THESlS

This is to certify that the

thesis entitled

A Study of Cozxnebacterium renale-like
Organisms Isolated from Cows

 

presented bg

Ganeswar Bi swal

has been accepted towards fulfillment
of the requirements for

M.S. degree in Animal Patholog’

 

Date August 23: 1950

 

0-169

 

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- 2 i,"

 

A STUDY OF CCRYNEBACTERIUM REHAEEgLIKE OI’AFISAS ISOLATED

FROM COWS

by

Ganeswar Biswal
‘

A Thesis

Submitted to tne School of Graduate Studies of
Michigan State Ccllege of Agriculture and
Applied Science in partial fulfillment

of the requirements for the degree
of

MASTER OF SCIENCE
Department of Animal Pathology
1950

TzLBLB OF CONTDITS

ENE
ACIQICNIEDGELEBT
INTRODUCTION - 1
. REVI Ed 0F LITERATURE 2
MA “2331an ALID i-ETiCDS 1 9
RESULTS 214
ms USSICN ug
CCI‘JCLUSION 5,2
SIEw'ARY 55
B I BL I OGRAPHY 53
ILLUSTRATIONS 58

2375”“

ACKNOWLEDGRIEN T

The writer expresses his sincere appreciation to Dr. Frank Thorp, Jr.
whose lectures, scientific conferences and seminars in research are re-
flected here. His broad view-point of the possibilities of research in
graduate study was a constant inspiration to me. The valuable advice

and encouragement generously given are respectfully acknowledged.

The writer expresses his thankfulness to Dr. R. A. Runnells, Head

of the Department of Pathology for the kind encouragement and guidance

given.

Appreciation is extended to Mr. M. L. Gray for the invaluable

help and suggestions in the preparation of the manuscript and photographs.

To Miss Sylvia Laine thanks are due for her aid in the technicological

work and for the artistic sketch of the mouse restraint device.

Cooperation of the ZOOIOgy Department in supplying the mice

is appreciated.

The assistance and suggestions of the members of the staff are

remembered.

Last but not the least the writer expresses his deep sense of

gratitude to the Governments of Otissa and India for their financial aid

Which enabled him to continue his study in this country.

INTRODUCTION

A study of ten strains of Corynobacterium renale-like organisms

 

isolated from the urine of ten cows suffering from pyelonephritis is re—
ported in this thesis. The organisms did not wholly fulfill the re-
quirements as outlined by Bergey (1948) to be classified as Q, ronale.
However, some of the characteristics such as morphology, growth in
culture media, biochemical reactions and pathogenicity to laboratory
animals were sufficiently similar to those of 2, renale that they

have been named 9, renale-like organisms. The Corynebacteria of animal
origin, however, have not been studied or classified adequately, although
they were known to cause both acute and chronic diseases of animals.
which were of great economic importance to animal health. Previous to
1946 there was a general opinion that laboratory animals were refractory
to infection by Q. renale. The primary objective of this investigation
was to determine whether or not these 9, Eggglgrlike bacteria would

produce lesions in laboratory mice and rabbits.

REVIEW CF LITERATURE

The etiology of pyelonephritis in cattle has been described as
poly-bacterial in nature. With few exceptions, however. the bulk of
the evidence supports the view that G. renale is the specific etiological

agent in infectious pyelonepnritis and cystitis.

Bruckmullerp(18b9) described enlargement and abscessation of the

 

kidney of an ox accompanied with dilatation of the renal pelvis.

Siedamgrots§y_(1875) described the lesions in the renal parenchyma

 

of a cow. Necrosis of the papillae associated with dilated pelvis
was the main characteristic feature. Bacteria were isolated from the

pus in the renal pelvis.

Pflgg (187b) observed a case of nephritis in a cow. The author
suspected that micro-organisms were responsible for the onset of pyelitis

and pyelo-nephritis.

Danmann (1877) described the gross and microsc0pic lesions in a
case of bacillary nephritis. Bacteria were demonstrated in the uriniferous
tubules in addition to the leukocytic infiltration in the renal inter-

stitial tissues. The affected kidneys increased in weight.

