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thesis entitled

EVALUATION OF I'IECXTYCIN SULFATE IN THE I‘RLLT. ENT

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.l“ ‘I

' OF BOVINE 1"'C‘TITIS

presented bg

has been accepted towards fulfillment

of the requirements for

Masters degree in Surgery and Lledieine

 

M E? ltji/L'v-I'rwh‘x

Major professor

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EVALUATION OF NEOMYCIN SULFATE IN THE

TREATMENT OF BOVINE MASTITIS

By

A. R. Drury

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Surgery and Medicine

1952

s

ACKNOWLEDGMENT

It is indeed regrettable that the late Dean C. S. Bryan
was unable to see the completion of this work which he did
so much to bring about. The author fully appreciates his
guidance and encouragement.

The author hereby expresses sincere thanks to all with
whom he came in contact and particularly Drs. B. V. Alfredson,
W. 0. Brinker and H. J. Stafseth for their aid in the prepa-
ration of this manuscript.

This work would have been impossible without the fine
cooperation of Drs. G. S. Stocking and J. L. Davidson of the
Upjohn130mpany in Kalamazoo, whose company furnished all the
neomycin used in this work in addition to technical assistance
in solving many problems concerning assay procedures.

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TABLE OF CONTENTS
Page
INTRODUCTION....................................................... 1
REVIEW OF LITERATURE............................................... 2
Neomycin...................................................... 2
Organisms Causing MastitiS.................................... 7

Local Irritation as Evidenced by Leucocyte Response........... 8

DiagnOSisooooooooo00.000000000000000.00..0.0000000000000000... 9

AssayOOOOOOOOOOOO00......OOOOOOOOOOOOOOOOOOOO00......000...... lo

WWHHMS1WDMEHWB.H.n.n.u.u.u.u.u.n.n.n.u.u.u.n. 13
Sensitivity Test.............................................. 13
Cultures................................................. 13
Standard Solution of Neomycin............................ 13
Method................................................... 13
Irritation Studies............................................ 1h
AnimalS.................................................. 1h
Sampling................................................. 15
Effect of the Drug on the Udder and Milk................. 15
Assay of Neomycin............................................. 1?
Animals Used and Administration of Drugs................. 17

SamleSOOOOOO0.00.0...OOOOOOOOOOOOOOOOO0......0.0.0.0...O 1?

Assayoooooooooooo.ooocooooooooooooooooooooooooooooo000.00 1?

MilKOOOOOOOCOOOOOOOOOOOOOO00.0.00...OOOOOOOOOOOOOOOOOOOOO 20

TABLE OF CONTENTS - Continued

DiagnOSiSOOOOOOOOOOOOOOCOOOOOOOOOOOOOO0.0.0... ..... 0.0.0.0....

Direct Microscopic Method for Diagnosis of Mastitis......
Identification of OrganismS..............................
Treatment of AnimalS..........................................
Acute Cases..............................................

Chronic Mastitis.........................................

RESULTS AND DISCUSSION.............................................
in 33333 StudieS..............................................
Irritating Effect on the Udder................................
Retention of Neomycin.........................................

Milk.....................................................
Blood....................................................
CLINICAL RESULTS AND DISCUSSION....................................
SUMMARY............................................................

BIBLIOWI‘HOOOOOOOOOOOOOOOOOOOOOO...IOOOOOOOOOOOOOOOOOOO0.0.000...

APPENDIX...OOOOOOOOOOOOOOOOOOOOOOO0.0.0.000...OOOOOOOOOOICOOOOOOOOO

Page

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56

INTRODUCTION

INTRODUCTION

Mastitis has been a problem to the dairyman ever since cows have
been kept for milk production. Antibiotics have brought many improve-
ments to the health and welfare of our society. Veterinarians have
always been eager to explore new treatments for mastitis. The results
of these investigations have created an immense volume of literature
concerning the relative merits of the various treatments.

One of the more recent antibiotics is neomycin. This drug has been
shown to be effective against a wide variety of organisms; therefore, it
is logical that neomycin be investigated for its effectiveness in the
treatment of bovine mastitis since there are several genera of causative
organisms involved in this disease.

In this study I have taken neomycin which had never been used in
dairy cattle and attempted to evaluate its safety, efficacy, and limi-
tations in the treatment of bovine mastitis.

The following points have been considered: (1) The in 31:33 effects
of neomycin on the causative organisms of mastitis. (2) Its effects on
the udder tissues and milk. (3) Retention of the drug when infused into
the udder. (b) Blood levels following a single intramuscular injection.
(5) Results of its use in hOS cases of mastitis based on bacteriological

findings.

REVIEW OF LITERATURE

REVIEW OF LITERATURE

I. NEOMYCIN

Neomycin as an antibiotic of therapeutic significance was first
brought to the attention of investigators in 19t9 by Waksman and
Lechevalier (l9h9a). They isolated the active principle from a soil

organism now listed in Bergy's Manual of Determinative Bacteriology as

 

Streptomyces fradiae. Neomycin sulfate is a basic compound. Originally
it was thought to be more active in an alkaline medium. This was later
shown to be inconsequential by the same workers (1950). It is soluble in
water, insoluble in organic solvents, thermostable, and active against
many gram.positive and gram negative bacteria. It was first mentioned as
an active agent against streptomycinpresistant bacteria, particularly
tubercule bacilli. Waksman listed among the desirable preperties of
preparations of neomycin: 1. Similar activity against both streptomycin-
sensitive and streptomycin-resistant microorganisms. 2. Considerable

activity against various forms of Mycobacterium tuberculosis and other

 

mycobacteria. 3. Limited or no toxicity to animals. h. Marked activity
against other bacteria in 3113, including gram negative and gram positive.
5. The deve10pment of resistance against neomycin among organisms sensi-
tive to it has not been reported.

Waksman et al (l9h9b) subsequently reported that neomycin is highly
effective against the ordinary pathogenic gram negative and gram positive

bacteria in a dose about 1/20 - 1/50 the toxic dose. waksman, Frankel

and Graessle (l9h9c) reported that a crude neomycin, varying in potency
from 30 - lOO units/mg, was effective in experimental infections of

Staphylococcus aureus in mice, Salmonella schottmulleri of mice and chick

 

 

embryos, Salmonella pullorum of chick embryos, and Eberthella typhosa of

 

mice. Neomycin was as effective against streptomycin-resistant strains of

Staph. aureus and S; schottmulleri as against the streptomycin sensitive

 

 

strains. They also recorded that neomycin exerted no serious toxic
effects when instilled into the eye of a rabbit which is a standard pro-
cedure to determine irritability of a substance.

Hobby, Lenert and Dougherty (l9h9) produced tables showing the anti-
bacterial spectrum of neomycin which reveals that it is highly active
against many gram negative microorganisms. It was less active against
gram positive organisms and showed no activity against twenty strains of
pathogenic fungi. They also reported that neomycin could be readily de-
tected in the serum of injected animals.

Harme, Bernstein and Donovich (1950) reported that a dose of neomycin
of 10 mg per egg prolonged slightly the survival time of embryonated eggs
infected with vaccina virus, but did not prevent their death. 'Warth,
Chandler and Bliss (1950) reported that neomycin was the only antibiotic
studied in their laboratory that was potent against both Proteus and

Pseudomonas. Compared with aureomycin in vitro, neomycin was just as

 

effective against gram negative bacilli of the coli-aerogenes group,

Hemophilus influenzae staphylococci, and gram positive bacilli. It was

 

more effective against strains of proteus and pseudomonas and was less
active in inhibiting the growth of streptococci. Neomycin was valueless

in infections With hemolytic streptococci and the pneumococcus.

Felsenfeld, Volini and Ishihara (1950) stated that the action of
neomycin is strong, both in vitro and in vivo against gram negative rods,

tubercle bacilli, Bacillus anthracis, Listeria monocytogenes and intesti-

 

nal protozoa. Its action on gram positive cocci was variable. Adequate
doses of neomycin.in‘zivg were able to prevent experimental salmonellosis,
cholera, tuberculosis, amebiosis and rickettsialpox.

A report of the committee on.Medical Research and Therapy of the
American.Medical Association (1950) stated that neomycin, which is active
against the tubercle bacillus in zitgg and in 3139, is too toxic for
treatment of human tuberculosis, even on an experimental basis. The
unfavorable reactions attributed to neomycin treatment are deafness and
renal impairment.

Welch,Reedy and Wolfson (1950) reported on nine different common
antibiotics when used against experimental typhoid in mice. They placed
neomycin in the middle and chloromycetin is the most successful of the
group in treating cases of typhoid fever in man.

Waishren and Spink (l9fl3) showed that neomycin was bactericidal in
concentrations equal to or only slightly higher than those in which it
was bacteriostatic.

Waksman, Katz, Lechevalier (195C), in an extensive study, showed a
very complete antibiotic spectrum for neomycin. Neomycin is active
against a variety of gram positive and gram negative bacteria, acid fast
bacteria and actinomycetes, but not against fungi and viruses. It has a
strong bacteriostatic and bactericidal effect upon various bacteria. The

amount of the antibiotic required for complete destruction of cells is

definitely correlated with the number of bacterial cells in a given
inoculum. No absolute resistance to neomycin has been observed. Neomycin
is highly stable; no activity was lost when a solution of neomycin was
adjusted to pH values ranging from 1.5 to 12 and kept at room.temperatuna
for twenty-four hours. Nor was there any loss in activity when a solution
adjusted to pH 2 was heated for ten.minutes at 60° and 100° C. A soluthan
with a pH of l or 10 lost hO per cent of its activity after ten minutes of
heating at 1200 C.
Felsenfeld, Volini, Young and Ishihara (1950) in a later report

stated that neomycin is very effective against tuberculosis, typhoid, and
other salmonella infections, Shigella, Vibrio cholerae, Pasteurella pestis,

Brucella, and Entamoeba histolytica. It was not effective against the

 

small sized viruses such as that of poliomyelitis. The same authors in
another paper (1950b) reported that in 127 patients, who received neomycin,
unfavorable sequelae develOped in two cases. There was impairment of hear-
ing in one case and kidney irritation in the other. They also stated that
oral routes are not satisfactory for systemic therapy.

