other hand, rabbits immunized with
Coagulase and later challenged with a coagulase negative

Strain showed no resistance and died 2.5 days after

36
inoculation. The same survival time was found in the control
group. Based on mice experiments, Boake (1956) found that
"in vitro" inhibition of coagulase activity appeared to
diminish the pathogenicity of micrococci.

Rammelkamp, Jr., a: ai (1950) were able to demonstrate
anticoagulase--an antibody specific for coagulase--in 11 of
17 monkeys injected with cell-free coagulase.

Although coagulase is an important factor in patho-
genicity, it does not appear to be the only mechanism for
this condition. There are authors like Deubler and Cole
(1956) who believe that coagulase and hemolysin are not
indicative of micrococcal pathogenicity. From 12 cases of
acute micrococcal mastitis they isolated 8 strains which
were both coagulase and hemolysin negative.

From a comprehensive review of the literature on
this subject, one can see that a definite method for
evaluating micrococcal pathogenicity has not yet been found.
Coagulase production, however, appears effective in this
respect than hemolysin activity. Analysis of studies in
this subject reveal that many controversies are brought
about because of the differences in the blood employed,
both their sources and concentration; the sources and dilution
of blood plasma and finally the different technique used.

Due to these difficulties other methods have been
suggested for indicating pathogenicity such as the number

of antigen-antibody floculation lines formed on a plate

37
(Howard--1954) and phage pattern (Fisk-~1942; Howard-~1954,
and Edwards and Rippon--1957). In this study, coagulase
production was used as an indicative test of the micrococcal
pathogenicity.

Zebovitz, Evans and Niven (1955) employed a Tellurite-
Glycine Agar medium to isolate coagulase producers micro-
cocci. They compare the new medium with Chapman-Stone
medium and Phenol Red Mannitol Salt Agar and reported signi-
ficantly superior results with the improved medium.

Innes (1953), using Ludlam's medium for plating milk
samples, reported the isolation of 5 coagulase positive
micrococci from 24 milk samples cultured. The same samples
plated on Blood-Agar yielded no coagulase-producers due to
the high interference of contaminanting bacteria. Samples
were enriched in special selective broth before incubation.

Velilla, Faber and Pelczar (1947) using Phenol Red

Ddannitol Salt Agar medium (PRMS) and Staphylococcus medium

 

110, were able to isolate 90 strains of micrococci on the

first medium and 137 Micrococcus strains on the latter medium,

 

from a total of 270 quarter samples. From 90 of those
strains isolated using PRMS, only 18 were coagulase-producing
Inicrococci while from the 137 isolated on the medium 110,
31 were coagulase positive.

The results found by using Tellurite—Glycine Agar
medium (83 per cent agreement) were superior to those ob-

tained by Innes (1953) on Ludlam‘s medium and by Velilla,

-:.__ L...7 g.

38

Faber and Pelczar (1947) on PRMS agar or Staphylococcus

 

medium 110.

The criterion used to identify pathogenic Micrococcus

 

taking its ability for coagulating blood plasma as a simple
and single test, is actually superior to the production of
hemolysins. Coagulase is found in almost all pathogenic

strains of Micrococcus pyogenes and it is believed to be a

 

specific characteristic. 0n the other hand, hemotoxin pro—
duction is a less constant occurrence in pathogenic micro-
cocci and it has been found to be elaborate by coagulase
negative strains. However, in this study, a total agree-

ment was found between alpha-beta hemolytic and coagulase-

 

producing micrococci.
By using the Tellurite-Glycine Agar medium, it is
possible to isolate a coagulase producers micrococci strain

in 24 hours.

Catalase production by cocci. By testing for catalase
in cultures of cocci, one has an easy way of screening out
certain streptococci (short chain formation). It is generally
agreed that catalase production is very constant in Mippp-
coccus, although Lucas and Seeley (1955) reported the
finding of a negative catalase strain which was strongly
coagulase positive.

The data obtained in this work confirms earlier ob-

servations on the catalase test.

