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, 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 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 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 percentagoeof 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 TfD 1 and TfD2.
Transcript
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
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
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). --
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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
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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.
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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
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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.
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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
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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).
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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
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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
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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
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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)
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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.
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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
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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
* 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
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
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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APPENDIX
Analysis of Variance of Mean. CMT Scores (/ x + i>
