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ABSTRACT
S. R. Ahmed M.Sc. Animal Science
COMPARISONS OF THE PATHOGENESIS OF BOVINE MASTITIS CAUSED BY VARIOUS BA.CTERIAL AGENTS WITH SPECIAL REFERENCE TO CELLULAR
RESPONSE
In all, 2,658 individual quarter fore-milk semples (IQFM)
from 68 freshly calved cows representing two experimental herds of
Macdonald College were tested. Out of these, 31.5~ were California
Mastitis Test positive (OMT(+». The average Total Somatic Cell Count
(TSCC) in CMT(+) quarters was 3.29 million cells/ml. In this count,
epithelial cells were 15.50%; neutrophils 44.98,%; lYmphocytes 39.21%
and eosinophils 0.31%.
The overall incidences of infection due to various organisms
were~. agalactiae 11.93%; other streptococci 0.34%; Staph. aureus
3.42%; micrococci 2.18%; non-hemolytic staphylococci 17.68%; coliforms
1.02% and mixed infection 34.95%.
The TSCC was highest in coliforms (10.15 millio~ml) followed
by Str. agalactiae (4.13 million/ml) and Staph. aureus (4.03 million/ml).
The percentages of neutrophils were also highest in the above infections.
The percentage of neutrophils ran almost parallel to the TSCO but was
interrupted by a high percentage of lymphocytes in the 1st, 2nd, 4th
and 5th lactations. The percentages of eosinophils were highest (5.14%)
in the 2nd lactation.
The average TSOC was higher (3.41 million/ml) in Ayrshire
than the Holstein breed. The percentagœof neutrophils were higher
(49.08,%) in the Holstein èows than in the Ayrshire cows. It was
observed that the percentages of neutrophils were highest in the
fall months and lymphocytes were highest in the spring months. The
neutrophil/lymphocyte (N:L) ratio was highest in serum transferrin
type TfEE (1:1.94). The highest percentage infection due to
Str. agalactiae was observed in Tf types possessing alleles TfD1
and TfD2.
Résumé
S. R. Ahmed M.Sc .• Science Animale
Comparaisons de la pathogénie de la mamite bovine causée par différents
agents bacteriens avec une attention speciale ~ la reaction de la cellule.
Un total de 2658 échanti~lons de lait prélevés individuellement pour chaque quartier avant la traite (ELITT) provenant de 68 vaches fraîchement Vêlées dans 2 troupeaux différents du Coll~ge Macdonald ont été analysés. De ceux-ci, 31.53% étaient positifs selon le "Calif'ornia Mastitis Test" (CMT(+». La moyenne du nombre total de cellules somatiques (NCST) pour les quartiers affectés selon le CMT(+) a été de 3.29 millions de cellules/ml. Ce nombre était composé de 15.50% de cellules epithéliales; 44.98% de neutrophiles; 39.21% de lymphocytes et 0.31% d'oesinophiles.
L'incidence totale de l'infection causée par les différents organismes pathog~nes ont été de 11.93% pour Str. agalactiae; 0.34%· pour les autres streptococcis; 3.42% pour Sta~ aureus; 2.18% pour mdcrococci; 17.68% pour staphylococci non hemolytique; 1.02% pour coliformes et 34.95% pour infections multiples.
Le NCST a été le plus elevé pour coliformes (10.15 millions/ ml) suivi par Str. agalactiae (4.13 millions/mi) et Staph. aureus (4.03 millionS/ml). Les pourcentages de neutrophiles ont été également les plus élevés pour les infections mentionnées antérieurement.
Le pourcentage de neutrophiles se comporte d'une façon paralléle au NCST ~s a,été in~errompu,par un pourcentage élevé de lymphocytes à la 1ere , 2eme, 4 eme et 5eme lactations. Les pourcentages d'eosinophiles ont été les plus élevés (5.14%) ~ la 2éme lactation.
La moyenne de NCST a été plus éleves (3.41 million~ml) pour la race Ayrshire que pour la race Holstein. Le pourcentage de neutrophiles ont été plus élevé (49.08%) pour les vaches de la race Holstein que celles de la race Ayrshire. On a abservé que les pourcentages de neutrophiles ont été les plus élevés ~ l'automne et que ceux des lymphocytes ont été les plus élevés au printemps. Le rapport neutrophile/lymphocyte (N:L) a été le plus élevé dans le serum type "transferrin" TfEE (1:1.94). Le plus élevé pourcentage d'infection causée par~. f6alactiae a été observé pour les types Tf possédant les all~les TfD et Tf'D2.
Suggested Short PitIe:
Cellular Response in Bovine Masti.tis
Ahmed
ACKNOWLEDGEMENTS
The author extends bis sincere thanks to Dr. H. C. Gibbs
for his inval.uable guidance and encouragement during the course of
this investigation. Special acknowledgements are due to
Dr. H. F. MacRae for his keen interest.
The author wishes to thank Dr. S. S. Malik for his valuable
suggestions througbout the study period and for transferrin pheno
typing of the cows under investigation.
Sincere thanks are also due to Dr. J. E.. Moxley for
providing computor facilities and to Mrs. M. Baker for programming.
Appreciation is extended to Mrs. M.. Mackie for her
tremendous support througnout the study period both in rendering
technical assistance as well as untiring efforts in t,yping the
manuscript.
Appreciation is extended to Mr. R. Channon for assistance
in photomicrography, to Dr,. L. J. Martin for assistance in collecting
blood samples and to the personnel of the Macdonald College farm for
cooperation and assistance duri~.g this investigation.
The author expresses appreciation for the timely co
operation of Mr. A. H. Javed, Mr. Q. Sheriff, Mrs .. Musht.er1 Begum,
Dr. R. P. Gupta and Mr. Arca Romeo.
The author is indebted to his brother, Mr. S. Su1aiman
for his moral support during the depressing moments of the study.
The financial assistance of the Quebec Agricul tural
Research Council is gratefully acknowledged.
Finally, l take this opportunity to thank: my wife,
Nasreen, who, with great patience, understanding and sacrifice
undertoOk the sole responsibility of bringing up the children,
Farooque, Rafat and Rehana while l was aWB:if on my "academic
pursuits."
TABLE OF CONTENTS
INTRODUCTION ••••••••••••••••••••••••••••••••••••••••••••••
REVIEW OF LITERATURE ••••••••••••••••••••••••••••••••••••••
A. General Review • ••••••••••••••••••••••••••••••••••••
l. Histo~ ••••••••••••••••••••••••••••••••••••••••
II. Pathology ••••••••••••••••••••••••••••••••••••••
1. Clinical Mastitis ••••••••••••••••••••••••••
2. Subclinical Mastitis •••••••••••••••••••••••
Interstitial Mastitis ••••••••••••••••••••••
4. Exudative Mastitis •••••••••••••••••••••••••
5. Suppurative Mastitis ••••••• ~ •••••••••••••••
6. Gangrenous Mastitis ••••••••••••••••••••••••
7. Fibrosis •••••••••••••••••••••••••••••••••••
III. Predisposing Factors in Mastitis •••••••••••••••
1. Anatomical Factors •••••••••••••••••••••••••
2. Physiological Factors ••••••••••••••••••••••
a) Age ••••••••••••••••••••••••••••••••••••
b) Stage of Lactation •••••••••••••••••••••
c) Milk Yield •••••••••••••••••••••••••••••
d) Hormones •••••••••••••••••••••••••••••••
3. Environmental Factors ••••••••••••••••••••••
a) Season and Weather •••••••••••••••••••••
b) Housing ••••••••••••••••••••••••••••••••
c) Grazing Land Topography ••••••••••••••••
Page
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TABLE OF CONTENTS
4. Heredî tary ••••••••••••••••••••••••••••••••••
5. Management Factors ..........•.........•..... a) Herd Size •••••••••••••••••••••••••••••••
b) Feeds and Feeding •••••••••••••••••••••••
c) Sanitary Procedures •••••••••••••••••••••
d) Milking Machine •••••••••••••••••••••••••
IV. Diagnosia of Bovine Mastitia •••••••••••••••••••
1. Barn Tests •••••••••••••••••••••••• ~ •••••••••
a) Physical Examination of the Udder •••••••
b) Physical Examinatioll of Secretion •••••••
c) Bromothymol Blue Test for Altered pH ••••
d) Whiteside Test ••••••••••••••••••••••••••
e) California Mastitis Test ••••••••••••••••
2. Laboratory Tests ••••••••••••••••••••••••••••
a) Chloride Test •••••••••••••••••••••••••••
b) Catalase Test •••••••••••••••••••••••••••
V. Somatic Cells and Mastitis •••••••••••••••••••••
1. Number of Somatic Cells •••••••••••••••••••••
2. Types of Somatic Cells ••••••••••••••••••••••
3. Origin and Function of Different Cell Types in Mîlk •••••••••••••••••••••••••••••••••••
a) Epithelial Cella ••••••••••••••••••••••••
i) Non-Vacuolated Epithelial Cells ••••• ii) Vacuolated Epithelial Cells •••••••••
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TABLE OF CONTENTS
b) Neutrophils •••••••••••••••••••••••••••••••
c) Lymphocytes •••••••••••••••••••••••••••••••
d)
e)
f)
g)
Monocytes •••••••••••••••••••••••••••••••••
Eosinophile •••••••••••••••••••••••••••••••
Colostrum Bodies ••••••••••••••••••••••••••
Erythrocytes ••••••••••••••••••••••••••••••
4. Ph;ysicaJ. Factors Influencing Numbers and Relative Proportion of Cells ••••••••••••••••
a) Different Fractions of Milk •••••••••••••••
b) Diurnal Variation •••••••••••••••••••••••••
c) Stage of Lactation ••••••••••••••••••••••••
d) Lactation Age •••••••••••••••••••••••••••••
5. Effect of Mastitis on Cell Count ••••••••••••••
B. THE MASTITIS COMPLEX ••••••••••••••••••••••••••••••••••••••
1. Infectious Agents •••••••••••••••••••••••••••••
a) StreptococcaJ. Mastitis ••••••••••••••••••••
i) ~. agaJ.actiae ••••••••••••••••••••••• i1) Non-agalact1ae Streptococci •••••••••••
b) Staph;ylococcaJ. Mastitis •••••••••••••••••••
c) Coliform Mastitis •••••••••••••••••••••••••
d) Other Organisms •••••••••••••••••••••••••••
i) ii)
1i1) iv)
Corynebacteria •••••••••••••••••••••••• Pseudomonas ••••••••••••••••••••••••••• Yeast ••••••••••••••••••••••••••••••••• Mycoplasma ••••••••••••••••••••••••••••
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TABLE OF CONTENTS
2. Pathogenesis ••••••••••••••••••••••••••••••••••
MATERIALS AND METRODS ••••••••••••••••••••••••••••••••••••••••
Herds ••••••••••••••••••••••••••••••••••••••••••••••••••• 0
Collection of Samples ••••••••••••••••••••••••••••••••••••
Preparation of Media •••••••••••••••••••••••••••••••••••••
a) Blood Agar ••••••••••••••••••••••••••••••••
b) Esculin-Ferric Citrate Blood Agar •••••••••
c) Coagulase Mannitol Agar •••••••••••••••••••
d) Ebsin Methylene Blue Agar •••••••••••••••••
e) Nutrient Broth ••••••••••••••••••••••••••••
Preparation of Stains ••••••••••••••••••••••••••••••••••••
a) Newman's Stain ••••••••••••••••••••••••••••
b) Wright-Leishman Stein •••••••••••••••••••••
Experimental Procedure •••••••••••••••••••••••••••••••••••
Mastitis Criteria ••••••••••••••••••••••••••••••••••••••••
1. Cultural Tests ••••••••••••••••••••••••••••••••••••
a) Blood Agar Tests ••••••••••••••••••••••••••
b) Christie Atkins and Munch-Petersen (CAMP) Test •••••••••••••••••••••••••••• e •••••••
c) Coagulase~annitol Agar Test ••••••••••••••
d) Eosin Methylene Blue Agar Test ••••••••••••
2. California Mastitis Test (CM~) •••••••••••••••••••••
3. Total Somatic Cell Count (TSCC) •••••••••••••••••••
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?;o., ,
TABLE OF CONTENTS
4. Differential Cell Count •••••••••••••••••••••••• 94
a) Preparation of Milk Samples •••••••••••••••• 94
b) Preparation and Staining of Milk Smears •••• 95
c) Interpretation ••••••••••••••••••••••••••••• 96
d) Appearance of Cell Types ••••••••••••••••••• 96
ig iii.) iv)
Neutrophils Lymphocytes Eosinophils
•••••••••••••••••••••••••••• ......................... _ .. ••••••••••••••••••••••••••••
Epithelial Cells •••••••••••••••••••••••
96 98 98 98
5. Statistical Analysis of the Data ••••••••••••••• 98
Blood Serum Transferrin Polymorphism •••••••••••••••••••••• 98
RESULTS ••••••••••••••••••••••••••••••••••••••••••••••••••••••• 105
1. Incidence of Subclinical Mastitis •••••••••••••• 105
2. Incidence and Type of Infection •••••••••••••••• 109
3. Influence of Age (Lactation Number) on Incidence of Mastitis •••••••••••••••••••••••• 124
4. Influence of Breed ••••••••••••••••••••••••••••• 130
5. Influence of Month of the Year on the Incidence and Type of Mastitis ••••••••••••••••••••••••• 137
6. Serum Transferrin (Tf) Type of Cows and Mastitis ••••••••••••••••••••••••••••••••••••• 146
DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••••••••• 155
SUMMARY AND CONCLUSION •••••••••••••••••••••••••••••••••••••••• 174
REFERENCES •••••••••••••••••••••••••••••••••••••••••••••••••••• 183
APPENDIX
Table
1.
II.
III.
IV.
V.
VI.
VII.
VIII.
IXa.
IXb.
X.
XI.
XII.
XIII.
XIV.
XVa.
LIST OF TABLES
Results of California Mastitis Test (CMT) on Individual Quarter Milk Samples for AlI 10 Tests •••••••••••••••••••••••••••••••••••• 104
California Mastitis Test (CMT) on Individual Quarter Fore-Milk (IQFM) Samples and Mean Total Somatic Cell Count ••••••••••••••••••••
Number a~d Percent of Quarters Having TSCC /,0.5x106 Per ml and 4(0.5x106 Per ml ••••••••••
Stage of Lactation, Mean CMT Scores (~ and Least Square Estimates (L.S.E.) ••••••••••
Incidence and Type of Infection Testwise •••••••
Distribution of the Type of Infective Organisme in AlI CMT Grades ••••••••••••••••••••••••••••
Type of Organisme and Their Relation to the
105
107
108
110
113
CMT Reaction ••••••••••••••••••••••••••••••.••• 114
The GMT Reaction of Strepag Infected Quarters ••
Strepag Infection and California Mastitis Test Index (CMTI) •••••••••••••••••••••••••••••••••
Analysis of Variance •••••••••••••••••••••••••••
Total Soma tic Cell Count and Differential Count in GMT(+) Quarters Testwise ••••••••••••
Neutrophil/Lymphocyte Ratios in AlI Tests • •••••
Total Somatic Cell Count and Differential Count in CMT(+) Quarters in Different Organisme ••••
NeutrophiljLymphocyte Ratios with Various
117
118
118
119
121
122
Organisms •••••••••••••••••••••••••••••••••••• 125
Total Somatic Cell Count and Differential Count in GMT (1+), (2+) and (3+) Grades ••••••••••••
Lactation Number and CaliforniaMastitis Test
126
(GMT) Index •••••••••••••••••••••••••••••••••• 128
Table
XVb.
XVI.
XVII.
XVIII.
XIX.
LIST OF TABLliS
Analysis of Variance ••••••••••••••••••••••••••••
Page
128
Relation of Incidence and Type of Infection to Lactation Periods ••••••••••••••••••••••••••••• 131
Total Somatic Oell Oount and Differentiel Count in CMT(+) Quarters in Different Lactations ••••
Neutrophil/Lymphocyte Ratios in All Lactations ••
Relation of Breed to Oelifornia Mastitis Test
133
135
Index (CMTI) •••••••••••••••••••••••••••••••••• 136
XX. Incidence and T,ype of Infection in Holstein and
XXI.
XXII.
XXIII.
XXIV.
XXV.
XXVI.
XXVII.
XXVIII.
XXIX.
xxx.
Ayrshire Cows ••••••••••••••••••••••••••••••••• 138
Total Soma tic Oell Oount and Differentiel Count in CMT(+) Quarters in Holstein and Ayrshire
Cows ••••••••••••••••••• 0..................... 140
Neutrophil/Lwmphocyte Ratios in Two Breeds ••••••
Month of the Year, Mean CMT Score ( "x + !) and Leest Square Estimates ••••••••••••••••••••
Monthly Incidence and Type of Lactation •••••••••
Monthly Percentage of CMT(+) Quarters, Total Somatic Oell Oount and Differential Count •••••
Monthly Neutrop~Lwmphocyte Ratios ••••••••••••
Frequency Distribution of Genotypes and Gene Frequency at Tf Locus •••••••••••••••••••••••••
Serum Transferrin (Tf) Types and CMTI's of
142
143
145
148
149
150
Cows •••••••••••••••••••••••••••••••••••••••••• 152
Incidence and Type of Infection in Transferrin Types ••••••••••••••••••••••••••••••••••••••••• 153
Total Somatic Oell Count and Differentiel Oount in CMT(+) Quarters in Transferrin Types (Tf) •• 154
LIST OF TABLE3
Table
XXXI. Neutrophi~Lymphocyte Ratios in Transferrin Types .#., .. '................................. 155
LIST OF FIGURES
Figure
"1. Relationship of CMT Grades and Mean TSCC ••••••••
2. Incidence and Type of Infection Testwise ••••••••
Total Incidence and Type of Infection in 68 Cows •••••••••••••••••••••••••••••••••••••••
4. Percentage of Infected Quarters Showing Reaction te CMT •••••••••••••••••••••••••••••••••••••••••.
TSCC and DC in CMT(+) Quartera Testwise ••••••••••
6. TSCC and DC in CMT(+) Quarters in Various Organisme •••••••••••••••••••••••••••••••••••••
TSCC and DC in CMT(1+), (2+) and (3+) Grades ••••
~
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115
120
123
127
8. Influence of Lactation Number (Age of Cow) on Mean GMTI ••••••••••••••••••••••••••••••••••••• 129
9. . Percentage of Infection in Different Lactation Periode ••••••••••••••••••••••••••••••••••••••• 132
10. TSCC and DC in Different Lactations • •••••••••••• 134
11. Percentage of Incidence and Type of Infection in Holstein and Ayrshire Cows ••••••••••••••••• 139
12. TSCC and DC in Holstein and Ayrshire Breeds ••••• 141
13. Influence of the Month of the Year on CMT Score (/ x + !) ................... ( ................ . 144
14. Relationship of the Month of the Year to the Type of Infection ••••••••••••••••••••••••••••• 147
/10
3. Interstitial Mastitis
Occurs as a subacute or chrome process and is charac
terized by focal and diffuse cellular infiltration of the inter
stitial tissue. The cells coneist mainly of reticulo-endothelial
cells and lymphocytes, with some fibroblasts, some plasma cells,
and occasionally a few neutrophile. In early and mild casee,
the lesions m~ consist of small foci or rather diffuse areas
of cellular infiltration. In older and more severe cases, there
is extensive diffuse cellular infiltration, and maqy very dense
cellular foci JD837' be noted. There may be some exudate in the
alveoli, made up of endothelial cells, epithelial cells, debris
and occasional neutrophils. As the condition progresses, there
is proliferation of connective tissue which ~ become very
extensive. There is no appreciable change in the appearance of
the secretion (Sholl, 1946).
4. Exudati ve Masti tis
This type is characterized by accumulation of exudate
in the alveoli and ducts, accompanied by degenerative and necrotic
changes :Ln the parenchyma. In mild cases, there ~ be only a
few scattered foci in which exudation of the neutrophils are
present. In acute cases, the alveoli may become packed with cells,
mainly neutrophils, and the alveolar epithelium may show de
generation, necrosis and desquamation. More severe cases JD837'
develop into suppurative mastitie with destruction of the
/11
pare.llchyma; the milk: lIIB\Y contain much exudate and may be watery,
stringy, fl~ or bloody in appearance. Advanced chronic cases
m~ show much increase of connective tissue (Sholl, 1946).
5. Suppurative Mast1tis
Suppurative mastitis may develop from the exudative
type or mBiY be primary. The a..;'fected part may appear enlarged
and nodular. This condition ~ become widespread and lIIB\Y result
in death of the anjma] from septicemia, or encapsulated abscesses
of various size and number. There is necrosis of the tissue,
purulent exudate, and proliferation of connective tissue in an
attempt to encapsulate the suppurating area (Sholl, 1946).
6. Gangrenous Mastitis
This type may develop as the result of entrance of
anaerobes such as Clostridium welchii. The affected part becomes
dark red, bluish or greenish in colour and is cold. Extensi va
necrosis of tissue results (Sholl, 1946).
7. Fibrosis
A type of change that ma.y develop as a sequel of both
the interstitial and the exudative form of mastitis~ The part
becomes firm and shows marked increase in connective tissue.
These areas are the indurations found in the udder on physical
examination, (Sholl, 1946).
/12
III. Predisposing Factors in Maetitis
The predisposing or contributory factors in aQy in
fectious disease are frequently as significant as the infecting
agents in altering the course of the disease. In mastitis, a
number of factors 1nvûlving the host and her environment are
considered to be the fundamental ressons why so many cows are
infected. These factors may be classified as anatomical,
physio1.ogical, envirollD1ental, heredi tary and management.
1. Anatomical Factors
Cows with pendulous udders are more susceptib1.e both
to udder infection and clinical mastitis. Large, l.ow-hanging,
pendulous and s1.ack udders, the teats of which frequently come
1nto contact wi th the ground end are resdUy injured, have an
increased susceptibi1.ity to mastitis compared with normall.y
developed mammar.y glands (Heidricb and Renk, 1967). The trans
mission, penetration and establishment of bacteria are favoured
by certain malformations of the udder, teat and teat canal.
With regard to the shape of the teats, it has been
estab1.ished that galactogenous infections of the udder occur
more frequent1.y in teats that are dished at the ends and in
those with funnel-shaped, dilated orifices than teats with more
rounded and tapered ends (Heidrich and Renk, 1967). The size
and shape of the teat, inc1.uding the length of the small duct,
apparently are of little significance in the incidence of
mastitis (Murp~ and stuart, 1955; Brown II &_, 1965) ..
/13
The teat sphincter is the main p~sicaJ. barrier to
infection as almost all organisms associated with mastitis gain
access to the udder via the streak canal.. The condition of the
teat canal is of primary significance in masti tis. A straight
and properly closing canal that is not too short and whose
epithelia1 lining is intact and undamaged constitutes an
effective barrier against the penetration of mastitogenic
bacteria (Heidrich and Renk, 1967) _ A wax-like mass 1s formed
by the stratified, actively desquamating epithelium of the streak
canal. Because of high free fatty acid content, this substance
(lactosebum) possesses definite bacteriocida1 properties,
organisme entering the teat orifices are caught up in it and
destroyed (Schalm, 1962).
Injuries resulting from cows stepping on teats, cuts
t'rom bariled wire, teat surgery, and lesions at the end of the
teat, destroy or impair a natural barrier to infection and may
facilitate the entrance into the udder of various kinds of
microorganisms present on the skin of' the teat. The variety of
organisms associated with teat in jury was shown by the f1ndings
of Ferguson (1944). Of 283 samples from cows with mastitis
following teat in jury, 24 percent contained~. agalactiae,
/14
23 percent ~. dysgalactiae, 15 percent ataphylococci, 6 percent
COrynebacterium pYogenes, 2 percent coliforms and 7 percent mixed
infections.
2. Physiological Factors
A number of physiological factors like age, stage of
lactation, milk yield, and hormones influences the susceptibilit,y
of cows' udders in the development of mastitis. A single factor
aJ.one is seldom invol ved.
a) Aae
Many workers have reported that an animaJ's suscept
ibility to mas titis infection is said to increase with age.
Seelemann (1932) reported an infection rate among 5,834 cows of
9 percent, 30 percent, 42 percent, 44 percent and 52 percent for
cows in their first, second, third, fourth and fifth lactations,
respectively.
Murphy and stuart (1953a,b) and Lancaster and Stuart
(1951) have shown that older cows are more easily infected than
heifers when the mammary gland is challenged with streptococci and
some staphylocDCci. Older cows are more likely to have contracted
udder infections because of the longer period of exposure to
organisme and the predisposing factors.
According to Little and Plastridge (1946), with few
exceptions, first calf heifers are free of ~. agalactiae at the
/15
t~e of parturition. The exceptions are due to infection
acquired during calfhood, when cal ves are fed infected milk and
allowed to suckle each other (SChalm, 1942; Schalm et al., 1971). --Murpby" (1947) made observations on a large herd over a 7-year
period indicating that an "age factor" independent of teat in jury ,
milking rate, prior sensitization, and degree of exposure 1s
involved. He related the rise and fall of infection to the
average age of the herd. He found this relationship was highly
significant statistically, and indicated that age or some function
of age was the major predisposing or l~ting factor in the spread
of streptococcal infection. Lancaster and Stuart (1949) supported
this explanation. They found that two of the seven first-calf
heifers, four of f1ve second-calf heifers, and six of six older
cows became in:fected wi thin a period of 15 weeks, when exposed
by milking them wi. th hands previously dipped in.§!!:. agalactiae
infected milk.
Observations of Ormsbee and Schalm (1949) and Spencer
and Kraft (1949) showed that the degree and extent of exposure
are major factors affecting the rate of infection in heifers as
well as the older cows. The incidence of infection varied with
different herds and appeared to be lower:in herds where management
practices tended to r.educe the intensity of exposure and higher
in herds where the opposite was true. Schalm and Woods (1953)
found in a large~. agalactiae-free herd that the incidence
of Micrococcus pyogenes infection increased with age, from 20
percent for first lactation animϝs to 74 percent for cows in
their eighth lactation.
b) Stage of Lactation
/16
Mastitis producing bacteria ~ enter during aIl stages
of lactation. The predominant times of entry appear to be when
the animals are in production, however, in some studies, one
fourth to one-third of all infections occurred during the dry
period (Brown ~~., 1965). In general, clinical mastitis ~
occur at any stage. ~. agalactiae infection does not appear
to be related to the stage of lactation, as indicated by the
find1.ngs of Plastridge n &. (1942). Of 157 heifers in six
~. ~ac~ infected herds, seven, eight and seven became
infected during the first third, second third, and last third
of their first lactaticn. Oliver n&. (1956) reported that the
ra~e of new infection with non~. agal8ctiae, streptococci
and staphylococci was higbest during the first month of lactation
and during the early dry period. About one-half of the latter
infections persisted until calving. It is known, however, that
the rate of infection during the dry period is high (Neave and
Oliver, 1962; Oliver ~&., 1962).
c) Milk Yield
There is some evidence of a relationSbip between high
milk yield and mastitis (Little, 1940a; McLeod and Wilson, 1951;
/17
Brodauf, 1963), al thougb. other workers have fai~ed to confirm
this finding (Ward, 1944; Watts, 1951; Murnane, 1940). Dodd and
Neave (1951) claimed that a higb. rate of milking was correlated
with susceptibility to infection and also that higb. lactation
yields were correlated with fast milking. Fell (1964) in his
review stated that milk yield could be considered only as a
possible factor af'fecting individual susceptibility to mastitis.