Clancy (1951) investigated the sensitivity of 171 strains of organisms
commonly encountered in a hospital diagnostic laboratory. He found neo-
mycin to be highly active against Aerobacter aerogenes, Klebsiella

pneumoniae and Micrococcus aureus variety pyosenes. All the gram positive

 

 

rods studied yielded except clostridia. Streptococci were rather resistant
when tested in a tube test. Many proteus and pseudomonas strains were

moderately sensitive to neomycin upon overnight incubation; bacteriostasis

was evident up to seven days and showed an unusual potentiality for the
development of neomycin resistance.

In zitgg work done by Prier (1951) with the seven commonest anti-
biotics showed terramycin to rank first and neomycin second in activity

toward four strains of Vibrio fetus.

 

waishren (1951) divulged that neomycin was used in two cases. In one
case before its toxicity was established, and in the second case, when
$2.2i352 tests showed it to be by far the most active agent against an
unclassified bacillus isolated from the blood of a critically ill child.
He felt the neomycin probably saved the life of the patient in both
instances but attributed the deafness that deveIOped in one child to a
harmful effect of the treatment.

Poth, Fromm and Wise (1951) felt that oral medication of neomycin
was an excellent intestinal antiseptic as it reduced the bacterial flora
of the stool to nil. They reported yeasts to be present. They also felt
certain that there was no influence on the blood clotting mechanism or
clotting time. They observed that neomycin.promotes tissue healing in
intestinal surgery.

When neomycin is administered parenterally it is found in the blood
and eliminated in the urine. The only assay procedures for neomycin are
those that Hanson and Collingsworth (19h?) adapted from procedures used

for streptomycin in blood and urine.

II. ORGANISMS CAUSING MASTITIS

According to the E. Munch-Petersen survey (1938) of the literature
on bovine mastitis, credit goes to Nocard and Mollereau (188h; 1885; 1887)
for the first description of a microorganism, and, more particularly, a
streptococcus as a cause of mastitis. No specific generic name was
assigned to the organism but it was listed as having the same biochemical

behavior as the organism that is now known as Streptpcoccus agalactiag.

 

Bang (1889) described an organism identical with that described by
Nocard and Mollereau as a cause of mastitis, (E. Munch-Petersen 1938).

In the book, Bovine_Mastitis_, by Little, Brown and Plastridge, (who),

 

Chapter V, it is stated that "Streptococci are regarded as responsible for
at least 85 per cent of the chronic mastitis observed in dairy cattle.
In about 80 per cent of these conditions the organism responsible is

Streptococcus agalactiae. Other streptococci in order of importance are

 

Str. uberis, Str. dyagalactiae and strongly hemolytic Group C and Group G

 

 

 

streptococci (Lancefield)."
The Opinions of the various workers differ as to the role of staphy-

lococci as the etiological agent in mastitis. In the book, Bovine Mastitis

 

it was stated: "While some reports in the early literature suggested
that staphylococci were normal inhabitants of the udder, others incrimi-
nated staphylococcus as a cause of mastitis. In 1889, Lucet reported the
finding of gelatin-liquefying micrococci in the secretion of 7 of 21
'animals affected with mastitis. Other early investigators, including

Guillebeau, Steiger, Savage, Jones, Carpenter, Hardenburg and Schlotthauer

found that staphylococci were present in a significant prOportion of
abnormal udder secretions in.which streptococci were absent."

Miscellaneous organisms causing mastitis, described in the textbook

 

Bovine Mastitis (19h6), are: Escherichia coli, Aerobacter aerogenes,
Corynebacterium pyggenes,.Pseudomonas aeruginosa, Pasteurella sgptica,

Actinomyces necrophorus, Clostridium perfringens and yeasts.

 

III. LOCAL.IRRITATION AS EVIDENCED BY LEUCOCYTE RESPONSE

Leucocytes are normally found in freshly drawn milk. However in
abnormalities involving the mammary gland, the leucocyte count is altered,
both in numbers and kind. Merely striking the udder with the fist will
cause an outpouring of leucocytes. They are a good indication of irri-
tation produced by certain substances in the udder, (Bryan 19h8). Even
reinjection of withdrawn milk will result in a temporary increase in the
white cell count. Authors do not agree as to what is a normal leucocyte
count in milk, but it is generally assumed that there are less than a
million leucocytes per ml in milk from.a healthy, normal udder of a cow
(Bryan 19b8). However, in an infected udder, there is not necessarily a
'high cellecount. Klein and Learmouth (1935) shOwed that irritants increase
cell counts which are a measure of the irritating qualities of a product.
This, then, to some degree, would be a toxicity determining factor. Many
drugs create a high cell count which persists for a.period of several
days. Others give only slight increases of only a few days' duration.

A cow will establish a pattern of numbers and will generally return to a

similar level after stimulation by an irritant.

IV. DIAGNOSIS

From the many articles that have been published dealing with diag-
nosis, it would seem that the direct microsc0pic procedure is the most
reliable. Particularly significant are the early reports of Plastridge
(1935) dealing with microscopic diagnosis. Murphy (1932), Hucker (l9h2,
Biddle (l9hh), Cone (19h0), Ferguson (19h0), Kelser (1950), Klien (1935,
1938), Palmer (l9h0), Schalm (l9hh), Slanetz (1939), and Udall (19h?) all
contributed to the development of mastitis diagnostic methods. Hotis
(1936) developed the test that bears his name. Kleckner (l9hl, 19h2a,
l9h2b) published a great deal on efficiency and practicability of various
procedures to diagnose mastitis.

Murphy (1939, l9h1, l9h3a, 19h3b) also published on the efficiency
and practicability of some methods. Bryan (19h?) modified the Hotis pro-
cedure to add convenience and accuracy. Several outstanding articles by
Bryan et al (1932, 1933, 19140, 19143, 191.7, 191:8) both of a review nature
and original contributions have appeared. The book of Little and Plastridge
(19h6) on bovine mastitis gives an excellent review of methods of diagnosis.
The recent bulletin by Merilan, Herman, Edmondson, Tallman and Crisler
(1950) from the Missouri Agricultural Experimental Station gives much
information on the evaluation of diagnostic procedures.

The publication of Brown and Bryan (1950) is very interesting. They
collected samples from a non-infected and an infected cow's udder every
day for six months. On the basis of microsc0pic examination of the speci-
- mens collected they demonstrated the reliability of the direct microscopic
procedure. As a matter of fact, they reported 100 per cent efficiency

for this method, the only one that is so reliable. The work of Brown and

10

Bryan showed blood agar and Edward's Esculin.Agar to be 96 per cent, the
Hotis test 85 per cent and chloride content percentages as only 35 per
cent accurate. The leucocyte count method was only 83 per cent accurate,
giving wrong results in both positive and negative cows. Thybromol tests
gave 11 per cent inaccuracies over all.

In the final analysis the results of Brown and Bryan were comparable
to the results published by Merilan et a1 (1950), Malcolm (l9b2), Fay

(1938), Roach (19th), Rowland (1939), Johns and Hastings (1938).

V . ASSAY

To learn something of the sojourn of a drug, in the body, assay
procedures for body fluids have been developed. These must be studied
when evaluating a new antibiotic. Detailed procedures for assay of

penicillin are discussed by Schmidt and.Moyer (l9hh). They described a

cylinder plate method and a serial dilution procedure using Staph. aureus.

 

Rosenblatt, Altun, herber, Kashdan and Loeuve (l9hh) gave a procedure
for the determination of penicillin levels in body fluids involving M1.
pyogenes with serial dilution techniques. g

Cooke (l9h5) deveIOped a simple clinical method for assay of
penicillin in body fluids. He first used a known concentration of the
antibiotic against the organisms to establish their sensitivity. He then
used the body fluids containing the unknown concentrations of penicillin
against these organisms to determine the amount of antibiotic present.

Kirby and Rantz (19hh) discussed the Oxford cup detection method and

attempted to establish the superiority of a turbidimetric method. This

presupposes that a clear solution is to be examined. Therefore, another
problem is encountered when milk is the fluid involved.

DeBeer and Sherwood (l9h5) evolved a paper disc agar plate method
for the assay of antibiotic substances. They used absorbent paper discs
and established curves with known amounts and plotted unknowns into a

curve on a graph using Bacillus subtilis as a test organism. They demon-

 

strated statistically the accuracy of this type of assay.
Price, Nielsen and welch (l9h6) adapted a procedure for estimation

of streptomycin in body fluids. They selected Bacillus circulans as the

 

test organism and adapted a suitable serial dilution technique.

Loo, Shell and Thornberry (19h5) published a procedure for assay of
streptomycin by the paper disc method. They used a susceptible strain of
B. subtilis. Standard curves were prepared from zones of inhibition on
Bacto-streptomycin assay agar using a Schleicher, Schnell disc containing
a volume of 0.08 ml of antibiotic delivered with a calibrated pipette of
this capacity.

Hirsch (1950), writing on the assay of the antibiotic nisen, stated
that because nisen does not diffuse quickly through the agar it must be
assayed by dilution methods. Also, because it is bactericidal and not
merely bacteriostatic it may be assayed by estimating numbers of surviving

bacteria. ‘With Str. agalactiae as the test organism, it may also be

 

assayed in terms of acid production.

Bond and Nook (19h8) could detect as little as 0.02 units per m1 of
bacitracin in blood serum, urine or saliva. They used a hemolytic strepto-
coccus in penicillin assay agar plates. Their method involved a standani

curve .

12

watts and McLeod (l9h6) suggested a way to estimate penicillin levels
in blood serum and milk of bovines after intramuscular injections by the
use of litmus milk and susceptible acid producing strains of bacteria in
various dilutions. Other outstanding articles have appeared by Foster and
hbodruff (19u5), Jackson and Finland (l9h5), Randall et a1 (l9u5) and
belch et a1 (19h5).