39

The streptococci. The selective medium of Edwards

 

was used to culture streptococci. From the color and size

of colonies, streptococci, Pseudomonas and coliform organisms

 

can be recognized but the micrococci were inhibited. In
this medium the selective agents are crystal violet and
esculin. Identification was made on the basis of the
following tests: CAMP test described by Murphy, Stuart and
Reed (1952), catalase production, esculin, mannitol and
inulin breakdown, hydrolysis of sodium hippurate and hemoly-
sin production.

The CAMP test is an application of the lytic pheno-
menon found by Christie, Atkins and Munch-Petterson (1944)

in which Streptococcus agalactiae, hemolytic or non-hemolytic,

 

secretes a lytic product that can totally lyse sheep or oxen

erythrocytes previous altered by a micrococcal papa toxin.
Wilson and Salvin (1950) included some micrococci

as CAMP positive but confirmed the fact that all the Strepto-

coccus agalactiae tested were positive.

 

However, Murphy, Stuart and Reed (1952) reported

that 96.6 per cent of 323 strains of Streptococcus agalactiae

 

produced CAMP reactions, whereas 85 per cent of 222 strains

of Streptococcus uberis and 100 per cent of 49 Streptppoccus

 

_gysgalactiae gave negative results.

*m—o—u-_m L‘4 —. - .

CHAPTER VI

SUMMARY AND CONCLUSIONS

The California Mastitis Text (CMT) was applied to
70 milk samples and its value as a new method for the
detection of bovine mastitis was compared with 7 well known
tests.

The CMT, which is an indirect test, compared favorably
to leucocyte counts, pH, Whiteside test, catalase test, and
Hotis test but not to the bacterioscopic method.

The bacteriological finding, which was used as the
standard test, was made on Edwards‘ medium and on.the new
Tellurite-Glycine Agar medium. This medium, which is a modi—
fication of Ludlam's medium, proved to be very efficient
for isolating coagulase producing strains of micrococci.

Coagulase and hemolysin were studied and their value

as indicators of micrococcal pathogenicity was discussed.

BIBLIOGRAPHY

BIBLIOGRAPHY

Backer, J. C. and Breed, R. S.--1920. ”The reaction of
milk in relationship to the presence of blood cells
and of Specific bacterial infections of the udder."
N. Y. Sta. Agri. Exp. Sta., Tech. Bull. 80.

Blackburn, P. 8., Laing, C. M., and Malcolm, D. F.--1955.
"A comparison of the diagnosis value of the total
and differential cell counts of bovine milk." J.
Dairy Res. 22 (l) : 37—42.

Boake, W. C.--l956. "Antistaphylocoagulase in experimental
Staphylococcus infections." J. Immun. 76 (2) : 89-96.

 

Boyd, H.-—l956. "An evaluation of lyophilized plasma in the
coagulase test for micrococcal pathogenicity."
Am. J. Med. Tech. 22 (4) : 232-236.

Brown, R., and Bryan, C. S.--l950. "Six months of daily
mastitis testing of an infected and a non-infected
cow.” Mich. Agri. Exp. Sta. Quarterly Bull. 32 (3)
322-327.

Bryan, C. S.--l935. "The microscopic diagnosis of infectious
mastitis." Vet. Med. 30 (4) : 149-155.

.--1941. "The microscopic detection of bacterial
defects of milk." Vet. Med. 36 (8) : 415-419.

 

Bryan, C. S., and Devereux, E. D.--l937. "A comparison of
the microscopic test, Hotis test—and blood agar plate
in detecting Streptococcus of mastitis in milk."
Cornell Vet. 27 (l) : 68474.

 

Chapman, G. H., Berens, C., Peters, A., and Curcio, L.--1934.
"Coagulase and hemolysin tests as measures of the
pathogenicity of staphylococci." J. Bact. 28 (4)

343-363.

Charlett, S. M.--l954. "An improved staining method for the
direct microscopical counting of bacteria in milk."
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“ ‘o-. k-»u—Pf
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_‘_

‘wf

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__ V
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G
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__._ -4 .. —r~.

 

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