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L L5 R A R Y
Michigan Sm:
University

 

A COMPARISON OF NITROFURANTOIN AND DIHYDROSTREPTOMYCIN
IN THE TREATMENT OF EXPERIMENTAL FELINE
ESCHERICHIA COLI UROCYSTITIS

by
DONALD LOSS PIEBMATTEI

AN ABSTRACT

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

MASTER OF SCIENCE

Department of Surgery and Medicine

I.
Approved; ,4 2/2/22 C (f.

1960

 

 

ABSTRACT

The cystitis-urolithiasis syndrome is an important pro-
blem in feline medicine. Infection plays a part in this syn-
drome. Urocystitis was experimentally induced in eighteen
cats by traumatizing the bladder mucosa and injecting a cul-
ture of g. 22;; into the bladder lumen. The cats were divided
into three groups of six, designated nitrofurantoin, dihydro-
streptomycin, and untreated control.

Treatment nith nitrofurantoin proved to be more effective
than dihydrostreptomycin in producing bacteriologically nega-
tive post-treatment urine specimens. The control cats were
not as severely affected and did not remain infected as long

as was anticipated. Suggestions for further study were made.

A COMPARISON OF NITROFURANTOIN AND DIHYDROSTREPTOMYCIN
IN THE TREATMENT OF EXPERIMENTAL FELINE
ESCHERICHIA COLI UROCYSTITIS

by
DONALD LOSS PIERMATTEI

A THESIS

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

MASTER OF SCIENCE
Department of Surgery and Medicine

1960

I.
II.

III.

IV.

TABLE OF CONTENTS

Introduction and Review of Literature

materials and Methods

A. Preparation of Experimental Animals

B. Selection of Test Organism

C. Infection Procedure

D. Treatment

E. Bacteriological Procedure

F. Necropsy Procedure

Results

A. Progress of the Infection

B. Bacteriologic Results and Statistical
Analysis

C. Necropsy Findings

Discussion

Summary and Conclusions

References

page

13
13
13
1A
15
15
17
18
18

19
20
22
26

35

LIST OF TABLES AND FIGURES
Table

I. Microorganisms causing urinary tract infections
in cats

II. Percent change in bladder wall thickness from
pre-infection (biopsy) to post-infection

(necrOpsy)

III. Bacteriological results and schedule of treat-
ment days

Figure

1. Normal bladder - cat
2. Normal bladder - cat

3. Increased density of the lamina propria and
submucosa -- post-infection, cat 633

a. Increased vascularity of the lamina propria --
post-infection, cat 209

5. Comparison of the number of bacteriologically
negative specimens occurring in each
experimental group

page

28

28

29

30
31

32

33

3b

ACKNOWLEDGMENTS

The help, guidance and encouragement of many people have
contributed to this work.

Sincere thanks is extended to Dr. w. V. Lumb for his
guidance, physical help, and stimulating encouragement in the
deveIOpment of this program.

I would also like to thank Dr. R. G. Schirmer, Director,
Small Animal Clinic, for arranging my work schedule so that
I might work on this project. Thanks are due also to my other
colleagues for carrying part of my share of clinic work while
the project was underway.

Dr. R. F. Langham has assisted in the pathological areas
of the problem, and Mrs. Marion Bennett has been invaluable in
making suggestions concerning the bacteriological aspects.
Their help is gratefully acknowledged.

To my wife, Joann, who has typed and manuscript and has

been a source of help in all matters -- my thanks.

INTRODUCTION AND REVIEW OF THE LITERATURE

The practice of feline medicine and surgery has attracted
increased attention and interest in recent years. The cat is
more and more becoming a family pet and cat owners are usually
quite willing to consult a veterinarian when the animal is
sick. Most practitioners agree that the cystitis-urolithiasis
syndrome is one of the most difficult problems encountered in
feline medicine. McClellan and McClellan (31) reported that
this condition was the most common medical problem of the uro-
genital system, and accounted for 13 percent of 700 feline
cases over a period of three years. Glover (18) reported an
incidence of four percent in #5? cases.

Cystitis in the cat may be a primary condition due to
bacterial infection or, in the male, may be associated with
urethral obstruction due to "calculi". These obstructions
are usually precipitates of magnesium ammonium phosphate (27)
. rather than true calculi. True cystic calculi have been re-
ported in the cat, however (1?). Cystitis without blockage'
of the urethra is a serious, although not often fatal, condi-
tion. With the presence of a urethral obstruction, however,
death due to uremia can result. Uremia, in this case, is a
consequence of kidney damage due to back pressure of urine in
the bladder. The entire complex of urethritis, cystitis,
bladder atony, acute urethral block and uremia are called the

cystitis-urolithiasis syndrome by Lumb (2?).

Case histories from the files of the Department of Sur-
gery and Medicine, College of Veterinary Medicine, Michigan
State University, were reviewed to gain information on this
condition. A review of 33 cases of cystitis or cystitis and
urolithiasis revealed the median age for affected animals was
three years. The sex distribution was 33 percent females,
six percent spayed females, 36 percent males, and 25 percent
castrated males. Sixty-seven percent of the affected animals
were domestic short-hair breeds, 30 percent were domestic
long-hair breeds, and three percent were purebred Siamese.
There was a greater incidence of these cases during the winter
and early spring months.