According to Oliver !Œ al. (1956) the percentage of quarters that
become inf'ected increaeed from 18 percent for cows that yielded
less than 7 lbs at their laet milking to 43 percent for those
that yielded 21 lbs.
It would seem that under naturel. conditions when cows
are not exposed to infection by stable confinement, under modern
dairy conditions and are not forced to produce ~ milk yield
~or 10 or more months of' each lactation, nature can preserve the
usefulness of the udder for the purpose for which it wae originally
developed. This suggests that the present demands placed on the
dairy cow m~ be conducive to certain physiologicel. changes which
render the gland more susceptible to infection (Little, 1940b).
d) Hormones
Another concept that has received some investigation
is the possibility that natural estrogens as well as estrogens in
some plants ~ stimulate bacterial miltip~ication within the udder
and cause clinicaJ. mastitis (Brown.!Œ.!l., 1965; Sc~.!Œ.!l., 1971).
/18
The high concentration of estrogen in late pregnancy m~ have
been responsible for the greatest occurrence of clin1cal mastitis,
associated wi th cal. ving. Frank: and Pounden (1961) studied the
association of clinical mastitis with the estrus cycle in three
herds on legume forages and revealed that more than half the 509
attacks of mastitis occurred in the period from 31 d~s post
partum to the 30th day of the next pregnancy, inclusive. Within
the first month post-partum, 82 attacks occurred and 139 other
attacks occurred during the rest of the lactation period or when
the cowa were dry.
Pound en and Frank (1961) reviewed 140 mastitis attacks
in relation to estrus in 64 cowa indicating the highest incidence
occurred between three and nine daya post-estrus. This period of
the estrus cycle was considered to repreaent the peak of estro
genic activity in the cow.
They alao stated that legumea in the fresh atate are
more likely to stimulate masti tis than silage. It has been
demonstrated that alfalfa cut in the bud or one-tenth bloom stage
of matur1ty was more eatrogenic than that cut at later stages of
matur1ty (Stob ~~., 1958). The secretory cella of the normal
mammary gland are only slightly permeable to circulating estrogen,
even when estrogen levels in blood are at their peak (Turner,
1958). However, maat1tic glands do exhibit increased permeabi1ity
for blood plasma factors which may include an increased
permeability for estrogens (Laemanis and Spencer, 1954).
It may be that the clinical attacks of mas titis that
have been attributed to effects of estrogens are more the result
of altering the balance between bacteria and defense mechanisma
in the milk through dilution rather than by a stimulating effect
of estrogens upon the organisme (Schalm ~!!., 1971).
3. Environmental Factors
a) Season and Weather
/19
According ta Brown~~. (1965) there ia no conclusive
evidence to indicate that the sesson per ~ influences the
incidence of udder infections and mastitis. The incidence of
masti tis and certain udder infections are higher during the first
month of lactation and the early dry period. The relation of
new Str. agalactiae infection to the sesson of the year was
determined in 14 herds in Scotland by Ineson and Cunningham (1949).
The number of new infections that occurred during the spring,
summer, fall and winter waa 54, 45, 75 and 38, reapectively. The
peak months of the year were October and April. In Great Britain
and Europe, mastitis caused by COrynebacterium BYogenes occurs more
frequently in summer and as a resul t bas been called "summer
mastitia" (Brown et al., 1965). --
Exposure of the udder to chilling probablY increases
inflammation in udders already infected, however, this has not
been determined exper~entally (Plastridge, 1958). Reid (1954)
observed an increased rate of clinical mastitis in the spring
and in the fall in herds in which ~s were left on pasture
overnight while the ground was cold, and in animals which were
exposed to drafts in poorly ventilated barns.
/20
MacLeod et~. (1954) studied the leucocyte counts of
1,707 platform samples of milk from 39 herds over a period of
11.5 menthe. They found the leucocyte counts were highest in the
summer and lowest during the spring and early summer.
Nelson ll!d: .. (1967) examined over 2,000 farm tank
milk samples. The results Showed a definite trend in leucocyte
count that was correlated wi th seasonal temperature changes. The
percentage of samples with more than a million leucocytes per ml
increased from about 10 percent during mild weather in March,
April and early MSiY to between 40 and 50 percent during the hot
season in July, August and September. Over 65 percent of the
semples had above a million cell count in late July when dailY
o temperatures ranged between 26.6 and 42.8 C. Most of this trend
was reversed with cooler weather in October.
b) Housing
Two factors relating to housing which are frequently
considered as contributing to the problem of mas titis are,
/21
udder and teat injuries and improper ventilation (Brown ~~.,
1965). A higb incidence of udder infection and maetitis follows
injury to the teat orifice and canal.. Heizer ~ &. (1953) found
that stepped on teats, more frequent in the pen type or loose
housing barn than in the conventional. stanchion barns. Never
theless, sufficient data are not available to indicate that one
type of barn results in a higher incidence of udder ~ection or
masti tis. However, wider stalls in stanchion barns have been
associated with a lower incidence of mastitis, thus adequate space
per cow is important in reducing teat injuries and mastitis.
Drafts have been reported as predisposing to mastitis bec3Use cows
housed in drafty parts of barns apparently developed more clinical
masti tis than their stablemates (Brown ~ .!!.., 1965). Cramped
cow sheds, damp and muddy litters, uneven and sharp-edged floors
favour the occurrence of mastitis (Heidrich and Renk, 1967).
c) Grazing Land TopographY
It mS1 so happen that cattle must move through wet
marshes and excessive vegetative growth. This mS1 subject teats
and udder to chapping, scratcbing, bruising and insect bites which
predispose to mastitis (Anonymous, 1957).
/22
4. Hereditary
Mastitis is caused by bacteria, however, a number of
reports on the inheritance of susceptibility or resistance of cows
to mastitis indicate that mastitis is infJuenced by heredity
(Zieger, 1932; Murphy .2.t &., 1944; Lush, 1950; Legates and
Grinnels, 1952; Reid, 1954; Young .2.t&., 1960; Schmidt and
Van Vleck, 1965; Lotan, 1967; Koch ll&., 1968; Malik ll&.,
1970). Zieger (1932) stated that "it is certain tbat the tendency
to a weakness of the udder can be transmi tted in a large per
centage of cases." MurphJr ll&., (1944) found tbat the infection
rate of three daughters and two grand daughters of a cow w.l.th a
high infection rate was considerably higher than tbat of four
daugbters and two grand daughters of another cow in which no
infections were noted during one lactation.
Lush (1950) obtained an intraherd daughter on dam
regreseion of 0.19 for cows showing abnormal quart ers or abnormal
milk by the time they reached eight years of age. Legates and
Grinnels (1952) reported a heritabilit,y estimate of resistance to
mastitis to be 0.27 :!: 0.10. Young ll&. (1960) found the heri
tabilit,y estimates of clinical maetitis, ae defined by the per
centage of monthe of the lactation dur~ which the cow had an
abnorma1 appearance of the udder or its secretion to be 0.06 :!:
0.18 and 0.79 ± 0.21 when calculated on a daughter dam regression
and paternal half sib correlation, respectively. Schmidt and
/23
Van Vleck (1965) reported within-herd heritabilit.y estimates for
daily milk yield to be 0,353 and tbat for the number of quarters
infected with~. agalactiae to be 0.196.
Inherited variations or genetic po~orphism in blood
serum pro teins of cattle was first described by Ashton in 1957
and by Smi thies and Hickman in 1958 uaing starch gel electro
phoresis. Genetic variations in the cattle serum (3 -globulin
fraction is often referred to as the transferrin or the Tf
system.
Blood serum contains two types of globular proteine
called albumin and globuline. In a normal electrophoretogram,
run at pH 8.6, all serum proteine m:J.grate towards the anode.
Albumin travels fastest followed by c:J:, 1-' ~ 2-' (3 - and ~ -
globuline (Dimopoullos, 1963). The f3 -globulin fraction is the
iron binding protein of serum and is often called transferrin.
Five phenotypes were recognized in the earliest
descriptions of cattle serum transferrina (Ashton, 1957; Smithies
and Hickman, 1958) and a sixth phenotype was added later (Ashton,
1958). A series of three allelomorphic genes at one locus, giving
three homo~gotes and three heterozygotes accounted for the six
phenotypes, namely, M, AD, AE, DD, DE and EE. A fourth F::i.lele
bas been recognized by Kristjeneen (1962) in European cattle and
four others in Zebu and Afrikana cattle (Ashton, 1959; Os terhoff ,
/24
1964; Ashton and Lampkin, 1965). Jamieson (1966) has presented
evidence of 10 phenotypes in cattle tested in Britain and
suggested that cattle transferrin polymorphism is controlled by
a series of at least eight Co dominant genes; Tfa1 , Tfa2, Tfb,
Tfd1
, Tfd2 , Tff , Tfe and Tfg at the Tf locus. The Tfa and Tfd
genes of Jamieson (1966) correspond to T~ and TfD genes
( d1 d2 deaignated by Gahne 1961). The Tf and Tf genes have been
equated to TfD1 and TfD2 alleles resolved by Kristjensen and
Hickman (1965).
Recently, Malik et~. (1970) studted 701 cows in two
Series. In Series 1 (105 cows), the percentage of Str.
agalactiae(+) and percentage of hemolytic staphylococci(+)
quart ers were significantly different from genotype to genotype
(P", 0.005) • In Series 2 (596 cows), only the percentage strepto
cocCi(+) quarters were significantly different between genotypes.
The TfD1D1 cows had a higher incidence of Str. agalactiae
infection in Series 1 and of streptococci in Series 2. TfD1D1
cows also had a significantly higher (P~0.05) mean CMT score and
mean total somatic cell count per cow in Series 1. TfEE cows,
however, were free of ~. agalactiae infection in Series 1 and
of streptococci in Series 2. TfEE cows also had a significantly
lower (P< 0.05) mean streptococcal score per cow in Series 2.
No information is available about the genetic influence
affecting cellular response in bovine mastitis.
/25
5. Management Factors
Any management factors which cause physiological stress
will lower the cow's resistance to disease. The stress may be
"internal" such as nutritional deficiencies or the stress of
high production or "external" such as mechanical injury to the
udder. Some management operations m~ be associated with mastitis
without causing stress by aiding the dissemination of organisme
(Fell, 1964). The factors within the framework of management are
herd size, feeds and feeding, sanitary procedures, milking machines
and some of the factors discussed earlier.
a} Herd Size
Numerous studies have shown a higher incidence of udder
infection and mastitis in large as opposed to small dairy herds.
The caliber of workers and the b~ of replacement cattle have
been reported as contributing to the problem of mastitis.
Consequently, one can speculate that these factors may contribute
to the greater incidence of mastitis in large herds because there
are more hired workers and more cattle brought in as replacements
(Brown et al., 1 965) •
b} Feeds and Feeding
Very little reliable information is available con
cerning the effect of "internal" stress on susceptibility to
mastitis (Fell, 1964). Some investigators have suggested that
/26
heal thy and well-nourished animals are less prone to infection
and mastitis (Motion, 1933; Australian Dairy Produce Board
Report, 1950; Trehane, 1951; No orlander, 1962). The possible
role of feed in mastitis has been a subject of debate for yeara.
Increasing the protein in the ration is intended to stimulate
milk secretion, if volume of milk ia increased, the equilibrium
between bacterial infection and hoat response migbt be altered
by dilution and lead to a flare-up of clinical masti tis. Pound en
and Frank (1961) claimed "that attacks of clinical masti tis
increased when cows were placed on pasture or fed certain forages.
There have been indications that the legume forages, alfalfa and
ladino clover in the fresh state or as silage, sometimes
stimulated the occurrence of clinical mastitis associated with
bacteria other than~. agalactiae. Reducing the amount of
protein concentrate in the ration tends to reduce clinical
mastitis in cows with a past history of mastitis and in cows with
udder infections (Udall and Johnson, 1931). Hotis and Woodward,
(1935) and Moore ~~. (1942) failed to show a relation between
the diets used and either the severity or rate of maatitis.
However, definitive exper1ments would be difficult to arrange
due to other factors that affect the establiShment of infection
and rate of clinical mastitis in infected quart ers (Plsstridge,
1958).
c) Sanitary Procedures
The purpose of any sanitar,y program 19 to prevent or
minimize the spread of organisme from infected to non-infected
cows and reduce the chance of infection by organisme inhabi ting
the immediate en\~ronment or ekin of the cow, milkers' bande,
mi.lking machines, contaminated floors and litter, all of which
serve as potentiel vectors of mastitis organisme (Harrison,
/27
1941; Fell, 1964). Disinfection of the teat cups, the teat skin,
and the milkers' bands, is usually attempted by using chemical
disi.n:f'ectants, of which the hypochlorites, quartemary ammonium
compounds, chlorhexidine and iodine compounds are the most widely
used.
The efficiency of any dis1nfectant is dependent upon
several factors - time, concentration, temperature, organic matter,
pH (acidi ty or alkalini ty), and hardness of water. Generally,
disinfectant is more efficient the longer it acts, the stronger
its concentration, the higner the temperature of the solution, and
the freer the surfaces and disinfectant solution are of organic
matter such as mi.lk and manure (Brown ~ !:!.., 1965).
There is no doubt that the machine can aet as a vector
during milking and facilitates the spread of organisme. A report
(Rev. Commonw. Bur. Anim. Hlth. Weybridge, 1944) shows that under
average conditions of milking, organisme are constantly transmitted
from udder to udder and can be recovered from the teats of all
cows in a herd. Any hygiene practice, therefore, that reduces
the transfer of infective organisme from one udder to the next,
will reduce the amount of infection and consequently the amount
of mastitis. This has been demonstrated in both ~.
agalactiae and Staph. aureus (FeIl, 1964). The difficulty,
however, has been to devise a practical and economic technique
for achieVing tbis.
/28
Proper stall hygiene is a prerequisite to udder hygiena.
This is accomplished by keeping the floors under the udder, clean,
dr,y and weIl supplied with planty of good litter. This reduces
the opportunity for congestion and injury of the udder. It
decreases the opportunities for dissemination if infectious
material and thus the possibility of infection occurring in
he al thy udders.
The physical cleanl.iness of the cow aids materially in
the maintenence of udder health, and in the production of low
count milk:. Bryan et al. (1 ~4o) showed that clipping the hair --from the udder and flanks reduced the bacterial count two to
four times in contrast to the case of anj mals on which no
clipping was practiced. They also showed the value of wiping
and disinfecting the udders and teats with chlorine solution or
some other suitable material.
/29
Washing of the udder and teats w1 th disinfectant wi th
free running tap water and drying with individual dispos able
paper towels before each milking, sterilization of the milking
unit by circulating hot water (930 C) for at least 30 seconds and
teat dipping in a disinfectant solution after milking, prevented
transmission of microorganisms from cow to cow, and decreased the
number of organisms on the teat skin (Hughes, 1953; Murnane,
1953; Davidson ~ .!1.., 1954; Wilson, 1955; Newbould and Barnum,
1956; Neave et al., 1966;1969; McDonald, 1970). --Neave ~!:l. (1969) stated that there are drawbacks to
hygiene. Even when an udder is washed wi th a sterile cloth wet
with disinfectant, pathogens may be distributed over the surface
of the udder and teats from infected teat lesions or orifices or
from infected milk. Omitting udder washing would prevent this,
but there would still be some transfer of bacteria by tail and
leg movements and oy flies. They also stated that even after
pasteurization of teat cups with hot water at 850 C circulated
for five to seven seconds, Staph. aureus could be recovered,
although in only small numbers from about five percent of
pasteurized teat cups. They suggested that the treatment could
be made effective by addition of a disinfectant, improving the
surfaces of the inflation (liner) and by prolonging duration of
heat treatment.
/30
Disinfectant of the env:l.ronment, like handa and
premises, ia of importance only in cases where the major source
of pathogenic organisme ia the interior of the inf'ected udder.
This ie evidenced by the fact that mastitis due to ~.
agalactiae has been eradicated merely by segregation and treatment
of inf'ected animaJ.e (Minett !Œ. al., 1933; Stableforth!Œ. &.,
1935; Plastridge et &., 1942; Cunningham et &., 1947).
Cows Bhare their environment with bacteria and it is
inev:l. table that these microorganisme will invade external body
openinge with no exception to the teat duct (Schalm!Œ.&., 1971).
d) Milking Machine
The milking machine is coneidered as an important
management factor affecting the cows l ausceptibility to mastitis.
There are two distinct rolee which the machine may ~lay in its
possible relationahip with udder heaJ.th. One concerna the machine
as a vector and the other refers to the possible traumatic
effecta of machine action (Fell, 1964).
The MOst obvioua vector acti vi ty of the machine con-
cerns the transmiasion of the 1nfective organisme between cows
and between quart ers of the same cow. One set C"f cups applied to
a non-1nfected cow immediately after removal from an infected cow
113 very likely to be associated with inter-cow epread of infection
(Fell, 1964). It bas been noted that transfer of milk t'rom one
teat cup into a separate teat cup is quite common in con
ventional Australian machines, this effects cross infection
between quartere (Whittlestone, 1962b; Fell, 1962). According
/31
to Fell (1964) the possibility existe of backflow of milk through
the streak: canal and thie mSiY' be an extremely cri tical. factor in
proViding organisme with access past the teat sphincter barrier.
Noorlander (1962) claims to have demonstrated backflow of water
with an artificial teat when the outlet tubes are crimped.
McE},van and Samuel (194é) appear to have shown that bacteria ~
enter the teat during the set of milking, possibly because of
this backflow.
It is weIl known that the machine can cause tra.uma cf
many kinds in the region of the teat. The machine has been
reported to obstruct circulation and cause congestion of the
peripheral. blood vessels in the teat (Leslie and Whittlestone,
1938). The tip of the teat ~ be p~ently red and inflamed
and petechial haemorrhages are qui te common at the teat apex.
The teat sphincter ~ be eroded in which case, the wax-like
layer of desquamated epithelial. celle on the liner surface is
damaged and partly removed. An obvious depression at the base
of the teat or a "pressure ring" can also occur as a resul t of
machine milking and this ~ be severe enough to resul t in the
sloughing off of the teat tissue (Neave, 1959; Whittlestone,
1962b; Noorlander, 1962).
/32
Vacuum stability can be considered as a theoretical
force whereby the machine causes trauma. The teat sphincter,
which responds to pressure differences across it by opening and
closing, could conceivably be damaged by rapidly fluctuating
vacuum conditions (Fell, 1964). In a review of machine mi~ing
and mastitis by Burkey and Sandere (1949), the data presented
suggested that a vacuum level of over 1 5 inches of mercury
tended to increase the incidence of mastitis. An increased
incidence of clinical masti tis and high leucocyte count in milk:
have been associated with vacuum fluctuations caused by lack of
vacuum reserve and by blocking air admission holes in the claw
(Brown et al., 1965). --
Any obstruction to milk: flow in the pipee or tubes
leading from the teat cup is known to cause serioue vacuum
fluctuations in the inner chamber of the cup (Noorlander, 1962).
Whittlestone (1962b) has observed outbreaks of mastitis apparently
precipitated by this effect.
Braund and Schultz (1963) found a significant corre-
lation between the extent of vacuum fluctuations and the per-
centage of CMT positive quart ers in a field survey. Stanley
~~. (1962) demonstrated that wider fluctuations caused an
increase in the GMT scores of a group of cows.
/33
Pulsation, by allowing the intermittent collapse of
the rubber ~lation, provides relief to the teat tissue, other
wise under constant vacuum. Wilson (1958) consid~red pulsation
rate a doubtful factor as a cause of mastitis and Neave (1959) in
a review, reported that there was little published evidence
associating pulsation characteristics with udder infection.
Some workers have reported that high pulsation rate
may be associated with udder infection. Bratlie ~~. (1959;
1962) reported an increase in teat damage and in the cell count
of the milk with a pulsation rate of 75 per minute, compared to
40 per minute.
Whittlestone and Olney (1962) recommended that the
pulsation rate should not be excessive (a tentative upper l1m1t
of 50 per minute is suggested) in order to avoid the possibility
of creating conditions suitable for backflow of milk through the
streak canal.
The phenomenon known as "cup crawling" is another means
by Which the teat cup ~ lead to udder trauma. Towards the end
of milking, as udder tissue becomes slack, the teat cups m~
"crawl" up the teat and draw in the lower portion of the udder.
This subjects the region of the annular fold to some maceration
when the tissues rub aga1net one another due to the movement of
the teat. Evidence ahowed that leaving the machine attached to
.. ,
the udder after milk flow has practically ceased, contributes
to mas titis (McEwen and Samuel, 1946; Burkey and Sanders, 1949;
Pier ~ .!1 .. , 1956).
Many workers have noted that different types of teat
/34
cups, partictü.arly inflation, have very different effects on the
teat and also vary in their milking efficiency. Wilson (1958)
considered that liner design and particularly the internal
diameter, tension and softness of the mouthpiece was one of the
major machine factors concerned with mastitis. Moulded, rather
than extruded, rubber liners have been commonly associated wi th
mas titis in the field (Leslie and Whittlestone, 1938; Watts,
1942; Gambrel, 1950; Neave ~ al., 1952; Wilson, 1952). Dodd
~ &. (1957) confirmed this result with a within-cow, half udder
trial involving 84 animals. The moulded liners resulted in 32.8
percent infected quart ers while the extruded liners were
associated with only 12.3 percent infected quartera. The same
authors, however, and Oliver !Œ~II (1957) found no difference
between a different type of moulded inf~ation and extruded liners.
Schalm and Noorlander (1958) and Maffey (1959) claimed that wide-
bore inflations greater than 7/8 of an inch were definitely
dangerous from a mastitis point of view.
IV. Diagnosis of Bovine Mastitis
1. Barn Tests
/35
a) Ph:sical Examination of the Udder:- May be defined
as a olinical examination of the mammary glands by Visual ob
servations and dig1 tal palpation. Prior to 1931 no acceptable
specifie outline for the clinical· examination was used. In 1931,
Udall and Johnson reported a practical systematic method of
classification of the udder, it was baeed on degree of tissue
changes in each quarter of the udder, together with additional
data concerning the an:imal·'s age, stage of lactation, stage of
pregnancy, and production and disease history of the individual
as well as of the herd as a whole.
Udall and Johnson (1933) believed that in the diagnosis
of mastitis the physical examination rated over aDY other single
method, they aJ.so emphasized the importance of including all
available data from other tests, auch as atrip cup, bromo~ol
blue (thybromol) test2, and the bacteriaJ. a~amination of the milk.
The physical examination bas alao proven valuable as a basis for
the rejection of cows in public health programs when the principal
aim is to prevent abnormal milk from entering the milk S'llPPly
(Tompkins .!1 al., 1946).
The physical examination of glands must be made on the
empty udder. The most opportune time, therefore, is immediately
after milking when the hormone stimulation bas ceased and the udder
/36
is completely relaxed. When the udder is distended with milk,
a thorough examination cannot be made because the tissues are too
firm to be palpated (Tompkins et al., 1946; Schalm et al., 1971). -- --It is known that various forma of mastitis - acute, rapidly
progressing, chronic and slow, and insidious - lead to diffuse,
focal nodular cord-like induration of the udder with consequent
replacement of secretor.y tissue. Heidrich and Renk (1967)
suggested that palpation of the udder for the detection of
indurations that are not sharply demarcated should be interpreted
with care. They also suggested that palpation could not be
utilized alone but must be correlated with bacteriological and
cytological examination.
Hucker and Udall (1933) attempted to correlate the
findings of the ph3'sical exam1nation wi th leucocyte count, and the
cultural examination. They concluded that udders free from in-
durations or scar tissue were free from demonstrable streptococci
and free from cells in exc~ss of 500,000 per cubic centime ter (cc)
and did not give a positive reaction to bromotbymol blue. Cell
counts greater than 500,000 per cc indicated that the milk had
been secured from an udder containing an appreciable amount of
fibrosis. When streptococci were found in milk aseptically
drawn from the udder, the quarter was al~s indurated, and all
indurated quart ers Showed streptococci or a significant number
of cells. In a survey, Rosell and Miller (1933) found that
/37
65.6 percent of diseased quarters co"uld be diagnosed by clini.cal
examinat10n alone.
b) Physical Examination of Secretion:- The strip cup
serves a number of useful purposes, namely; a) the detection of
abnormal milk, b) removal of the first milk aids in stimulating
for milk let-down, and c) discarding the fore-milk leads to a
lowering of bacteria count since the first streams of milk
commonly have the largest number of bacteria per ml (Schalm
~~., 1971). To be effective as a means for the detection of
masti tis, the strip cup should be used at every milking as an
integral part of the milking routine.
Abnormal changes in colour and consistency of milk,
however, were readily detected when tubes containing these samples
were compared with tubes of normal milk when allowed to stand.
In ~ instances, abnormal changes in the secretion in the absence
of infection suggested a marked pe~eability of the udder to the
constituents of the blood (Tompkins et al., 1946). --c) Bromothympl Blue Test for Altered pH:- The pH of
normal milk may vary between 6.5 and 6.8. Colostrum 1s more acid
(6.4 or below) and milk near the end of lactation is more alkaline
(6.8 or greater) than milk produced during mid-lactation (6.6 to
6.7). The indicator dye bromocresol purple (BCP) was first used
/38
by Baker and Van Slyke (1919), and Baker and Breed (1920) for
mastitis detection and to detect changes in the pH value in milk.
Normal fore-milk is a dove grey colour wi th BOP and a
change toward the purple is indicative of increasing alkalini. ty
as seen normally in late lactation milk. In mastitis, as lactose
production decreases and alkaline salts from the blood enter the
milk, the milk becomes more alkali.ne. Thus, increasing alka-
li.nity of the milk is characteristic of a progressive mastitic
condition. Mastitic milk drawn from the teat m~, on rare
occasions, be acid and is yel.low with BOP. Str. agalactiae when
mu1tipl.ying rapidly witbin the udder, m~ convert lactose to
lactic acid. This is, however, an extremely rare occurrence
(SchaLm et al., 1971). -- .
In routine use of col.orimetric teste, bromotbymol. bl.ue
bas l.argely repl.aced bromocresol. purpl.e (Udall. and Johnson, 1931;
H~den and Johnson, 1934; F~ et ~., 1938), becauee the shades
of green that develop wi. th alkaline milk are more easily graded
than purple.
d) Whiteside Test:- Wh1teside (1939) reported that on
the addition of 2 cc N NaOH to 10 cc of masti tic milk and eub-
sequent beating of the mixture wi. th a gJ.ase rod, a viecid mass
was formed. The reaction took place immediately and at room
temperature. Murphy and Hanson (1941) modified the test and they
auggested the designation "modif'ied Whiteaide test." Later,
Schalm ~~. (1955) developed a field Whiteside test for use
in the barn on fora-milk of individual mammary quartera.