The Federal Security Commission (1950) through the Food and Drug
Administration maintains and publishes up-to-date information on methods,

criteria and regulations pertaining to the assay of antibiotics.

MATERIALS AND METHODS

13

MATERIALS AND METHODS
The following procedures were used in performing the investigation.
I. SENSITIVITY TEST

A. Cultures

Cultures of Staph. aureus 2g, Str. agalactiae‘lg, Str. dyagalactiae

 

 

 

23, Str. uberis 22, E, coli 29 and Pg. aeruginosa.33 were used. These

 

 

were from a stock accumulated from cases of infectious mastitis by various
members of the staff of Bacteriology and Surgery and Medicine over a period
of many years. They have been maintained by monthly transfer on tryptose
agar slants and stored in a refrigerator at 38° - LOO F.

The growth phase of these cultures was stimulated by daily transfers
for several days. This tends to stimulate a constant, rapid, uniform and

more vigorous growth when used in a sensitivity test.

B. Standard Solution of Neomycin

 

One quarter of a gram of neomycin sulfate was weighed and dissolved
in 25 ml of sterile distilled water; therefore each ml contained .01 gm.
Ten ml of the solution was combined with 90 m1 of sterile distilled water.

Thus, eachrul contained 0.001 gm.

C. Method
1. A row of 25 sterile 6 inch tubes was placed in a rack.
2. One ml of Difco's nutrient tryptose broth was placed in all but

the first tube of the above series.

3. Using a one ml sterile pipette, one m1 of neomycin containing
0.001 gm (Step B) was placed in tubes 1 and 2. The contents of tube 2
was mixed and one ml was transferred into tube 3 and then mixed. One ml
was removed from tube 3 and mixed with the contents of tube h and so forth,
resulting in tube 1 containing .001 gm per ml; tube 2 .0005 gm per ml ard
tube 3 .00025 gm per m1 and so forth.

h. Each series of tubes was then inoculated with a standard lOOpful
of the 2h hour tryptose broth culture of each bacterium.

5. The tubes were incubated at 37° C. for forty-eight hours.

6. The end point was considered to have been reached in the tube
which first showed growth. If, in the series, the tube to first show
growth was tube 3, .0005 gm.per ml was the lowest concentration of neo-

mycin to inhibit that organism.

II. IRRITATION STUDIES

A. Animals

Normal, healthy cows were used. The animals were housed in separate
box stalls, fed a good quality hay and grain ration. They were hand
milked at 7:30 in the morning and h:30 in the afternoon by the same care-
taker.

Each quarter of the udder of a cow is a separate functional unit as
shown by the work of Turner and his group (19h8). Bryan (19h8) also
showed that reSponses to irritation with resulting changes are confined

to the local area or quarter involved.

15

A period was Spent establishing normals for each quarter of an udder
by regular collection of milk samples and determination of their constitu-
ents. Varying amounts, as indicated in the results, were infused aseptic-
ally into the quarters.

In preparation for infecting a quarter with S. agalactiae, the same
strain used in the sensitivity procedure was transferred in broth for
several days. Two m1 of a 2h hour broth culture was introduced with a
teat canula. The udder was roughly handled to insure establishment of
the infection. Daily samples were collected to insure continued infection

of the quarter.

B. Sampling

Samples were collected at 8:30 A.M. and h:00 P.M. An observation
period of one week was used during which regular samples were collected.
A strip cup was used in order to observe the gross appearance of the milk.
The ventral aspect of the udder and particularly the teat orifices were
then treated with a solution containing hOO P.P.M. available chlorine.
Streams of milk were drawn directly into a hO ml test tube and the samples
were immediately taken to the laboratory. Sampling continued at regular
intervals until the milk was normal in all respects. Observation and

palpation of the udder were made whenever samples were collected.

0. Effect of the Drug on the Udder and Milk

 

1. Leucocyte Count: The procedure used was that of Bryan, Mailman

 

and Turney (19h?) devised for counting bacteria in raw milk. In brief,

a flamed loop with an outside diameter of h mm, which was made of a 2h

16

gauge German silver wire, bent around a 6 penny box nail, was immersed
and withdrawn from the sample of milk. The milk was Spread over an area
h mm x 8 mm on a grease-free glass slide. The film was air dried and
stained by the three jar procedure of Xylol, 95 per cent alcohol and
methylene blue, one minute in each. The slide was washed on the reverse
side of the film to remove the excess stain. After the slide was dry, the
smears were examined under an oil immersion objective with a calibrated
microscope. The microsc0pe used has a factor of 300,000 so if each field
has an average of one leucocyte, the milk has a count of 300,000 per m1.

2. The Methylene Blue Test: The procedure used was described in the

 

American Public Health Association Standard Methods for the Examination

 

of Dairy Products 9th edition. This test, when used on freshly drawn

 

milk, is a measure of leucocyte concentration and not bacterial reduction
in milk (McClement 1939 and McBride 1951) .

3. The Resazunin.Test: This test is a determination similar to that

 

of the Methylene Blue Test. The procedure was described in the A.P.H.A.
Standard Methods for the Examination of Dairy Products, 9th edition.

h. Hydrogen ion concentration, pH: A model H-2 Beckman pH meter was

 

used to determine the pH of the milk. The meter was standardized prior
to use with a buffered solution of pH 7.

5. Chloride content: The procedure was that of Cone (l9h0), using

 

.2 ml of milk placed in a test tube. Five tenths cc of KgCr03 and .5 m1
of AgN03 was added. 'With a 1 m1 pipette with .1 graduations, milk was

added by .1 m1 quantities until a constant 1emon.yellow color deveIOped.

17

From the amount of milk needed for the titration the percentage of chloride
in the milk was determined by reference to a chart.

6. Standard Plate Counts: The procedure used was that described in

 

the 9th edition of the A.P.H.A. handbook for milk analysis.

III. ASSAY OF NEOMYCIN

A. Animals Used and Administration of Drugs

 

Healthy cows maintained under optimal conditions of herd management
were used. The neomycin was dissolved in sterile distilled water. The
injection site was cleansed with liquid germicidal detergent. A steam
sterilized needle and syringe were used to inject the material in the

gluteal muscle.

B. Samples

The jugular furrow was rubbed with an antiseptic. California bleed-
ing needles that were steam sterilized were used to pierce the jugular
vein and 10 ml of blood was allowed to flow into a sterile Kahn vial con-
taining 3 crystals of sodium citrate (C.P.). The vials were gently shaken

to prevent clotting.

C. Assay
This procedure was deveIOped by Hanson and Collingsworth (l9h9) and

is reproduced with their permission. The neomycin assay method follows:

1. Preparation of Plates: The plate medium contains 25 gm of Difco

 

streptomycin assay agar per liter. The medium can also be prepared from

18

the ingredients listed in the reference cited above. The pH of the medium
should be 8-0.1 0.1.

After sterilization the medium is cooled to 600 C. and about 7 ml.
of the E. subtilis Spore suspension is added per liter. The volume of
Spore suSpension to be used is indicated on each bottle of spores. The
medium is shaken thoroughly and allowed to set until no air bubbles are
present.

Five ml of inoculated agar is transferred to each flat bottom Petri

dish and allowed to solidify. It is important to prepare the plates on a

 

level surface to obtain a uniform thickness of the agar layer. The agar
plates should be stored at refrigeration temperatures until used.

2. Addition of NaOH to Blood: The pH of blood samples and normal

 

blood for diluting is adjusted by adding 0.1 ml of 0.1 molar NaOH to 5 ml
of blood. About LO ml of normal adjusted blood is required for the prepa-
ration of standard solutions and dilution of samples.

3. Preparation of Standards: An aqueous solution containing 1 mg of

 

neomycin.per ml is prepared, and 0.1 ml of this solution is added to 6.15
ml of adjusted normal blood. This blood contains 16 micrograms of
neomycin.per m1 and this is the highest level used for the standard curve.
Serial dilutions with adjusted blood give standards containing 8, b, 2, 1
and 0.5 micrograms of neomycin per ml.

h. Preparation of Samples: Blood samples obtained after treatment

 

are adjusted with NaOH as described above. If levels higher than 16
micrograms per ml are anticipated, serial dilutions are prepared using

adjusted normal blood. Several dilutions of each sample are set up on

19

the assay plates so that inhibition zones of at least one dilution will
be within the range of the standard curve.

5. Plating the Standards and Samples: Paper discs, having a diameter

 

of l/h inch, are saturated by dipping them into either standards or
samples. Excess solution is removed by touching the discs to the edge of
the sample bottle.

For setting up the standard curve, discs containing the six standards
are distributed on a plate and eight replicate plates are made. A disc
for each of five blood samples and of the 2 micrOgrams per ml standard is
placed on a plate and four replicate plates are prepared. The plates are
placed in an incubator at 30° C. for 16 to 20 hours.

6. Estimation of Potency: After incubation, the diameters of the

 

zones of inhibition are measured in.mm and diameters of replicate zones
are averaged. Zone diameters for the six standards are plotted against
micrograms of neomycin.per ml to obtain the standard curve. Typical read-

ings are given below:

 

Neo cin Zone Diameter

micrograms ml. mm.
16 1h .1

8 12.6

h 11 .6

2 10.2

1 8.6

0.5 7.h

Zone diameters for the blood samples are converted to micrOgrams
per ml by using the standard curve. An example of a typical result is

given below:

 

 

 

Potency of Potency of
Sample Dilution Zone Size Dilution Sample
mm. micrograms/m1. micrograms/ml.
1 hr. 1:2 11.8 5.0 10.0
l:h 10.8 2.8 11.2
D. Milk

1. The preparation of the plates was identical with the procedure
used for blood.

2. Preparation of Standards: An aqueous solution containing 1 mg of

 

neomycin was added to 6.15 ml of skim milk. (This skim milk had been
boiled for one half hour before use.) Milk with a normal pH of 6.? was
chosen. This milk sample then contained 16 micrOgrams of neomycin.per ml
and this was the highest level used for the standard curve. Standards
with Skim milk were prepared by serial dilution containing 8, b, 2, l and
0.5 micrograms of neomycin per ml.