Guillebeau and Hess (1888 to 1892) described the symptomatology,

 

postomortem pathology and pathogenicity of the organisms isolated from
the urine and the renal pelvis of affected animals. The authors could

not produce the disease in cows, oxen. goats and pigs either by in-

travenous or intravesical inoculation with urine or cultures.

Guillebeau (1888) found non-motile. slender bacilli in the

 

renal abscesses of cows affected with pyelonephritis.

Bang (1889) and Schmidt (1890) observed bacilli in the lesions

of the kidney of affected cattle.

Friedbeggeg (1890) in a study of pyelonephritis found clumped

 

gram-positive bacilli occurring in the kidneys of affected cattle.

Hoflich (1891) described the gross and microscopic lesions of
the bacillary pyelonephritis in the ox. Dilatation and leukecytic in-
filtration in uriniferous tubules associated with increase in amount of
interstitial connective tissues of the kidney were noticed. Organisms
were isolated from the renal abscesses, the pelvic pus and the urine.
The organisms were small gram positive reds, measuring 1 to 3 x 0.7 u.

He named the organism the "bacillus pyelonephritidig_boum'.

 

Enderlen (1891) isolated a small gram positive bacillus
measuring 2.1 micra to 2.8 micra in length by 0.7 micra thick from
cases of pyelonephritis in cattle. He was able to cultivate the organism
in a pure state and also produced typical lesions of pyelonephritis
in two uroter-ligatured rabbits following intravenous inoculation.
This disease could not be produced by other methods. It was stressed

that the infection was of hematogenous type.

Bollinger (1891) supported the work of Enderlen in that the in-
fection was of hematogenous type. There was no generalization. The

lesions were located for the most part in the renal pelvis and parenchyma.

Lucet(1892) found the bacillus pyogenes bovis as the cause of
pyelonephritis in cattle. These were isolated from renal abscesses and
were thought to be the same organism as previously identified by Hoflich

in 1891e

Kitt (1893) described bacillary pyslonephritis in cattle. The

 

author could not demonstrate the bacillus pyelonephritidis boum as the

specific etiological agent in the causation of the disease. Diseaseig
processes of varying degrees were observed in kidneys, ureters and

bladder.

Mollereau and Porcher (1895) described the same type of bactehia
as previously discovered by Hoflich and Enderlen in 1891, in the renal

tissue of cattle affected with pyelonephritis.

Bartels (1897) described pyeloaephainephritis in cattle due

to bacillus pyelonephritidis.

Albrecht (1900) described the preponderance of the bacillus of

pyelonephritis in the affected kidneys of cattle.

Ernst (1905) studied the histopathology of bovine pyelonsphritis.
The organitm could not be isolated in pure state. In 1907. he5>laced
this organism in the group of corynebacteria and called it the ”coryno—
bacterium renalis bovis”. It measured 2 to h micra x 0.5 to 0.b micra.
The bacteria occurred in clumps. Bacteria were present in the necrosed
collecting tubes of the papillae. There was marked cellular infiltration
into these tubes. With the degree of advancement of the disease a

marked increase in the interstitial connective tissue was noticed. The

5

predilection of the organism was for urine. This was substantiated by

observing the increased growth in agar medium containing urine.

Ritzenthaler (1910) described the microscOpic findings in 28 out

 

of a total of 88 cases encountered. The essential lesions in the renal
parenchyma consisted of necrosis of lobules, dilatation of the pelvis,
leucocytic infiltration and thickening of the mucous membrane of the
pelvis. The author believed that the type of infection was an ascending

0116.

9213 (1918) described a bacterial pyelonephritis in a cow. Organ-
isms isolated from the urine were aerobic, gram positive, non-motile
rods, 2 to M micra long by 0.5 micran thick. They were pleomorphic and
showed deeper staining at the ends and middle. Bacteria occurred in
clumps in smears made directly from the urine. This clumping’was a
special characteristic in diagnosis of the organism which he called

"corynobacillus".