According to Astermark et al. (1969) the underlying --mechanism of the Whitesi1e test is not completely clear. Dunn
~al. (1943) reported that leucocytes are directly or in
directly responsible for reaction. He postulated that protein
/39
materi&! of leucocytea in mastitic milk reacted with NaOH to form
a gelatinoua mass similar to that which is formed by the action
of Na0H on nucleic acid from animaJ.. cella. Petersen.!Œ !J:..
(1950) stated that the reaction was csuaed by adsorption of fi brin
onto the white cella in the milk. SchaJ.m and Noorlander (1957)
found that fat content of the milk could al.so have an effect,
while Kastli (1963) stated that the clot formation arises from a
reaction between sodium and calcium ions and the cell albumen.
Negretti (1959) reported that the use of antiformin to replace
NaOH resulted in greater accuracy, and recorded 13.2 percent more
positive testa with the antiformin test than wes obtained with the
original. Whiteside teat.
e) Californ1a Mast! tis Test:- In the course of in
vestigations with the Whiteside test, Scha1m and Noorlander (1957)
found that the addition of anionic detergents (such as a1kyl
sulphates or sulphonates or alkylaryl sulphates or sulphonates) to
/40
mastitic milk resulted in the formation of typical gel streaks or
clumps, according to the degree of abnormality of the milk. The
cell count of the milk was reflected by the degree of pre-
cipitation or gel formation. The extent of depression of milk
secretion was indicated by colour reaction with the bromocresol
purple in the formula due to alkalinity.
Investigations into the nature of the positive OMT
reaction strongly indicated that the active principal in mastitis
milk is deoxyribonucleic acid (DNA) originating DlB1nJy ill the
nuclei of cells constituting the inflammator,y exudate (Carroll
and Schalm, 1962). The addition of DNA-ase but not RNA-ase or
Trypsin to CMT positive reactions resulted in almost instantaneous
dissolution of the gel, this ~ be the probable cause for ra-
vers al observed in Trace and OMT 1+ reactions probably due to the
action of natural DNA-ase released from the ruptured soma tic celle
(Schalm et al., 1971). --6 6 Cell counts of 0.0-0.2 À 10 ; 0.15-0.5 x 10 ; 0.4-1.5
x 106; 0.8-5.0 x 106 and over five million cella/ml have been
classified as CMT reactions of Negative, Trace, 1, 2 and 3,
respectively, by Haller ~ al. (1964) and Schalm~~. (1971).
Appleman ~.!!.. ( 1964) and Schalm n.!!... ( 1971) gave the
values of polymorphonuclear cells (PMN) to be 30-40 percent, 40-60
percent, 60-70 percent and 70-80 percent of the cells/ml of milk
for Trace, 1, 2 and 3 CMT reactions, respectively.
/41
Krieger (1961) made a detailed study of the CMT reactions
obtained with colostral milk and compared them with those obtained
w1 th true milk. In milk from certain cows posi ti ve reactions were
observed up to the fifth d~ after calving and in exceptional
cases, such reactions could persist up to nine days post-partum.
Acclording to Heidrich and Renk (1967) the test should not be used
w1 thin the first three dB3"s after parturition or at the end of the
lactation period, because the physiological increase of cells at
these t1mes could lead to an incorrect evaluation ..
Schalm and Noorlander (1957) recommended the test not
only for quarter milk but also for testing combined quarter
samples.. It is common practice to conduct a CMT on the first
streams of milk (fore-nlk) at milking t1me as a measure of the
health status of separate quart ers of the lactating cow.. In some
instances, fore-milk may be CMT negati ve but strippings milk ~B3"
be CMT positive.. This type of reaction is more commonly found
among young cows and may mean that mastitis has not yet involved
large aresa of the gland parenabyma (Schalm.!!i al., 1971) ..
Gray and Schalm (1960) have reported that a CMT score
of 1 in an indiv1dual cow bucket semple may be regarded as in
dicative of mastitis.. They also stated that a CMT score of
"Negative" in a tank sample did not imply complete freedom from
mastitis because of dilution of mastitic milk with milk from
normal glands.
/42
Schalm and Noorlander (1957) stated that although the
CMT was a sensitive indicator of the presence of inflammation in
the udder, it could not distinguish the causes of inflammation.
:Wesen ~.!1.. (1968) made bacteriologiCal. tests on strippings
milk and compared them with the GMT scores on these semples. They
found at least one type of potential pathogen was reported in 6.0,
6.5, 27.3, 64.7 and 71.3 percent of the quartera secreting milk
showing Negative, Trace, 1, 2 and 3 CMT scores, respectively. Non
pathogens were found more frequently in quartera with low GMT
reactions, namely, Negative, Trace and 1, than in quartera with
the hi8her CMT scores of 2 and 3.
Bar-MoShe (1969) concluded that as the percentage of
positive CMT findings in a herd increased, there was a
corresponding rise in the rate of infection. Malik (1968)
reported the distribution of the types of infective organisme in
the five GMT grades. The percentage of infected quart ers in
Negative(-), Trace, 1, 2 and 3 grades was 91.6, 98.4, 98.0, 97.4
and 94.0, respectively, as compared to the overall percentages of
93.4 for intected quart ers in all the quart ers tested for CMT
and infection.
Daniel ~ al. (1966) showed the relationShip between
CMT scores and monthly milk production, they predicted the average
decrease in monthly milk production to be 5.3 percent for each
one unit increase of CMT score. Althougb various investigators
/43
have differed in the1r approach for determjnjng milk loss as
correlated with CMT score, e.g., bucket milk versus paired
quart ers compar1sons, the data revealed a significant drop in
production wi th increasing CMT score (Schalm et al., 1971). --Heidrich and Renk (1967) considered the value of the
CMT as an aid in the diagnosis of masti tis to be strictly limi ted,
owing to the fact that a considerable number of false reactions
were encountered and infection of the udder leading to little
or slight inflammation was inevitably missed. The CMT is not
capable of replacing or numerically reducing the bacteriological
and cytologie al examination of milk in the diagnoeis of mastitis
and one cannot advocate the institution of antibiotic therapy
of a quarter on the basis of a positive CMT reaction alone and
without the benefit of a bacteriological examination of the
secretion. Despite these limitations, the CMT remains a valuable
aid in practice, a rapid means of assessing the heaJ.th of the
udder.
2. Laboratory Tests
a) Chloride Test:- Normal milk containe ODOS to 0.14
percent of chloride. Abnormal milk contains greater quantities
of chlor1de because of the presence of an exudate resulting from
the inflammatory reaction (Coles, 1967). Some of the earliest
investigators observed that milk from mast1 tis udders often had
a salt Y taste, and chlorides in excess of 0.14 percent
indicated abnormal milk (Tompkins!:!i al., 1946). Sharp and
De'Tomasi (1932).. described a procedure f'or determining the j
chloride content of' milk. The test was more sensitive than the
bromothymol test. According to Scbalm et al. (1971) an inverse
relationship exists between the concentrations of' lactose and
sodium chloride in milk. Both the stage cf' lactation and
/44
mastitis influence the lactose:chloride ratio. They also stated
that the chloride content of' colostrum was high but decreased
rapidly as secretion of' normal milk replaces colostrum, and then
rose rapidly as late lactation approached. Caulf'ield and Riddell
(1935) f'ound that average values f'or chlorides in the f'ollowing
breeds were: Holstein 0.139 percent, Ayrshire 0.126 percent,
Jersey 0.125 percent and Guernsey 0.123 percent. No signif'icant
dif'f'erences were observed between the dif'f'erent breeds.
b) Catalase Test: - Catalase is an enzyme f'ound in
cells of' plants and animals. The catalase content of' the milk
increases in mastitis (Schalm et al., 1971). The catalase test
is based upon the f'act that blood cells,and in particular
leucocytes, produce catalase, an enzyme that liberates oxygen
f'rom hydrogen peroxide. Increased liberation of' oxygen af'ter
the addition of' hydrogen peroxide to milk indicates an increase
in cell content (Heidrich and Renk, 1967; Schalm et al., 1971).
This test was f'irst described by Trommsdortf' (1906). Measurement
of' catalase activity bas been a popular laboratory screening test
/45
for diagnosing mastitis. The catalase activi.ty of a milk sample
can be correlated with the leucocyte level since the enzyme
catalase is found in increasing concentration in milk as the
mastitic severity increases (Luedeke et al., 1967). --Leucocyte count and cultural tests are important
laboratory tests used for diagnosis of mastitis. Both these
tests are dealt with under aomatic cella and mastitis, and
mastitis complex, respectively.
v. Somatic Cells and Mastitis
Freshly drawn cow's milk containe relatively large
numbers of cells consisting mainly of pOlymorphonuclear leu
cocytes and lymphocytes as well as some epithelial cells. The
number and types of cells in milk vary under physiological and
pathological conditions. Epithelial cells are derived from the
local tissue as a resul t of physiological wear during milk
secretion or as a result of tissue injury. Leucocytes are
derived from blood in response to tissue injury, irrespective of
cause.
1. Humber of Somatic Cells
Enumeration of somatic cells is receiving considerable
attention as a means of detecting abnormal milk, whether due to
mastitis or other causes, as a standard of milk quality, and in
mastitis research. This m~ be done by a direct counting method
(microscopie or electronic) or by any of the indirect teats
(Schalm and Lasmanis, 1968; Schalm et !:l., 1971). Currently,
/46
the terme cell count and total cell count are being replaced with
the term "aomatic cell count" (Sub Committee on Screening Tests,
National Mastitia Council, 1968). The term "leucocyte count" is
reatricted to those inatances when neutrophil leucocytes are
counted. According to S.cha.lm and Lasmanis (1968) the term
leucocyte means a white cell of blood origin. Leucocptea in
milk, therefore, are derived from bloodr Whereas, other cells
occurring in milk are tissue cells from the secretory epithelium
of the mammary gland.
According to Schalm.!1'&. (1971) milk from a perfectly
heal thy bovine udder ahould not contain any somatic cells since
the gland is not a holocrine secretory organ. Such milk can
hardly be found, therefore, the presence of a certain number of
somatic cella in milk has been universally accepted as normal.
Severa! workers have proposed a range or maximum permissible level
of total cell content per ml of milk to be considered normal.
While a number of workers have accepted up to 500,000 cella/ml
as a maximum for normal, others have suggested a much lower
(20,000/ml) or higher (1,500,000/ml) level (Scbalm~!:l., 1971).
Cell counts of as low as 100,000/ml in quarter milk were thought
to be too high and unacceptable according to Chu (1949). He
found that whenever the total cell count in milk was above
/47
20,000/ml. there was al.ways histological. ev:i.denoe of in:flammatory
Changes in the udder.
According to Smith and Schultze (1964) an average oell
oount of 765,000/ml. was repreaentative of normal milk. The
British Veterinar,y Association (1965) atated that quarter sample
oounts of 500,000 cella/ml. or over are generally indicative of
subclinical. masti tis. A t the International. Dairy Federation (IDF)
meeting held in Munioh in 1966, the National Committees of IDF
from 14 countries proposed less than 300,000 cells/ml. of milk ae
being normal.. A threshold value of more than 500,000 cells/ml.
of milk was accepted as indicative of mastitis (Kastli, 1967).
Ruffo (1968) made suggestions that a quarter be con
sidered normal. in the following three cases: a) when the
bacteriologicsl test was negative and the oell count under
100,000 oells/ml; b) when the bacteriological test was positive
for StaphYlococcus aureus and the cell count under 100,000 celle/ml
and c) when the bacteriological. test was negative and the cell
count was over 100,000 cells/ml provided that the neutrophil count
was under 12 percent. Cole!! &. (1965) cl.assified milk as
mastitic if any of the following conditions were fulfilled:
1) presence of ~. agalactiae (regardless of oell count); 2) eny
other pathogen slong with a cell count greater than 500,000 cells/
ml and 3) 1,000,000 cells/ml with or without organisme.
~,
i
Strokes and Wegefarth (1897) first deVised a method
for enumeration of the cells. Doane (1905), Savage (1907) and
Hewlett ~ al. (1909) improved the method by centrifuging the
milk, and examining the deposit in a haemocytometer. Prescott
and Breed (1910) introduced their method which gained general
/48
acceptance and has been used with minor modifications ever since.
It consists of placing 0.01 ml of fresh milk on a slide and
spreading i t over an area of one square centimeter. The film is
dried, fixed, defatted and stained. The cells are counted in a
number of fields and the total count determined by mul tiplying
the figure by a constant. The constant is derived from the area
which is covered by the microscope field with the particlùar lens
system in use. According to Schalm.!!!!:!.. (1971) cell counts
obtained by the Breed method should be considered estima tes
rather than absolute counts.
The actual counting of the cells involves long periods
of very tedious work at the microscope. To speed up the work,
some laboratories reduce the number of fields cOUllted, but this
increases the error enor.mously (Cullen, 1966). According to
Schneider and Jasper (1966) the precision of the direct micro
scopic cell count (DMCC) depends upon the working factor (WF)
and the number of cells in a sample. The smaller the WF the
greater the precision. The higher the cell count the more
accurate will be the estimate using a constant WF.
/49
Errors can occur in determining cell counts by the
Breed method, . as the method has distinct limitations. Therefore,
accurate, rapid and lesa tedious methoda for enumerating somatic
cells in milk have been developed usimg a millipore membrane or
an electronic counter. In the millipore membrane the cells in
whole milk are stained in a test tube and the milk is passed
through a millipore fil ter of a 0.65 pore size wbich concentrates
the cells on the fil ter. The fil ter is cleared with immersion
oil before exam1nation (Duitschaever and Ashton, 1968). Cell
counts obtained by this method were found to be higher than those
obtained by the direct microscopie method.
The system of electronic particle counting allows one
to ascertain a representative number of' particles according to
size. Cullen (1965) investigated the use of an elect~onic
counter deaigned for blood cell work with a view to finding a
more accurate and less laborious method for milk. The main
problem in the electronic counting of oells in milk consists of
treating the samples in a way that the cells remain the only
particles of their size range in suspension. Fat globules in milk
range from 1 to 15".IU. in diameter and thus overlap the size range
of the cells (Tolle ~~., 1966). The presence ~f bacteria is of
no consequence as their size is below the lower threshold,
setting used for counting cells. Cullen (1965) spun the milk
in special centrifuge tubes to remove fat globules. He also
diluted the milk to 1: 30 to prevent cells rising wi th the fat.
Phipps and Newbould (1966) later published details of eomewhat
similar methods. Efforts are underway to develop a simple
electronic cell counting method that would be generally
applicable.
2. Types of Somatic Cells
/50
Cullen (1966) cited Savage (1906) as being the firet to
describe three types of cells in milk. The firet type he called
polymorphonuclear cells (polymorphs) and they were 7.5 ft to 10.0...u.
in size. The second he called "large celle" about 15.0 A to
24.0.Al in size. His third group was comprised of lymphocytes which
were usually 5.0...u. to 7.0 p. in size. Savage (1906) regarded the
polymorphs and lymphocytes as identical with the correeponding
cells of blood. However, Hewlett ~ al. (1909), althougn
describing three similar groups of cella, were of the opinion that
none of them were blood celle. Thus, Prescott and Breed (1910)
wrote of body cells rather than leucocytes. However, later Bréed
(1914) described the cells in the milk of healthy cows as two
kinds, polymorphonuclear leucocytes and epithelial cells. Holm
(1934) found that small and large lymphocytes were the only
leucocytes of normal milk. Polymorphs were not a constituent of
normal mi.lk, and the preeence of even a few was indicative of
mastitis. Christiansen (1929) divided the celle into lymphocytes,
large mononuclear cella, transitory forms, eosinophils, basophils
and neutrophil leucocytes. Bachmann (1932) described small and
large lymphocytes, ordinary and large monocytes, neutrophils,
eosinophils and basophils, mononucleosides containing fat, and
epithelial cells.
/51
Varrier-Jones (1924) gave a very thorough and accurate
description of different cell types. He described finely granular
eosinophil cells, coarsely granular eosinophil cells, large
mononuclear leucocytes, lymphocytes, basophil cells, large neutro
phil cells, red blood corpuscles, large epithelial cells and mono
nuclear eosinophil cells. Zlotnik (1947) divided the epithelial
cells into six types. Some of these descriptions ~ go beyond
the limit of practicality, especially if large numbers of samples
are being examined (eullen, 1966).
Blackburn and MacAdam (1954) used a special staining
technique which clearly differentiated granulocytes from
agranulocytes. The non-granular cells they found were lymphocytes
5-9~in diameter, vacuolated epithelial cells (with large nuclei)
9-24..,u. in diameter laden wi th fat. Schalm and Lasmanis ( 1968)
described five types of leucocytes: 1) pOlymorphonuclear (PMN)
leucocytes; 2) eosinophils; 3) basophils; 4) lymphocytes, and
5) monocytes.
Okada (1960) made a cytological stuqy of milk from
mares, cows, ewes, goats, pigs, dogs, cats, rabbits and mice.
/52
The same cell types were seen in the milk samples but the
differential cell picture varied from species to species.
Wahby (1954) studied the cells of buffalo milk and found only
epithelial cells and lJï~phocytes. No polymorphe or monocytes were
present, although the total counts of these animaJs were not low,
the average being 240,000 cells/ml.
3. Orig:i.n and Function of the Different Oell Types in Milk
a) Epithelial Oells:- These are derived from the acini
and ducts of the mammary parenchyma. They cannot be differentiated
as to their specifie location. Zlotnik (1947) attempted to
differentiate between cells derived from acini and those from ducts.
He was unsuccessful as cells from all parts varied greatly in
Bize and shape. He described six groups of epithelial cella. These
cella were cast off from acini. Preaumably, thia waa a normal
process of wear and replacement in a hard-worked tissue. Towards
the end of lactation, they were shed in greater numbers on account
of involution of the gland (Blackburn and MacAdam, 1954). Schalm
~~. (1971) deacribed two t,ypea of epithelial cella on the basia
of their cytoplasmic ataining character.
i) Non-Vacuolated Epithelial Oella:- These cella varied
in aize and were usually comparatively amaller than the vaculated
epithelial cella. The nucleus was large, well-defined and spherical
or eliptical. The cytoplasm favoured a narrow to broad zone and
~
1 t
stained sligntly or moderately basophilie. The cytoplasm was
non-granular and devoid of fat globules, but sometimes appeared
fo~.
ii) Vacuolated Epithelial Oells:- These cells varied
from medium size to the largest eells found in milk films. The
nucleus waa large, well-defined and spherical, eliptical or
irregular in outline. The cytoplasm varied in amount, contained
few to numerous fat globules of various sizes, stained slight to
dark blue (basophilie) or pink (acidophilie) and mignt eontain
reddiSh-purple granules. Degenerating forma of vacuolated
epithelial cells reported by Zlotnik (1947) gave rise to pseudo
polymorph and micropseudo cells.
b) Neutrophils:- Polymorphonuclear leucocytes (PMN),
polymorphs, are derived from blood and retain almost all of their
charaeteristics (Schalm and Lasmanis, 1968; Schalm!Œ~., 1971).
They are round, elliptical or irregular in outline. The nuclei
stain moderate to deep purple and are eharacteristically lobu
lated. Fine filaments connecting the lobes of the nuelei ~ be
evident in some cells. The cytoplasm is clear and contains few
to Many distinct or indistinct small pink granules giving the
cytoplasm a pinkish hue when stained with Wright - Leisbman stain.
Neutrophils are produeed in bone marrow by the process
of extravascular granulopoiesis and have a maturation time of
/54
about six d~s in the cow (Kaneko ~~., 1964), ~ain entrance
into the circulation by diapedesis and have a circulating time of
five to six hours, and half-life of about one to two weeks.
According to Schalm et~. (1971), neutrophils em1grated into
the mammary tissue tbrougb the capillaries then passed through the
parenchyma and alveolar or ductal epithelium to lie free in the
acini or ducts. The mode of passage across the alveolar or
ductal epi thelium was not defi.n1 tely known. The drawing of PMN
leucocytes from the blood stream into an area of injury was called
chemotaxi s. The PMN leucocytes engulf bacteria and tissue debris
by the act of phagocytosis. En~es within leucocytes digest
and destroy the engulfed bacteria. In the process, the leuco
cytes become degranulated, releasing chemical substances which
specifically affect capillary walls, increasing permeabili ty and
allowing escape of fluid and proteine of the blood with the
tissues (Jain' ~ al., 1968). This action of PMN leucocytes 1s
respons1ble for the intense swelling of the gland, characterist1c
of acute mastitis. The purpose of exudation of PMN leucocytes
~to milk in mast1tis is to destroy the irritant and permit
repa1r to take place.
Neutrophils provide a sign1ficant barr1er to exper1mental
infections of the bovine mammary gland wi th several organisme
including: E. coli, ! .. aerogenes, .§.E:. agalactiae, Pseudomonas
aeruginosa and Staph. aureus (Jaïn et !!.._, 1968). Schalm and
Lasmanis (1963) reported the effect of pre-existing leucocytes
on the development of experimental ~. ~ masti tis. Animals
with low cell counts were easily infected w:l.th milk doses of
about 10 organ:l.sms of ~..~.. When a leucocytosis of about
/55
five million cells/ml was produced by infusing the quarter w:l.th a
Seitz-filtered Staph .. pyogenes culture, nine quarters resisted
repeated inoculation with ~ .. ~. Schalm et & .. (1964b) showed
that pre-exist:l.ng leucocytes would protect quarters from experi
mental infection with~ .. aerogenes, even though the same quart ers
were susceptible when challenged alter the inflammatory reaction
had subsided.. Further experiments by these workers suggested
that a level of 200,000 cells/ml was sufficient to give good
protection against artificial inoculation with A. aerogenes.
Blobel and Katsube (1964) were able to induce a
pronounced leucoc~osis in milk by infusing sterile 0 .. 14M of
sodium chloride solution into the quarter. Untreated quarters of
the same cow showed no rise in cell count. The cows were then
challenged by intramammary inoculation with various organisme,
each cow received the same number of organisme in treated
(leucocytic) and untreated (normal) quarters. All untreated,
normal quarters developed mastitis with the appropriate organism ..
Three out of four treated quart ers were protected against the
streptococci ..
Blobel and Katsube (1964) further investigated this
action in vitro. They found that most of the leucocytes were,
in fact, polymorphe, and they readily ingested Staph. pyogenes,
str. agalactiae, E. coli, !. aerogenes and coagulase-negative
stapbylococci. Nearly all the Staph. pyogenes and Â. aerogenes
survived the phagocytosis. There are maQy factors which govern
the migration of leucocytes, phagocytosis and the fate of the
organisme within the leucocytes (Zweifach et al., 1965). --
/56
c) Lymphocytes:- ~phocytes are spherical cells with
moderately or darkly stained nuclei. The cytoplasm stains light
to dark blue and an occasional cell may contain few azurophilic
granules. The cytoplasm is sparse in small lymphocytes in which
the nucleus almost fills the cell, and forma a narrow to broad
zone in large lymphocytes. Lymphocytes in milk are derived from
blood. They are produced in lymph nodes and other lymphocytic
tissues in the body and enter the blood via lymph vessels.
Unlike neutrophils, some lymphocytes ~ re-circulate along with
the lymph (SChalm.!1 ~., 1971). According to Doughterty and.
White (1947) lymphocytes are continually losing cytoplasm to the
lymph stream, without death of the nucleus. This cytoplasm
containe gamma globulin, which in immun1zed animals, is a specifie
antibody. The rate of production of the material is under the
control of the pituitar.y and adrenal cortical secretion.
Lymphocytes in milk may be contributing gamma globul.ine, wbich
may be immunologically specifie under certain conditions
(Cullen, 1966).
/57
d) Monocytes;- These cells are occasionally found in
milk and resemble those present in the blood. They are round,
oval or elongated. The nucleus is large, oval, kidney-shaped
or deeply constricted and may almost fill the cytoplasm. Their
identification in milk film stained with Romanowsky stains is
somewhat difficul. t (Schalm II & .. , 1971).. Monocytes in milk are
probably derived fram the blood or originate from local connective
tissue elements (histiocytes) (Cullen, 1966).. They infiltrate
tissues during inflammation and characteristically more numerous
in chronic inflammation ..
e) Eosinophils:- These cells in milk are derived fram
blood. They m~ be round or irregular in outline. The cytoplaem
1s clear and contains many discrete round acidophil1c granul.es.
The nucleus may be spherical or lobulated. This cell does not
commonly occur in milk (Schalm!.i al., 1971). Eosinophils
accumulate at the site of antigen and ant1body reactions and it
bas been suggested that they inactivate histamine or histamine
like toxic materials. They increase in a situation involving
decomposition of bOdy protein and tbis reflects a function of
detoxification (Zweifach et ~., 1965). Studiee of Litt (1963)
/58
provide convincing evidence that antigen and antibody complexes
attract eoeinophils. Accumulation of eosinophils is used as an
index of rapidit,y of antibody production.
f) Colostrum Bodiesj- A special type of cell found in
milk a few da.ys post-partum. These are larger cells, 9./1l- to
40 ft in diameter and are often capable of active amoeboid movement.
They usually have one small nucleus, and are filled with numerous
small, and a few large, fat droplets. They usually show signs
of degeneration (Cullen, 1966). According to Maximow and Eloom
( 1948) they ma.y be transformed epi thelial. glandular celle which
are filled with the production of secretion and become detached
but are more likely to be wandering lymphoid cells which have
ingested fat droplets by phagocytosis.
g) Erythrocytes: - Ma.y also be found, particularly in
centrifuge deposits.
4. PbysiCal. Factors Influencing Numbers and Relative Proportion of Cells
a) Different Fractions of Milk
A small sample of milk obtained wi thout discarding a
single stream is cal.led strict for~lk, whereas, if the first
tbree or four streame are discarded, the sample taken next i8
called fore-milk:. A sample obtained during milking, approximately
mid-wa.y, is referred to as middle milk, and the sample collected
/59
when a milkLng is completed constitutes strippings milk. A
sample of the total milk of an udder is caJ.led bucket milk. The
soma tic cell content of milk varies among the different fra.ctions
of' milk obtained. The total cell count in fore-milk is usually
lower than that in strippings milk and the counts in middle milk
are the lowest. Sometimes fore-milk counts may be higher, but
this m~ be a reflection of a les10n in the teat rather than being
a normal occurrence. Bath PMN leucocytes and mononuclear cell
counts are greater in ètrippings milk than in f'ore-milk (Schalm
.!i &., 1 971 ) •
b) Di~ Variation
Diurnal variations in soma tic cell counts in milk have
been observed (Gradient, 1954; Cullen, 1967; Schalm, 1967;
Smith and Schultze, 1967; White and Rattray, 1965). White and
Rattray (1965) took milk sampleseach hour from 6.00 a.m. until
10.00 a.m. the following d~ from three cows. They plotted mean
log 10 cell count Talues for total count and polymorphonuclear
count, against time. The shape of the plot was remarkably
similar for each cow and quarter, and showed a marked rise in
cells during and after milking with a subsequent f'all before
the next milking. The total count and polymorph count showed
the same pattern of variation and the percentage of pOlymorphs
remained fairly constant whether the total count was high or low.