3. When levels greater than 16 micrograms per ml were anticipated,
dilutions using sterile distilled water were made.

h. The balance of the procedure was the same as steps 5 and 6 as

used for assay of blood.

IV. DIAGNOSIS

A. Direct Microscopic Method for Diagnosis of Mastitis

 

Samples were never collected within two hours after a complete milkp
ing. The ideal time as reported by Bryan (19b?) was prior to a milking.
A strip cup was used to discard the first streams of milk and remove the

seal from the teat orifice. This also permitted a gross examination of

21

the milk. The cow's udder was then washed with a chlorine solution.

A sample was collected in a sterile container and brought to the labora-
tory. Where plating and culturing was indicated, plates were made at
this point. ‘Where only direct microscopic results were indicated, samples
were incubated fifteen to eighteen hours at 37° 0.; films were prepared
as mentioned for leucocyte counts. These films were then examined by
microsc0pe using the oil immersion objective. If any chains of five or
more elements were noted, the sample would be considered infected with
streptococci. If there was a high cell count and clusters of micrococci,
a diagnosis of staphylococcal infection was made. Also needed was an
accompanying history of some gross abnormality (Bryan l9h7). When short
rods and cells plus a clinical history of acute onset of infection was
noted and a subsequent transfer of milk to brilliant green broth resulted
in gas formation upon incubation, a diagnosis of coliform infection was
made. If no gas formation occurs, a diagnosis of pseudomonas infection
was tentatively made and confirmed taxonomically. In the cases showing
gross abnormality of the udder with no bacteria demonstrable, a diagnosis
of non-infectious mastitis was made. When no bacteria or high cell count
or history of abnormality was present, the cow was considered negative,
(Bryan 19h7). A cow that shows non-pathogenic bacteria at 21 days was

considered cured, (Federal Security Agency 1950).

B. Identification of Organisms

 

Primary culture was made on 5 per cent blood tryptose agar and

Edward's Esculin agar. Suspicious colonies were picked and grown in

22

tryptose broth and then classified according to Breed's (l9h8) book

Bergey's Manual of Determinative Bacteriology.

 

V. TREATMENT OF ANIMALS

A. Acute Cases

 

The majority of these animals were in the college herds. Excellent
c00peration existed between the departments of Dairy Husbandry and Surgery
and.Medicine. *Whenever an animal showed any abnormality involving the
udder or milk a call for service was immediately made. Such cases were
attended before four hours had elapsed from the previous milking. When
an acutely affected animal was first seen by the veterinarian the tempera-
ture was taken, the udder and milk examined, and samples were collected.
The involved quarter was milked dry.

If there was a fever, sulfamethazine was given orally at the rate of
1 grain.per pound of body weight. If the cow remained febrile the next
day, the dose was repeated. When a cow remained dull and there was atony
of the rumen, ruminatorics were administered. If an animal returned to a
normal temperature and did not eat on the third day, a rumen liquor
transfer was administered from a rumen fistula cow on the farm.

Local treatment of the udder consisted of removal of the milk, wash-
ing the teat orifice and infusion of 0.5 gm neomycin sulfate dissolved in
10 ml of water. If the condition of the animal remained unsatisfactory,

medication of the udder was repeated. Whenever material was placed in the

udder, it was followed by massage to facilitate more extensive permeation

23

of the drug. Samples of the milk were collected at intervals and examined
for the presence of bacteria following treatment. A re-check was always

made at an interval of 21 days after treatment.

B. Chronic Mastitis

Only cows known to be infected were treated. All four quarters were
always treated and quarter samples were collected at the time of treatment
to determine the quarters actually infected, and, when cows were retested,
quarter samples were collected from treated cows and subsequent treatments
were administered to infected quarters. Cows were treated at a time when
there would be at least a lO-hour time lapse before a milking. The amount
of the aqueous solution used varied from 5 ml to 30 ml in which 0.25 to 1
gm of neomycin was dissolved. The ointments contained 0.5 gm of neomycin
per 3.5 gms of vehicle.

The udder and teat orifice were always washed with a bactericidal

solution.before anything was placed in the udder.

RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

I. _I__N_ VITRO STUDIES

‘When the organisms from infectious mastitis were exposed to the

dilutions of neomycin as outlined in the procedure, Staph. aureus showed

 

no growth until tube 20; therefore, 0.000000039 gm or 0.039 micrograms

of neomycin sulfate per ml inhibited Staph. aureus strain 26 for h8 hours.

 

Str. agalactiae grew first in tube 21 so 0.000000020 gm (0.02 micrograms)

 

per m1 inhibited its growth. Str. gyagalactiae grew first in tube 21 but

 

failed to grow in tube 20, so in this case 0.02 micrograms inhibited this

organism. Str. uberis was less vigorous as it failed to grow in tube 21

 

but grew in the remaining tubes. Thus 0.010 micrograms inhibited this
strain of the organism, E. coli #30 was less susceptible; showing growfll
from tube 17 on, therefore 0.31 micrograms of neomycin was needed to

inhibit the growth of this organism, Pg. aeruginosa grew from tube 15 on,

 

therefore 1.25 micrograms per ml was needed in this determination.

This series was duplicated using the same procedure and only the end
point of E, 2211 varied, which went out another tube. A third determina-
tion was made using 5 less tubes and diluting stock solutions of neomycin
1 ml to 99 ml or 1-100 instead of the 1-10. The results are set forth in
the Table I. All discrepancies can be attributed to normal variations

in the dilution technique. These results show the small concentration of

neomycin that was sufficient to control the growth i2 vitro of the

principal organisms which are at present known to be the etiological agents
of infectious bovine mastitis. Since some of the organisms are gram
positive and some gram negative it shows the effectiveness of the drug for

both groups.

II. IRRITATING EFFECT ON THE UDDER

In eventual acceptance of a drug for mastitis treatment, little or
no irritating effect on the udder is of prime importance. Changes in the
milk with respect to gross appearance, pH, chloride content and leucocyte
counts are indications of the irritating effect of a product when.p1aced
in the udder, (Jackson 1950).

Table II shows leucocyte counts, chloride content, pH, resazurin
class and standard plate counts respectively for an animal. The milk from
this cow was normal in appearance on strip cup examination at all times.
Normal leucocyte counts were from 60,000 per ml to 300,000 per ml before
any material was introduced into the udder. The animal was free from
bacterial infection. The cow was a grade Holstein, giving about 20 pounds
of milk a day. Her butterfat test was 3.3.

After injection of: one gram neomycin in the right front quarter,
0.5gmxin the left front, 0.25 gm in left rear and 10 ml of water in the
right rear quarter which was the vehicle used for all infusions. The
leucocyte counts seven hours after treatment were 15 million, 15 million,
10 million and 100 thousand respectively. Twenty-four hours later counts

were still high, showing 10, 5, 3 millions and 100 thousand respectively.

26

TABLE I

Number of Micrograms of Neomycin Sulfate Required to Inhibit
Mastitis-causing Organisms for h8 Hours 12 vitro

 

*- ~— .,

Test Organism lst_Trial 2nd Trial 3rd Trial

Str. agalactiae #15 0.02 0.02 0.039
Str. dyagalactiae #22 0.02 0.02 0.02
Str. uberis #29 0.01 0.01 0.01
Staph. aureus #26 0.0).; 0.0L. 0.01:,
E. coli #30 0.31 0.155 0.31
Ps. aeruginosa #33 1.25 1.25 1.25

27

By 96 hours the cell counts were nearly normal. At no time was there any
gross abnormality of the milk.

During the period preceding injections the chloride content was less
than .1h per cent. The samples collected seven hours after infusion showed
a chloride content of over .20 per cent. There was a gradual return of
the chloride content until a normal was reached after 96 hours.

The pH of the milk showed a pattern very similar to the chloride con-
tent in terms of hours before returning to normal.

The resazurin class of the milk returned to one after 72 hours.

The standard plate counts of the milk during the observation period
varied from a low of zero to a high of lh0 bacteria per ml. The loga-
rithmic average would be 50 per ml. The counts on milk from treated
quarters showed no bacteria at 2b and h8 hours and only 10 per ml at 72
hours.

There did not appear to be any regularity in the response to the
various dosage levels in the different quarters.

On a second.cow, Table III, 5 grams, 3 grams, 2 grams and 1 gram of
neomycin in 10 ml of sterile distilled water were placed in the quarters
of the udder. The 1 gram dose in the RR quarter was repeated 2h hours
later. The milk from all quarters remained normal in appearance as viewed
in a strip cup. ‘When judged by the leucocyte count, pH, resazurin class,
chloride content and standard plate count, the quarter receiving 5 grams
was normal 10h hours later. The quarter receiving 3 gms was normal at
96 hours as was the one receiving 2 gms. The quarter receiving 1 gm and
another gm 2h hours later was normal on the morning of the fourth day from

the last treatment.

28

The preceding experiments indicate that no lasting harmful sequelae
followed the infusion of amounts of neomycin ranging from 0.25 gm to 5 gns.
In order to determine if an infected udder would respond, a cow was experi-

mentally infected with the test organism, Str. agalactiae 15. In Table IV

 

there is a complete summary of the status of this animal before infection,
during infection and following treatments.

Six days after artificial infection, treatment was instituted by
infusing 10 m1 sterile water solutions containing 0.25 gm, 0-5 gm and 1
gm of neomycin into the RF, LF, and LR reSpectively and the 0.5 gm dose
repeated 2h hours later in.RR. ReSponse was favorable in all the quarters.
The favorable reSponse in treatment was attributed to the fact that they
were administered soon after infection took place and there was limited
induration of the udder. Some retention data were also assembled from
this series of treatments and indicated that neomycin remained in the
milk in detectable amounts for 72 hours.

All quarters were negative at the next milking and remained so for
the next 21 days. The irritation from the treatment was not as severe_
as the infection when judged on the basis of the leucocyte count, chloride

content, pH and resazurin class.