 

Bgyd (1918) described pyelonephritis in two cows - one a Guernsey,
the other a Holstein. The kidneys were enlarged and the cortex was studded
with numerous abscesses. The pelvis was dilated with yellow brown fluid
which contained blood clots, some calcarebus materials and tissue threads.
The ureters were enlarged to the size of a man's middle finger. The
bladder wall was thickened. HistOpathologic examination of the affected

kidney revealed cellular infiltration and club shaped bacteria in the cortex.

Bacteria could not be isolated.

Boyd ££_gl (1918) observed a case of pyelonephritis in‘a sheep.

Eberson (1918) in a review of the bacteriologic study of the
diptheroid organiSAs stated:

"higula (1900) described tne B. renalis bovis.

Rods with thickened ends non-motile, gram-

positive. In broth, a granular urecipitate

is formed, the medium remaining clear. 0b-

ll;?tr aerooe no not grow at room temperature.

The organism grows poorly as compared with

the d'pntneria bacterium. Babes-Lrnst gran lws

appear much latcl. It is club-Shaped, lacet—

like or cylindrical. E3 acid is produced in

dextrose and glycerine, no spores. Pathogenicity
appears doubtful. Ernst was unabl» to ceno1<trate
virulence for guinea pigs or rabbits by means

of intraperitoneal or intrapulmonary inoculations.
Concludes that the organism is not etiologic for
pyelonepnritis, since he could not recover the

organism from the lesions nor roproducu the

disease."

Jones and Eittlfi_(l925) stated: "Joest (1929) considers pyelo-
nephritis a specific infectious disease of cat‘le and called attention to

the fact that tiers is no disease having similar etiology and pathology."

From the protocols of 13 cattle autopsied and studied, it was
evident that there were lesions in ens or both kidneys and ureters. The
bladder wall was tnicxened up to one to two cm. Bacteria were present in
the urinary sediment in large numbers. When preparations from the pelvis
of the infected animals were made and stained, bacteria were found in
large number. An attempt was made to diagnose the infected animals by the
agglutination test. However the titers of the healthy and the infected
animals were about the same. No recognizable allergic reaction in the in-
fected animals was observed when the skin and cornea vere injected with

he filtered culture.

Jones and Little (lBEb), on the basis of previous findings, made

a bacteriologic and animal inoculation study. The organisms were non-

motile, gram positive slender rods, two to three micra in length,

£0

Plemorphism in artificial culturemedia was pronounced. The bacteria
grew in ordinary laboratory media. There was bacterial sedimenttin broth
cultures. 0n serum medium there were raised, grayish white, dry colonies
which did not digest the serum. Blood was not hemolyzed nor was gelatin
liquefied. There was reduction of litmus in lihnus milk, the casein
coagulated at the bottom, leaving the upper stratum blue. Only dextrose
was fermented with a final pH of u.9 to 5.0. The 26 strains studied were
immunologically the same. These cultures were obtained from five herds.
In a study of pathogenicity for laboratory animals it was fouid that
white mice, rabbits and guinea pigs were resistant to infection. No

toxin was demonstrated.

Renal lesions failed to develcp in cattle following intravenous
inoculation of 5 ml and 10 ml amounts of broth cultures of the organism.
Cows failed to develop lesions in the bladder, when swab cultures were
introduced into the vagina. On two instances when cows were fed large
quantities of freshly isolated cultures, one develoPed cystitis and the
other remained normal. When this part of the experiment was repeated
the results were negative. Fecal examination of cows artificially fed adfi.
pure cultures, revealed no organisms. The introduction of broth cultures

into the bladder produced the disease whicn pointed to an ascending type

of infection.

Udall (l92b) observed pyelonephritis in a 9 year old Jersey cow

and a bacteriological examination revealed ”B. pyelonephritis bovis -

 

35 A, Hagan” from the left kidney and B. pyelonephritig and many cocci

 

from the right kidney.