Considerable variations were seen between morning and evening
semples, especially when irregular milking intervals were in
affect (Schalm et al., 1971). --Cullen (1966) suggested an explanation for the diurnal
/60
variations in cell counts. He has proposed that cyclic pressure
changes in the alveoli, due to the quantity of milk in the udder,
could influence passage of cells into the acini. When pressure
was reduced towards the end of milking, large numbers of cells
would pass into the acini, but this migration would gradually
slow down as pressure buil t up again towards the next milking.
c) Stage of Lactation
It has been recognized that the somatic cell count is
high during the first week of lactation, then soon decreases and
remaina low for several weeks after which a gradua! increase
occurs until the end of lactation (Schalm ~~., 1971; Cul 1 en,
1966). For the first few d~s of lactation, the cell count is
abnormally high.. According to Savage (1912) this is mair,ly due
to lymphocytes, although colostrum bodies are characteristically
present at this time. As the milk volume decreases in the latter
part of lactation, an apparent increase in cell numbers occure
which 1e different from mere concentration of celle in the smaller
volume of milk (Schalm et &_, 1971). Natural drying off of
normal glands occure gradually and the increasee in cell numbers
1s mainly due to ep1thelial celle (Johneon and Trudel, 1932).
/61
Drying off occurring as the resuJ. t of a severe masti tis is
characterized by the presence of variable numbers of neutrophils
in the secretion. Therefore, cell counts are higher in intected
quart ers than in normal quart ers (Kaiser, 1965). MacLeod and
Anderson (1952) found that the ce11 count fe11 during the first
three weeks of lactation from 1,000,000 cells/ml to 70,000
cells/ml in a study of heal thy cows. Wai te and Blackburn (1957)
found that the average count was lowest and least variable from
the 70th to the 130th d~ of lactation.
d) Lactation Age
Age affects the milk leucocyte count in heifers. They
usually have lower countsthan older cows. Mochrie et ~ ..
(1953) found that 94 percent of heifers had less than 300,000
cells/ml of milk. The average count was 446,000 cells/ml for
cows whose lactation average was 2.4. Blackburn (1968) noted that
the average total cell count of s8Dlples from cows in their second
lactation (0.37 million ce11s/ml) was almost double that of
samples taken from cows in their first lactation (0.19 m111ion
celle/ml). He also noted 'that in subsequent lactations, there was
a graduel rise in the average total cell count until the seventh
lactation when it reached 0.67 million cells/ml. He concluded
that the increase in the average total cell count in the second
lactation was due mainly to a rise in the number of polymorphe
and to a lesser degree, to a rise in the number of cells other
/62
than polymorphs, whereas, the increase in subsequent lactations
was due entireJ.y to a rise in the number of polymorphe.
Blackburn (1966) found that in the infected and uninfected
samples together, the rise in the average total cell count from
one lactation to the next was due mainl.y to an increase in the
number of pOlymorphs.
5. Effect of Mastitis on Cell Count
Mastitis is inflammation of the udder, and inflammation
is always accompanied by a raised cell content. The effect of
mastitis is to raise the cell count and also to alter the
differential cell count. Tapernoux (1931) found that normal. m:Llk
had a leucocytic formula eimilar to that of blood, i.e., a
leucocyte:polymorph ratio of about 2:1. In maetitic milk he
found nearly all leucocytes were polymorphe. Bachmann (1932)
had similar findinge.
Cell typee in normal and maeti tic milk JI18iY fluctuate
daily. Their proportion in masti tic milk varies wi th the total
cell count. Neutrophils are the first to appear in the milk in
eignificant numbers at the beginning of the mastitic process even
though there may not be a significant elevation in total cell
count in the fore-milk. Some workers have regarded the presence
of even a few neutrophils in milk as an indication of masti tie,
whereas, others consider up to 44 to 70 percent neutrophile as
normal (Schalm.!!1!:!.., 1971). According to Galli (1965) the
/63
presence of less than 12, 13 to 20 or greater than 20 percent
neutrophils in milk samples bas been taken as indications of
he al thy , suspicious and infected quarters, respectively. Their
proportion increases with the severity of inflammation and the,y
m~ constitute as much as 90 to 95 percent of the cells in
masti tic milk. Other typee of cells may aleo increase in
mastitis, but their proportion would var,y with duration and
eeverity of the inflammator,y process. The epithelial cells might
be shed in large numbers in mastitie, but their relative pro-
portion etill remains low due to an overwhelming number of
neutrophile infiltrating from the blood. Mononuclear celle may
be seen in loarge nUl4bere in chronic udder infections, but
neutrophile are predorn; nant (Schalm ~ &_, 1971).
Under pathological conditione, the higher the cell
count the higher the percentage of polymorphs, up to a maximum of
about 90 percent. The increase in numbers of celle is thought to
be due to chemotactic stimuli, which resulte fram the preeence of
microorganisme in the udder. The degree of cellular reeponse i8
likely to be proportional to the eeverity of infection, both in
terme of numbere of organisme, and of the degree of tissue in
vasion (Pattieon and Smith, 1953; Klaetrup, 1956). Blackburn
~~. (1955) coneidered that the differential cell count can
give very ueeful information, particularly in evaluating slightly
raised counte and for counte of bulk milk. Gua.llini and Vallie
/64
(1957) oompared counts of milk from heal thy udders wi th milk
from Str. agalactiae infected udders. They suggested that total
counts of 1,000,000 cells/ml should be classified positive for
mastitis, 300,000 to 1,000,000 cells/ml suapicioua, 80,000 to
300,000 cells/ml dubious and less than 80,000 cells/ml as
negative. They found the differential count was much more
informative and suggested that a ratio of lymphocytes/polymorphe
resulting in a value of < 1.0 indicated mastitis.
B. THE MASTITIS CO.MPLEX
1. Infectious Agents
Mastitis is a complex disease. Inherent physiological
factors make the bovine udder susceptible to invasion by a wide
range of microorganisms which vary in pathogenici ty. In addition,
numerous environmental factors discussed earlier influence the
severity of udder infections. Haï (1957) has listed bacterial
species belonging to 25 different genera as being the cause of
mastitis. Of these, streptococci and staphylococci are the
prinoipal organisme responsible. The specifie infectious agents
in this section are grouped as streptococci, stap~lococci,
coliforms and other microorganisme.
a) Streptococcal Mastitis
Prior to 1940, it was not uncommon for investigators to
state that 90 peroent of chrome mastitis was caused by strepto
cocci. With the emergence of the antibiotic era, streptococcal
/65
mammary infections were reduced to much lower levels (Stableforth,
1950; Berger and Francis, 1951; Wilson, 1952; Schalm ~~.,
1971). The three main.species of streptococci involved in
m.a.sti tis are~. agalactiae, ~. d,ysgalactiae and.§.k.. uberis.
Udall (1947) reported 90 percent of cases were due to ~.
agalactiae at that time. In India, 76 percent of the infections
were due to streptococci (Indian Farming, 1962). Malik (1968)
reported that eight percent of 3,326 quartersamples screened
with the CMT were infecte1 with Str. agalactiae. Slanetz and
Nagbski (1940) studied the incidence of infection by species and
revealed that among 680 cultures from milk containing 500,000 or
more oells/ml, the distribution was ~. agalactiae 84.2 percent,
Str. uberis 12.2 percent, .§.!!:. dysagalactiae 2.2 percent and
Str. fecalis 1.3 percent.
A random survey on masti tis in Canada during 1945-46
showed streptococcal infection to be 20 percent (Barnum, 1953).
Barnum and Newbould (1961) reported the incidence of Str.
agalactiae to be 12.9 percent out of 2,122 quart ers tested.
Williams ~~. (1966) have reported the incidence of streptococci
(Str. agalactiae in parenthesis) to be 27.62 (8.83), 37.4 (9.8)
and 26.7 (7.8) percent, respectively.
i) ~. agalactiae:- Belongs to Group B of Lancefield's
(1933). Mastitis due to this organism is contagious. Str.
/66
agalactiae is generally recognized as an obligate parasite,
al though i t bas been shown by Chodhowski (1949) that the organisme
can remain viable for 20-26 d~s outside the host on the skin of
the udder or in parts of the building. As an obligate parasite
of the mammary gland, it is, therefore, susceptible to complete
eradication from a dairy herd. Minett n &. (1933) were the
first to demonstrate the possibility of assembling and main
taining dairy herds free of infection with~. agalactiae.
Francis (1962) bas pointed out that a single treatment with an
appropriate preparation of penicillin could eliminate this
organism from at least 90 percent of infected quarters, thus
illustrating its susceptibility to antibiotics.
As an obligate parasite and due to its susceptibility to
antibiotics, this' would suggest that mastitis due to~.
agalactiae could be completely eradicated or reduced considerably
(Stableforth ~&., 1949; Francis, 1949; Wilson, 1952;1958;
Livoni, 1953). The incidence of ~. aga!actiae in heifers at
parturition is influenced by the degree of infection in the herd
and the method of management of the calves (van Rensburg, 1942).
In a~. agaiactiae infected herd where raw milk was fed to the
calves and suckling of ~ture teats between calves was not
prevented, 5.7 percent of the heifers were shedding ~.
agalactiae at parturition (Schalm et &., 1971).
/67
Stableforth et al. (1935) noted that there was a gradua!
increase of Str. aga!actiae infection with succeeding lactations.
They reported 18.4, 47.6, 64.6 and 67.3 percent in first, second,
third and fourth lactations. White.!!.!!.. (1937) reported that
in the initial stages of ~. agalactiae infection, the decrease
in milk yield might be negligible, not exceeding five to six
percent, but with each additionai mammar,y quarter becoming in
fected the decrease in production bècame successively greater
attaining 15 to 20 percent. McLeod and Wilson (1951) and Shaw
and Beam (1935) reported losses of 22 to 24 percent in milk
secretion due to~. agalactiae mastitis.
Althougb~. ag!lactiae is dependent upon the mammar,y
gland for its perpetuation in nature, the organism may survive
for vary~ periods of time on objects, with which it has come
into contact. Chodhowski (1949) reported the recovery of patho
gens in tests on 38 percent teat skin, 38 percent of milkers'
bands, 20 percent of clothes and in 22 percent of tests of the
air, utensils, fioora, stools and brooms. The bands of the milker
might pl~ an important role in the spread of Str. agalactiae
when cowa are hand milked rather tbSn when machine mi1ked (Spencer
II al., 1946).
ii) Non-agaJ.actiae Streptococci:- The term "non
agalactiae" is employed to include all streptococci other than
Str. agalactiae whethe: aaprop~tic or pathogenic that m~ appear
/68
f'rom time to time in milk samples. This designation is used
in order to indicate that the problem of' epizootiology and
control is dif'f'erent f'rom the chronic mastitis caused by ~.
agalactiae. ~. dysgalactiae and~. uberis are potentially
pathogenic f'or the udder and occur f'requently enough to be
encountered in ~ dairy herd. ~. zooepidemicus, ~. pyogenes
of' humanorigin and Lancef'ield's Group G and L streptococci have
been described as causing individual cow or herd infections in
a limited number of' herds. In addition, certain "atypical"
streptococci ~ appear in milk samples as contaminants or even
reside temporarily wi thin the streak canal or teat cati ty. In
general, the "atypical" streptococci are non-pathogenic or onl.y
mildly pathogenic f'or the bovine mammary gland (Little, 1940b).
To distinguish~. agalactiae f'rom non-agalactiae
streptococci, particularly ~. uberis and ~. dysgalactiae,
the Christie, Atkins, Munch-Petersen (CAMP) test (Christie et
~., 1944) bas been widely used.
b) StaPBvlococcal Mastitis
Staphylococcus (Staph. aureus or Micrococci pyogenes)
is associated with mild and severe f'orms of' acute and chronic
inflammation of' the udder, and may &lso be excreted in milk of
healthy udders. Staph. epidermid1s which 1s commonly regarded
as non-pathogenic, 1s associated with the bovine mamma.ry gland.
It has been reported to be capable of' causing a mild, subclinical
form of mastitis (Brown ~~., 1967). The latter species
together with other non-pathogenic organisme of the family
Micrococcaceas are commonly referred to as"Micrococci." Bang
(1889) and Lucet (1889) first demonstrated the presence of
staphylococci in the secretion of inflemed udders of cows.
Jones (1918) reported that the streptococci and micrococci have
been the next MOSt frequent group of organisMS isolated from the
inflamed udder. Staphylococcal mastitis bas received more
attention than any other form in the past decade or so, mainly
because of the increasing incidence of staphylococcal mastitis
(Schalm and Lasmanis, 1957; Plastridge, 1958; Neave, 1959).
/69
Frost (1962) observed that 46.8 percent of infections
were due to Staph. aureus in South Eastern Queensland. In Sweden,
80 percent of the cases observed were associated with staphylo
cocci (Rendel and Sundberg, 1962). Dhanda and Seth! (1962) in
India, reported the incidence of Staph. aureus to be 41.2 percent
in mastitic cows. In New Zealand, incidence of Staph. aureus
infection bas been given as 34 percent (Wilson and Brookbanks,
1967). Malik (1968) examined 3,329 quarter semples from 133
cows and reported that 69.1 percent of the quart ers were infected
with staphylococci. According to Newbould (1968) the incidence of
infection in clinical mastitis due to Staph. aureus varies
considerably from area to area.
/70
Spencer and Lasmanis (1952) studied the incidence of
micrococci in the environment and on the bodies of 62 cows.
Hemolytic staphylococci were isolated from the skin of 77 teats,
from 40 percent of the teat cups before disinfection and from
the hair of the flanks of the cows. Milk samples collected
aseptically yielded hemolytic staphylococci in 59 cases. They
concluded that the principal extra mammar,y reservoirs for
coagulase-producing hemolytic staphylococci were the skin of the
teats and the teat cups of the milking machines.
These organisme have been isolated from several body
tissues in cattle, e.g., teats, udder external orifices of the
a.n:i.ma.ls, vagina, from skin of the cow and the milker, and lDELD3'
other parts of the environment including buildings (Francis,
1941; Davidson, 1961a,b). The resistance of staphylococci to
antibiotic therapy is a commonly reported phenomenon. Frost
(1962) noted that 33 percent of the staphylococcal cases observed
were resistant to penicillin. This was due to the inaccess
ibili ty of the orga.n:i.sm when i t is established . in the udder
(Murnane, 1964). Due to the above facts, the eradication of the
organism becomee virtually impossible and therefore, eeverely
limite the simple mastitis control techniques of isolation and
therapy. It is obvious that very complicated measures will be
required to control this disease when the causal organism :i.s not
an obligate parasite and partially or highJ.y resistant to anti
biotics.
/71
A great dea1 of research has been carried out on the
microbiological characteristics of staphylococcal organisme
causing mastitis, much of this referring to bacteriophage typing
of the organisme. Edwards (1961) detected 13 phage types in
staphylococci isolated from dair,y herds, but never more than two
or three at a time. Davidson (19619) determined 70 phage types
in strains of staphylococci obtained from different parts of the
cow's body and from the environment. Loken and Hoyt (1962)
observed that mastitis staphylococci were mainly lysed by certain
phage types (e.g., 42D Group IV). Bacteriophage typing has re-
vealed that in most dairy herds where typing was employed one or
two strains of staphylûcûcci predominated (Schalm et ~., 1971).
Heidrich and Renk (1967) stated that phage typing is clearly
of value in determining the source of infection and the manner in
which infection bas spread. Various workers have attempted to
utilize the tox1genicity of staphylococci as a criterion for their
pathogenicity (Scbalm and Lasmanis, 1957; Obiger, 1961). According
to Plastridge (1958) the coagulase test 1s the most efficient
method of differentiating between pathogenic and non-pathogenic
groups of staphylococc1.
c) Coliform Mastitis
Col1form organisme may cause either a mild mast1tis or a
severe acute mastitis with systemic symptoms (Barnes, 1954;
Easterbrooks and Plastridge, 1956; Plastridge, 1953; Schalm and
/72
Woods, 1952). The onset of acute coliform mastitis is sudden.
Affected cows usually appear normal at one milkipg and show the
following symptoms by the next milking: nearly complete cessation
of udder secretion, loss of appetite, depreaaion, a temperature
of 104-10SoF, and one or more swollen quartera (Plastridge, 1955).
The coliform group of organisme include a number of
apecies belonging to the genera Escherichia, Aerobacter,
Klebsiella and Paracolobactrum. These bacteria are ubiquitous in
the en~ronment of the dairy cow and are commonly present in the
large intestine and the colon. Its presence in milk, water, etc.,
indicates fecal contamination and only ver,y rarely can E. ~ be
demonstrated in milk aseptically drawn from healthy udders
(Heidrich and Renk, 1967). Nevertheless, Schalm and Woods (1952)
observed latent udder infections that resulted in the excretion of
coliform bacteria in the milk over many months without any
apparent derangement of secretion of tissue changes. Malik (196S)
reported that 4.7 percent of the 3,329 individual quarter fore
milk samples representing 133 cows revealed the presence of coli
form organisme. At times, coliform infection leads to mild,
intermittent catarrhal mastitia with temporar,y changes in the
secretion (Plastridge, 1955).
Coliform mastitis ia a disease of the gland uninfected
with other pathogens (Schalm et ~., 1971). Coliform organisme
/73
are not br'ought to the mammary gland by the blood but invade
througn the teet orifices. Coliform mastitis is a single quarter
disease. This form of mastitis may be expected to occur with
increasing frequency in herds in which mastitis control programs
result in an ever-increasing number of older cows free from the
more common forms of mastitis (Schalm and Woods, 1952).
d) Other Organisme
i) Co~!ebacteria:- Diphtheroid organisme are commonly
encountered in milk and with the exception of COrynebacterium
pyogenes, are generally regarded as harmless (Little et al., 1946).
Q. Blogenes causes a severe acute mastitis which ~ become
gangrenous (Schalm, 1944). This form of mastitis is not common
in the United States (Plastridge, 1958). In England, a condition
known for many years as "summer masti tis" occurs fairly frequently
during the summer months, affects dry cows more often than those
in milk and is caused by Q. pyogenes (Plastridge, 1953). Udder
infections and masti tis due to Q. bovis and Q. ulcerans have been
reported (Cobb and Walley, 1962; Bourland ~~., 1967; Higgs
~ aL, 1967).
ii) Pseudomonas: - Masti tis due to Pseudomons aeruginosa
is usually sporadic but in a few herds, may reach serious pro
portions (Burkey and Bouma, 1955; Tucker, 1950). The onset is
usually sudden and accompanied by clinical symptoms of relatively
short duration.
/74
Tucker (1954) found that 20 to 40 percent of the cows in
45 percent of 3,000 dairy herds examined had established ~.
aeruginosa infeotion. This organism bas i ts natural habitat in
soil, water and se~6ge and in the environment of cattle. They are
h1ghly res1stant to d1sinfectants, to penic111in, streptomycin,
chlortetracyline and oxytetracyline (stewart ~~., 1955;
Tucker, 1950; SOhalm, 1948;1950; Barnes, 1955).
111) Yeast:- Klimmer and Fle1scher (1930) were the
f1ret to describe a case of bovine parencbymatous maetitis
associated with a particular yeast. Murphy and Drake (1947) re
ported masti tis associated wi th yeast-like fungi. The most
pathogenic of these is C;yptococcus neoformans.
Pounden~~. (1952) reported 106 cases in a herd of
235 cows in the course of a year. Simon ~~. (1953) diagnosed
the condition in 50 cows in a herd of 280. The major1ty of
olinically affeoted animals suffered severe attacks with marked
swelling of the udder and decreased milk production. In recent
yeare, masti t1s due to yeasts and yeast-like organisms have been
reported quite frequently (Pounden ~~., 1952; Simon et al., 1953; Tucker, 1954; Prasad and Prasad, 1966). The usual anti
biOtics and sulfonamides do not appear to impair the growth of
yeasts; indeed, some may even promote their growth (Heidrich and
Renk, 1967).
/75
iV) Mycoplasma:- Mycoplasma mastitis is usually acute,
but subclinical and chronic forms also exist. Mycoplasmas are
the smallest f'ree-li Ving microorganisme, lack a cel1 wall and
are higbly pleomorphic due to the effects of' enViromnental
physical f'orces on the cell (Schalm ~~., 1971). Alstrom
(1955) had been cited by Schalm!Œ~. (1971) as being the first
to isolate ~coplasma from the milk of' both normal and mastitic
cows from herds af'f'ected with chronic mastitis. In New York
State, as maqy as 77 percent of' the cows in a herd had to be
slaughtered because of' lD3'coplasma masti tis and on an aver88e, one
third of' the cows in 15 herds was slaughtered (Carmichael .!i~.,
1963). During an epizootic of mycoplasma,mastitis in 2,800 cows
in four herds in Calif'ornia, about 280 cows were sold because of'
this infection (Jasper ~~., 1966).
2. Pathogenesis
The term pathogenesis (Gk pathos + genesis) means the
production or development of' disease. Most f'requently, organisms
that cause mastitis reach the udder through the streak canal, the
cistern, and lactiferous ducts and eventually reach the secretory
end pieces (Heidrich and Renk, 1967). Normally, the streak canal
is closed between milkings thus preventing the entrance of'
organisme. Kitt (1921) pointed out that at times, the capillary
crevices in the walls of' the streak canal are moist wi th milk and
that when the cistern is full, a drop of' milk may bang from the
/76
teat orifice. Accordingly, it was assumed that rapidly multi
plying, mobile organisme could penetrate and pass through the
streak canal. According to Johnston (1938) the intermittent
pressure of the arteries enables the column of milk in the teat to
be moved up and thus aspirate bacteria. Little (1937) as weil as
McEwen and Samuel (1946) demonstrated that the aspiration of
organisme is possible during the act of milking by band or machine.
Once the organisme have passed thr.ough the streak canal, they mave
upward into the mammar.y gland by the effect of pressure on the
cistern and udder when the cow lies down or by differential
pressure arising during some phase of the milking act (Heidrich
and Renk, 1967; SChalm~~., 1971).
The development of mas titis or inflammation of the udder
can be explained in terms of three phases, namely, invasion,
infection and inflammation (Murpby, 1945). Invasion is the passage
of organisme into the interior of the udder. In invasion, the
organisme multiply and invade the mammary tissue. In the in
flammator.y phase, the tissue reacts to injur,y by the organisme or
their products. It is in this stage that clinical signa of
maetitis appear. The factors affecting bacterial invasion of the
udder are teat patency, erosions of the teat orifice, byper
sensitivity, Virulence of the organisme and period of exposure
to infection (plastridge, 1953;1958; NewbOuld, 1964; Heidrich and
Renk, 1967; Edwards, 1968; Scha1m~ al., 1971).
Bacteria that reach the glandular tissues do not
necessarily cause masti tis. The intact epi thelium of' the milk
cistern and lactif'erous ducts may prevent the establishment of'
infection. Once the inf'ecting organisme have entered the teat
/77
duct and reached the teat cistern, they meet one of' two
situations. Either they meet the def'ence mechanism of' the udder
and do not establiSh or they overcome the inf'lammatory resistance~
mul tiply and invade the mammary gland. The intramammary
resistance to inf'ecting organisme may be the phagocytic action of'
leucocytes present in the milk, inhibitory f'actors in milk and
immune substances present in milk and blood (Edwards, 1968).
It is generally accepted that infection with ~.
aga!actiae occurs via the teat canal (galactogenous infection).
Str. agalactiae characteristically lives in the milk channels and
rarely penetrates beyond the surface lining of' these channels. The
irritant, whether it be lac tic acid or a toxin, accumulates in the
milk and sets in motion an exudation of' leucocytes and blood
plasma factors into the alveoli and ducts. Acute f'lare-ups lead
to filling of' the alveoli with leucocytes and sloughed epithelial
cells. Clots and bacteria may form plugs in smaller ducts leading
to rapid involution of the secretory tissue. The mastitis is
caused by an ascending infection of the ducts (Schalm ~ al.
1971). The primar,y response to infection by Str. agalactiae is
exudative and f'ocal in nature. This is f'ollowed in time by a
reaction in the interstitial tissue leading to fibrous and
atrophJr. Usually,.§.E.. a.ga.lactiae infection is mild and slow
and do es not lead to readily detectable inflammation of the
udder tissue or obvious changes in the secretion.
/78
Jones and Little (1934) observed that frequent repeated
inoculation of the udder with small numbers of Str. agalactiae
were required before an experimental infection could be
established. This led some investigators to postulate that prior
sensitization of the udder to the organisms is a pre-requisite
for permanent colonization.
Staphylococci enter a mammar,y quarter througb the teat
orifice. The organisms eventually enter the large ducts and
progress onward to the smaller ducts and alveoli. The response
of the tissues to the presence of stapbylococci will depend upon
the rate of their multiplication, the type and concentration of
toxine, and the effectiveness of the cows' defence mechanisms
(Schalm .!1 !!.., 1 971 ) •
Staphylococcal toxins produce injur,y to ductal epi
thelium resulting in release of chemotacti~ substances from the
cells which attract blood leucocytes into the area of injur,y.
Bernheioner and Schwartz (1964) stated that the stapbylococcal
toxine cause release of leucocyte lysosomes increasing the injur,y
to the mammar,y epithelium and leading to exudation of blood plasma
and massive infiltration of the area by additional neutrophil
leucocytes. The plasma factors and cell masses lead to the
formation of clots that occlude the ducts and pre vent drainage
of milk from the lobes and lobules (Pattison, 1958). Leuco-
cytes engulf staphylococci but substances elaborated by the
organisme m~ protect them from being destroyed by the
digestive enzymes of leucocytes. The survival of stapbylococci
within some leucocytes leads to death of the leucocytes and
further multiplication of staphylococci. This phenomenon was
/79
shown by Newbould .!i&., (1966) with five strains of pathogemc
staphylococci and was produced by leucocytes fram the milk of
Dine cows. Rapid growth of staphylococci with production of
high concentrations of CIC -toxin can resul t in a constriction
of the arterioles with interference in blood flow and a rapidly
developing gangrene.
The beginning foci of inflammation, whether amSll or
large, are alwe.vs acute in nature. If the process remains con-
fined, the neutrophilic infiltration is suapended by the cellular
changes in the stroma which suggests a chromc pro cess. The
epithelium of ducts, consisting normally of a single l~er of
cells, becomes stratified through metaplasia, and as the in-
flammatory process becomes chrome, ~he stroma becomes infiltrated /
by lymphocytes and plasma cells (S halm.!i.!!.., 1 971 ) •
J
In coliform mastitis, the natural infection commonly
occurs via the teat canal. Coliform organisms responsible for
acute mastitis differ from ordinary strains by possessing a
distinct capsule (P1astridge, 1958). Systemic signs of the
disease are due to absorption of endotoxin rather than bacter
emia (SChalm.!i!.!.., 1971).