III. RETENTION OF NEOMXCIN

Milk
Specific indications of concentration and persistence of neomycin in

the milk of a normal healthy cow were obtained. The subject was in the

TABLE II

Irritation Determinations

 

 

 

(Cow‘ H60)
* Leucocyte—Counts Chloride Content
Date RF LF LR RR RF LF LR RR

3-6-50 100T 300T 300T 200T .1h .lh .1h
3-7-50 200T 200T 300T 100T .1A .1A .1h
3-8-50 9 A.M. 100T 100T 200T 100T .1h .1u .1u
3-8-50 h P.M. 60T ACT 100T 60T .1b .1h .18
3-9-50 9 A.M. 100T 200T 300T 200T .lb .1h .18
3-9-50 9:15 A.M.* - - - - - '- -

3-9-5’0 h P.M. 15M“ 157M“ 101%: 1001‘“ .20 .20 .20
3-10-50 9.AJM. 10M 5M 3M 100T .20 .18 .18
3-10-50 h P.M. 2M 3M 1M SOT .18 .19 .18
3-11-50 9.AJM. 1M 1M 1M 100T .16 .16 .16
3-11-50 u P.M. 1M 1M 1M 100T .17 .16 .16
3-12-50 10 A.M. 1M 1M 1M 100T .16 .16 .16
3-13-50 9.AJn. 1M 600T 1M 100T .16 .15 .16
3-13-50 h P.M. 300T 300T 720T 200T .1h .1u .1h
3-lh-50 9 A.M. 800T hOOT 300T 100T .15 .15 .lh
3-1h-So u P.M. 100T 100T 1001‘ 601‘ .11; .1h .1h
3-15-50 9 AJH. 200T 200T 300T 100T .18 .1h .1L
3-15-50 L P.M. 100T 100T 100T 100T .15 .15 .18
3-16-50 9 A.M. 200T 100T 200T 100T .1h .lh .1h

 

* One gram RF, .5 gm LE, .25 gm LR in 10 m1 of water for each
and 10 ml water in.RR.

** Samples collected before milking.

TABLE II A

Irritation Determinations

 

 

 

(Cow H60)
pH Resagbrin.Class

Date RF LF LR RR RF LF LR RR
3-6-50 6.7 6.7 6.7 6.7 1 1 l 1
3-7-50 6.7 6.75 6.7 6.65 1 1 1 1
3-8-50 9 A.M. 6.7 6.7 6.7 6.7 1 1 1 1
3-8-50 h P.M. 6.6 6.7 6.7 6.7 .
3-9-50 9 A.M. 6.7 6.7 6.7 6.7 1 1 1 1

3-9-50 9:15 A.M.*

3-9-50 h P.M.
3-10-50 9 A.M.
Baosoupm.
3-11-50 9 A.M.
3-11-50 A.P.M.
3-12-50 10 AJM.
3-13-50 9 AM.
3-13-50 h P.M.
3-lh-50 9 A.M.
3-1h-50 h P.M.
3-15-50 9 A.M.
3-15-50 8 P.M.
3-16-50 9 A.M.

61: 6.145 6.14 6.65 h h h 2
6.h . 6.5 6.5 6.7 h h h 1
6.5 6.55 6.5 6.7

6.6 6.6 6.6 6.65 3 2 2 1
6.6 6.65 6.65 6.7.

6.6 6.7 6.7 6.7

6.7 6.65 6.7 6.65 2 1 1 1
6.6 6.7 6.7 6.7

6.7 6.75 6.7 6.7 1 1 1 1
6.7 6.? 6.7 6.7

6.75 6.75 6.75 6.75 l l l 1
6.7 6.7 6.7 6.7 '

6.7 6.7 6.7 6.65

 

* One gram.RF, .5 gm LF, .25 gm LR in 10 ml of water for each
and 10 ml water in RR.

** Samples collected before milking.

31

 

 

 

TABLE II B
Irritation Determinations
(Cow H60)
Standard Plate Counts
Date RF LF LR RR
3-6-50 120 180 60 0
3-7-50 20 100 80 to
3-8-50 9 A.M.** 80 10 O 20
3-8-50 8 P.M.m
3-9-50 9 A.M. 60 to 30 80
3-9-50 9:15 A.M.*
3-9-50 8 P.M. o 10 o to
3-10-50 9 1114. 0 0 0 60
3-10-50 h P.M.
3-11-50 9 A.M. 2o 0 10 110
3—11-50 h P.M.
3-12-50 10 AM.
3-13-50 9 AM. 10 30 20 to
3-13-50 8 PM.
3-1h-50 9 AN. 20 0 to o
3-1L-5o h P.M.
3-15-50 9 A.M. 60 7o 20 80

3-16-50 9 AM.

 

* One gm RF, .5 gm LF, .25 gm LR in 10 ml of water for each

and 10 ml water in R.
‘H Samples collected after A.M. milking.

a.” Samples collected before P.M. milking.

32

 

 

 

TABLE III
Irritation of the Udder with Neomycin
(Cow H70)
Leucocyte CoDELQIII_ pH
Date RF LF LR RR RF LF LR RR

h-2-51.AJM. 200T 100T 60T 100T 6.7 6.7 6.65 6.7
h-2-51 P.M. 300T 200T 100T 100T 6.75 6.7 6.7 6.7
h-3-51 A.M. 200T 100T 100T 200T 6.7 6.7 6.7 6.7
b-3-51 P.M. 200T 50T “50T 100T 6.7 6.7 6.65 6.7
h-h-Sl A.M. 100T 100T 7ST 200T. 6.7 6.7 6.7 6.7
u-6-51 A.M. 60T 100T 100T 100T 6.7 6.7 6.7 6.7
h-9-51 A.M. ‘lOOT 200T 100T 200T 6.7 6.65 6.7 6.7

Animal infused with neomycin after A.M. milking with 5 grams RF, 3 grams

LF, 2 grams LR, 1 gram RR. Each was contained in a 10 ml solution. The

1 gram was repeated the next morning for RR.

h'9-Sl P M o
h-10-51 A.M.

h-1o-51 P.M. .

h-ll-Sl AJH.
u-11-51 P.M.
h—lz-Sl AJM.
h-12-51 P.M.
h-13-51 AJM.
h-13-51 P.M.
h-lh-Sl A.M.
D-15-51 A.M.
8-16-51 A.M.

20M

20M

. 15M

10M

10M

500T
200T
100T
200T

100T

20M
20M
15M
10M
8M
5M
1M
200T
100T
200T
100T

200T

20M
'20M
15M
@10M
8M
2M
500T
300T
6OT
100T
60T

100T

15M
20M
15M
10M
7M
1M
500T

hOOT

100T
hOT

100T

6.6
6.5
6.h
6.5
6.6
6.5
6.7
6.7
6.75
6.7
6.7
6.7

6.6
6.5
6.h
6.5
6.6
6.65
6.7
6.7
6.7
6.8
6.7
6.7

6.65
6.5
6.h
6.6
6.6
6.7
6.7
6.7
6.7
6.7
6.7
6.7

6.6
6.5
6.5
6.5
6.6
6.5
6.7
6.7
6.7
6.7
6.7
6.7

 

33

 

 

 

 

TABLE III A
Irritation of the Udder with Neomycin
(Cow H70)
Regazurin Test-H‘C'Elfiride Contgnt— Standard Plate —=
Date Count
RF LF LR RR Rg_ LF __LR _ RR RF LF LR RR
h-2-51 AJM. 1 1 1 1 .18 .18 .18 .18 8o 20 Do 100
8-2-51 P.M. 1 1 1 1 .18 .1h .18 .18
u-3-51 A.M. 1 1. 1 1 .18 .18 .1h .1h 60 90 7o 80
h-3-51 P.M. 1 1 1 1 .11 .1h .15 .1h
h-h-51 A,M. 1 1 1 1 .18 .1h .lh .1h
h-6-51.AJK. 1 1 1 1 .1h .1h .1h .1h 100 20 8o 20
h-9-51.AJH. 1 1 1 1 .1R .18 .1h .18 Do 60 30 80

Animal infused with neomycin after A.M. milking with 5 grams RF, 3 grams

LF, 2 grams LR, 1 gram RR.

1 gram was repeated the next morning for R.

h-9-51 P.M.

14-10-51 AM.
1-10451 P.M.
h-ll-Sl AM.
h-11-51 PAM.
142-51 Am.
h-12-51 P18.
h-13-51 AM.
h-13-51 PJM.
h-lh-Sl A.M.
h-15-51 AM.
h—16-51 A.M.

h
h
h
h
h
h

2

HHH

h

mwrt‘c‘t’

|-‘

h

mwn'trt'

Each was contained in a 10 ml solution. The

L») 1:” 1T 5’ I?

.20
.20

.20

.20

.20

.20

.111

ho

2O

10

30

10

20

80

20

10

20

ho

3O

 

3b

powwow mom mmflvooHpca o
pcmmmpa mOHoOHoHpc¢ *

 

 

 

 

H H H H 6.6 5:6166.6 5.611 EH. HH. HH. EH. 566 HOOH HOQH e66 . u n u Hmu5nm
H H H H 6.6 5.6 5.6 5.6 4H. mH. 4H. 4H. HOOH 5665 Boom 506m u u n a Hmnamue
H H H H 6.6 5.6 5.6 m5.6 4H. mH. mH. RH. HCOH eooH eooN 5664 u u - . Hn-mmue
H H H N 6.6 5.6 5.6 6.6 4H. mH. mH. 6H. 5666 2H 2H 2H m m m .m HmuHmna
H e H a e.6 6.6 ©.6 e.6 ON. ON. ON. ON. aw 26H Se 25 .m .m _m _m Hm-6mue
a a a a m.6 5.6 w.6 w.6 ON. em. 65. cm. 26H 2e 26 26H m m. m m HmueHua
e e a a 6.6 6.6 m6.6 6.6 ON. ON. ON. om. 26H zNH 26H zNH .m .m m _m HmumH-e
a e a a 6.6 6.6 6.6 6.6 5H. ON. ON. oN. 26H 26H 26H EmH .m .w .m .m Hm-5H-e
.eoeoz eoHHHeeHo oHHeoeo H2 OH eH mooHeooneH HHa
.Hma5Hne am eH eoemoeoe me so m. mmH aw H meH ea m. Hem eHoesooo em mm. Hmu6Haa
a a a a m®.6 e.6 5.6 w.6 ON. ON. 5H. 65. em 26H 2m 26H + + + + Hmn6H-6
a a H a w.6 6.6 m5.6 5.6 ON. 6m. ON. ON. SEH zmH 24H emH + + + + Hm-mH-e
H a a H 5.6 5.6 5.6 m6.6 ow. mH. ON. ON. emH eNH zmH gem + + + + Hm-HH-a
mam owwpomHWMm .wpm mo whopaoo opopo poo: gm mo as N pmpomncw moeppmsv Haw Hmloau:
H H H H 5.6 5.6 m5.6 5.6 HH. 4H. HH..eH. HOOH HOOH Hoes 566m - a u - HuuoH-a
H H H H 5.6 5.6 6.6 5.6 4H. HH. 4H. 4H. HCOH eooN Boom HOOH - n n u Hmu5ua
mm mmmmmw mm mm mama 59 mm pMWpomW mmwoowmo mmpcamw opmwoooMM .mwamwmmemm open
saweummmm pomawa

 

 

 

.‘n'llr'h'l '1'... 1"." I: llll'll‘ll

'8

 

111'“ I‘ll"

mmlw 300v

oeeeHom oHo.