Symptoms observed are dependent on the type of condition
present: cystitis alone, or cystitis and urethral obstruction.
In uncomplicated cystitis, the constant symptoms are hematuria
and dysuria. The cat makes frequent trips to the litter-box
and voids small quantities of urine at a time. The bladder is
usually contracted and thickened. Abdominal palpation is
often painful to the patient. The body temperature may be
elevated or normal, depending upon the severity of the in-
fection.

In urethral obstruction, the first symptom noted is dys-
uria, with urine voided a drop at a time or in short spurts.
As the bladder continues to fill, the cat becomes more irri-
tated and restless. The animal will spend several minutes in
the litter-box and strain in a manner that suggests constipa-

ilion to the average owner. Priapism may be noted at this time,

and if the obstruction is near the urethral orifice, the tip
of the penis distal to the obstruction may be inflamed and
discolored due to licking of the area and interference with
circulation. If the obstruction continues for several days,
uremia develops.

Much of the literature available on this subject is con-
cerned with possible causes of this syndrome. Bloom (A) has
listed as predisposing causes for urocystitis the following:
trauma to the mucosa, cystic calculi, tumors, parasites of
the bladder, obstruction of the urethra due to calculi and
inflammation, obstruction of the prostatic urethra due to

prostatitis, and neurogenic disturbances resulting in bladder

paralysis. Smith (#6) theorized on possible general causes

and predisposing factors. He thought that females were as
often affected as males and noted that closely confined house
cats were more often affected than cats that were allowed
complete freedom. The incidence of this syndrome was increased
during the colder seasons of the year. Inactivity due to clos-
er confinement and consequent urine retention were considered
to have increased during these seasons. Other possible reasons
listed for the seasonal incidence were decreased fluid intake
during cold weather and lack of opportunity for varying the
diet by hunting.

Possible avenues for infection of the bladder have been
discussed by Bunce (7). These included bacterial infection
from kidneys, from the intestines via local lymphatic channels,
and ascending infection from the urethra. Trauma from instru-
lnentation and surgery was noted to facilitate infection of the

bladder.

The normal pH of cat urine is slightly acid. Bacterial
infection with urea-splitting organisms.causes an increase in
the pH of the urine (A). The end product of urea-splitting
is free ammonia, which is very irritating to the vesical mu-
cosa (#6). Nielsen (37) concluded that most cases of uro-
lithiasis in farm mink were associated with infection of the
urinary tract. The most common infection was a urease posi-
tive, hemolytic Micrococcus pyogenes Egg. glbgg. Mosier and
Coles (36) reported a series of urine cultures from cats with
urinary tract infections. Their findings are summarized in
Table I. Many practitioners feel that bacterial infection
is present in the usual case of acute urethral blockage, as
antibiotic therapy is a routine part of their treatment. How-
ever, Fishler (1“) has called attention to the fact that peni-
cillin and streptomycin have been much less effective in the
treatment of this syndrome in recent years. He speculated
that antibiotic residues in foods, particularly in dairy pro-
ducts, might be causing an increase in the number of anti-
biotic resistant bacteria.

Concerning possible causes of calculus formation in the
bladder and urethra, Bunce (7) noted that bacteria, mucus,
and desquamated epithelial cells from the bladder or kidney
may serve as a nucleus for stone formation. Thus, chronic
cystitis could influence the growth of stones by furnishing
many of these nuclei.

A high-ash diet, as is provided by many commercially
available cat foods made from whole fish, has been considered

as a possible cause of calculus formation. Stansbury and

Truesdail (47) have stated that they could find no evidence
of increased vesical calculi formation as a result of feeding
this type of diet for 33 months to a group of cats. Dickin-
son and Scott (11) attempted to produce calculi in kittens by
feeding high levels of bone meals, but the kittens showed no
abnormalities of the urinary tract at the conclusion of the
experiment.

Armistead (1) has considered several factors related to
the formation of uroliths. Those listed included avitaminosis
A, urinary tract infection, urine pH, urine volume, lack of
protective colloids in the urine, and the anatomy of the male
cat.

Urinary colloids were considered by Butt and Hauser (9)
to be the basis of the problem of urolith formation in man.
They stated that the protective action of urinary colloids
is of major importance in preventing precipitation, agglom-
eration and conglomeration of urine crystalloids. Boyce gt
ggl. (6) determined that the total quantities of proteins and
other particles of crystalloid size in the urine of humans
'with calculi were increased three to thirteen times above nor-
Inal. Butt and Mauser (9) also pointed out the increase in
'urinary colloids in humans following subcutaneous injection
of hyaluronidase. Puntriano (hi) thought hyaluronidase had
ti possible use in preventing urolithiasis in livestock. There
liave been reports related to the successful use of hyaluroni-
tiase in the treatment of canine urolithiasis.(19, 48).

Vitamin A deficiency has been demonstrated by Higgins (20)

'to cause inflammation of the urinary tract of rats and dogs.

This deficiency caused abnormal keratinization of the epithe-
lium and predisposed these tissues to infections. Higgins
also demonstrated that a diet low in vitamin A predisposes
rats and dogs to calculus formation and, in the absence of
infection, administration of vitamin A will cause dissolution
of some stones.