Boyd (1927) described five clinical cases of pyeloneohritis and

stated that the disease was usually urc‘e-ous in origin, but might at
certain occasions be hematogenous, especially when occurring after par-
turition. In one case the affected left kidney, ureter and bladder
weighted bu oz. The ureter was u cm in diameter and one cm thick. The
mucous membrane of the bladder was deeply furrowex aid the wall was

one cm thick. The lesions were conIined mainly to the ureters. There

were also abscesses ir the kidneys.

Cellular infiltration in the cortical zone and necrotic areas
in the renal papillae were observed. Large amounts of erythrocytes,
white blood cells, fibrin, dipntneroid bacteria and unrecognizable material
were found in the calyces. Degeneration and desquamatiOn of ureteral
epithelium was accompanied by fibrosis. Fibrosis also was seen in the

bladder wall. The urethra was apparently normal.

Jones and Little (1928) described the symptomatology of 20 head

of cattle from three herds. Urine analysis of these cattle and many herds
in New Jersey revealed white blood cells, erythrocytes, round cells,

epithelial cells and masses of gram positive diphtheroids in the sediment.

The gross lesions found in the bladder were a hemorrhagic, swollen
mucosa thrown into folds and accompanied by necrosis. The pelvis of

the kidney was filled with yellow, purulent exudate. Most often the

lesions were confined to the pelvis. When the involvement was diffuse
in nature the enlargement of the kidneys was noticed. The capsule was ad-
herent to the cortex. The microscOpic findings seen in the renal parenchyma

were fibrosis and leucocytic and round cell infiltration.

Cystith and pyelonephritis were found to be related processes of

the same disease.

9
The disease was an ascending type attacking first the bladder and
then the kidneys through the ureters. This was substantiated by producing
cystitis after the introduction of small quantities of a broth culture via
the urethra but no lesions were found in the kidneys when five to ten cc of
a broth culture was given intravenously. Furthermore, in the earlier
cases bladders were alone involved. In the chronicity of the disease

lesions were seen in the bladder, ureters and kidneys.

The diptheroids normally present in the urine of cows and bulls
were not identical with the pyelonephritis bacillus. Vaginal swabbing
with the cultures of the organism failed to transmit the disease. however,
infection was produced in two cows which were groomed and brushed after

infected animals.

The disease seemed to have no relation to the diseases of the genital
tract. It occurred independently without genital infection. The onlyllocus

of the organism was the urinary tract of an infected animal.

McFadyean (1929) in a discussion of nephritis in animals described
a pyelonephritis in which the pelvis and medulla of the kidney were the
main sites of the disease. One or both kidneys might be involved. Even
the extent of invasion in the latter case was not to the same degree in

the two organs. It was stated that the hematOgenous theory of infectLon

was inadequate as the main sites of lesions were in the pelvis and medulla.

The macroscopic lesions observed were enlargement of the kidney and
increase in weight - ”up to 8-9 lbs”. The lesions located in the pelvis varied
from slight congestion to ulceration. In some cases the inflammatory process

extended to the papillae of the medulla.

10
The microscopic alterations of the tissues were degeneration,
destruction and desquamation of the epithelial lining of the tubules
accompanied by polymorphonuclear leucocytic infiltration from the neigh-
boring capillaries in the medulla. As the invasion of the bacteria was
through the uriniferous tubules. recent exudation and cellular infiltration
were in the cortex. He stated ”Bollinger thought the pyelonephritis was

the third most frequent disease of cattle in Germany.”

19.9.9.9. and Little (1930) isolated 128 strains of diphtheroids from
the gsnito-urinary tract (sheath, urethra, bladder, urine and vagina) of
3H calves from a herd in which infections cystitis and pyelonephritis
were present. They placed these strains into five cultural groups on the

basis of morphology, biochemical and immunological reactions.

Olafson (1930) recorded a case of pyelonephritis and cystitis in

a two-year-old English Shepherd female and was able to demonstrate
”9, renalis” as the etiological agent. The organism isolated was identical

with the bovine strains.