/so
C01iform mastitis is characteristically peracute or
acute (Schalm and Woods, 1952). The serous phase is f0110wed by
a massive movement of leucocytes into the gland so that somatic
ce11 counts readily attain 1evels in excess of 100 millio~m1.
The c1inical signe are referab1e to the re1ease of endotoxin
from the bacteria as they are destroyed by the leucocytes and
humoral factors from the blood (SchaIm et al., 1964a,b,c). The
udder may return to secretion of normal mi1k within one to two
weeks. If the initial inflammator,y reaction reduces the c01i
form population wi thin the gland to mi nj maJ numbers but fai1s to
destroy al1 of the organisms, the inflammator,y reaction subsides
and coliform organisms begin to mu1 tiply rapidly again 1eading to
another acute flare-up of mastitis. A chronic infection ~ become
established which is characterized by periodic subacute to acute
flare-ups (Schalm!1!.!.-, 1971)_
Co;ynebacterium bovis is generally be1ieved to be a
common saprop~te in aseptically drawn mi1k associated with bovine
/81
masti tis (Bourland ~ &., 1961). .2.. pyogenes ms\y gain access
to the udder via the streak canal, through defects in the skin
of the teats and udder, or via the blood stream from other foci
of disease. C. pyogenes cause a severe acute mastitis. The
udder secretion in the beginning is serous, later the exudate
becomes purulent, greenll.sh in colour. The exudate bas a
characteristic foul odor due to the presence of an anaerobic
organism, Micrococcus indolicus, cammonly associated with Q.
pyogenes. Both subacute and chronic forma are also seen
(Schalm et al., 1911). --Pseudomonasaeruginosa mas titis ~ be chronic, sul>-
acute, acute, local. or systemic. The chrome cases are chara-
cterized by intermittent flare-ups. The virulence of the organism
de pends both on the tox1genicity and the ability to grow in the
blood serum of the animals (Liu and Mercer, 1963). Since the sera
of many animals contain antibodies to various serologie al t,ypes
of !.. aeruginosa, a gi ven strain can show virulence to a particular
animfÙ when its serum doee not contain sufficient antibodies to
inhibit the growth of the organism and then only if the organism
is capable of producing extracellular toxic substances. Acute
infections ~ become chronic or septicemia m~ develop leading to
localization of the organism in other tissues and death (SchaLm
et al., 1911). --
/82
Infection of the udder with yeast and yeast-like
organisms may occur in! tially via the streak canal and spread
from there to the udder tissue via the lactiferous duct system.
The investigations of Kauker (1955) showed that the vitamin A
content in the host animal is reduced by protracted administration
of large doses of antibiotics. The result of thia deficiency is
epithelial injury to the udder, which facilitates invasion by
the blasto~cetes. The cellular reaction of the disease and
clinical signs of yeast masti tis vary greatly and are net
diagnostic.
"'-.."
MATERIALS AND METHODS
Herds
Two experimental herds Nos. 1 (Ayrshire and 2 (Holstein)
of Macdonald College were selected for study. A total of 68 cows
were studied, 47 COWB (1,818 quartere) from the Holstein herd and
21 cows (840 quarters) from the Ayrshire herd. The study was
spread over a period of one year from January, 1971, to December,
1971, and cows were included in the study as they calved. Each
cow was tested for 10 consecutive periods. The first test was
done between the 3rd and 7th d8iY af'ter calVing and the
successive tests were done at 14-d8iY intervals.
Collection of Samples
IndiVidual quarter fore-milk (IQFM) sampI es were collected
aseptically from each quarter before the afternoon milking. The
udders were washed with disinfectant solution and dried with paper
towels. The tips of the -teats were swabbed w:i th 7~ alcohol and
a squirt or two of milk was discarded. Ten to 20 ml of milk were
collected in sterilized specimen bottles of 15 to 30 ml capaoity.
The samples were then transferred to the laboratory for examination.
preparation of Media
a) Blood Agar:- Fort y g of dehydrated Blood Agar Base
Infusion (Fisher, J-1009-C) were suspended in 1,000 ml of dis
tilled water, mixed thoroughly to obtain an even suspension, and
/84
heated to boiling to dissolve completely. The rehydrated medium
was then sterilized in the autoclave for 15 minutes at 15 lbs
pressure (121 0 C). The medium was allowed to cool to 480 - 450 C
in a water bath. Sterile, defibrinated sheep blood was added at
the rate of 5% and the flask containing the medium rotated gently
untll uniform mixing was accomplished. The medium was then
dispensed into sterilized disposable petri dishes to provide an
even 1/4 to 3/8 inch thick layer of agar. One or two blood agar
plates were tested for sterility by incubating at 370 C for 24
hours and the rest of the plates were stored inverted in the
refrigerator until further use.
b) Esculin-Ferric Citrate Blood Agar:- This medium was
prepared in the same way as the Blood Agar except that Esculin
and 1% Ferric Citrate solution WBS added at the rate of 0.1% and
1.0%, respectively. The pH of the medium was adjusted to 7.2
before sterilization.
c) Coagu!sse Mannitol Agar:- This medium was prepared
by dissol ving 11.75 g of Coagulase Manni. tol Agar Base (BBL) in
250 ml of distilled water. The medium was soaked in cOld,
distilled water for 15 minutes and then sterilized by autocle,vj.ng
at 15 lbs pressure (121 0 C) for 15 minutes. The medium was then
o 0 allowed to cool to 48 -45 C and reconstituted by adding Bacto-
Coagulase Plasma. (Difco, 0286-83) at the rate of 12%. The
medium was gently mixed by rotating the flask and then dis-
pensed into sterilized petri dishes, pouring 10 to 12 ml per
plate. One of the poured plates was tested for sterility by
° incubating at 37 C for 24 hours.
/85
d) Eosin Methylene Blue Agar: - This medium was pre
pared by adding 9 g of Eosin Methylene Blue Agar (EMB) (Difco,
0076-02) to 250 ml of distilled water, mixed until the suspension
was uniform, heated with frequent agitation and boiled for about
one minute, and then sterilized by autoclaving at 15 lbs pressure
(121 0 C) for 15 minutes. The medium was allowed to cool to
o 0 48 -45 C, mixed by rotating the flask and then dispensed into
sterilized petri dishes, pouring 10 to 12 ml per plate. One of
the poured plates was tested for sterility by incubating at 37°C
for 24 hours.
e) Nutrient Broth:- Eight g of dehydrated Bacto Nutrient
Broth (Difco, 0003-01) were rehydrated in 1,000 ml of distilled
water and dispensed into 150 x 15 mm bacteriological tubes at
10 ml per tube. The tubes were capped with metal caps and
sterilized by autoclaving for 15 minutes at 15 lbs pressure (121 0 C).
The sterilized nutrient broth tubes were then stored in the re-
frigerator until use.
~.
1
/86
Prepara tion of Stains
a) Newman' s Stain:- This stain was prepared by adding
54 ml of 95% Ethyl alcohol to 40 ml of tetrachloroethane
(technical), and heated in a water bath to 600 C. The mixture
was added to 1.0 to 1.2 g of Methylene Blue (certified) powder,
and Shaken until the dye was completely dissolved. After cooling,
6 ml of acetic acid (glacial) was added very slowly with con
tinuous shaking. The stain was filtered through coarse filter
paper and stored in a tightly stoppered bottle.
b) Wright-Leishman Stain:-
Solution I:- This solution was prepared by placing
0.6 g of powdered Wright's stain (Fisher, 702003), 5 ml of
glycerine, and 300 ml of absolute methyl alcohol (acetone-free)
into ~ 500 ml pyrex flask. The mixture was heated gently, using
constant agitation until the solution reached a temperature just
below the boiling point. Caution wes observed to avoid ignition
of the Blcohol. The solution was allowed to cool and the heating
process was repeated three or four times. The solution was then
cooled to room temperature and filtered. The solution was stored
in a tightly stoppered brown bottle.
Solution II:- This solution was prepared in the
same manner as Solution l except that 0.6 g ofLeishman stain wes
substituted for the 0.6 g of Wright's stain.
/87
A final solution was prepared by mixing three parts of
Solution l with one part of Solution II.
Experimental Procedure
The IQFM samples were thoroughly mixed and loopful was
streaked onto the blood agar plates immediately after they were
received in the laboratory. The plates were transferred to the
incubator to be incubated at 370 C for 24 to 48 hours. The smears
for cell counts were made, stained and left over to be examined
later. The milk semples were then tested by the California
Mastitis Test (CMT), (Schalm and Noorlander, 1957). The CMT(+)
IQFM samples were centrifuged and smears were prepared from the
sediment. The smears were stained the following day. All the
IQFM samples, whether CMT(+) or CMT(-) were incubated overnight
and transferred to the refrigerator. The semples Showing growth
at 24 to 48 hours after incubation were examined, the results were
recorded and then the semples were discarded. Those showing no
growth after incubation of plates for 48 hours and CMT(+) , were
recultured on fresh blood agar plates after their overnight
incubation.
The blood agar served as a primary medium for isolation
of the causative organism. The colonies present on the blood
agar were classified as streptococci, staphylococci, coliforms
and corynebacteria, on the basis of colony characters. The
/88
doubtful colonies were stained by Grams (BDH) method of staining.
The morphology of the organisms and their reaction to Gram's
stain was observed. The streptococci and staphylococci are Gram
positive and coccoid, whereas, coliforms are Gram-negative and
are rods. Corynebacteria are Gram-positive, diphtheroid organisme.
In some cases, a smear from the nutrient broth culture of the
suspected colony was examined by this technique.
The representative streptococcal colonies, irrespective
of hemolysis, were tested for their "CAMP" reaction and esculin
fermentation, and classified into~. agalactiae and other
streptococcie The staphylococcal colonies were differentiated
as hemolytic or non-hemolytic on the basis of hemolysis of red
blood cells on blood agsr. The representative hemolytic colonies
of staphylococci were plated on coagulase-mannitol agar. On the
basis of mannitol fermentation and coagulase production, they were
classified as Staph.aureus or hemolytic micrococci.
The representative colonies of coliforms were plated on
Eosin Methylene Blue agar. On the basis of sugar fermentation and
appearance of metallic sheen, they were differentiated as E. coli
or Aerobacter.
The analys:es of the serum samples for transferrin types
of the cows were done by Dr. S. S. Malik, as given in Malik et al.
(1970).
/89
Mastitis Criteria
1. Cultural Tests
a) Blood Agar Tests:- Each blood agar plate was divided
into four quarters by marking on the bottom of the petri dish with
a wax pencil. One plate was ueed for each cow. The milk samples
to be cultured were mixed with the Vortex Jr. Mîxer (Scientific
Industries Inc., Springfield, Maas., U.S.A.) to disperse the cre am
evenly and inverted four to five times immediately before ino
culation. The inoculation loop (internal diameter approximately
4 mm to deliver approximately 0.01 ml of milk) was sterilized in
an. open flame of a Bunsen burner and âLlowed to cool. A loopful
of the mixed milk sample was streaked onto a quarter of the blood
agar plate. The four individualquarter milk semples from each
cow were streaked on the four quartere of a blood agar plate,
clockwise, in the order, right front (RF or A), right hind (RH or
B), left hind (LH or C) and left front (LF or n). The inoculated
plates were allowed to dry, inverted and incubated for 24 houre
o at 37 C. The plates were then examined for colonial characteristics
and hemolysis. If no coloniee were present, the plates were in-
cubated for an additional 24 hours.
Streptococci appeared as small colonies having varying
degreee of hemolysie - alpha, beta or gamma. Staphylococcal
colonies were larger than streptococci and were either hemolytic
or non-hemolytic. Colonies of coliforms were circular, convex,
smooth, moist, mucoid, somewhat translucent and tended to
coalesce if incubation was prolonged. Corynebacterial colonies
/90
were pinpointed, resembled streptococcal colonies, were hemolytic
or non-hemolytic and appeared mostly after 48 hours of incubation.
b) Christie Atkins and Munch-Petersen (CAMP) Test:- Tr~s
test was performed as described by Christie ~ al. (1944) on the
esculin-ferric-ci trate-blood agar. A culture of Staph. aureus
capable of producing a large zone of beta hemolysis was streaked
across an esculin-ferric-citrate-blood agar plate with a wire loop
using an aseptic technique. The streptococci were streaked at
right angles to it on both sides. The inoculated plates were then
o incubated at 37 C for 18 to 24 hours. The typical reaction
produced by CAMP positive streptococci was a semi-circular area
of complete hemolysis in the partial zone of hemolysis produced
by the staphylococcal culture. Streptococci were classified as
~. agalactiae if they were CAMP positive and esculin negative,
if not, they were classed as other streptococci.
c) COagulase~annitol Agar Test:- This test was performed
in a manner similar to that given by the BBL Manusl ("i968). A
loopful of a hemolytic staphylococcal colo~ to be tested was
spotted using one plate for five to seven isolates. The plates were
then incubated for 18 to 24 hours and the isolates examined for
mannitol fermentation and coagulase production. The mannitol
/91
fermentation was indicated by change in colour of the medium
from red to yellow, whereas, the coagulase production was
accompanied by a white halo around the colony. The isolates
producing coagulase were classed as Staph. aureus, if not, they
were classed as micrococci.
d) Eosin Methylene Blue Agar Test:- The coliform colonies
in question were streaked on EMS agar in a zig-zag manner. The
o inoculated plates were incubated at 37 C for 12 to 18 hours, and
examined for appearance and persistance of metallic sheen. If the
metallic sheen appeared and persisted for 48 hours, it was classi-
fied as~. coli. if the metallic sheen did not appear or did not
persist, it was classified as Aerobacter.
2. California Mastitis Test (CMT)
The CMT, as developed by Schalm and Noorlander (1957) was
conducted on IQF.M samples immediately after they were transferred
to the laboratory. A plastic paddle with four receptacle cups
marked A, B, C and D, and a CMT solution (Pi tman Moore) containing
balanced proportions of bromocresol purple and alkyl-aryl sodium
sulfonate in aqueous solution, were used for the test. Two to
3 ml of milk from each of the four quart ers was transferred into
the four cups of the paddle in such a manner that milk from the
right front (RF) quarter was in cup A; from the right hind (RH)
in cup B; from the left hind (LH) in cup C and that from the left
/92
front (LF) quarter was in cup D. An equal amount of CMT solution
was added to the milk samples in the cups by squ1rting it from a
polyethylene waeh bottle. The paddle was then gently rotated to
completely mix the sample .and the reagent. The reaction was graded
while the paddle was being rotated. Both the colour and the
viscosity of the sample-reagent mixture was obeerved. A deep blue
or purple colour indicated excessive alkalinity and a yellow colour
indicated abnormal acidity. The precipitate or gel formation wae
indicative of the inflammation of the udder. The reactions were
graded as suggested by Schalm and Noorlander (1957) into the
following grades:
Negative (-)
Trace (T)
Weak Po si tive (1+)
Distinct Positive (2+)
Strong Positive (3+)
- The mixture remained liquid wi th no evidence of formation of a precipitate. 0 to 200,000 cells/ml of milk; 0 to 25 percent PMN.
- A slight precipitate which tended to disappear with continued movement of the paddle. 150,000 to 500,000 cells/ml of milk; 30 to 40 percent EMN.
- A distinct precipitate but no tendency toward gel formation. 400,000 to 1,500,000 cells/ml of milk; 40 to 60 percent PMN.
- The mixture thickened immediately wi th a sugges,tion of gel formation. As the mixture was swirled, i t tended to move in towards the center, leaving the bottom of the outer edge of the cup exposed. When the motion was stopped, the mixture levelled out again, covering the bottom of the cup. 800,000 to 5,000,000 cells/ml of milk; 60 to 70 percent P.MN.
- A distinct gel formed which tended to adhere to the bottom of the paddle and during swirling, a distinct central peak was formed. Over 5,000,000 cella/ml of milk; 70 to 80 percent PMN.
The two grades based on the eolour change, namely,
alkaline milk (+) and acid milk: (Y), were seldom used.
3. Total Somatie Cell Count (TSCC)
/93
The Direct Microscopie Somatic Cell Count (DMSCC) method
of Prescott and Breed (1910) was used for estimating the TSCC per
ml of milk with modifications (Brazis, 1965).
The IQliM semples were mixed thoroughly by means of a
Vortex Jr. mixer so as to disperse the leucocytes and cream
throughout the specimen. The samples were then allowed to stand
at room temperature until the froth on the milk surface had dis
persed. Immediately before sampling, the sample bottle was
inverted four to five times slowly to thoroughly remix the milk.
A portion of milk (0.01 ml) was drawn from the sample wi th
a standard inoculating loop (4 mm inside diameter, FiSher Scientific
Co., Montreal) and delivered onto a cleaned microscope slide and
spread evenly over a one square centimeter area using a Breed
Guide Plate (7 .. 5 x 5 cm with 15 1 sq.cm aresa). The smear was dried
at approximately 400 C over a mieroslide warming table (Eberach
Corporation, Michigan, U.S.A.).. The dried smear was dipped in the
Newman's stein for one minute then drained and dried thoroughly
before being dipped in water to remove exeess stein. The atained
slide was drained and air dried.
/94
Four quarter samples from the same cow were smeared on
one slide in the order, RF, RH, LH, LF. The standard inoculating
loop was sterilized over a Bunsen burner flame between samples.
The smears were prepared on the same dey the samples were received
in the laboratory.
The stained milk smears were examined under an oil
immersion lens running completely aoross the smear starting about
midway on one side and two to three fields from the edge. The cells
were differentiated as epithelial cells and leucocytes, and counted
separately using a laboratory counter (Clay Adams Inc., New York).
Epithelial cells and leucocytes were added to get the TSCC. After
counting 10 fields, if the average number of TSCC per field was
less than six, TSCC in 40 or more fields were counted, otherwise
TSCC/ml was oalculated as follows:
TSCC/ml = No. of cells (epithelial + leucocytes) in all fields
No. of fields examined xMF
The microscopie factor (MF) was calculated by multiplying
the number of sq mm in a sq cm with the number of 0.01 ml portions
in 1.0 ml of milk and dividing the product by the area of the micro
scopie field of the microscope used which was 0.08 mm, the MF was
found to be 500,000.
4. Differential Cell Count
a) Preparation of Milk Samples:- Ten ml of IQEM showing
CMT(+) was transferred into a conical graduated centrifuge tube.
/95
A drop or two of 40 percent formaldebyde was added. The semple
was allowed to settle at room temperature. The sample was then
centrifuged at 900 r.p.m. for 10 minutes using an International.
Centrifuge, Univers al Model UV (International. Equipment Co.,
Boston). The top fat l~er and a small portion of the milk were
removed by suction through a water vacuum tap. Pat adhering to the
exposed inner sides of the tube was wiped with a cotton swab and
suction was re-applied to remove all but 0.2 to 0.5 ml of the
remaining milk depending upon the GMT grades. The sediment in
the remaining milk was gently mixed, first with a smooth-tipped
glass rod and then by tapping the end of the tube.
b) Preparation and Staining of Milk Smears:- Approxi
mately 0.01 ml portion of the suspended sediment was transferred
to a clean microscope slide by means of s pasteur pipette. The
drop of suspended sediment was spread evenly in a smear 1 cm wide
and 2.5 to 3.5 cm long lJJ3T means of a thin wire 1 cm long bent st
right angles. o The smear was dried at approximately 40 C over a
microslide ~ table. The dried smears were stored to be
stained for the next d~. The dried smears were fixed and defatted
in acetic aCid/tetrachloroethane/etlwl aJ..cohol mixture (4 ml of
glacial acetic acid; 44 ml of tetrachloroethane and 52 ml of
etlwl alcohol) for 2 minutes. The smears were then rinsed in 95%
alcohol, shaken to remove excess alcohol and allowed to dry. This
defatted and fixed smear was placed on a stednjng rack, and stained
~.
\ 1
/96
with 8 to 10 drops of Wright - Leishman stain to cover the emear.
The undiluted stain was allowed to react for 1 'minute, then an
equa! amount of distilled water (8 to 10 drops) was added and
mixed thoroughly by blowing on the slide until a metallic sheen
appeared on the surface of the mixture. The diluted stain was
allowed to react for 8 to 10 minutes and then poured off. The
smear was then washed in distilled water. Care was taken to
avoid washing off the smears, to accomplish this, the distilled
water was squirted from a polyethylene bottle onto the top end
of the slanted slide, so that the water flowed slowly onto the
surface of the emear. The slide was then drained and dried.
c) Interpretation:- The smear was examined under oil
immersion lens and 100 cells were counted across the length of the
smear in the center. If required, additional rows above or below
were also examined. Three types of cells - polymorphs, lympho-
cytes and eosinophils were counted.
d) Appearance of Cell ~pes:-
i) Neutrophils:- They were round, eliptical or irregular
in outline. The nucleus stained moderate to deep blue and was
characteristically lobulated, fine filaments connecting the lobes
were evident in some cells. The cytoplasm was clear and contained
few to Many distinct or indistinct small, pink granules giving
the cytoplasm a pinkish hue (Photograph 1). A variable proportion
of cells had one or more fat vacuoles in the cytoplasm.
">c-, 1
/97
:,.'~t. ,'f. . ..... /
Photograph 1. Showing Neutrophila.
Photograph 2. N = Neutrophila; L = Lymphocyte; E = Epithelial Cella.
Photograph 1. Showing Neutrophils.
" . (.
/97
, ...... " "
- ,r··;, "
,iF
Photograph 2. N = Neutrophils; L = Lymphocyte; E = Epithelial Cells.
/98
ii) ~ymphocytes:- These cells were generally spherical
in shape and ranged in size from small to large. The cytopl.asm
stained light to dark blue. The cytoplasm was sparse in small
lymphocytes and the nucleus al.most filled the cell and fonned a
narrow to broad zone in large lymphocytes (Photograph 2).
1ii) Eosinophils:- These cells were round or irregular
in outline. The cytoplasm was clear and contained many discrete
round acidophilic granules. The nucleus might be spherical or
lobulated (Photographs 3 and 4).
iv) Epithelial Oells:- These cells were large, in
distinct cells approximately 10 to 20.Jl.l. in diameter and were
generally of irregul.ar shape. They were usually without a well
defined nucleus, which, if it was apparent, was stained blue
(Photograph 2).
5. Statistical Analysis of the Dat~
Blood Serum Transferrin Polymorphism
The gene frequencies and the expected genotype frequencies
were calculated as suggested by AShton (1958). Since there were
four codominant Tf alleles in the population, calculations were
modified accordingly (Malik et ~., 1970). The gene frequencies
for the four genes at the Tf locus were calculated as follows:
/99
Photograph 3. L = Lymphocytes; N = Neutrophile; Eo = Eoeinoph11.
Photograph 4. L = ~phocytee; N = Neutrophile; Eo = Eosinophil.
/99
Photograph 3. L = Lymphocytes; N = Neutrophils; Eo = Eosinophil.
i.
• '.
Photograph 4. L = Lymphocytes; N = Neutrophils; Eo = Eosinophil.
/100
a = 2(TfAA) + TfAD1 + TfAD2 + TfAE /2N
d1 = TfAD1 + 2 (TfD1D1)0+ TfD1D2 + TfD1E /2N
d2 = TfAD2 + TfD1D2 +2(TfD2D2) + TfD2E /2N
e = TfAE + TfD1E + TfD2E + 2(TfEE) /2N
when TfAA, TfAD1, etc., represent the numbers of animaIs of these
phenotypes observed in a herd, breed or a population under study;
N is the total. number of an:i.maJ.s in the population under
study; and
a, d1 , d2 and e are the frequencies of the genes T~,
TfD1, TfD2 and TfE, respectively.
The expected genotype frequencies were calculated from
gene frequenc~es as given below:
Genotype
Homozygotes
TfAA
TfD1D1
TfD2D2
TfEE
Heterozygotes
TfAD1
Tf.AD2
TfAE
TfD1D2
TfD1E
TfD2E
Expected Frequency
(a)2.N
(d1)2. N
(d2)2. N
(e)2.N
2(a.d1)·N
2(a.d2) .N
2(a.c) .N
2(d1·d2)·N
°2(d1 .e) .N
2(d2 ·e).N
Note: The mUltiplier, 2, in heterozygotes was ueed because the expected number of heterozygotes in genotype x genotype matings in a four-codominant-allele theory is twice the number of homozygotes.
/101
The difference between the observed and expected genotype
frequencies were tested for their significance by the chi-square
test (Steel and Torrie, 1960) as follows:
2 2 ~ (0 - E)
~ n-2 = E where
n was the number of genotypes in the population
o and E were the observed and expected frequencies of a genotype,
and ~ indicates sum of all (0 - E)2 values for n number of genoE
types ..
For the purpose of statistical analysis, the five CMT
grades were given numerical scores for negative (-) 0; (Trace) 1;
(1+) 2; (2+) 3; and (3+) as 4.. The total numerical scores of each
cow for each test were tranaformed as ( vi x + i) for the purpose
of analysis of variance, x being the score before transformation ..
The linear addi ti ve model was used to analyze the numerical
scores:
where Yijklmn is the nth observation of the kth cow with jth geno
type in ith breed, and was tested in the lth month at the mth
stage of lactation
}J.. = is the population Mean
Bi = ith breed
Gij = jth genotype in ith breed
Cijk = kth cow in jth genotype in the ith breed
Ml = lth month of the year
Sm = mth stage of lactation
/102
e1jklmn = random error nor.mally and independently dis-
tributed with mean zero and variance
11) Duncan's New Multiple Range Test
The comparisons between means were made by using Duncan's
New Multiple Range Test (Steel and Torrie, 1960). The standard
error, Sx, for compar1ng the means was obtained as follows:
Sx = JRrror Mean Square/no·
where, no 1e the representative number of replications per
group, was calculated
= .1 n:T
as given by Snedecor :E n":2
(~ni- ---=.), ~n
n = the number of gr-oups
(1959) as follows;
~ = the number of observations in each group
1ii) Cal1fornia Mastitis Test Index (CMTI)
The GMTI of a cow was calculated by adding numerical CMT
scores of the four quarters for 10 tests and d1Vi.ding by 10.
R.E5ULTS
1. Incidence of Sub-C1inical Masti tis
The California Mastitis Test (GMT) and the Direct Micro
scopie Somatic Ce11 Count (DMSCC)were used as screening tests
for subc1inical mastitis on 2,658 individual quarter fore-m1lk
(IQFM) samp1es from 68 cows. The results of these tests are given
in Tables l and II.
The incidence of CMT positive cows and quart ers was
64.32% and 31.53%, respective1y. The percentage of positive cows
varied from 53.73% in the 4th test to 85.29% in the 1st test.
The variation in positive quarters was 23.99% in the 3rd test to
55.35% in the 1st test. The distribution of CMT grades was
57.41% for negative; 11.06% for Trace; 16.25% for 1+; 5.72% for
2+ and 9.56% for 3+ (Table 1).