The importance of adequate fluid intake in cats that are
subject to calculi formation was cited by Lieberman (25). He
was of the opinion that increasing the fluid intake prevented
the formation of calculi in most cases. The significance of
urine volume as related to fluid intake was reported by Grove
‘gt gl. and cited by Lumb (27). Small zinc discs were surgi-
cally placed in the lumen of the bladder in a group of rats.
The discs acted as foreign bodies which enhanced stone forma-
tion. Intake of large amounts of water was encouraged by
adding glucose to the drinking water.* This procedure resulted
in a five-fold increase in urine volume. None of the rats
that received this treatment developed stones, whereas 63 per-
cent of those fed stock diet and 78 percent of those fed stock
diet with glucose powder developed stones. The effect of urine
pH on stone formation was also demonstrated in this experiment.
Some of the animals receiving the glucose in water developed
urinary tract infections with urea-splitting organisms. The
pH of the urine rose above 7.5 in these rats and stones formed
despite diuresis.

There are many reports concerning the clinical management

of the cystitis-urolithiasis syndrome. Most of these are de-

voted to the relief of acute urethral block, and prevention
of subsequent stone formation. The patient with acute ure-
thral obstruction must be provided immediate relief. Occa-
sionally the only procedure necessary is the application of
pressure over the distended bladder, this pressure being suf-
ficient to eXpel the obstruction from the urethra.

Usually anesthesia is required to permit the necessary
treatment and ultra-short-acting barbiturates or ether are
recommended.(ZU). Following induction of anesthesia, the
penis is extruded and an attempt is made to break down soft
calculi by massaging the penis. This is usually followed by
irrigation of the urethra with saline or mineral oil. A blunt
20 gauge needle attached to a syringe is introduced into the
urethra and the penis and urethra are compressed around the
needle. By applying pressure on the syringe, it is usually
possible to flush the calculous material back into the bladder.
This procedure has been described by several authors (5, 24,
A9, 50). It may be necessary to repeat this treatment daily
for several days. If the urethra cannot be immediately opened,
it is necessary to empty the bladder by paracentesis. Leigh-
ton (Zh) has stressed the necessity of using a needle no lar-
ger than 22 gauge to minimize the possibility of leakage
through the hole produced in the bladder wall.

Fishler (14) and Burroughs (8) have advocated the use of
indwelling catheters of polyethylene tubing. These catheters
were inserted after the urethra had been cleared and were left
in place for several days. Free flow of urine was thus in-

sured during this period. Burroughs also stressed instilla-

tion of local anesthetics into the bladder as a part of his
treatment. He believed this technique provided relief from
urethral spasm present in most cases of obstruction. If the
urethra cannot be cleared, Crawford (10) has recommended
urethrotomy above the penile bone. Since the obstruction is
usually found in the penile bone, the opening produced allows
free flow of urine. Beamer (2) has described a procedure for
ureterocolostomy and urethralcolostomy. This procedure was
recommended for relief of intractable urethral obstruction.
He found urethralcolostomy to be the more successful tech-
nique.

Schein (Ah) and Hoffman (21) have reported good clinical
results with the use of an anti-spasmodic drug to aid in the
treatment of urethral block. They recommend injection of a
deproteinated nitrogenous extract of pancreas(a). This mate-
rial caused relaxation of the urethral wall, and thus allowed
for easier catheterization and passage of calculous material.
Irwin, Clark, Konde and Taylor have been quoted by Lumb (27)
as having successfully used methyliso-octenylamine‘b) for its
antispasmodic effect.

Following relief of the obstruction, many medical treat-
ments have been recommended. Bloom (5) and Fishler (it) have
used urecholine to stimulate contraction of the bladder. They
both stressed the necessity of having the urethra open before

this powerful parasympathetic stimulant is used.

 

(a)Depropanex (R), Merck, Sharp and Dohme, Philadelphia, Penn.
(b)0ctin (R), Knoll Pharmaceutical 00., Orange, New Jersey

 

.II... n 1 II, III)“. (In.

Changing the pH of the urine, if it proves to be alkaline,
has been advocated. This was done by changing the diet to
fresh meat (5, A5) or by administration of acidifying agents.
Sodium phosphate, five grains three to five times a day, was
recommended by Bloom (5). Frick (15) used methenamine, five
grains a day. The use of ethylenediamine dihydrochloride‘c)
or ammonium chloride was also reported (27). Administration
of therapeutic amounts of vitamin A (1, 5) and testosterone
therapy (45) have also been advised.