\

omz ape: popwmpa one
max mmflpowaome .opm spa: umpoomoH haampomermaxm_soo mo mmcoammm HmoHEonoOHm one Hmoawoaoflm

>H mamas

35

middle of her lactation period, giving approximately 15 pounds in the
morning and 8 pounds in the evening. The quarters were infused with 1 gm
of neomycin in 10 m1 sterile distilled water in the RF; 0.5 gm in LF; 0.5
gm ointment in LR and 0.25 gm ointment in RR. Milk was collected and
assayed at 7, 2h, 32, h8, 55, 72 and 80 hours. The peak concentration
was found at 7 hours and progressively decreased to 72 hours; when only
a small zone of inhibition was evident from foremilk. At 80 hours there
was a discernible ring of inhibition but it was not measurable. The 7
and 2h hour assays were made on complete milkings, the remainder only on
foremilk samples. The results are summarized in Chart 5A.

A second cow, Table V B, was infused into the udder with 0.25 gm
neomycin sulfate in 20 m1 sterile distilled water in RF; 0.5 gm neomycin
sulfate in 20 ml sterile distilled water in LF and 0.5 gm neomycin in 20
m1 aluminum monostearate in LR. Samples of foremilk were collected at
l, 2, h, 8, 8:10, 12, 2h, 28, 32, h8 and 72 hours. A high concentration
was evident 1 hour afterward. This was followed by a rapid decrease in
neomycin concentration in both quarters that received the aqueous solu-
tions. The quarter receiving the aluminum monostearate never did reach
levels as high as the quarters that received the aqueous infusion. The
length of time during which the drug was detectable was somewhat less in
this quarter (L8 hours), whereas, detectable amounts were seen in the
other two quarters at 72 hours. It is rather curious that a detectable
zone of inhibition appeared in the RR quarter at the 2 and h hour col-
lections, since this quarter received no neomycin. The last few m1 of

milk at the 8 hour collection time had less antibiotic per m1 than the

 

36

foremilk collected at the same time or the foremilk taken four hours
later.

The udder of a third cow, represented in.Table V C, was infused with
the same amounts of neomycin sulfate dissolved in only half the volume of
vehicle used in the previous experiments. The data in the table show
that the first sample collected after treatment contained substantially
greater quantities of neomycin. This can be explained by the fact that
since dilutions were so high, more of the residual antibiotic in the teat
canal was removed in taking the first samples. Subsequent samples showed
nearly the same potency as those in the previous experiments. Levels
were nearly as high and lasting when either volume was infused.

Table V D shows results from a S, agalactiae infected cow infused

 

with 0.5 gm neomycin in 10 ml water. Retention was evident at 60 hours
but not at 72. This cow was in a herd milked by machines, while all other

animals were hand milked. The animal remained negative for S, agalactiae

 

following this treatment.
From the amount of neomycin.per m1 required to inhibit the growth of

S. agalactiae in the test tube and the results showing the concentration

 

of neomycin in the udder at various time intervals, it seems that should
a follow-up treatment be indicated the time to give it is after a 88 hour

period.

Blood
A total of four cows received the antibiotic by intramuscular in-

jection and their blood was assayed for neomycin at 1, 2, 3, h, 5, 6, 7,

37

8, 9, 11 and 2h hours. Results are shown in Table VI. One animal (J25)
was a Jersey cow weighing 800 pounds. This subject received two gms of
neomycin sulfate in 10 ml of sterile distilled water injected into the
gluteal muscle. A detectable level persisted for 8 hours. Another animal
was a Holstein cow weighing 950 pounds, which was given 2 gms in 10 ml of
aluminum monostearate by the same method of injection. The levels did not
reach as high and did not remain any longer than in the cow injected with
neomycin in an aqueous vehicle. Two additional animals (H75 and H80)
received only 1 gm of neomycin, the former in water and the latter in
aluminum monostearate. Lower levels of neomycin were attained in both
instances and did not persist in the blood, for as many hours. One animal
was killed one week later and no abnormal macrOSCOpic findings were evident
in the carcass at the site of the injection.

From these limited studies, it would appear that if systemic blood
levels are desired for a prolonged period, the drug apparently must be
injected at eight hour intervals when used in aqueous solvents. Milk was
collected several times during these trials and did not show amounts of

neomycin sufficient for detection.

38

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CLINICAL RESULTS AND DISCUSSION

CLINICAL RESULTS AND DISCUSSION

These studies on neomycin show that the drug was: (1) effective in
ip_zit£g studies against organisms causing mastitis, (2) innocuous to the
udder as judged by irritation trials in healthy cattle, (3) successful in
the treatment of experimentally infected animals, and (L) retained in the
udder for 72 hours. ‘With this information at hand, it seemed logical to
extend these studies to cows in the field.

Throughout this work all preliminary diagnoses were based on direct
microsc0pic examination of aseptically collected, and incubated milk
samples. Retests were made at 7 to 10 days and 21 to 30 days. If an
animal or quarter was positive seven days after the last treatment, the
infected quarter of the udder was retreated at this time. If the quarter
of the udder was negative, it was retested 1b to 20 days later and if
still negative, considered cured. The word cured is used with qualificar
tions. In chronic streptococcus infections, "cured" means that the cow
was no longer shedding streptococci of five elements or more. In most
instances the streptococcus infections treated were in the chronic stage.
Some of the cows were not economically practical subjects for treatment
as judged by a physical examination of the udder. However, in this study
these were also included.

Some cases were not treated with a sufficient dose in the early phase

of the treatment. Many chronic cases were cured by one or more treatments.

Also a few cases did not respond to three treatments with neomycin but did

L2

reSpond to other antibiotics. Conversely, some cases that were refractory
to other antibiotics, responded to neomycin therapy.

In instituting therapy the ideal time to administer treatment is
shortly after a complete milking. Cows were never treated unless a mini-
mal time period of three hours could elapse before the next milking.
Generally a 10 ml amount of sterile distilled water was used as a vehicle
to facilitate placement of .5 gm of neomycin in each quarter. In treating
chronic mastitis, all four quarters of the udder were infused. 'When a
quarter was retreated, two half-gram doses, 36 or h8 hours apart, were
given. Cows that did not reSpond to a series of three treatments were
treated with other antibiotics or removed from the milking line, either
for slaughter or to serve as nurse cows. Some quarters were dried up.

A summary of results of treatments for various infections is provided in
Table VIII.

A total of 261 cases of streptococcus mastitis were treated with
aqueous neomycin sulfate. Two hundred and eight eventually were cured
with neomycin. Of these, the first five were treated with only 0.25 gm
of neomycin and none responded. All were later subsequently treated with
0.5 gm and they reSponded. Twenty-nine were treated on successive days
with 0.25 gm, 2h of these treatments proved successful. Of the remaining
227 treated, all were given an initial treatment of 0.5 gm. One hundred
and seventy-seven of these were negative at the 7 - 10 day test period and
still negative at the 21 - 30 day test period. Of the remaining fifty,
eight were treated with 0.5 gm at 7 to 10 days. Retest results showed

cure in three cases. The five remaining cattle were retreated with 0.5 gm

143

on two successive days. Two of these responded favorably and three did
not. These three were treated on successive days with 100,000 units of
penicillin. One reSponded favorably and two did not. These two were
treated with aureomycin and subsequently with tyrothrycin to no avail.
They were finally given up as incurable. One animal was lactating in
only two quarters; the others were lost, presumably, as the result of
earlier infections. There was a great deal of induration evident in all
quarters. The reason for this cow still being available was that she was
a foundation cow of a herd and had produced some outstanding progeny.

The second cow was just one of those cows which some farmers seem to keep
as her milk seldom showed any abnormality.

The twenty-six cows treated for staphylococci infections are diffi-
cult to discuss except from the standpoint of individual case history.
The generalizations that can be drawn are: All cows had shown an acute
syndrome along with the presence of the organisms and a high cell count
in the excretions from the udder. A half gram of neomycin was instilled
into an affected quarter as early as possible. Confirmation of the type
of infection was gained later. When the cows had an elevation of tempera-
ture, supportive treatment involving sulfamethazine orally or intra-
venously was utilized, When the cow was off feed ruminatorics were ad-
ministered. ‘When the cow remained dull and showed evidence of atony of
the rumen for a couple of days, rumen liquor transfers were effected.

In some instances penicillin was given intramuscularly. Of this group
only three cows died. It is not known how many lost the function of the

afflicted quarter but some did. There was also some impairment of udder

hh

function in some cows that were considered cured. This cannot easily be
measured.

The encouraging results in these cases of Staphylococci infections
may have been due in part to early medication. Also, since staphylococci
vary so greatly in pathogenicity, the possibility of a chance encounter
with a high percentage of strains of low pathogenicity should not be over-
looked.