In Table II, the mean total somatic ce11 count (TSCC) in
fi ve GMT grades are shown. The mean TSCC increased as the GMT
grade increased (Figure 1). The numbers of quarters having more
than o. 5 x 106 TSCC/ml and 1ess than 0.5 x 106 TSCC/ml are shown
in Table III. In Table IV the mean GMT score ( "x + i) and
1east square estimates (L.S.E.) for 10 tests (stage of lactation)
are shown. The mean and L.S.E. was found highest (2.62) in the
1st test as against the mean 1.90. The differences between the
means were found significant at the P < 0.01 1eve1 (see Appendix).
Table l. Resulte of California Mastitis Test (CMT) on IndiVidual Quarter Milk Samples for All 10 Tests
Test No. of GMT Grades % of Positive** % of Poaitive*** No. Cows Quartera T 1+ 2+ 3+ Quarters Cows 1 68 271 80 41 72 14 64 55.35 85.29 2 68 271 160 43 36 12 20 25.09 55.88
3 68 271 179 27 34 12 19 23.99 54.41
4 67 267 172 28 31 13 23 25.09 53.73
5 67 267 166 28 35 13 25 27.34 64.18
6 66 263 167 23 40 15 18 27.76 63.64
7 66 263 159 27 35 19 23 29.28 60.60
8 66 263 156 20 49 17 21 33.08 63.64
9 66 263 143 26 50 20 24 35.74 72.73 10 65 259 144 31 50 17 17 32.43 69.23
Total 667* 2658* 1526 294 432 152 254 838.00 429.00 Percent 57.41 11.06 16.25 5.72 9.56 31.53 64.32
* The two totale indicate the number of tests performed on cowa and quarters, respectively.
** CMT(+) quartera 1+ and over.
*** Cows showing positive GMT in one or more quarters at one or more tests.
"-~ o ~
.. J'
Table II. California Mastitis Test (CMT) on Individual Quarter Fore-Milk (IQFM) Samplea and Mean Total Somatic Cell COlUlt
/105
GMT No. of TSCC/ml o:f'6 Av. TSCC/ml g:f' Grade Quartera ~ Milk ~1x10 l Milk ~1x10 l
1526 57.41 74.36 0.05
T 294 11.06 75.64 0.26
1+ 432 16.25 365.10 0.85
2+ 152 5.72 424.82 2.79
3+ 254 9.56 1968.45 7.75
Total 2658 2908.37 Average 1.09
\0 o
/106
10·0
8·0
0.0 L..======::;:::::.... ___ ..... _____ ..... _____ ..-_ T 1 2 3
GMT GRADES
Figure 1. Relationsh:i.p of CMT Grades and Mean TSCC.
Table III. 6 6 Number and Percent of Quartera HaVing TSqC > 9_.5xl0 ~e~ II1l_~d __ < 0.5x10 per &
No. of Quartera with No. of Quartera with
Teat Total No. of CMT(+) TSCC 0.5x106/ ml TSCC 0.5x106/ml
No. Quartera Teated Quartera No. % No. % 1 271 150 139 92.67 132 100.00
2 271 68 63 92.65 208 100.00
3 271 65 57 87.69 214 100.00
4 267 67 62 92.54 205 100.00
5 267 73 67 91.78 200 100.00
6 263 73 63 86.30 200 100.00
7 263 77 72 93.51 191 100.00
8 263 87 78 89.66 185 100.00
9 263 94 85 90.43 178 100.00
10 259 84 72 85.71 187 100.00
Total 2658 838* 758 90.45 1820** 100.00
* GMT 1+, 2+ and 3+.
** GMT (-) and (T).
~ o ~
Table IV. Stage of Lactation, Mean GMT Scores (../x + i) and Leest Square Estimates (L.S.E.)
Test No. of Cows No. Tested L.S.E. Mean
1 68 +0.72 2.62d
2 68 -0.13 1.77ab
3 68 -0.22 1.66a
4 67 -0.15 1.7Sab
5 67 -0.06 1.84abc
6 66 -0.11 1.79abc
7 66 -0.03 1.87bc
8 66 -0.02 1.aabc
9 66 +0.07 1.97c
10 65 -0.03 1.87bc
Total 68 1.90
a,b,c,d = Means with the same superscript are not significantly different as tested by Duncan's New Multiple Range Test (p <0.05).
/108
/109
2. Incidence and Type of Infection
The incidence and type of infection are given in Table V.
The incidence of ~. agalactiae was highest in the 6th test and
lowest in the lst test. The percentage of Staph. aureus infection
was highest in the 3rd test and lowest in the 1st test. The
percentage of non-hemolytic staphylococci affected quarters were
more in the 1 st test and least in the 4th test. The mixed in
fection due to more than one organism having less than 10 colonies
was highest in the 8th test (Figure 2). The percentage incidence
of coliforms was highest in the lst test and zero in the 7th test.
The overall incidence of infection due to various organisms
was streptococci 12.27%; hemolytic staphylococci 5.60%; non
hemolytic staphylococci 17.68%; coliforms 1.02%; and mixed infection
34.95%. The percentage of quart ers showing no growth on blood agar
plates was 28.48 (Figure 3).
The distribution of the type of infective organisms in the
five CMT grades are given in Table VI. The percentage of infected
quart ers in negative, Trace, 1+, 2+ and 3+ grades was 62071; 76.87;
83.56; 90.79 and 86.61 percent, respectively. The overall infection
was 71.52 percent in all the quarters tested.
The type of organisms and their relation to the CMT
reaction are given in Table VII, and Figure 4. Out of 1,901
infected quarters, 37.8~ were CMT(+). The percentage of CMT(+)
Table V. Incidence and TYpe of Infection Teatwise
Test No. of Strepag Other Strep Staph. aureus % of Quartera No. Quartera Infected*
1 271 8.49 0.37 2.21 2.60 24.35 2.21 32.47 27.30 72.70
2 271 10.70 0.37 2.95 2.21 21.10 1.11 33.58 27.68 72.32
3 271 10.70 0.37 6.27 2.58 14.76 0.74 33.58 31.00 69.00
4 267 11.61 0.37 4.12 2.62 13.11 0.37 34.08 33.72 66.28
5 267 13.11 0.37 3.37 0.37 16.10 0.75 35.21 30.72 69.28
6 263 14.07 0.38 2.28 2.28 13.69 0.76 36.12 30.42 69.58
7 263 13.31 0.00 3.80 1.90 15.59 0.00 34.98 30.42 69.58
8 263 12.55 0.00 3.04 1.52 20.15 1.14 38.41 23.19 76.81
9 263 11.79 0.76 2.66 3.80 19.40 1.14 38.02 22.43 77.57
10 259 13.13 0.39 3.47 1.93 18.15 1.93 33.20 27 .. 80 72.20
Total Quartera 2658 317 9 91 58 470 27 929 756 1901
% Quartera Infected 11.93 0.34 3.42 2.18 17.68 1.02 34.95 28.48 71.52
* Quarter samplea showing baterial growth on blood agar.
Strepag = Streptococci agalactiae Mixed = Mixed infection due to more than one Other Strep = Streptococci other than Strepag type of organiam having 10 colonies Staph. aureus = Staphylococcua aureue per type, including cor,ynebacteria. Micrococci :: Hemolytic ataphylococci coagulase negative NG :: No Growth ![. Staph = Non-hemolytic ataphylococci Coli :: Col1forma
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/112
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/113
Table VI. Distribution of the Type of Infective Organisms in All CMT Grades
Type of No. of Quartera in CMT Grades Total No. of Organism T 1+ 2+ 3+ Quartera
Strepag 8 13 77 91 128 317
Other strep 1 1 5 1 1 9
Staph.. aureus 35 8 26 8 14 91
H. staph 45 5 4 3 1 58
NH. Staph 285 71 80 11 23 470
Col.iforms 2 2 5 2 16 27
Mixed infection 580 126 164 22 37 929
No growth 569 68 71 14 34 757
Total 1526 294 432 152 254 2658
Total inf'ected 957 226 361 138 220 1901
% infected 62.71 76.87 83.56 90.79 86.61 71.52
.c...
/114
Table VII. TlEe of Organisme and Their Relation to the CMT Reaction
Type of GMT Positive* GMT Negative** Organism Quarters No. % Quarters No. % strepag 296 93.38 21 6.62
Other strep 7 77.78 2 22.22
Staph. aureuB 48 52.75 43 47.25
Micrococci 8 13.80 50 86.20
NH. Staph 114 24.26 356 75.74
Coliforms 23 85.19 4 14.81
Mixed infection 223 24.00 706 76.00
No growth 119 15.72 638 84.28
Total 838 31.54 1820 68.46
Total Infected 719 37.82 1182 62.18
* GMT 1 + and over.
** GMT (-) and (T).
ID J.4 Q) .p
J ~ 0
i ()
J.4 Q)
l1t
'0
---80-1 - ------- --- -.- -- --60~
- -- -- -- -- -- -- -- -- -- -- -- -- -- -- ---- --- -- --- --- -- -40 -- -- -- --- ---- -- --. -- -- ---; -20 -1 - .- .- -= -- --- -
o Strepag. Other Strep· Staph'Aureus Micrococci NH.Staph.
TYPE OF ORGANISM
Figure 4. Percentage of Infected Quartera Showing Reaction to CMT.
== CMT == Il CMT +
-------------------------------.----
----------------== ----------------------------------
~ .... \11
J
quart ers amongst quart ers infected with~. agalactiae, Staph.
aureus and coliforms was 93.38; 52.75 and 85.19, respectively.
/116
The relation of ~. agalactiae infection to the GMT
reaction is shown in Table VIII. The reaction varied from 100%
in the 10th test to 86.21% in the 2nd test. The Mean California
Mastitis Test Indices (GMTI) of the~. agalactiae infection are
given in Table IXa. The Mean CMTI's of the testwiee infection
varied from 2.69 in the 2nd teet to 3.53 in the 1et test. The
differences in the meane were not significant (P~0.05) (Table
IXb).
The TSCC and differential count (DC) in CMT(+) quarters
are shown in Table X. The average TSCC and epithelial cells were
higheet in the 1et test (Figure 5). The highest percentage of
neutrophile, lymphocytes and eosinophile were found the 4th, 1st
and 10th teets, respectively. Overall, 3.29 million TSCC/ml,
15.5% epithelial celle, 44.98% neutrophils, 39.21% lymphocytes
and 0.31% eoeinophile. The neutrophil and lymphocyte (N:L) ratio
is Shown in Table XI. The ratio varied from 1:1.62 in the 1st
test to 1:0.47 in the 10th test.
The TSCC and DC in CMT(+) quart ers infected with various
organisme are ehown in Table XII and Figure 6. The average TSCC
wae higheet in quarters infected with coliforme (10.15 million/ml)
followed by those infected with~. agalactiae (4.13 million/ml)
/117
Table VIII. The CMT Reaction of StrePag. Infected Quarters
Test No. of Quartera No. Inf'ected Poaitive* % Negative** %
1 23 22 95.65 1 4.35 2 29 25 86.21 4 13.79
3 29 27 93.10 2 6.90
4 31 29 93.55 2 6.45
5 35 32 91.43 3 8.57
6 37 34 91.89 3 8" 11 7 35 32 91.43 3 8.57
8 33 32 96.97 1 3.03
9 31 29 93.55 2 6.45
10 34 34 100.00 0 0.00
Total. 317 296 93.38 21 6.62
* CMT grade 1 + and over
** CMT (-) and (T).
/118
Table Ixa. Strep:t. Infection and California Mastitis Test Index CMTI)
Test No. of No. Quarters
1 23
2 29
3 29
4 31
5 35 6 37
7 35 8 33
9 31 10 34
Total 317
Table IXb. Analysis of Variance
Source of Variation
Between test
Within test
Total
NS = Not Significant.
d.t
9
308
317
Total CMTI 81
78 88
97 104 106
100
106
97 98
955
S.S.
13.75
330.20
343.95
M.S.
1.53
1.07
Mean CMTI
3.53 2.69
3.04
3.13 2.97 2.86
2.86
3.21
3.13 2.88
3.01
F.cal..
Table X. Total Somatic Cell Count and Differential Count in CMT{+) Quartera Teatwiae
Av. Leucoc;!tea
Teet No. of CMT{+) Av. TSCC/ml Av. ~ithelial Celle Neutro;ehils Lymehoc;y:tee Eoelino;ehile 6 6 6 No. (lx106) No. Quartera (lxl06) No. (lxlO ) % No. (lxl0 ) % No. ( 1x10 ) % %
1 150 4.94 0.80 16.19 1.58 31.98 2.55 51.62 0.01 0.21
2 68 3.27 0.45 13.76 1.52 46.48 1 .. 29 39.45 0.01 0.31
3 65 3.15 0.50 15.87 1.34 42.54 1.29 40.95 0.02 0.64
4 67 3.44 0.52 15.12 1.94 56.39 0 .. 96 27.91 0.02 0.58
5 73 3.01 0.40 13.29 1.62 53.82 0.98 32.56 0.01 0.33
6 73 2.39 0.42 17.57 1.13 47.28 0.83 34.73 0.01 0.42
7 77 3.34 0.39 11.68 1.43 42.81 1.38 41.32 0.14 4.19
8 87 2.30 0.37 16.09 1.12 48.70 0.80 34.78 0.01 0.43
9 94 2.84 0.42 14.79 1.43 50.35 0.98 34.51 0.01 0.35 10 84 2.86 0.28 9.79 1.67 58.39 0.78 27.27 0.13 4.55
Total 838
Average 3.29 0.51 15.50 1.48 44.98 1.29 39.21 0.01 0.31
~ .... \0
J
1.0 o ,...
....
5·0
3·0
1·0
0·0
/120
TSCC
• \ • \ • \ .~ . . ..
• •• e.
\ •• •••• • •• NeutroPhils •••••• ~. ~ e. • •••
........
...... ..e e.. ... . ... \ . ••.• • .~ •. e. • •• __ a, e.._ ,.. . .... . .. -.. ........ • .... e. .-
~ . /' . .-.,._........~. '. ..~ ' .. , ~ .. ~ .. ,
... LYmphocytes
.......... ----_.--------- ............ -------- .. _ ........ -- -........ -. -- --_.- .. -. -" .. -.. EPi.Cells
1 2 3 4 5 6 7 8 9 1·0
TFST NUMBER
Figure 5. TSCC and DC in CMT(+) Quartera Teatnae.
/121
Table XI. Neutrophil/Lymphocyte Ratios in all Tests
Test No. of Quarters Neutrophil/Lymphocyte No. Infected Ratio
1 150 1:1.62
2 68 1:0.85
3 65 1 :0.96
4 67 1:0.50
5 73 1 :0.61
6 73 1:0.74
7 77 1 :0.94
8 87 1 :0.72
9 94 1: 0.69
10 84 1:0.47
Table XII. Total Somat1c Cell Count and Different1al Count in CMT(+) Quartera in Different Organiams
No.. of CMT~+)Quartera Av. TSCC/ml Av. ;§e1. Cella
No .. (1x106) % Organism Quartera No .. ~ (lx106)
Strepag 317 296 93.38 4.13 0.63 15.25
Other Strep 9 7 77.78 2.21 0.38 17 .. 19
Staè. aureus 91 48 52.75 4 .. 03 0 .. 55 13 .. 65
M1crococci 58 8 13.79 2.32 0.50 21 .. 55
NH Staph 470 114 24.26 2.19 0.42 19.18
Coliforms 27 23 85.19 10.15 0 .. 61 6 .. 01
Mixed 929 223 24.00 1.88 0.34 18.09
No growth 757 119 15.72 3 .. 41 0.54 15.84
Total 2658 838
31.53 3.29 0.51 15.50
(Key to organisme - see Ta,ble V).
Av. Leucocltea NeutroEhila IurmEhocltes Eoaino]2h1ls No.(1x106) % No. (1x106) % No. (lX106) %
2.06 49 .. 88 1.42
0.97 43.89 0.86
1.95 48.38 1.51
0.89 38.36 0.93
0.51 23.29 1.26
7.01 69.06 2 .. 51
0.60 31.91 0.93
1.42 41.64 1.44
1.48 44.98 1.29
34.38 0.02
38.92 0 .. 00
37.47 0 .. 02
40.09 0.00
57 .. 53 0.00
24 .. 73 0.02
49.47 0.01
42.23 0.01
39.21 0.01
0.49
0 .. 00
0.50 0.00
0.00
0 .. 20
0.53
0.29
0.31
~ 1\) 1\)
~ TSCC
s.0 - Neutrophi Is
~:§:~! EPi. Cell s :::.:.:::~
Il LYmPhocytes
4·0
~ 3'oi 1 1 1 Q)
~~ 2oO~ 1. 1 1.. 1 1 ~ 1 1·0
0.0 ••
TYPE OF ORGANISM
Figure 6. TSOO and DO in OMT(+) Quartera in Various Organisme.
10·0
S·O
.. 1 1 ~6'0 ~m Il ~4.oi
......... .. 1\) \.).l
..f
/124
and Staph. aureue (4.03 million/ml). The average percentage of
neutrophile was also highest in quarters infected with coliforms.
The percentage of lymphocytes was highest in quarters infected
with non-hemolytic staphylococci. The percentage of eosinophils
was highest in mixed infection.
The N:L ratios for various organisms are shown in Table
XIII. The ratio was highest in quarters infected with non-hemolytic
staphylococci (1:2.44) and lowest in those infected with coliforms
(1:0.36).
The mean TSCC and De in three different CMT(+) grades are
shown in Table XIV. The mean TSCC, and the percentage of epi
thelial cells, neutrophils, lymphocytes and eosinophils increased
as the CMT grades were increased. The mean TSCC, epithelial cells,
neutrophils, lymphocytes were plotted on log paper against GMT
grades (Figure 7). The plots were found to be linear.
3. lnfluence of Age (Lactation Number) on Incidence of Masti1l.s
The influence of lactation number and CMTI for different
lactations wi th standard error are gi ven in Table XVa. "The mean
GMTI varied from 1.14 in the 1st lactation to 7.04 in the 7th or
over lactation group. The differences between the me ans were
observed to be highly significant (p ~ 0.01) (Table XVb). The
mean CMTI increased from the 1st lactation to the 4th lactation,
there was a decrease in the 5th lactation and there was an increase
from the 5th to the 7th lactation (Figure 8).
/125
Table XIII. Neutrophil/Lymphocyte Ratios with Various Organisme
No. of Quarters Neutrophil!Lymphocyte Organism Inf'ected Ratio
Strepag 296 1 :0.69
Other Strep 7 1 :0.89
Staph. aureus 48 1 :0.77
Mi.crococci 8 1:1.04
NB Staph 114 1: 2.44
Coliforms 23 1 :0.36
Mixed inf'ection 223 1:1.56
No growth 119 1:1.01
Table XIV. Total Somatio CeU Count and Differentia! Count in CMT (1+), (2+) and (3+) Grades
Av. Leucocytes CMT Total No. of Av. TSCC/ml Av. Epithe~ial Cells Neutrop~ls LocmPhoc~tes EbSinOp~lS Grade Quartera (1x106) No.(1x10 ) % No.(lx10 ) % No.(1x10) % No.(lx10) %
1+
2+
3+
432
152
254
Total 838
Average
0.85
2.79
7.75
3.29
0.17
0.52
1.07
0.51
20.00
18.63
13.81
15.50
0.34
1.23
3.56
1.48
40.00
44.09
45.94
44.98
0.34
1.03
3.09
1.29
40.00
36.92
39.87
39.21
0.00
0.01
0.03
0.01
0.00
0.36
0.38
0.31
~ 1\) (TI
~ CD r-I r-I Q) 0
\0 0 or-
H or-
10·0 9·0
7·0
5·0
3·0
2·0
1·0 0.9 0·8 0·1
0'6
0.5
0·4
0.3
0.2
0.1
/127
NeutroPhi/s
CMT 1 + CMT2+ CMT3+
Figure 7. TSCC and DC in CMT (1+), (2+) and (3+) Gradee •
..
/128
Table XVa. Lactation Number and California Mastitis Test (CMT) Index
Lactation No. of Mean CMT Standard Error No. Cows Index of the Mean
l 10 1.14 0.81
II 12 3.14 0.74
III 9 4.68 0.85
IV 7 5.16 0.97
V 13 3.32 0.71
VI 7 4.64 0.97
VII & over 10 7.04 0.81
Total 68 4.02 0.36
Table XVb. Analysis of Variance
Source of Variation d.f. S.S. M.S. F. cal.
Between lactation 6 205.38 34.23 5.22**
Within lactation 61 400.39 6.56
Total 67 605.77
** Highly significant (P<0.01).
/129
S·O
6·0
2·0
0.0 ------~------,_------r_----~------,_----~~----_r-------1 2 345
LACTATION NUMBER
6 7 OR OVER
Figure 8. Influence of Lactation Humber (Age of Cow) on Mean CMTI.
/130
The percentage of incidence and type of infection, as
related to lactation, is shown in Table XVI. On a quarter basia,
51.63% were infected in the lat lactation compared to 85.00% in
the 4th lactation. The percentage of ~. agalactiae infection
waa highest in the 7th lactation and zero in the lst lactation.
In mixed infections, a peak was observed in the 6th lactation
(Figure 9). The coliform infection was highest in the 7th
lactation.
The TSee and De in CMT(+) quart ers are shown in Table
XVII and Figure 10. The average TSee was found highest (4.03
million/ml) in the 7th and over lactation cows and lowest (1.02
million/ml) in the lst lactation cows. The percentage of
neutrophils, lymphocytes and eosinophila were higheat in the
7th, let and 2nd lactation cowa, reapectively. The average TSee
increased as the lactation number increased except in the 4th and
5th lactation. The neutrophil and lymphocyte (N:L) ratio ia shown
in Table XVIII. The N:L ratio was highest in the lat lactation
and loweat in the 7th lactation.
4. Influence of Breed
The influence of breed and CMTI are given in Table XIX.
The mean CMTI was lower (2.52) in the Ayrshire breed than the
Holatein breed (4.69). The difference between the means were
observed to be highly significant (P~O.Ol) (see Appendix).
Table XVI. Relation of Incidence and TYpe of Infection to Lactation Perioda
Strepag Lact. No. of No. of % Other Stal!h Micro- NH No. Cowa Quartera Strel!% aureus% cocci% Stal!h% Coli%
1 10 400 0.00 0.00 1.75 1.75 25.56 0.26
2 12 480 8.12 1 .. 25 3.54 1.88 17.50 0.21
3 9 308 7.14 0.32 4.87 1.30 17.54 0,,32
4 7 280 21,,78 0.00 0.71 1,,79 21,,43 1.79
5 13 520 13.46 0.19 1.73 3.08 15.38 0.38
6 7 270 11.11 0,,37 5.93 4.07 15" 19 0.00
7 & over 10 400 23.75 0.00 6.25 1.50 12.25 4.25
Total 68 2658 11.93 0.34 3.42 2.18 17.68 1.02
Total Quartera Infected 317 9 91 58 470 27
* % of quarter samples Showing bacterial growth on blood agar.
Mixed%
22.31
35.83
31,,17
37.50
39.82
45,,92
34.00
34.95
929
Total %* No Quartera Growth% Infected
48.37
31.67
37,,34
15.00
25.96
17,,41
18.00
28.48
756
51.63
68.33
62,,66
85,,00
74,,04
82.59
82.00
71.52
1901
~ ~ .....
100·0
80·0
i .... 0 Q)
~ H
ID ~ 60·0 Q) .... J CH 0
11 4000 0 ~ Q)
Pot
20·0
1 2 345
LACTATION NUMBER
/132
6 7 OR OVER
Figure 9. Peroentage of Infeotion in Different Lactation Periode.
Table XVII. Total Somatic Cell Count and Differentia]. C,9unt in CMT(+) Quartera in Different Lactations
Lactation No. of
!!2.:-. _ Cows No.
l 10 38 9.50 1 .. 02 0.30 29.41 0.18 17.65 0.54 II 12 127 26.46 2.14 0.34 15.89 0.79 36.91 0.90
III 9 100 32.47 3.59 . 0.49 13.65 1.63 45.40 1.39
IV 7 111 39.64 3.10 0.57 18.39 1.45 46.77 1.07
V 13 138 26.54 '.12 0.48 15.38 1.22 39.10 1.41
VI 7 101 37.41 4.11 0.56 13.62 1.75 42.58 1.78
VII & over 10 223 55.75 4.03 0.51 12.66 2.08 51.61 1.43
Total 68 838 31.53 3.29 0.51 . 15.50 1.48 44.98 1.29
52.94 0.00 42.06 0.11
38.72 0.08
34.52 0.01
45.19 0.01
43.31 0.02
35.48 0.01
39.21 0.01
0.00 5.14
2.23
0.32
0.33
0.49
0.25
0.31
~ \,).l \,).l
._.1'
5·0
~ 3'0 m r-I r-I Q) C)
1.0 o
1·0
0·0 1
/ Î'~,-,---,/ /
/ /
/134
,---TSCC
., .,
Neutrophi Is
•• Lymphocytes
--- -- -- ---- - -- -- - --- - - - - -----.----- ------------ Epi .Cells
2 3 4 5 6 7 OR OVER
LACTATION NUMBER
Figure 10. TSCC and DO in Different Lactations.
Çc,., \
/135
Table XVIII. NeutrophiblLymphocyte Ratios in all Lactations
Lactation No. of' NeutrophillLymphocyte No. Quart ers Ratio
l 38 1:2.96
II 127 1:1.15
III 99 1 :0.85
IV 111 1:0.74
V 138 1:1.16
VI 101 1:1.02
VII & above 223 1:0.69
/136
Tabl.e XIX. Relation of Breed to Cali.fornia Masti tis Test Index (CMTI)
Breed
Holstein
4 r s hire
Total
No. of Cows
47
21
68
Mean CMTI**
4.69
2.52
** The differences between the mean CMTI's were highly signii'icant (p < 0.01) •
<;;0:., \
/137
The incidence and type of infection in the Holstein and
Ayrshire cows are shown in Table XX. ~. agalactiae infection
was higher in the Holstein than in the Ayrshire breed. Overall
infection was higher (75.69%) in the Holstein cows compared to
62.50% in the Ayrshire cows. The percentage of quarters showing
no growth on blood agar were higher (37.50%) in Ayrshire cows
than Holstein cows (24.31%) (Figure 11).
The TSCC and DC in CMT(+) quart ers in two breeds are shown
in Table XXI and Figure 12. The average TSCC was higher (3.41
million cells/ml) in the Ayrshire breed than the Holstein breed
(3.26 million cells/ml). The percentage qf neutrophils was higher
in the Holstein cows, whereas, the percentage of lymphocytes was
higher in Ayrshire cows (Figure 12). The N:L ratio was 1~0.73
in Holstein and 1:1.87 in Ayrshire cows (Table XXII).
5. Influence of Month of the Year on the Incidence and TyPe of Mas titis
The mean GMT score ( -.Ix + 'i) and least square eetimatee
are given in Table XXIII and Figure 13. The differences between
the means were significant (P~0.05) (see Appendix). However, the
mean GMT score ( Jx + !) and least square eetimate was 2.22 in
the month of December as against 1.90 during the study period of
12 monthe in all 68 cowe.