Cystitis without obstruction has been treated mainly with

antibacterial agents. Bunce (7) stressed the need for anti-

biotic sensitivity tests to choose the most effective agent.
Leighton (2h) advised chloramphenicol(d) and nitrofurantoin(e)
as the most effective drugs. Piermattei (39) has found nitro-
furantoin to have the broadest spectrum of in 11259 effect
against microorganisms found in feline urinary tract infec-
tions. ,0xytetracyc11ne‘f) was recommended by Armistead (1)
and a combination of sulfadiazine and streptomycin by Schnelle
(45). '

Prevention of recurrence of the cystitis-urolithiasis
syndrome has been approached from several viewpoints. Lieber-
man (25) recommended increasing the fluid intake by salting

the food enough to cause the cat to drink appreciable quanti-

 

(°)Chlorethamine (R), Pitman-Moore Co., Indianapolis, Indiana
(d)Chloromycetin (R). Parke-Davis & Co., Detroit, Michigan
(e)Furadantin (R), Eaton Laboratories, Inc., Norwich, New York

(f)Terramycin (R), Pfizer Laboratories, Brooklyn, New York

10

ties of water. Sternfels (49) advocated that the cat receive
only distilled or boiled water. He thought minerals in the
drinking water were a factor in the formation of calculi. He
also recommended a low-ash diet and the use of a urinary anti-
septic for six months after the occurrence of a urinary stop-
page. Despite the work of Higgins (20), Bloom (5) has not
been impressed by the effectiveness of vitamin A in prevent-
ing calculus formation in the cat. Bloom recommended a meat
diet as the most satisfactory long term prophylaxis. Leigh-
ton (24) advised the use of meat such as hamburger and liver,
fresh cooked fish, and the prescription diet C/D(8).

Despite all of these measures, many cats have recurrent
attacks of cystitis-urolithiasis which often lead to their
death. This thesis presents the results of an experiment de-
signed to increase the knowledge of one phase of this pro-
blem -- namely, efficient treatment of infection of the uri-
nary bladder. Specifically, this research was undertaken to
compare the efficacy of treatment of bacterial urocystitis
with nitrofurantoin and dihydrostreptomycin. A secondary aim
of the experimental work was to find a means of instituting
and maintaining a lower urinary tract infection.

Nitrofurantoin (5-nitro-2-furfurylidene aminohydantoin)
.is a synthetic antibacterial compound of the nitrofuran group.
Tfl1e mode of action of the nitrofurans is considered to be in-
terference with carbohydrate utilization by bacterial cells

(:38). Nitrofurantoin has been extensively employed in human

 

(é;)(R), Hill Packing Co., Topeka, Kansas

 

“If. (IIIIII‘AI‘IIIII‘J'I‘III. :).III.I. iii

 

11

medicine for treatment of various urinary tract infections
such as pyelonephritis, pyelitis and cystitis (26, 29, 32,
51). Infections due to the following organisms are reported
(30) to be particularly sensitive to nitrofurantoin: Egghgg-
Aghig‘gglg, Aerobacter aerogenes, Proteus ggttgggi, ngtggg
morganii, and Staphylococcus aureus. Nitrofurantoin has also
been reported as effective in the treatment of chronic puru-
lent rhinitis refractory to the usual antibacterial therapy
(28).

In veterinary medicine, nitrofurantoin has been used for
treatment of epizootic tracheobronchitis (kennel cough) in
dogs (3, 13, 34, 35, 42) and urinary tract infections in both
large and small animals (3. 30, 36). On oral administration
to dogs and cats, about 40 percent of the drug is excreted in
the urine (3). The recommended dose for urinary tract infec-
tions in small animals is one to two milligrams per pound of
body weight three times daily (3).

Streptomycin is biosynthesized by several species of act-
inomycetes, belonging principally to the genus Streptomyceg
or related genera. Dihydrostreptomycin is produced by hydro-
genation of the parent compound streptomycin. Both products
have equivalent antibiotic activity (23). In human medicine,
streptomycin and dihydrostreptomycin are used in the treatment
of tuberculosis, urinary tract infections, tularemia, undulant
fever, pulmonary infections, and various enteric infections
(40).

In veterinary medicine, these drugs have been used in the

treatment of pneumonia, leptospirosis and cystitis, as well as

12

gastrointestinal, ocular, and wound infections (23). The
aqueous sulfate salt is the most commonly employed form of
dihydrostreptomycin. The minimum dose recommended by Jones
(22) is five milligrams per pound of body weight intramuscu-

larly at twelve to twenty-four hour intervals.

II
MATERIALS AND METHODS

A. Preparation g§_§éperimental Animals

All cats in this experiment were procured in the vicin-
ity of East Lansing, Michigan. Most of them were from farms,
although a few had been city pets. Ages varied from young
mature (eight to ten months), to rather aged animals. All
cats were given two doses of feline distemper vaccine(h) at
seven to ten day intervals. They were also given one injec-
tion of feline pneumonitis vaccine(1) and wormed with Vermi-
Plex (R)(J). Urine samples were bacteriologically examined
to insure all cats were negative for bacteriuria at the start
of the experiment. Three cats were rejected because of bac-
teriuria on initial cultures. During the experiment, the
animals were given an exclusive diet of C/D and water. The
cats were housed in individual stainless steel cages and were

:furnished a plastic pan filled with wood chips for their elim-

inations.