In the course of these investigations, eighteen cases of coliform
mastitis were encountered. Treatment consisted of infusion of 0.5 gm
quantities of neomycin into each quarter on succeeding days until external
evidence of acute inflammation had subsided. Animals were always febrile
so supportive treatment with sulfamethazine or penicillin was given
parenterally, When indicated, rumen stimulants were freely used. Cases
were always treated prior to specific diagnosis and confirmations was
made later from the samples collected at the time of treatment. The major-
ity of the cases were treated early in the course of the infection. This
may partially explain the excellent record of only one death in the entire
series of coliform infected animals. Several cows were sold for salvage
after recovery because they would be uneconomical producers. In many
cases the return to normal pro-infection milk production and udder con-
sistency was slow.

Fourteen cases with Pseudomonas infection were encountered. With

 

one exception, these were in herds treated by the farmers with agents
obtained from sources other than veterinarians. Animals infected with

pseudomonas were given 0.5 gm of neomycin on two successive days. Milk

b5

was usually thick and stringy at the time of treatment. This condition
persisted for a considerable period of time following treatment. Eventu-
ally eight animals became useful producers again, and six were removed
from the herds as incurable cases.

Neomycin was the most effective in treating this infection. The
drugs used included penicillin, aureomycin, streptomycin, bacitracin,
tyrothrycin and sulfa preparations.

During the course of this study only four cases of yeast infections
were encountered. These were all in one herd of thirty Holsteins. The
herdsman had noted the irregular appearance of flakes in his strip cup.
Analysis of milk samples showed nothing significant except a possible con-
tamination with yeast cells. The owner had previously treated all animals
showing flakes in the milk. He had used penicillin but to no avail.
Subsequently, carefully collected milk samples disclosed two cows to be
shedding yeast cells. Treatment by infusion of 0.5 gm of neomycin cor-
rected the condition permanently in these two cows. Later a diagnosis
of yeast infection was made on two more cows in the same herd and thought
to be successfully cured with the same treatment. The owner had changed
from chlorine to quaternary ammonium compound for dairy sanitation prior
to his difficulties. After going back to chlorine, following the last
appearance of yeast cells in the milk, he has experienced no such diffi-
culties.

0n initial call, there have been 22 instances in which clinical cases
of mastitis had been treated. Subsequent laboratory findings failed to

show any known pathogenic organism. All such cases eventually recovered.

A6

Sixty-five cases of mastitis were treated with a.preparation of
Teatube-Neomycin which is neomycin in a water-miscible base, furnished
in a tube with attached canula.

The results of hO cases of streptococcic mastitis showed that twenty
reSponded to one treatment of 0.5 gm neomycin in 7.5 gms of vehicle.
Fourteen others, receiving a series of treatments of 0.5 gm h8 hours
apart, also recovered. Of the six that did not respond, three yielded
to penicillin therapy and three did not.

Eight cases of staphylococcus infection were also treated with
Teatube Neomycin. All animals survived and ceased to shed the organisms
in the milk. However, three of these failed to milk from the involved
quarter during that lactation period. It was not possible to observe
whether this condition persisted in subsequent lactation periods.

Six cases of coliform infection were encountered. All were treated
and survived. Udder secretions became negative for the organism. Four
returned to nearly normal milk production during the lactation period.
Two cows dried up in the affected quarter.

The eleven cows that turned out to have non-infectious mastitis
eventually recovered.

Because of the problems inherent in clinical results, one must follow
the case history of a few cows listed in the appendix in order to gain

full appreciation of the meaning of the results.

b7

TABLE VII

Summary of the h05 Quarters Treated with Neomycin Sulfate

 

v~-_ — - —

 

 

 

 

 

Type of Number of Number in Which
Infection Cows Treated Infection Overcome Died Remarks
Streptococci 261 208 O
Staphylococci 26 23 3
Coliform l8 l7 1
Pseudomonas 1h 8 0 6 sold for
' beef
Noninfectious 22 22 0
Yeast h h 0
Total 315 _ 282 h
Summary of Cows Treated with Teatube Neomycin
Streptococci hO 3h
Staphylococci 8 8 3 lost function
of quarter
Coliform 6 6 1 lost function
of quarter
Noninfectious 11 11

 

Total 65 59

 

SUMMARY

LB

SUMMARY

Neomycin 12:11332 at very low levels inhibited the growth of the
six common causative microorganisms associated with infectious bovine
mastitis.

The results of this study with neomycin indicate a minimum of local
irritation in the udder as judged by leucocyte counts, chloride content,
'resazurin class, pH and strip cup observation.

The agent was found to remain in the udder for 80 hours or longer
when an aQueous solution was infused.

Neomycin when injected intramuscularly was found to give demonstra-
ble blood levels for at least 8 hours.

The results in treating h05 cases of mastitis indicate this drug to
be very effective against infections caused by streptococci, staphylococci,
coliforms and a yeast. Neomycin was also of value in eight of fourteen
pseudomonas infections.

There appeared to be no difference in the effectiveness of neomycin

when employed in either an aqueous or water-miscible base.

BIBLIOGRAPHY

A9

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Bacillus. Proc. Soc. Exper. Biol. and Med. 7h:35-h0.

haishern, B. A. 1951. Bacteremia Due to Gram Negative Bacilli Other than
the Salmonella. Arch. Int. Med. 88:h67-h88.

 

Earth, P. T., C. A. Chandler and E. A. Bliss. 1950. The Antibacterial
Action of Neomycin and Furadroxyl in Vitro and in Vivo. Bul. of
John Hopkins Hosp. 86:167-178.

 

 

Matts, P. S. and D. H. McLeod. 19h6. The Estimation of Penicillin in
Blood Serum and Milk of Bovine after Intramuscular injection.
J. of Comp. Path. and Therap. 56:170-179.

 

5h

welch, H., R. P. Herwidk and A. 0. Hunter. l9h5. Properties and
Standardization of Penicillin. J. of Am. Pharm. Assoc. 6:93-96.

belch, H., R. J. Reedy and S. W. hblfson. 1950. A Comparison of the
Effect of Nine antibiotics on Experimental Typhoid Infections in
Mice. J. Lab. & Clin. Med. 35:663-666.

 

waksman, S. and H. C. Reilly. l9hh. Bacterial Action of Antibiotic
Substances. J. of Inf. Diseases. 75:150-159.

 

haksman, S. and H. A. Lechevalier. l9h9a. Neomycin, A New.Antibiotic
Active Against Streptomycin-Resistant Bacteria. Sci. 109:305-307.

 

haksman, S. A., J. Frankel and 0. Graessle. 19h9b. The In Vitro
Activity of Neomycin. J. of Bact. 58:229-237.

Maksman,:3. A., H. A. Lechevelair. l9h9c. Neomycin, A New Antibiotic
Active Against£3treptomycin-Resistant Bacteria. Sci. 109:305-307.

Eaksman,:3..A., E. Katz and H. Lechevalier. 1950. Antimicrobial
PrOperties of Neomycin. J. of Lab. & Clin. Med. 36:93-99.

APPENDIX

56

 

omen exec boncwpcoo

amphmdv

memo Om“: pm em mm. o>mm
Om“: pm mcflpmm mm: 300 .ha
Ipasopm pmpmopp .z.< Onaw pm
popeboomfiv I mm “mm emu aw

omueue pone edeecoe
exec pm .mon ems ©.HOH
.Qama .oHE a pooan
.pmmp no .mo: #39
.mbmpamsd Ham Hwahocbw

Emflcmwno wsflppozm eon HmEHGm .moz

Emflcmmeo wcflcpmzm HwEHcm mfiam #

mcfleno poo .o.eo~
.gEmB pmmnmw ecmaaozm

44
h

 

hammoam mafia : omnmaum mm. mg qaohEom: Em m. m>mo nmpumsc mg l Omlzatm snowflaoo ommno omugalm
mcaeoo .N.Hoa .eEoe .z.a oo.mn.z
Heston mcfien5pma .< omum 20pm as mama
acetone .Hmeeoe ease .z.e cmuw pm me oenaaoo o.moa .eEoe .ooaaosn em
omlmaum mHQEmm .mmz nfiobsom: aw m. o>mw «mafiampms enmppwdv mm Shogfiaoo ommlo omuaalm
omlqan omlmnm cfiohsooa aw mm. escapmmpp mdow>opm
coo om-aaum .moz cm-mcm eHoAEooo em mm. song head Haaem .peoeem ma-o ouua-m
ounnu: omnmlm aflohsoo: aw mm. newspmopp m50fi>mam
one omsaau .moe omumum deanooe em mm. song mead Haaem .eeotem Hmmno cmnaum
omlnn: pm omlmum aw mm. pamEpwopp macabopm
one emuafium .moz + omumsm Em ma. gone head Haaem .edooem mem-o omtaum
newness gown
omlwlm :0 mafia opmmfidm :HohEoo: em mm. xomno hagpcoz .pmmppm mmlo omlmlm
unnamed some
omtwnm no mean opwmadm :Hohaomz em mm. xoono mampcoz .ummppm Hmmao omlwlm
poppmdu some
poem: pmaafipmflc mafihmpm He came mmmaaoo
Omlwtm no means 0a mpmmasm caohsoo: Em mw. so xomno hanpcoz .pmoppm Nomlo omnwlm
mpHSmmm @ examamm uncapmmea muopmfim mfimomwmfia .300 open

 

 

 

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IF
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oenaaom_ceoe2ooz roe; oopeoee nonoo massacre anooaraooH ooeooaom

57

omma exec Umsafipcoo

soapospoaa has“
09 pocadpmp Zoo Omubao

adamao omlmlm poEHSm pm

ommH o>m0

.pmppd GOfimzmca

omuoalm .Omumlm no whophmdc

 

 