The incidence and type of infection observed during the
12 monthe of the study are gi ven in Table XXIV. The incidence of
<;oc., \
TABLE XX. Incidence and Type of Infection in Holstein and Ayrshire Cows
StaEh. Micro-No. of No. of
Breed Cowa Quartera
Holstein 47 1818 15.84 0.28 3.36 2.31
Ayrahire 21 840 3.45 0.48 3.57 1.90
Total 68 2658 11.93 0.34 3.42 2.18
* % of quarter samples Bhowing bacterial growth on blood agar.
No Mixed Growth
15.90 1.37 36.63 24.31
21.55 0.24 31.31 37.50
17.68 1.02 34.95 28.48
Total %* Quartera !nfected
24.31
62.50
71.52
~ VI <Xl
_J
'0 CD ..., o
~ H
;
30
..., 20
l tH o
11 o ~ P4
10
' ... ~ .. ~ 1:::-::::: HOLSTE 1 N t::!:::::
~ AYRSHIRE
0 ..... ----::.:.: St rep. H.Staph. NH.StaPh. Coliforms Mi xed NG
TYPE OF INl!'OOTION
Figure 11. Percentage of Incidence and Type of Infection in Holstein and Ayrshire Cows.
"---VI \.0
J
Table XXI. Total Somatic Ce11 Count and Differentia! Count in CMT(+) Quartera in Holstein and Ayrsbire Breeds
Breed
Holstein
Ayrshire
Total
Av.
No. of Cowe
47
21
68
No.
673 37.02
165 19.64
838
31.53
Av.
No.
3.26 0.49 15.03 .1.60 49.08 1.16 35.58
3.41 0.58 17.01 0.98 28.74 1.84 53.96
3.29 0.51 15.50 1.48 44 .. 98 1 .. 29 39 .. 21
0.01
0.01
0.01
0.31
0.29
0.31
~ ~ o
.J
~ 11)
M M Q) ()
\0 0 ..-><
..-
2-5
1-5
,-
0-
0-0 HOLSTEIN AYRSHIRE
/141
~ Epi_ Cells
Il Neutrophils
~ LymPhocytes
Figure 12. TSCC and DC in Ho1stein and Ayrshire Breeds.
/142
Table XXII. Neutrophil/HYmphocyte Ratios in Two Breeds
Breed
Holstein
Ayrshire
No. of Quarters Inf'ected
673
165
Neutroph1l/~phocyte Ratio
1 :0.73
1:1.87
/143
Table XXIII. Month of the Year, Mean GMT Score ( .jx + i)and Leest Sguare Estimates
Month of No. of Cows Mean CMT the Year Tested L.S .. E .. Score ( -:Lx + Il January 6 -0 .. 28 1.62a
February 21 +0.19 2.09de
March 47 -0 .. 01 1.89bC
April 77 -0.12 1.78ab
May 83 -0.12 1.78ab
June 88 -0.13 1.77ab
July 100 +0.05 1.95bcd
August 72 -0.03 1.87bC
September 61 +0.11 2.01 cd
October 54 +0.03 1 .. 93bcd
November 33 +0.03 1.93bcd
December 25 +0.32 2.22e
Total 68 1.90
a,b,c,d,e = Means with the same superscript are not significantly different as tested by Duncan's New Multiple Range Test (P< 0.05)
31
~2 ......,
Q)
F-t o ()
CI)
~ ~ :l 1
o Jan.
/~ ~~
Feb. March
,~~-----, .~ ......... ~~~~
APril May June July Aug. Sept.
MONTH OF THE YEAR
Figure 13. Influence of the Month of the Year on GMT Score (...,,/ x + f).
Oct. Nov. Dec.
-:::.. ~ ~
.f
Table XXIV. Monthly InCidence and Type of Infection
No. of Other Staph. Micro- NH No Month of Cows No. of Strepag Strep aureus cocci Staph Coli Mixed Growth Total % of the Year Teated Quartera 2f ~ ~ ~ 2f ~ ~ ~ Quarters Infected
January 6 24 16.67 0.00 4.17 8.33 4.17 0.00 25.00 41.66 58.34
February 21 84 9.52 0.00 2.38 7.14 14.29 0.00 38.10 28.57 71 .. 43
March 47 188 12.23 0.53 3.19 3.19 22.87 0.53 39.89 17.55 82.45
April 77 306 11.44 0.65 2.94 0.98 13.40 0.00 49.35 21.24 78.76
May 83 330 11.52 0.00 2.42 1.52 17.27 0.00 47.88 19.39 80.61
June 88 350 14.00 0.28 5.43 1.43 15.14 1.43 34.86 27.43 72.57
July 100 397 14.86 1.01 2.52 2.77 15.87 1.26 35.01 26.70 73.30
August 72 287 12.20 0.00 3.48 2.09 19.16 0.70 19.51 42.86 57.14
September 61 244 11.48 0.00 6.56 2.46 19.26 1.64 7.38 51.23 48.77 october 54 216 9.72 0.00 3.24 0.93 26.39 0.46 33.33 25.93 74.07
November 33 132 6.82 0.00 1.52 4.55 15.15 2.27 40.91 28.79 71.21
December 25 100 8.00 1.00 1.00 0.00 21.00 6.00 46.00 17.00 83.00
Total 68 2658 11.93 0.34 3.42 2.18 17.68 1.02 34.95 28.48 71.52
Total Quartera Infected 317 ~ 91 58 470 27 929 756 1901
"---.j:>. \.TI
.J
infected quarters was highest in the month of December (83.00%)
and lowest in the month of September (48.77%) as compared to the
other months of the year. The percentage of quarters showing no
bacterial growth on blood agar was highest (51.23%) in the month of
September. The mixed infection group showed two peaks, one in the
/146
month of April and the other in the month of December. The Strepto
coccal infection was highest in the month of January (16. 67%) and
July (15.87%). Hemolytic staphylococcal infection was highest in
the month of Januar,y. The non-hemolytic staphylococcal infection
showed two peaks, one in the month of March and the other in the
month of October (Figure 14).
The TSOO and DO for the 12 months are gi ven in Table XXV.
In the month of December, the neutrophil percentage was highest
(69.01%) and the lymphocyte percentage was highest (55.56%) in the
month of April. ~e percentage of CMT(+) quarters was highest in
the month of December (38.00%) and lowest in the month of November
The monthly N: L ratios are shown in Table XXVI'. The ratio
was lowest in the month of December and highest in the month of
April.
6. Serum Transferrin (Tf) Type of Oowa and Mastitis
The frequency distribution of genotypes and gene frequency
at the Tf locus in 68 cows are shown in Table XXVII. The gene w:l. th
the highest frequency was T:f'D2 (0.34) and the one with the lowest was
TfE (0.15).
ç"., \
L.
§ ..-1 .p
50
40
g 30
\1 H
CH o
i (.) 20 ~ Pi
10
o
/ ..... 1 ........... • 1. 1 \ . ~ 1 \ b.~'
• ~ oi-.
e.'
1 \ V .......' " .",-- \ ;# , • ;# ,. \ # . '_.-'- "
l " . \." 1 ~ 1 . \. , ~ 1 . \,
\ lA
A \ /\ / , ~_-/. \ ~'«oyl
1 \ ~""-.......... '. l \ :k:§Y
. /" _ \ 1 ~-r ~. V \ 1
•
~ '1 .' ." .... .- \"""
• ' ". • •• ' ". Ç,o,,' ••• ' •••• ..' .' ". "'It
'. ..' '. •• ," •• o~n ••• • •••••••• •••••••• •• •• u~' .. - ...... . .. ....... .... . .. ..... . .. .... .. -.. .-.•••...•....•...•.••. ...........••. _-..... -•.•.••••.•••.•..•••........... i
Jan. Feb. March April May June July Aug. Sept. Oct. Nov. Dec.
MONTH OP THE YEAR '--"
Figure 14. Relationship of the Month of the Year to the Type of Infection. !i
Table XXV. Montbly Percentage of CMT(+) Quartera, Total Somatic Cell Count and Differentia! Count
Month of No. of Cows Year Teated No.
January 6 8 :33.33 6.90 1.78 25.80 2.63 38.11 2.49
February 21 32 38.10 2.46 0.58 23.57 1.04 42.28 0.84
March 47 62 32.98 4.82 0.97 20.12 1.50 31.12 2.34
April 77 93 30.39 2.97 0.36 12.12 0.96 32.32 1.65
May 83 88 26.67 3.70 0.51 13.78 1.69 45.68 1.48
June 88 114 32.57 2.76 0.49 17.75 1.19 43.12 1.06
July 100 146 36.78 3038 0.41 12.13 1.65 48.82 1.31
August 72 84 29.27 3.19 0.38 11.92 1.49 46.71 1.31
September 61 78 31.97 3.12 0.43 13.78 1.60 51.28 1.08
October 54 64 29.63 2.76 0.36 13.04 1.52 55.07 0.87
November 33 30 22.73 3.61 0.47 13.02 2.06 57.06 1.08
December 25 38 38.00 3.13 0.32 10.22 2.16 69.01 0.65
Total 68 838 Ave. 31.53 3.29 0.51 15.50 1.48 44.98 1.29
36.09 0.00
34.15 0.00
48.55 0.01
55.56 0.00
40.00 0.02
38.41 0.02
38.76 0.01
41.07 0.01
34.62 0.01
31.52 0.01
29.92 0.00
20.77 0.00
39.21 0.01
0.00
0.00
0.21
0.00
0.54
0.72
0.29
0.30
0.32
0.37
0.00
0.00
0.31
~ ~ (Xl
-_ .J
Table XXVI. Monthly Neutrophil/Lfmphocyte Ratios
Month of No. of CMT(+) Neutrophil/~hocyte the Year Quart ers Ratio
January 8 1 :0.94
February 31 1 :0.80
March 62 1 :1.56
April 93 1:1.72
M~ 88 1 :0.88
June 114 1:0.88
July 146 1 :0.80
August 84 1 :0.88
September 78 1 :0.68
October 64 1:0.58
November ;0 1 :0.52
December 38 1 :0.30
/149
'ii=-, \
/150
Table XXVII. Frequency Distribution of Genot,ypes and Gene Frequency at Tf Locus
Serial Tf Observed Expected (DeViation2 2
No. Tue Freg,uencl Freg,uencl Dev:i.ation Exp. Freg,uencl
1 .AA 6 7.41 -1.41 0.27
2 AD1 3 8.08 -5.08 3.19
3 AD2 24 15.26 8.74 5.01
4 AE 6 6.73 -0.73 0.79
5 D1D1 4 2.20 1.80 1 .. 47 6 D1D2 4 8.32 -4.32 2.24
7 D1E 9 3.67 5.33 7.74 8 D2D2 9 7.86 1.14 0.17
9 D2E 1 6.94 -5.94 5.08
10 EE 2 1.53 0.47 0.14
Total 68 68.00 0.00 26.10
X2
= 26.10***, 8 d.f. (P<0.005) •
Gene frequency:
A = 0.33 D1 = 0.18
D2 = 0.34
E = 0.15
The Tf type and GMTI of cows with their standard errors
are shown in Table XXVIII.. The difference between mean GMT· scores
( ~x + 't) were found to be significant (P<.0.05) (see Appendix).
The highest GMTI of 6.07 was observed in TfAD1. The lowest (1.70)
was found in type T:fD2E.
The incidence of type of infections in Tf type are shown
in Table XXIX. The overall infection was highest (95.00%) in
TfD2E type. The incidence of Str. agaJ.actiae infection was highest
in TfAD1 (24.17%) and lowest (zero) in TfD2E. The incidence of
Staph. aureus was highest in TfD1D1, and TfD2E. The incidence of
coliform mastitis was highest (1.78%) in TfAD2.
/151
The TSee and De in CMT(+) quarters are shown in Table XXX.
Average TSee was highest in TfD1D1 type. The peroentage of neutro
phils and lymphocytes was highest in TfAE, and T:fD2E, respecti vely.
The peroentage of eosinophils was highest in TfAD1.
The N:L ratio is shown in Table XXXI. The ratio was
highest in T:fD2E type and lowest in TfAE type.
/152
Table XXVIII. Serum Transferrin (Tfl TlEes and CMTI' s of Cows
Serial Tf No. of Cows Total Mean Standard Error of No. ~;ee Observed CMTI CMTI the Mean
1 AA 6 18.60 3.10 + -1.13 2 .AD 1 3 18.20 6.07 + -4.29
3 AD2 24 88.00 3.44 ~.57 4 AE 6 29.20 4.87 + -1.28
5 D1D1 4 16.80 4.20 + -1.58
6 D1D2 4 21.90 5.48 :t2.06
7 D1E 9 25.30 2.81 ~.86 8 D2D2 9 47.60 5.29 + -1.11
9 D2E 1 1.70 1.70 ~.OO 10 EE 2 6.10 3.05 + -1.45
Total 68 273.40 3.94 ~.34
Table XXIX. Incidence and TlEe of Infection in Transferrin TYPea
No. of Other §.t,aph. Micro-Tranaf errin No. of Quartera Strepag Strep aureus cogei T;Œe Cowa Teated ~ ~ ~ AA. 6 240 18.75 0.00 0.83 0.83
AD1 3 120 24.17 0.00 0.00 2.50
AD2 24 956 6.80 0.52 4.08 2.62
AE 6 210 18.57 0.00 2.86 3.33
D1D1 4 160 1.86 0.63 7.50 0.00
D1D2 4 132 22.73 0.00 0.76 3.03
D1E 9 360 8.33 0.00 2.22 1 .. 94
D2D2 9 360 20.83 0.83 5.55 1.67
D2E 1 40 0.00 0.00 7.50 2.50
EE 2 80 1 .. 25 0.00 0.00 3.75
Total 68 2658 11.93 0 .. 34 3.42 2 .. 18
Total Quartera Infected 317 9 91 58
NIf Staph Coli Mixed ~ ~ ~
9.58 0.83 38.75
15.84 0.83 33.33
16.42 1.78 38.28
13.33 1.91 28.57
24.38 0.63 36.25
15.15 0.00 36.36
21.67 0.28 33.06
18 .. 06 0.28 28 .. 06
40.00 0.00 45.00
31.25 0.00 32.50
17.68 1.02 34.95
470 27 929
No Growth ~
30.42
23.33
29.50
31.43
28.75
21.97
32.50
24.72
5.00
31.25
28 .. 48
756
Total % of Quartera Infected
69.58
76.67
70.50
68.57
71.25
78.03
67 .. 50
75.28
95 .. 00
68.75
71.52
1901
~ \J1 VJ
.J
Table XXX. Total Somatic Cell Count and Differential Count in CMT(+) Quartera in Transferrin Types (Tf)
Transferrin No. of e Cowa No.
M 6 63 26.25 2.77 0.49 17.69 1.03 37.18 1.25 45.13
ADl 3 61 50.83 3.01 0.49 16.28 1.34 44.52 1.16 38.54
AD2 24 282 29.50 3.46 0.51 14.74 1.60 46.24 1.34 38~73
AE 6 77 36.67 3.16 0.44 13.92 2.00 63.29 0.71 22.47
D1Dl 4 51 31.88 4.69 0.66 14.07 1.91 40.72 2.10 44.78
D1D2 4 41 31.06 3.47 0.71 20.46 1.40 40.35 1.36 39.19
D1E 9 74 20.56 3.51 0.57 16.24 1.28 36.47 1.64 46.72
D2D2 9 154 42.78 3.22 0.51 15.84 1.48 45.96 1.23 38.20
D2E 1 5 12.50 1.55 0.24 15.48 0.19 12.26 1.12 72.26
EE 2 21 26.25 1.76 0.33 18.7~ 0.48 27.27 0.94 53.41
---Total 68 838
Ave. 31.53 3.29 0.51 15.50 1.48 44.98 1.29 39.21
0.00
0.02
0.01
0.01
0.02
0.00
0.02
0.00
0.00
0.01
0.01
0.00
0.66
0.29
0.32
0.43
0.00
0.57 0.00
0.00
0.57
0.31
........ -0
lJ1 .{:>o
_f
/155
Table xx:xr. Neutrophil/L,ymphocyte Ratios in Transferrin Types
Tranaferrin No. of CMT(+) Neutrophil/~hocyte Type Quartera Ratio
li 63 1:1.22
AD1 61 1:0.88
.AD 2 282 1:0.87
AE 77 1:0.35
D1D1 51 1: 1.10
D1D2 41 1: 0.97
D1E 74 1: 1.29
D2D2 154 1:0 .. 83
D2E 5 1: 5.82
EE 21 1:1.94
DISCUSSION
In the present study, 68 freshly calved cows were studied
from the two experimental herds of the Macdonald College Farm, 21
cows from Herd No. 1 (Ayrshire) and 47 cows from Herd No. 2 (Holstein).
The study was spread over a period of one year from January, 1971,
to December, 1971, and the cows were included as they calved. Each
cow was tested 10 times. Cows numbered 45RH, 75H and 97H were only
tested for 3, 5 and 9 tests, respectively, as they were shipped to
market before the 10 tests were completed. The first test was done
between the 3rd and 7th d~ after calv.Lng and the successive tests
were done at 14-d~ intervals. The last test was carried out
between the 130th and 134th d~ of lactation so each test is in
dicative of a stage of lactation.
Subclinical mastitis is inflammation of the udder with no
gross signa. One of the basic host responses to a bacterial infection
is the infiltration of leucocytes from the blood into the area of
injury. It is evidenced by an increased number of cells in mil.k
from quart ers infected with infective organisms. Frequently, latent
infection exists in quarters which ~ not result in an increase in
the cell count of the milk.
In this study, the California Mastitis Test (CMT) and the
Direct Microscopie Somatic Cel1 Count (DMSCC) were used as screening
tests to estimate the Total Somatic Cell Count (TSCC) of the individual
quarter fore-millt (IQJ!M) samples. In all, 2,658 IQl!M samples from
68 cows were subjected to the GMT and DMSCCO' While emphasis was
placed on GMT, the mscewas used to supplement the information and
to differentiate between leucocytes and epithelial cells.
The mean TSee within each GMT group were 0.05 for negative,
0.26 for Trace, 0.85 for 1+, 2.79 for 2+ and 7.75 million cells/ml
for GMT grade 3+ (Table II). These values were within the range of
those: 0-200,000 for negative, 150,000-550,000 for Trace, 400,000-
1,500,000 for 1+, 800,000-5,000,000 for 2+ and more than 5,000,000
million cells/ml for 3+ (Schalm and Noorlander, 1957). As the
severity of the CMT reaction increased, the mean TSCC Blso increasedO'
This is in agreement with the findings reported by other workers
(Barnum and Newbould, 1961; Leudecke .!1.!:lO" 1967; Miller and Kearns,
1967; Malik, 1968). The relationship between the GMT grade and the
mean TSeC was found to be curvilinear (Figure 1).
The distribution of the TSee within each GMT group and the
distribution of GMT reactions are summarized in Table II. The
percentage distribution of 2,658 IQFM samples from the 68 cows
studied was 57.41, 11.06, 16.25, 5.72 and 9 .. 56 percent for CMT
negative, Trace, 1+r 2+ and 3+, respe~tively. The results given in
Table III illustrate that 838 (310'53~ quart ers showed CMT positive
(1+, 2+ and 3+), 1,820 (68O'47%) quart ers showed CMT negative (-,T).
In the present study, the IQ1!N samples showing CMT Trace reaction
were included along with GMT negative samples because due to low
/157
TSOO's of these samples, DO's were not perfor.med. Among the CMT(+)
quarters, 90.45 percent of the samples contained more than 0.5x106
cells/ml, while 100 percent of the CMT(-)samples contained less than
0.5x106
cells/ml. This is in agreement with Daniel ~~. (1966).
Out of the 838 CMT(+) quarters, 80 quarters (9.55%) had
less than 0.5x106 cells/ml and more than 0.4x106 cells/ml. None
of the quart ers of CMT(-) and (T) showed more than·0.5 x 106
cella/ml
(Table III). This is contrary to the results of Barnum and Newbould
(1961) and Malik (1968). Miller and Kearns (1967) concluded that
interpreting all quarter milk semples wi th a CMT score of 0
(including GMT-Trace) as containing less than 500,000 leucocytes/ml
yielded correct interpretation 89.9 percent of the time. Conversely,
if all quarter milk samples wi th a CMT score of 1, 2 or 3 are
interpreted to contain over 500,000 leucocytes/ml, the interpretation
will be correct 83.8 percent of the time.
The overall incidence of CMT(+) quarters and cows were
31.53 percent and 64.32 percent, respectively. These percent~s were
highest in the firat test compared to the other tests (Table I). This
m~ be reasoned as due to the physiological increase of cells in the
early part of the lactation.
The physiological factors affecting the number and relative
proportions of cells are age of the cow, stage of lactation,
variations in different portions of a milking, milking practices,
/158
certain management practices and conditions of stress (Cone, 1944;
Mochrie ~~., 1953; MacAdam, 1954; Waite and Blackburn, 1957;
_ . "..rich and Renk, 1967; Schalm ~ al .. , 1971).
M~ workers have attempted to establish a level of cells
representing a dividing line between normal and abnormal milk.
Hucker (1933) regarded cell counts more than 500,000 cells/cc as
indicative of abnormal or pathological condition in the udder. The
British Veterinary Association (1965) etated that quarter sample
counte of 500,000 celle/ml or over are generally indicative of sub-
clin1cal mastitis although some workers accept a lower number as
indicative of udder damage.
The incidence of microorganisme found in milk of various
CMT grades was 62.71 for negative, 76.87 for Trace, 83.56 for 1+,
90.79 for 2+ and 86.61 percent for 3+ (Table VI). The overall
incidence of infected quarters in CMT tested quarters was 71.52
percent. These results were lower than those of Malik (1968). He
reasoned that the commercial herd samples were not refrigerated
duringtransi t which might have resul ted in a higher number of
bacteria of not much significance for mastitis but resulting in a
higher incidence of infection.
Out of the 2,658 IQFM samples tested bacteriologically, the
overall infection due to various organisms was ~. agalactiae,
11.93 percent, other streptococci, 0.34 percent, Staph.aureus, 3.42
/159
percent, micrococci, 2.18 percent, non-hemolytic stap~lococci
17.68 percent, coliforms, 1.02 percent, and mixed infection, 34.95
percent. 28.48 percent of the quart ers showed no growth on blood
agar plates (Table V). In this study, the blood agar plates were
incubated for a period of 48 hours so the chances of missing in
fective organisme were very rare.
A comparison of bacteriological results and their reaction
to the GMT are presented in Table VII. Of the 317 ~. agalactiae
infected quarters, 93.28 percent were OMT(+); 9 quart ers infected
with other streptococci, 77.78 percent were CMT(+); 91 quart ers
infected with Staph. aureus, 52.75 percent were CMT(+); 58 quarters
infected with Micrococoi, 13.80 peroent were CMT(+); 470 quarters
infected with non-hemolytic stap~lococci, 24.26 percent were CMT(+);
27 quart ers infected with coliforms, 85.19 percent were CMT(+) and
929 quart ers infeoted with mixed infection, 24.00 percent were
CMT(+).
Of the quart ers containing~. agalactiae, other strepto
cocci, Staph. aureus and coliforms, 6.62 percent, 22.22 percent,
47.25 percent and 14.81 percent, respectively, yielded CMT grades
of negative or Trace. In all probability, these organisme were
either contaminants that entered the semples during collection or
they were present in the udder wi thout hav:l.ng caused pathologicaJ.
alterations. Micrococci, non-hemolytic stap~lococci and mixed
infection (mainly corynebacteria) organisme were frequently cuJ.tured
/160
from milk samples and are commonly regarded as non-pathogens or
their pathogenecity is very low. These non-pathogens wer~ isolated
more frequently from quart ers with low CMT reactions (negative and
Trace) than from quart ers with high test reactions (1+, 2+ and 3+).
This is probably due to an increased phagocytosis that occurred in
the ones with high GMT grades. In the current study, the so-called
non-pathogens also created a mild irritation which could account
for some of the high CMT reactions (Table VII). Moreover, 15.72
percent of the culturally negative quarters produced CMT(+) milk.
Philpot (1969) also reported 13 percent of the culturally negative
quart ers which yielded CMT(+) milk. This m~ be reasoned due to the
entering of milk samples from cows treated with antibiotics or due
to some mechanical injury by machine milking without infection.
Marshall and Edmondson (1962) and Wesen.!l!!:! .. (1968)
reported that a relationship exists between the CMT and the bacterio
logical results from quarter samples. Since the number of leucocytes
is an indication of inflammation, it seems probable that both the
leucocyte numbers and the types of bacteria should be considered in
diagnosing mastitis.
The incidence of .§E. agalactiae was lowest (8.49%) in
the first test and found highest (14.01,%) in the 6th test (Table V).
This is in agreement with the observations of Seelemann (1954).
He reported that mastitis caused by §~. agalactiae occurs as
frequently in the middle of lactation as it does shortly after
calving. Heidrich and Renk (1967) claimed that mastitis due to
~. agalactiae occurs mainly during lactation at the peak of
production.
/161
The mean California Masti tis Test Index (CMTI) was caJ.
culated for ~. agalactiae infection. It was found highest (3.53)
in the first test as against the average (3.01, Table rxa). The
differences between the means were not significant (p c( 0.05;
Table IXb).
out of 149 quart ers inf'ected wi th hemolytic staphylococci,
91 (61.07%) quarters were inf'ected with mannitol positive and
coagulase positive (Staph. aureus) organisms. The rest of the 58
quart ers (38.93%) were in:f'ected with coagulase negative (micrococci)
organisme. The incidence of hemolytic staphylococci was highest
(8.85~ in the 3rd test and lowest in the 5th test (3.74%) (Table
V). According to Kastli (1951) micro cocci that are excreted in
apperently normal, heal thy milk can usually be classified according
to biological criteria for pathogenicity as apathogenic.
The incidence nf coliform mastitis was highest in the 1st
test and zero in the 7th test. The incidence of non-hemolytic
staphylococci and mixed infection was highest (24.35%; 38.02%) in
the 1st and 9th test, respectively. In this study, infection caused
by more than one type of organism having less than 10 colonies per
/162
type on blood agar and colonies of corynebacteria all alone or
with other organisms, were inoluded under mixed infection category.
The incidence of mixed infection was higbest throughout
the study period and non-hemolytic staphylococci infection was second
to it (Figure 2). Corynebacteria and non-hemolytic staphylococci
inhabit the papillary duct as non-pathogenic organisme (Diernhofer,
1958). These organisme occurred more frequently When the samples
were incubated for 48 hours.