£1. Selection‘gf Test Organism

 

The first consideration was selection of a representative

midcroorganism to be used in the experimental infection of the

 

(tL)Fromm Laboratories, Inc., Grafton, Wisconsin
(1)1’neumovax (R), Pitman-Moore 00.. Indianapolis, Indiana

(‘1 )Pitman-Moore Co. , Indianapolis, Indiana

14

animals. On the basis of a report by Mosier and Coles (36)
that g, 22;; was the most frequent microorganism found in
feline urinary tract infections, a hemolytic strain of g.

ggli isolated from a clinical case of feline urocystitis was
selected. This organism was sensitive to chloramphenicol,
dihydrostreptomycin, neomycin, polymyxin B and nitrofurantoin,
and resistant to chlortetracycline, penicillin, oxytetracy-
cline, tetracycline and novobiocin, as determined by the paper
disc method. Commercially prepared discs (k) were used in the
following strengths: dihydrostreptomycin and erythromycin,
two micrograms; chlortetracycline, oxytetracycline, tetracy—
cline, chloramphenicol, neomycin and novobiocin, five micro-
grams; nitrofurantoin, 100 micrograms, penicillin, two units;

and polymyxin B, 50 units.
9, Infection Procedure

Eighteen cats were divided into three groups of six each,
with males and females equally distributed. Exact ages were
not available; however, their approximate age was estimated.
They were classified as young (under two years), middle-aged
(two to five years) and old (over five years). The age
brackets were equally represented in the groups. Weights
were recorded to the nearest ounce before the experiment
started and at its conclusion. These three groups were des-
ignated as nitrofurantoin, dihydrostreptomycin and untreated

control, respectively.

 

Tk(Sensindiscs (R), Baltimore Biological Laboratories, Balti-
more, Maryland

15

A cystotomy was performed and the epithelial lining of
the bladder was abraded over a one to two cm. square area.
This abrasion was done with the scalpel blade. A specimen
of the bladder wall was also taken during the operation for
microscopy. After the bladder incision was sutured, two ml.
of a 24 hour broth culture of the bacterium (9.06 x 108 or-
ganisms/ml.) were injected through the bladder wall into the
lumen. Feed and water were withheld for 48 hours after sur-
gery to subject the animal to stress and to concentrate the

urine 0
2, Treatment

A cat was considered infected and treatment was started
if the urine culture was positive on the eighth postoperative
day. Nitrofurantoin in tablet form was given per os at the
rate of 10 mg. at eight hour intervals. This represented a
range of 1.17 to 2 mg. per pound of body weight. Dihydro-
streptomycin was given intramuscularly, as the aqueous sulfate
solution, once a day at the rate of 20 mg. per pound. Treat-
ment was continued for 24 hours after two consecutive daily

urine samples were bacteriologically negative.
E. Bacteriological Procedure

Urine samples were collected by catheterization daily
during the treatment period and every fourth day thereafter.
Catheterization was performed while the cat was under very

(1)

light thiamylal sodium anesthesia . Size four French, woven

 

(1)5urital (R), Parke, Davis and Co., Detroit, Michigan

16

nylon ureteral catheters with a whistle tip were used. They
were cut to a length of ten inches to simplify handling.
Sterilization was accomplished by autoclaving at 15 pounds
pressure for 18 - 20 minutes, and no chemical disinfectants
were used at any time on the catheters. Males were catheter-
ized by extruding the penis from the sheath and inserting the
catheter directly into the urethra. The procedure was slight-
ly more involved in the female and required greater care to
avoid contamination. The cat was placed on her right side,
and the assistant grasped the skin lateral to the vagina.
While the assistant pulled this skin laterally, the operator
grasped the ventral tip of the labia and pulled posteriorly.
The combined traction dilated the vestibule and allowed vis-
ualization of the urethral orifice on the floor of the vagina.
The catheter was then inserted in the urethra, with care taken
to avoid touching the vagina with the catheter.

Whenever possible, a ten m1. urine sample was obtained
of which five ml. was centrifuged. If less than five ml. was
obtained, the entire amount was centrifuged. The sample was
spun at 2500 r.p.m. for twelve to fifteen minutes. The super-
natant was then decanted or pipetted off and a loopful of the
sediment was streaked on a tryptose agar plate enriched with
five percent defibrinated bovine blood. The agar plate was
incubated at 37° C. for 18 to 24 hours, at which time it was
observed for growth. The specimen was considered bacterio-
logically positive and uncontaminated if all the colonies were

of the same typical gross appearance as the stock culture. If

17

more than one type of colony was observed, a representative
of each type was picked and identified by biochemical reac-

tions.
3. Necropsy Procedure

On the 43rd day following the start of treatment (5lst
post-infection day), the cats were sacrificed and a necropsy
was performed. Bladder and kidney specimens were collected
for histopathological examination. Kidney tissues and urine
were examined bacteriologically.

The tissues collected were fixed in formalin prior to
sectioning. Sections were cut at right angles to the vesical
mucosa. Sections to be stained with hematoxylin-eosin were
out six microns thick, and those to be stained by the Gram-
Wiegert technique were out four microns thick. Changes in
thickness of the various cell layers of the bladder wall were
determined by measuring the thickness of the cell layers at
several areas in each biOpsy and necropsy section for each

cat. Measurements were made with an ocular micrometer.

III
RESULTS

5. Progress g£_the Infection

Of the 26 originally infected cats, three died post-
operatively within the first 48 hours. It was considered
that one of these cats died from bacteremia due to excessive
abrasion of the bladder epithelium. This cat had a high
fever the first postoperative day but showed no evidence of
peritonitis at necropsy. The other two cats developed peri-
tonitis from leakage of the bacterial culture through the
bladder incision. These three cats had fevers of 103 to 106
degrees Fahrenheit and showed signs of depression. The re-
maining 23 animals showed great variation in response, with
temperatures ranging between 101 and 105.2 during the first
week. The degree of depreSsion and anorexia varied consist-
ently with the temperature. Hematuria and dysuria present
for the first two or three days disappeared rapidly in most
cases and by the eighth day only a few showed gross hematuria.
Five cats showed no bacteriuria on the eighth post-infection
day and were therefore withdrawn from the experiment.