.omlmfllm :o .moz Haw caohEom: Em mm. m>mo m.:oa .Qan epmmemo .nampm mmmao omsmlm
omlouo paw omaomlm pcmspmmpp mdoa>mum
.omnmanm co .moz om-a-m gm m. oeoo theme head Haaem .edoeem cmsem omuaum
outmlm am mm. m>wo
omumum no head .Omumsm gm mm. deco scone aHesse: .oeohom cmuem omuaum
. . cutout
mmuomlm omnmaum omlmalm aw mm. mpmu pcmspwoup m50fi>mha
omuma-m co .moz omnea-m aw mm. memo .oeaoanod Haaem .oeoeom meano omueanm
omum-m en mm. oeeo
omumaum co node omueum em mm. ores eooao aaeoeoz .eeoeem moano omumum
omuwaum :aohfiooc
soapospopm poem Op Sm m. mm o>mm .omlmaam ha
accesses zoo n ouumnm ufleeo omnaaue eosaon em oooa cane
.omlwmna “omnmau: m>mm .mm Em m. o>wm .mm and namHHozm nephews mm
,eomlwalz .moa mmz .mq_aaohsom: Em mm. m>wo .Ummm mmo .4.moa .msme .zmwpm muno omawaln
omuaals
omam omnqmum Go .moz hoppwsd some afiohaom: Em m. xoozo hazpsoz .pmohpm untam omnmaum
cmumanm
omuam-m no head cm-aaum an mm. crow doom noxeae ma .eooeem mam omraaum
aflomEoom
omuoaum no head omumaum no an m. oeeo coo canon co noxeaa as .eeocem mam om-aa-m
prSmmm ¢.mxmeom mmoapmmpe myopmam ”mmmdll. open

 

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mameMMfiQ

58

mmma Ham: pmscapcoo

 

.mma .mmc .moc .mm:

 

«OmImalw expwamm
.moc deQ .mo: .mo: pound
«omI0mIo mamppwsw Ham .EHomEomE Em m. pEmEpmmhp poscfipsoo .>mpa mom wam OmIoNIo
E0H9w95pcfi poxnmE oflconzo mdam
mambhmdc mm .deQ :q «mafia mg
mead mead mead .mmc Ham omImHIo cHomEomE Em m. “mafia mm I hephdm whom .paoppm HOmm omImIo
coapmnfipca pothE canoeso mafia
mumpamsc mm .mdam x4 “0 mg
.mm: mafia 0 mean Ham omImHIo :fiohEoo: Em m. .mdam mm I he>hdm chem .paoppm Hmom omImIo
mean
whopumdv mm .wdaa x4 «woe EA
.wmc mafia moan msaa Haw omImHIo EfiohEomE Em m. .deQ mm I hm>hsm vpom .pmonpm mwom omImIo
mafia
whoppmsw mm .mdag m4 «mafia mu
.mme mafia deQ mafia Haw onImHIo cfiohaoo: Em m. emdaa mm I Ambasm them .vmmppm mmom omImlo
.Mm:
myopnmsu mm .mmc x4 «mos mg
.mm: deQ .mma .mm: Ham omINHIo cfioono: Em m. «mafia mm I hm>hdm who: .pamhpm Homm omImIo
.woc .moc .mmc .mo: . .wmc
mm mg mg mm mpmpnmdd mm ewes x4 «woe mg
omImHIo Haw omINHIo :Hoonm: Ew m. .deQ mm_he>h:m chem .pdonpm mmmm omImIo
.mm: mafia .
mm .mdaa mg «mafia mpmpnmdd mm «mafia mg .mEHQ mg
mg «mm: mm omImHIo Haw omINHIo EfiohEomE Em m. .mo: mm I hm>hsm chem .pamhpm wmmm OmImIo
mpfismmm a mxamEmm Amepmope mmopmflm mamoEMMHm zoo meme

 

 

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4"!“ RI «1‘

4‘, F‘ E. ’IQ —

S9

omwm paw: bmdqflecoo

 

.edotem
omImHIm use meoE
coo omummua .moz eaoaaooe an m. noaoam mm noooone ame-o omuamuo
OmINHIm xomzo hanpcoE mmEoE
new omeme .moz cfiomEooE Em m. hoptmdv mm .pmmpmc Iopdmmm mmmIo omIHmIo
Efioonoa Em .H mpmppmdv mafia Ham umpmmpa «omIMIN
mSHQ mdaa mafia .mmc
omImHIm ethEom
mdam mdaa mafia .mo: pops:
omIOMIo myopnwsu Ham .EfiohEomc Em m. pEmEpmopp Umscfipcoo .>oam mom Homm OmIoNIo
.mo: .moq 0 mafia
omumalw exmemm
.moE .mm: 0 mafia pound
omIOMIo mpmpnwsv Haw eqaohEoms Em m. ecoEpwopp Umdawpcoo .>mpm mom .Hmom omIONIo
.mmc mafia .mon mafia
omImHIw mxmeom
.mm: mafia .mo: mafia genes
omIOMIo mpmppedv Haw .cfiohEoo: Em m. pEmEpwopp umdcfipcoo .>ohm mew mmom omIONIb
.mmE .mon .moc .moc
_ OmImHIN ethEom
.mo: .mmE .mw: mafia pecan
omIOMIo mampgmdv Haw .cfiohEom: Em m. pEmEvmoup Uodsflpcoo .>opm mom mmom omIONIo
.moc .mo: .mo: .mmE
omumaua
.mmn .mo: .mm: .mmc mxnmEmm
mm ma mg mm pops:
OmIomIo mumpamdw Haw .Eflomfiomc Em m. pamEpmohv pozcfipaoo .>onm mom Homm omI0mI©
mpHfimom a memEmm pEmEpmona .anopmHm mfimoqmmfia .300 mamm

 

 

60

mmma yams Emscflpcoo

m>HpHmog mpoppmdv mo nobEsz #

 

 

omumanoa
omImmua .moz omumaum esoEEooc m. acoEo EHEoEoE «Amy Sachem Nomuo omnmaum
omINHIOH
omImmua .moz omImHIm eaoaaooc m. eooEo Easecoz AAHV emotem ENEIO omumaum
omINHIOH .ot OmImHIm
omummua ecz cmanIa choEEooc m. moose adheooz rAHV ecohem mamuo omImHIa
omImHIOH . omImHIm
omummum moz omnmaua cHoAEooc m. mooao EHEeEoz rfiav ecotem Hmeuo OxImHIa
omumaua
was omIHIm .moz mbmpamso Ham cfiohEer m. xoono hazpcoz pmeppm mEHQ HMMIU Omnmmnw
omImaIm ado afiapm co mommam
use omIHIm .mmz mampemsv Ham EfiohEooe m. I moose hanpcoz pop mean mwlo QmImme
omumaum
was omIHIm .mmz mpmapmdd Ham aflohEomE m. moose hazpcoz emmppm mafia mquo omImNIN
OmlsHIm mmom
was OMIHIm .moz mpophmdw ads EHohEomE m. moose mazpaoz.pamnpm mafia pom omnquN
omIJHIm N04
use omIHIm .mmz numbness Ham cfiohEome m. moose hazpcoz pamppm mafia hnaoo Omlnmuw
mflpflpme
pmammdammflp mommam afiohEomE m. ado dfiapm so mommam pmmmzfiIcoc ommIo omIHNIo
mwpfipmsE
pohmmaammap momem EfiohEom: m. ado mauve co mmxmam pmmmuwIEo: oan OmIHNI©
measmmm m expeEmm pEoEpmoEa whopmwm mHmoEMeHD zoo mpmml

61

mmmm exec pmpdaocoo

mbfipAmom muopEMSu Ho EmbEsz *

 

 

omImHIoa
omuNNIa .moz omuoaum :HoEEooo m. motto eaceooz redeem moan eoeaao omueaum
omIQHIOH _
omImNIm .moz omImaIm Emoono: m. xomzo manpcoz.pmmppm usam among omIJHIm
omnoaum
omIaanoH .moz cmumaua caoEEooc m. rooco Afleecoz eeotem moan mecca omueaua
om-aauoa omISHIa
onumwna .moz omamaua caoEEooo m. score Aficeoozneootem neat denom omueaua
omumHIOH omucaIm as
omumm-a .moz omImHIa caoEEoco m. moose afieecoz eooeem mead Haom omIEHsm
omIAHnoa opoaoo omueaua me
each I omumw-a omamflua eHoEEooe m. scone a32oz eeocom noae Eeeoem omuaHIa
omumauoa omsoanm mm .eg
one omINNIm .moz omumaua esoaeooe m. moose essence anthem cede goo omueaum
omINNIm pEmEPMopp .>th anm
omumfiuoa .moz cmuomua eaoEEooo m. noecoeo oeo head Haaem *AHV poocom aeHIO omIeHIa
omuma-oa .moz omImHIa
coo omumNIm mafia omIMHIQ esoEEooc m. moose e.2221 eamv peonem moHIo cmumaua
ouumfiuoa omnmana eaoEEoee m. score afieecoz *AHV odotem aemuo omumHIa
omammum .moz omnmaua cHoEEooe m. Eooao aaeeeoz *AHV pooeem mmmuo omumanm
mpHdwmm w mthEmm pEmEpwopH hmopmflm mHmoqmeQ ,300 opmml

 

 

62

 

.moc omuaIHH

omIONIHH

omdw omImIHH Emma
pooab use .oflE pomp
Iflp ow m>HpmmoE mm:
300 Ahmpma .3: 3
0m“: em o.NoH dame

omIHIHH Efiohfioma Em m.

OmIHMIHH :HoEEom:

Em m. copmmaom .hHHmEo me
IHdm mawmhm omma emote: opcw
commsmafi have; pmaaapmac
mafinopm HE OH :HomEoo: Em m.

xomno hHEpEoz

hEdEom
:oaamh EHHE .Emaaozm
popgmsv mm m.©0H @Ema

oooeem

eeocem

moldm

momc

o

OmIHmIOH

OmIOmIoH

 

wmmmhom e wxEmEmm

pEmEpmeB

Eeoenfim

mfiwocmwwa

too

open

“.1 53

View"

WOO“ USE ONLY”

 

 

HICHIGRN STQTE UNIV.

lllllll H||| WW II llll
3

LIBRQRIES

 

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