In the present investigation, all CMT(+) IQFM semples were
subjected to a differential cell (De) count. The average TSCC in
CMT(+) quarters was found to be higbest (4.94 million cells/ml)
in the 1st test. In this TSCC, 51.62 percent were lymphocytes,
31.98 percent were neutrophils, 16.19 percent were epithelial cells
and 0.21 percent were eosinophils (Table X). The percentages of
lymphocytes and epi thelial cells were found to be highest in the 1 st
test compared to other tests. It is apparent that in the first few
dB\Ys of lactation, the cell count was abnormally high and this was
mainly due to lymphocytes. ~his is in agreement with the observations
made by Savage (1912). It is generally agreed that the TSCC :i.s higb
during the first week of lactation (Schalm ~~., 1971). The
increased number of epithelial cells in the early part of lactation
is due to glands resuming functional activity after a dormant period.
/163
It was found that the average TSCC was lowest and least
variable in the last four tests of the study. Waite and Blackburn
(1957) also found that the average cell count was lowest and least
variable from the 70th to the 130th d~ of lactation.
The percentage of neutrophils fluctuated throughout the
test period and was found highest (58.39%) in the 10th test and
lowest (31.98%) in the 1st test (Table X). There was an increased
influx of neutrophils from the blood into the mammary gland during
inflammation and there may be some differential chemotactic effect
with different infections. This ~ be the reason for the
fluctuations. It was found in this study that the highest percentage
of eosinophils (4.55%) occurred in the 10th test and 4.19 percent
in the 7th test. The studies of Litt (1961;1962) provided con-
vincing evidence that antigen-antibody complexes attract eosinophils.
Eosinophils probably occurred in the latter part of the tests after
the formation and accumulation of antibodies in the mammary gland.
It was found that the neutrophil/lymphocyte ratio was
highest in the 1st test (1:1.62) and lowest in the 10th test (1:0.47)
(Table XI). However, the ratio fluctuated throughout the stuQy
period. Neutrophils predominated in all tests except the 1st test.
Tapernoux (1931) found that normal milk had a leucocytic formula
similar to that of blood; a lymphocyt~polymorph ratio of about 2:1.
In mastitic milk, he found nearly aIl the leucocytes were polymorphs.
=.., \
/164
i~e average TSCC increased as the GMT grades increased. The
percentage of neutrophils and eosinophils also increased with the
GMT grades. Convers ely , the epithelial cells and lymphocytes de
creased with the increase of GMT grades. When these data were plotted
on log paper, this relationship was found to be linear except for
eosinophils (Figure 7). It is eVident that the increase of TSCC was
mainly due to neutrophils as the GMT grades increased (Table XIV).
In CMT(+) quarters, TSCC and DC for different organisme
were calculated and summarized in Table XII. The average TSCC was
10.15; 4.13; 4.03; 2.32; 2.21; 2.19 and 1.88 million cells/ml for
coliforms; ~. agalactiae; Staph. aureus; micrococci; other strepto
cocci; non-hemolytic staphylococci and mixed infection, respectively.
In bacteriologically negative samples, the mean was 3.41 million
cells/ml. Different authors reported different values of TSCC for
various organisms. The degree and the nature of the cellular
responses are likely to be proportional to the severity and duration
of infection, both in terms of the number and the Virulence of
organisme and the degree of tissue invasion (Schalm ~~., 1971).
It was found that the percentages of neutrophils were high
(69.06%; 49.88%; 48.38%; 43.89%) in coliforms, ~. aga!actiae,
Staph. aureus and other streptococci organisme. Conversely, the
percentages of lymphocytes were high (57.2~; 49.47%; 40.09.%) in
non-hemolytic staphylococci, mixed infection and micrococci organisme.
In bacteriologically negative samples, the percentage of lymphocytes
/165
was highest (42.23%; Table XII). From this study, it 1s very
difficul t to conclude whether this high lymphocyte count was in any
way respons1ble for the absence of bacteria. It appears from this
study that the organisme which produce toxine attract more neutro-
phils. When antigen injures tiesue cells, chemical substances are
released which initiate and perpetuate the inflammatory reaction.
Leucotoxine increases capillary permeability and by chemotaxis, it
attracte neutrophile into the area of the injured cells. A leuco-
cytosis-promoting factor also produced from injured celle 1s carried
by the blood stream to the bone marrow where i t etimulates granulo
poiesis, thereby increasing the supply of neutrophils (Schalm, 1962).
OtGairbhidh~ ..21 &~1970) reported that Staph. aureus in m:l.lk fram
an ~ndotoxin-treated quarter exhibited a marked chemotactic stimulus
for bovine neutrophile.
The percentages of eosinophils were 0.53; 0.50; 0.49; 0.20;
in mixed infection, Staph. aureus. ~. agalactiae and col1form
organisme, reepectively. In the bacteriologically negative samples,
it was found to be 0.29 percent and zero in the m:l.crococci and non-
hemolytic etapbylococci infected quarter eamplee, respectively.
Depending upon the antigen and antibody reaction, the eosinophils
are attracted to the injured sites. It has been suggested that they
inactivate histamine or histamine-like toxic materials (Vaughn, 1953).
They increase in situations involving de composition of body protein
and this may reflect a function of detoxification.
<;;:..., \
/166
The neutrophi~lymphocyte ratios were low (1:0.36;
1:0.69; 1:0.77; 1:0.89) for coliforms, ~. agalactiae, Staph.
aureus and other streptococci. Conversely, these ratios were high
in non-hemolytic staphylococci, mixed infection and micrococci
(Table XIII).
The mean GMT score ( "';x + i) and least square estima.tes
(LSE) for stage of lactation was 2.62 in the 1 st test as against the
mean 1.90 during all other tests (Table IV). The dLfferences
between the means were found significant (P"" 0.01; see Appendix).
The mean CMTI was seen to increase as the number of
lactations increased up to the 4th lactation and there was a decrease
in the 5th and 6th lactations (Figure 8). In the 7th lactation, it
was found to be 7.04 ~ 0.81 as against the mean 4.02 ~ 0.36 (Table
XVa) • This f1nding is not in agreement wi th that reported by
Marsh.a1.1 and Edmondson (1962) and Malik (1968), who reported a
decrease in CMT average for cows in the 9-year group. It appears
that once infection 1s establ1shed, it causes the seme amount of
inflammation in a cow regardless of age. Braund and Schultz (1963)
and Schalm and Ziv-S11berman (1968) have shown that the CMT score
increased as the lactation n~ber increased but they grouped the
lactations into first, second, third, fourth and fifth and over and
did not report the older cows as separate lactation groups.
The mean CMTI was calculated for the different lactations.
The differences between the means were higbly significant (P<O.Ol;
Table XVb).
/167
The incidence of ~. agalactiae infection was firet ob
served to reach a peak, on a quarter basis, during the fourth
lactation, and then in the seventh lactation (Figure 9). The number
of quart ers infected in cows in the first lautation was zero, whereas,
in cows in the seventh lactation or above, the percentage of in
fection was high (23.75%; Table XVI). An increase in the incidence
of infection due to age was reported by Seeleman as early as 1932.
Reifers were significantly more resistant to infection with ~.
agalsctiae in the present etudy. Other workers, (Ormsbee and
SChalm, 1949; Spencer and Kraft, 1949) also reported a low incidence
of infection in first-calf heifers.
This increased incidence of ~. agalactiae infection from
one lactation to another ~ be reasoned as due to damage to teats,
milk yield, previous sensitization, or the degree of exposure to
infection wi th the incresse of age.
The incidence of Staph. aureus was lowest in the 4th
lactation (0.71%) and highest (6.25%) in the 7th or over (Table XVI).
It is, therefore, seen that Staph. aureus infection does increase with
age of the cow and a higher incidence occurred in the aged cows.
The incidence of coliform mastitis was found to be highest
in the 7"J!l lactation or above. It was se en that there was no
incidence of infection by this organism in the cows in their 6th
lactation. The overall infection increased with age of the cow.
/168
The TSee and De in CMT(+) quart ers in different lactations
are caJ.culated and aummarized in Table XVII. The average total cell
count of the cows in the first lactation was 1.02 million cells/ml
and in aucceeding lactations, it increased to 2.14, 3.59, 3.10,
3.12, 4.11 and 4.03 million cells/ml for the second, third, fourth,
fifth, sixth and seventh or above lactations. There was a decrease
in TSee in the fourth and fifth lactations as againat the average
for all lactations (3.29 million cells/ml).
The percentage of neutrophils was lowest in the first
lactation and there was a gradual increase up to the 7th lactation
or above. When this was plotted, it was found that the graph of
neutrophils ran almost paraJ.lel to that for the average TSee but was
interrupted by higber percentages of lymphocytes in the 1st, 2nd, 4th
and 5th lactations (Figure 10). This means that the increase in TSee
from lactation to lactation was not solely due to an increase of
neutrophils. These results are contrary to the findings of
l3lackburn (1966). He found that the increase in cellcount from
lactation to lactation due mainly to an increase in the number of
polymorphs. The average' epitheliaJ. cells showed ver,y little increase
from the 2nd to the 7th or above lactations.
The percentage of eosinophils was found to be higbest
(5.'14%) in the 2nd lactation and zero in the 1st lactation. This
is further eVidence that these cells are associated with antigen
/169
antibody reactions probably of a bypersensitive nature. As lactation
p~ogressed, the animals were exposed more frequently to infection.
In the 2nd lactation, antibodies presumably had been formed. These
antigen'and antibody reactions served to attract the eosinophils.
The neutrophil/lymphocyte ratio was lowest (1:0.69) in the
7th lactation or over, and highest (1:2.96) in the 1st lactation
cows (Table XVIII). This might resul t in a loss of phagocytosing
ability with age. It is evident that the infection level in the 1st
lactation was low compared to the cows in the 7th lactation or above.
The older cows were more susceptible to disease compared to younger
cows in the 1st lactation.
In this present study, it was found that the Ayrshire
breed had the lower (2.52) GMTI compared to the Holstein breed (4.69)
(Table XIX). The GMT score ( Jx + i) for the two breeds was found
to be highly significant (P< 0 .. 01; see Appendix). The overall
infection wes higher (75.6g;b) in Holstein cows compared to Ayrshires
(62.50%) (Table XX). The~. agalactiae, micrococci, coli:form and
mixed infection were higher in Holsteina compared to Ayrshire cows.
Conversely, in the ~rshire cows, infection due to other strepto
cocci, Staph. aureus and non-hemolytic stap~lococci was found to be
higher than in the Holstein cows (Figure 11).
In this study, i t was found that the Ayrshire breed was
less susceptible to mastitis but it is ver,y difficult to assess the
/170
effect of breed factors individually on the cows t susceptibility
to mastitis when other factors could have been equally responsible
for the causation of the disease. Narayanan and Iya (1953)
reported that the breed of animal did not in any w~ influence the
incidence of the disease in the herds examined by them.
The average TSCC was higher (3.41 million cells/ml) in
Ayrahires compared to Holsteins (3.26 million cella/ml) (Table XXI).
The percentage of neutrophils and eoainophila were alao higher in
the Holstein breed. Conversely, th.e percentage of epitheliaJ. cells
and lymphocytes was higher in the Ayrshire breed (Figure 12).
The neutrophil/lymphocyte ratio was lower in Holsteins
(1:0.37) compared to Ayrahires (1:1.87) (Table XXII).
The average monthly GMT score ( ..Ix + i) and least
square estimate (LSE) was found highest in the month of December
(mean 2.22; LSE = +0.32) as against the general mean of 1.90 for
the atudy period (Table XXIII). In the months of February, July,
September, October, November and December, it was above average and
in the other months, it was below average. The differences between
the mean GMT scores ( "x + i) for the month of the year was
significant (P<0.05) (see Appendix). The lowest GMT score was
Qbserved in the month of January. This is in agreement wi th the
observations of MaJ.ik (1968).
<;<:., \
/171
Fluctuations were seen in the monthly percentage incidence
of infection due to various microorganisms (Table XXIV). Overall
infection was high in the months of December (83.00%), March
(82.45%), May (80.61%), April (78.76%), July (73.30%), June (72.57%)
as against the average 71.52 percent throughout the year. According
to Brown ~~. (1965) there is no conclusive evidence to indicate
that the season per ~ influences the incidence of udder infection
and mastitis. However, the incidence of infection during the
spring, summer, fall and winter was 79.69, 67.67, 64.68 and 73.81
percent, respectively.
Exposure of the udder to chilling increases inflammation
in udders already infected (Plastridge, 1958). This is probably
the reason for the high percentage of infection in the month of
December.
The incidence of Str. agalactiae infection that occurred
during the spring, summer, fall and winter was 11.48, 13.68, 12.01
and 11.61 percent, respectively.
The incidence of Staph. aureus infection was highest in
the month of September (6.54%) followe.d by 5.43 percent in the month
of June. Mixed infection showed two peaks, one in the month of
April and another in the month of December (Figure 14). There was
a drastic fall in mixed infection in the month of September. The
incidence of infection due to coliforms was lowest in the months
s.:., \
/172
of August and October, and highest in the month of December. These
results were not in agreement with the observations of Kudelka and
Holec (1960), who observed coliform infection to be highest in
August and lowest in the winter. It appears from these observations
that sesson of the year does influence the fluctuation of the
incidence and extent of mastitis in cows. A number of factors
probably responsible for these observations are the environmentat
temperature, the effect of warm climatic conditions on cows, the
effect of higher temperatures on the growth of microorgam sms and
variations in the freshening pattern to the age of cows. It is
very difficult to weigh the importance of each of these factors
in the present study.
In the CMT(+) quarters, the average TSCC was highest
(6.90 million/ml) in the month of January (Table XXV). The
differential count fluctuated throughout the study period. The
percentages of epithelial cells, neutrophils, lymphocytes and
eosinophils were high in the montlB of January, December, April and
June, respectively. It was observed 'tha.t in the months of September,
October, November and December, there was a gradual increase in
percentages of neutrophils. The neutrophil/lymphocyte ratio was low
atl through the study period except in the months of March and April
(Table XXVI).
The differences between the CMT scores ( ~x + f) of
various Tf types of COWB were significant (P",0.05) (see Appendix).
The highest mean CMTI was obtained for TfAD1 followed by Tf.D1D2
and the lowest mean CMTI was obtained for TfD2E (Table XXVII).
/173
The percentage infection due to~. agalactiae was studied
among the 10 Tf types. ~. agalactiae is an obligate parasite
and is, therefore, probably more closely aesociated with the genetic
mechanisms of reeietance in cows than other types of organisme.
The percentage of quarters infected with this organism was TfAD1
(24.17%), Tf.D1D2 (22.73%) and TfD2D2 (20.83%) (Table XXIX). It was
observed that the incidence of this organism in Tf types possessing
aJ.leles T;>1 and T~2 was high. This ia in agreement wi th the
findings of Malik (1968; 1971).
The overall infection was found highest in TfD2E (95.00%).
Unfortunately, there was only one cow in this type and so no con-
cluaion could be drawn. With the elimination of this one cow, the
next highest was Tf.D1D2 (78.03%), TfAD1 (76.67%) and TfD2D2 (75.28%).
In the rest of the Tf types, the infection was lower than the average
(71.52%) for all 10 types (Table XXIX).
In the CMT(+) quarters, the TSCC and DC was calculated for
the 10 Tf type cows. The average TSCC wes found to be highest in
Tf.D1D1 (4.69 million cells/ml) followed by D1E (3.51 million cells/ml);
TfD1D2 (3.47 million cells/ml) and TfAD2 (3.46 million cells/ml). In
the rest of the Tf types, the TSCC was lower than the average
(3.29.million cells/ml) for all the Tf types (Table XXX).
/174
The percentage of neutrophils was found highest in TfAE
(63.29%) followed by TfAD2 (46.24%) and TfD2D2 (45.96%). Conversely,
the percentage of lymphocytes were highest in TfD2E (72.26%), TfEE
(53.41%), TfD1E (46.72%), TfAA (45.13%) and TfD1D1 (44.78%). It is
contended here that a severe host response in terms of high somatic
cell counts in milk results from extensive injur,y to the marnrnary
tissue caused by a severe infection. It does appear that neutrophils
play a considerable role in resistance or susceptibility to infection.
Phagocytosis by leucocytes has been suggested to be the phenomenon
responsible for protection against infection (Blobel and Katsube,
1964; Schalm II !:!.., 19 64a) • The phagocyti c acti vi ty of milk
leucocytes also appears to vary from cow to cow and from quarter to
quarter in the same cow (Wisniowski, ll~" 1967).
Out of the four homoz.ygous Tf types (TfAA; TfD1D1; TfD2D2;
TfEE) present in the population, the neutrophil/lymphocyte (N:L)
ratio was highest in TfEE (1:1.94) and lowest in TfD2D2 (1:0.83).
The highest N:L ratio in type TfEE was associated with the lowest
percentage incidence of ~. agaiactiae, Staph. aureus, coliforms
and overall infection. (Table XXIX). This observation is in
agreement with Malik II al. (1970) who observed TfEE cows to be free
of Str. agalactiae infectinn in their Series l cows and the lowest
streptococcal score was in their Series II cows.
/175
Amongst heteroz.ygous Tf t,rpes (TfAD1; TfAD2; TfAE;
TfD1D2; TfD1E; TfD2E), the neutrophil/lymphocyte ratio was lowest
in TfAE (1:0.35) and highest in TfD2E (1:5.82) (Table XXXI). Type
TfAE had a high percentage incidence of infection with ~.
agalactiae and coliforms presumably resulting in a high neutrophil
count which, in turn, resulted in a low N:L ratio. The type TfD2E
group had only one cow and this cow was free from streptococci and
coliform infection.
As already mentioned, a high lymphocyte count appears to
be associated with a low incidence of infection, and it appears from
the present study that gene TfE in a homozygous condition is most
resistant (or least susceptible) than the remaining homozygotes.
Since the Tf type frequencies of some of the types were
very low, i t is difficul t to draw a cl.ear-cut con-èlusion. Young ~
~. (1960) obeerved that estimations of the heritability of leucocytes
in the milk indicated that ma.ny of the genes influencing clinical
mastitis also influence leucocyte counts.
:
SUMMARY AND CONCLUSIONS
Studies were conducted to determine the cellular response
to non-clinicaJ. masti tis caused by various organisms in cows. The
influence of predisposing factors such as stage of lactation, age
of cow (lactation number), breed of the cow, month of the year and
serum transferrin type, on the incidence and type of mastitis, and
cellular responses were studied.
The two experimental herds of Macdonald College were used
for the study. The first herd consisted of Ayrshire cows and the
second was of the Holstein breed. The study was spread over a period
of one year from January, 1971, to December, 1972, and cows were
included in the study as they calved. Each cow was tested 10 times.
The first test was done between the 3rd and 7th dey after caJ.ving
and the successive tests were done at 14-dey intervals.
The fore-milk samples were collected aseptically from
each quarter separately. In all, 2,658 IQFM samples from 68 cows
were tested. Out of these, 1,818 were from the Holstein herd and
840 were from the Ayrshire herd. A total of 2,658 IQFM samples were
tested for the CaJ.iforn1a Mastitis Test (GMT) reaction and Direct
Microscopie Somatic Cell Count (DMSCC). Although the main emphasis
was on the GMT, the :wrSCC was used to supplement the information and
to differentiate between leucocytes and epithelial cells.
/177
The distribution of CMT reactions was 57.41; 11.06;
16.25; 5.72; and 9.56 percent for CMT negative, Trace, 1+, 2+ and
3+, respectively. The mean Total Somatic Cell Count (TSCC) within
each CMT group were 0.05, 0.26, 0.85, 2.79 and 7.75 million cells/ml
for negative, Trace, 1+, 2+ and 3+, reapectively. The relationship
between CMT grade and the mean TSCC was found to be curvilinear.
The overall incidence of CMT(+) quarters and cows were 31.53 percent
and 64.32 percent, respectively.
An overall incidence of infection due to various organisms
was found in 71.52 percent of the quartera. Out of this, !E:..
agalactiae represented 11.93 percent; other streptococci 0.34 percent;
Staph. aureus 3.42 percent; micrococci 2.18 percent; non-hemolytic
staphylococci 17.68 percent; coliforms 1.02 percelt and mixed infection
34.95 percent. The quarter samples that showed no growth on blood
agar plates represented 28.48 percent.
The incidence of microorganisms found in milk for various
CMT grades was 62.71 for negative, 76.87 for Trace, 83.56 for 1+,
90.79 for 2+ and 86.61 perc~nt for 3+. Of the q~ters containing
~. agalactiae, other streptococci, Staph. aureus and coliformsg
6.62, 22.22, 47.25 and 14.81, respectively, yielded CMT grades of
negative and Trace. In all probability, these organisms were either
contaminants that entered the semples during collection or they
were present in the udder without having caused pathological
alterationa. Micrococci, non-hemolytic ataphylococci and mixed
/178
infection (mainly corynebacteria) organisms were frequently
cul tured from milk samples and are commonly regarded as low-grade
or non-pathogem.c.
The mean CMTI for~. agalactiae was not significant
(P..(0.05) • Out of 149 quarters inf'ected wi th hemolytic staphylococci,
61.07 percent were infected with Staph. aureus and 38.93 percent were
infected with micrococci.
The average TSCC in CMT(+) quart ers was 3.29 million
cells/ml. In this count, 15.50 percent were epithelial cells; 44.98
percent neutrophils; 39.21 percent lymphocytes and 0.31 percent
eoainophile. The average TSCC vias found to be highest (4.94 million
cells/ml) in the first test. It waa observed that the TSCC was
abnormal.ly high in the first test and this was mainly due to
lymphocytes. In the CMT(+) quarters, the percentages of neutrophils
and lymphocytes fluctuated throughout the study period depending
upon the type and severi ty of infection. It was found that the
highest percentage of eosinophils occurred in the 7th and 10th tests.
The average TSCC increased as the CMT grades were increased.
The percentage of neutrophile and eoeinophils also increased.
C~~versely, the average percentage of epithelial cella and lymphocytes
decreased wi th the increase of CMT grades. The average TSCC was
10.15, 4.13, 4.03, 2.32, 2.21, 2.19 and 1.88 million cells/ml
for colifor.ms, ~. agalactiae, Staph. aureus, micrococci, other
/179
streptococci, non-hemolytic staphylococci and mixed infection,
respectively. The percentages of neutrophils were highest in coli-
form Str. agalactiae, Staph. aureus and other streptococci
infections. Conversely, the percentages of ~phocytes were highest
in non-hemolytic staphylococci, mixed infection, micro cocci and
bacteriologically negative samples. The organisms which produce
toxine seem to attract more neutrophils. The mean CMT scores
( ~x + i) for stage of lactation was found to be significant
(p~ 0.01).
The influence of lactation number (age) on GMTI was
studied in 68 cows. + The mean GMTI for these cows was 4.02 - 0.36.
The GMT scores ( ~x + i) for the different lactations were sig
nificant (P", 0.01). The incidence of ~. agalactiae infection wae
highest in the 7th lactation and zero in the first lactation. This
increased incidence from one lactation to another may be due to
damage to teate, increased milk yield, pretioue sensitization or
the degree of exposure to infection with the increase of age.
The overall incidence of infection was found to be higheet in the
4th lactation.
The average TSCC increased from the 1 st lactation to
the third lactation, decreased in the fourth lactation and again
increased in the eixth and seventh lactation. The percentage of
neutrophils ran almost parallel to the TSCC but was interrupted by
high percentages of lymphocytes in the 1st, 2nd, 4th and 5th
<;;:-.., ,
/180
lactation. The percentage of eosinophils was found to be highest
5.14%) in the second lactation cows. This m~ be due to formation
of antigen and antibody reaction which attracts the eosinophils.
This study showed that the cows of the Ayrshire breed had
a lowerOMTI than those of the Holstein breed. The GMT scores
( .jx + !) for the two breeds was found to be highly significant
(P~0.01). The overall incidence of infection was higher in Holstein
cows as compared to Ayrshires.
It was interesting to note that the average TSCC was
higher in Ayrshire breed than Holstein breed. The percentages of
neutrophils and eosinophils were higher in the Holstein breed.
Conversely, the percentage of epithelial cells and lymphocytes was
higher in the Ayrshire breed.
The average monthly GMT scores ( vix + !) for the month
of the year was f'ound to be significant (p.(. 0.05) • Fluctuations were
observed in the monthly percentage incidence of infection due to
various microorganisme. The overall infection was highest in the
month of December (83.00%). The incidence of infection during the
spring, summer, fall and winter was 79.69, 67.67, 64.68 and 73.81
percent, respectively. The incidence of ~. agalactiae infection
was highest in the summer season. It was observed that the season
of the year does influence the fluctuations of' the incidence and
extent of mastitis in cows, due probably to the effects of
ç,:,.., \
/181
environmental temperature on the growth of microorganisms. The
variations in the freshening pattern to the age of cows ~ al.so
have influence on the final outcome. It is very difficult to
weigh the importance of each of these factors in the present study.
It was observed that the average TSCC and percentage of
epithelial cells were higbest in the winter months. The percentages
of neutrophils and lymphocytes were higbest in fall and spring,
respectively.
The mean CMT scores ( ~x + !) for various Tf types of
cows were significant (P<.0.05). The highest CMTI was obtained for
Tf type AD1 and lowest in TfD2E.
The percentage of Str. agalactiae infection found in the
various Tf types was TfAD1 (24.17%), TfD1D2 (22.73%), and TfD2D2
(20.83%). It was found that in Tf types possessing alleles ~fD1
and TfD2, the percentage incidence of quart ers infected with this
organism was high.
The highest overall incidence of infection was found in
TfD2E (95.00%). Unfortunately, there was onlyone cow in this
type, so no conclusion could be drawn. With the elimination of this
one cow, the next higbest type was TfD1D2 (78.03%), followed by
TfAD1 (76.67%) and TfD2D2 (75.2e.%). In the rest of the Tf types,
the infection incidence was lower than the average (71.52%) for aIl
10 types.
ç,:.. \
/182
The average TSCC was found to be highest in TfD1D1 (4.69
million celle/ml) followed by TfD1E (3.51 million celle/ml),
TfD1D2 (3.47 million celle/ml) and TfAD2 (3.41 million cells/ml).
In the ~eet of the Tf typee, the TSCC was lower than the average
(3.29 million cella/ml).
The percentage of neutrophile was fOtUld higheet in type
TfAE followed by TfAD2, and TfD2D2. Conversely, the percentage of
lymphocytee were highest in TfD2E, TfEE, TfD1E, TfAA and TfD1D1.
It does appear that neutrophils pl~ a considerable role in the
resietance or eusceptibility to infection. Since the Tf type
frequencies of some of the types are very low because of the sample
size, it is difficult to make a clear-cut conclusion.
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i'.
APPENDIX
Analysis of Variance of Mean. CMT Scores (/ x + i>
Source of Variation d.f.
Bree4 1
Transferrin Type 15
Cow 51
Month of the Year 11
Stage of Lactaiion 9
Error 578
* S1gnif1cant at P < 0.05.
** S1gn1ficant at P (0.01.
S.S. M.S.
37.34 37.34
8.64 0.58
321.66 6.31
5.45 0.50
38.53 4.28
131.43 0.23
F. cal..
162.35**
2.52**
27.43**
2.17*
18.61**