Cats in the infected untreated control group lost an
average of 1.6 ounces in body weight, and those in the dihy-
drostreptomycin group lost an average of 5.6 ounces, whereas
those in the nitrofurantoin group had gained an average of

1.8 ounces at the end of the experiment.

19

B. Bacteriologic Results and Statistical Analysis

 

The results of urine cultures and a notation of the num-
ber of days each cat was treated are compiled in Table III.
Comparison of the numbers of bacteriologically negative urine
in each group of cats is demonstrated graphically in Figure 5.
The nitrofurantoin group was treated an average of eight days
and the dihydrostreptomycin group an average of nine days.

Statistical analysis for significance was directed to a
comparison of the percentage occurrence of bacteriologically
negative post-treatment urine specimens in each experimental

group. The number of cultures that could be considered for

each animal was determined by the number available from the
animal that had been treated the longest (cat 634, Table III).
since all cats were terminated on the same day. Therefore,

a series consisting of six urine cultures was compared for
each cat. This series for the treated groups consisted of
cultures taken at four day intervals beginning with the fifth
to the eighth day following cessation of treatment. The aver-
age series of cultures from the two treated groups covered the
period from the 15th to the 35th day from start of treatment.
Since the control group was not treated, the same period was
used to obtain the sample series from this group.

Using this sampling plan, there were 36 urine specimens
for comparison in each group. The occurrence of bacteriologi-
cally negative specimens among the experimental groups was:
control, 66.66 percent; dihydrostreptomycin, 66.66 percent;

nitrofurantoin, 88.88 percent.

20

Comparison of these percentages for significant differ-
ence was accomplished by ”Student's” t-test (12). The occur-
rence of sterile urine cultures in the nitrofurantoin group
was significantly higher than in either of the other groups.
The test showed a P value between .05 and .02 (95 to 98 per-

cent level) for significant difference.
9. Necropsy Findings

There were no gross changes noted on necropsy except in
the urinary bladders and surrounding tissues. Adhesions of
omentum and mesentery to the bladder surface in the incision
area were usually observed. In a few cases, the bladder was
adherent to the ventral abdominal wall, with no significant
variation among the three groups. The epithelial lining of
the bladder generally showed scattered areas of hemosiderosis
and in several cases (three control, one dihydrostreptomycin,
and three nitrofurantoin), small vesicles one to two mm. in
diameter were noticed within the epithelium. These vesicles
were filled with a clear, serous fluid. In most cases, the
epithelium was intact and free from any breaks in the surface.
Cat 633 (dihydrostreptomycin) had a shallow ulcer of the mu-
cosa, five mm. in diameter, at one side of the incision, and
cat 635 (dihydrostreptomycin) showed a similar, although
healed, area.

Comparison of the biopsy and necropsy bladder sections
revealed changes in the character and thickness of certain

cell layers. In a normal bladder<m> (Figures 1 and 2), the

 

Tm)Bladder section from normal cat. Anatomy Department, College
of Veterinary Medicine, Michigan State University, East
Lansing, Michigan.

21

demarcation between the lamina propria and the submucosa was
easily distinguishable, since the submucosa was less dense
than the lamina propria. In most of the post-infection blad-
ders, this differentiation was much more difficult, mainly
because of an increase in density of the submucosa (Figure 3)
as a result of hyperplasia of connective tissue. Capillary
proliferation in the lamina propria, and to a lesser extent
in the submucosa, was marked in these bladders. Figure 4
shows the increased number and size of blood vessels in the
lamina propria.

As shown in Table II, there was a fairly consistent in-
crease in thickness of both the lamina propria and the submu-
cosa. These changes were less in the nitrofurantoin group
than in either of the other two groups, with no appreciable
difference between the control and the dihydrostreptomycin
group. Following infection, the transitional epithelium was
consistently reduced in height in all three groups, and the
cells of this layer appeared shrunken in comparison to those
of the normal bladder.

Examination of Gram-Wiegert stained bladder and kidney
tissues failed to demonstrate any bacteria in situ and post-
mortem cultures of kidney tissue did not reveal any ascending

infection.

IV
DISCUSSION

The technique used in this experiment to establish and
maintain infection in the bladder was not entirely adequate.
Evidence of infection was lacking in 22 percent of the infected
cats by the eighth day. Urine from control cat 557 was bac—
teriologically negative after the 15th day (seventh post-
treatment day, Table III) and two others (174 and 209) were
negative after the 23rd day (15th post-treatment day, Table
III). A means to prevent spontaneous recovery from experi-
mental infection was needed. The method of Miller gt.§;.
(33), in which a sterile zinc disc was dipped into a culture
of the test organism and then surgically placed in the lumen
of the bladder, may fulfill this purpose. The disc would act
as a source of irritation to prevent spontaneous recovery from
the infection.

As originally conceived, urine specimens were to be ob-
tained by paracentesis. Unfortunately this could not be ac-
complished during the first few days following surgery because
the bladder was very thickened and the cats were voiding urine
frequently. Thus, there was never a very large quantity of
urine present. As a result, it was necessary to resort to
catheterization to obtain the necessary urine samples. Con-
trary to common belief, the collection of urine samples by
catheterization proved to be a bacteriologically acceptable

technique. Contamination of the urine was virtually nil.

23

Of the first 143 specimens collected, only two were bacterio-
logically contaminated. However, careful collection technique
was necessary, particularly in the female.

It was thought that the infection should have a chance
to become established before any treatment was initiated. The
period of eight days was arbitrarily selected. It is interest-
ing to speculate on the possible results had treatment been de-
layed several weeks.

The dose range of nitrofurantoin used is now considered
to be fairly low. The latest labeling information on Fura-
dantin advises a minimum dose of two mg. per pound at eight
hour intervals. Emesis following the oral dose of nitrofuran-
toin was observed in one animal (209) for the first two days
of treatment. Feeding a small amount of food with the medi-
cine seemed to prevent this. The problem solved itself by
the fourth day, and no further feeding was required with the
medication.

Treatment was continued in both groups for a total of 72
hours after the urine first became negative. It is a general
concept of chemotherapy that treatment should continue for 48
to 72 hours after disappearance of the cardinal sign or symp-
tom. -The fact that three animals (066 and 633 - dihydrostrep-
tomycin, and 634 — nitrofurantoin) showed positive cultures
within three to four days following cessation of treatment
may indicate a need for more prolonged treatment.

The average weight changes in the three groups presented

a confusing picture. Although it might have been expected

24

that the untreated control group would lose weight, the reason
for an even greater loss in the dihydrostreptomycin treated
group was puzzling. Perhaps this was a manifestation of toxi-
city of the antibiotic. The author can present no reason for
the weight gain in the nitrofurantoin group other than that
this group apparently was less severely affected by the infec-
tion and recovered more quickly. Evidence of these two points
has been previously mentioned under "Results".

The most striking fact noted in this study was the ex-
treme inconsistency in bacteriological results. As noted in
Table III, cat 774 (dihydrostreptomycin) had a series of nine
consecutive negative samples over a period of 36 days. On
the 43rd day, however, the culture was positive. Other exam-
ples of this tendency were noted in animals 560, 074 and 195.

During the late stages of the experiment, an attempt was
made to determine the cause of inconsistent urine cultures in
certain animals. Out of curiosity and not as an integral part
of the experiment, certain urine samples were cultured using
an enrichment technique. The sample was inoculated into brain
heart infusion broth containing 0.5 percent agar and incubated
24 hours prior to streaking on blood plates. In some cases,
growth was obtained by this procedure when none was found by
the previously outlined method. All of the urine samples in-
cluded in the results were cultured in the same manner, how-
ever, and the method of comparing the occurrence of sterile
samples remains valid.

It has been strongly suggested that the only valid cri-

terion of a cure in urinary tract infections is the absence

25

of bacteriuria, and this can be determined only by culture
(43). The author also considered this fact of primary im-
portance and constructed the statistical evaluation on this
basis. There seems to be no other way to evaluate a problem
of this nature. McCrea gt_gl. (32) have stressed that cre-
dence cannot be placed on the routine urinalysis, and inter-
pretation based on the presence or absence of leukocytes in
the sediment can be in direct opposition to the true bacte-
rial picture.

An experiment of this nature would probably be more sig-
nificant if the animals could be kept for a longer period of
time. As evidenced in Table III, many of the cats were in an
equivocable position in regard to status of infection when the
experiment was terminated. Ideally, the animals should be
held long enough to establish a definite pattern for each ani-
mal. McCrea 22 gl. (24) have not considered their human pa-
tients cured until they were clinically free of symptoms and
had five consecutive negative post-treatment cultures taken

at weekly intervals.

SUMMARY AND CONCLUSIONS

Urocystitis was induced in 18 cats by traumatizing the
bladder and introducing a culture of g. ggll. Nitrofurantoin
was given orally to six cats at a dose of 1.17 to 2 mg. per
pound of body weight at.eight hour intervals, for an average
of eight days. Dihydrostreptomycin sulfate was given intra-
muscularly to six cats at a dose of 20 mg. per pound of body
weight once a day, for an average of nine days.

There were significantly more bacteriologically negative
urine specimens following treatment with nitrofurantoin than
with dihydrostreptomycin. There was no difference in the oc-
currence of bacteriologically negative urine specimens between
the dihydrostreptomycin group and the control group.

Inflammatory reaction of the bladder wall to the experi-
mental infection was considerably less in the nitrofurantoin
treated cats than in either the dihydrostreptomycin treated
cats or the infected untreated controls.

The methods used in this experiment to produce and main-
tain infection in the urinary bladder were not entirely satis-
factory. Twenty-two percent of the cats originally infected
had sterile urine cultures in eight days, and the untreated
controls did not stay as uniformly infected as was expected.
In future experiments, it would seem useful to provide means
of continued bladder irritation in order to prevent spontane-

ous cures of experimental infections.

 

27

Nitrofurantoin was well-tolerated by five of six cats.
One cat required feeding before medication to prevent vomiting.
Bacteriuria was not always detected by the usual bacte-
riologic laboratory procedures. A cultural method to increase

the probability of detecting bacteriuria was discussed.

TABLE I

MICROORGANISMS CAUSING URINARY TRACT INFECTIONS IN CATS
Mosier and Coles (36)

 

 

 

Bacteria Cases* Percent of Total
Proteus sp 1 3.9
Streptococci 6 23.1
Staph. aureus 3 11.5

Total 26 100.0

 

*Dual infections were present in 3.7 percent of these cases.

TABLE II

PERCENT CHANGE IN BLADDER WALL THICKNESS FROM PRE-
INFECTION (BIOPSY) T0 POST-INFECTION (NECROPSY)

 

Cell Layers

 

 

Transitional Lamina
Group Epithelium Propria Submucosa
percent percent percent
Control -23.5 +60 +46
Dihydrostreptomycin -26 +33 +74

Nitrofurantoin -22.5 -2 +19

 

TABLE III

BACTERIOLOGICAL RESULTS AND SCHEDULE OF TREATMENT DAYS

 

 

 

 

 

 

r( I I I + I I I I + I I I I I I I I I
3'
a + I I + I I I I I + + I I I I I I I
vw I I I + I + I I I I + I I I I I I I
m
H + I I + I I I + I + + I I + I I I I
r1
u- I I I + I + I I I + I I I I I + I I
N
F) I I I I I I I I I + + + I I I + I I
N
ex + I I + I I I I I I I + I I I I I I
H
a) a
H
p *l
at?
m
80 *I
43.4
ovy + + + + I I + I I + + I I I I *+ I I
I-u—I
E!
.3 *I
6041-!
e * e+
ad
001 t *I #l *+
3;“
H + I I + I I + *l I I *l *l I + *I #I I I
5..
o
no *I *I III-I- ¢+ III-I-
me.
In
a t in *+ *+ *+ *+
8
a: *I *+ *+ *+ *+ *+
N + X I I + +*I*+ I I*I*+ I I*+ *4- #I I
~o *+ *+ *+ *+ *+ *I #I
VI + + I + + +vtl *+ + *+ *+ *+ I I *I *+ *I I
3‘ + + I + + + *+ *+ *+ *I *+ *+ ex ex *+ *l *+ *I
ry + + I + I + *l s+ *I *I *+ *+ *I #I *+ t+ *+ *l
N + + I + I + *+ *+ *I *+ t+ *+ *l *I *+ *I a+ *I
H + + + + + + *+ *+ *+ *+ *+ t+ *+ *+ *+ *+ *+ *+
o.
:3.
In .3 o~.3 a. o ~o .3 .3 b— «x :1 O\ mI~o .3 «x N
HI In a. o a. W\‘O ~o a. a. c> ax a) ox :3 co cx I»
g «I HI oI W) V\ w( 0’ o h- N ~o ~o H H N ~o n.~o
H m“— * ~'
‘g STOUINOO GRIORRNI NIOXNOIdHHISOHGIHIG NIOINVHRJOHIIN

- = Urine Culture Negative;

+ = Urine Culture Positive
X = No Sample; * = Treated

O
0

Key

 

30

Figure 1
NORMAL FELINE BLADDER

X90

TRANSITIONAL
EPITHELIUM

LAMINA PROPRIA

  
 

) SUBMUCOSA

 

MUSCULARIS

31

Figure 2
NORMAL FELINE BLADDER

X235

RANSITIONAL
EPITHELIUM

LAMINA PROPRIA

 

SUBMUCOSA

 

32
Figure 3

INCREASED DENSITY OF TH

LAMINA PROPRIA AND
CAT 633

7“
N
H

SUBMUCOSA - POST-INFECTION.

X235

LAMINA
PROPRIA

SUBMUCOSA

 

. at. . (.

 

33
Figure 4

CULARITY O'

INA PROPRIA

‘ ‘ If.
A.

V I 1
Trig. i...

,2
A.

a
‘

VA:

'T-INFECTICN.

POI:

 

Number of sterile urine samples

4:-

U

N

 

34

Figure 5
COMPARISON OF THE NUMBER OF BACTERIOLOGICALLY
NEGATIVE URINE SPECIMENS OCCURRING
IN EACH EXPERIMENTAL GROUP

 

 

4 A .‘ 3 A
// \ l‘ .. 01’" I\\ II
I, \\ I \.‘ i. \ I \ I
/ I \ . \ I /
I ’ \ ' " I \ I
/ I '\ , . I \
‘ / i I I I
I' I \ ' \ I ..I.. ....
I . \ I ' \ I I I
I \ I \ I \ I
I \ I \ ’ \ I
I \ I \I \I
.. , , V V v
, O
I I a
’ \ I .
’ I I
II ‘I I . .
I
I- ’ ‘, I
0.! ‘ I
I" ‘ I,
I
I . \ '
I 3., I
.. I, .11.“..?-!
; Nitrofurantoin
J """ Dihydrostreptomycin
0"
'/ ~°"---Control
fl 1 l- I l . I f 1 I L 1 l ,1
1 2 3 4 5 7 11 15 19 23 27 3f 33 5933

Days from start of treatment

1.

9.

10.

11.

12.

13.

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