Palmer (1931) observed three cases of specific infectious pyelonephritis.
Gram positive diptheroids were isolated from two cases. The lesions were

alike in all cases.

Merchant (1935) in a study of the corynebacteria associated with

the diseases of domestic animals stated that 9, renalis isolated from

the renal exudates were bacillary-shaped in all media and had a striated

appearance in stained smears. Metachromatic granules were observed tn

the organisms when grown on Loeffler's serum medium. They occurreéin

clumps, were gram positive and non-acid fast. In solid media, the culture

became drier with age. Nine of the 20 strains caused an alkalinity of

ll

litmus milk with digestion of milk casein but no coagulation. There was
no digestion of curd. There was no action on gelatin nor on solidified
serum. The majority of the cultures fermented glucose, a few levulose

and mannose and only one strain attacked dextrin. All the strains were

not agglutinated by sera prepared against three strains.

Boyd and Bishop (1937) recorded eight cases of pyelonephritis in
horses (seven mares and one stallion). E, renale was isolated from the
urine of all animals. Pathologic findings were practically the same as

observed in cattle.

McIntosh (1938) reported four clinical cases of pyelonephritis

in cattle at the Ontario Veterinary College Clinic. The clinical symptoms

were described, cornyebacteria were isolated and identified from two cases.

Palmer (1938) reported on a purebred Holstein-Friesian cow. At
autopsy, in addition to the presence of pathologic changes in the urinary
organs, gram positive diphtheroids were isolated from the kidney pus.

These were thought to be the specific etiological agent of the disease.

Doll (1942) in an unpublished study found typical pyelonepnritic

lesions in the kidneys of 13 rabbits intravenously inoculated with E, renale.

Morgan (19u2) mentioned an atypical 9, renalis culture which did

not ferment dextrose.

Thorp £3 a; (19u3) reported six cases of pyelonephritis among
five cows and one male calf. C. renale was isolated from the cows and a

diphtheroid from the calf. The organisms were non-motile, non-spore-

forming, gram positive rods, occurring in palisade groups showing the

beaded staining properties.

12
The bacteria fermented glucose, hydrOgen sulfide and indole were
not formed and nitrate was not reduced. Urine analysis indicated the
presence of albumin, sediment, bacteria, erythrocytes, alkaline pH and

decrease in creatinine and non-protein-nitrOgen.

The affected kidneys were enlarged. In cnronic cases the capsule
was thickened and adhered to the cortical substance. They showed mottling,
and grayish yellow foci on the lobes. On the cut surface necrotic exudates
containing blood and pus were observed. Fibrosis developed in chronic
cases. Necrosis, suppuration, sloughing and disappearance of some portions
of the medulla were seen. The major and minor cal’ces were invariably dis-
tended with inflammatory and degenerative exudates. Numerous inflammatory,
edamatous, hyperplastic and focal degenerative changes were observed in

the bladder and ureters.

Histopathologic changes were characterized by cellular infiltra-
tion and connective tissue proliferation in the interstitial tissues.
Atrophy and cellular casts in the tubules were observed. The renal corpuscles
showed various inflammatory and regressive changes leading to loss of

architectural design. The medulla showed necrosis, suppuration and leuco-

cytic infiltration.

The renal arteries displayed arteritis, thickening of the intima,
necrosis and fibrosis in the media and numerous thrombi. The ureters
showed inflammatory and degenerative changes. The ureteral epithelium
showed metaplasia simulating stratified squamous epithelium. Varying de-
grees of congestion, edema, necrosis and desquamation of the bladder

epithelium were noticed.

15
Coffin (l9u3) described 9, renale as a gram positive pleomorphic
rod occurring in palisade groupings. The banded appearance of the organism

was seen with methylene blui staining.

91351 (19u3) reported a case of pyelonephritis in a Holstein-
Frisian cow. The hemoglobin content was 7.5 mg, the erythrocyte and
leucocyte counts were b.15 millions and 6.b thousands mm3 respectively.
The urine was positive for albumin and blood. Grossly the kidneys were
enlarged and weighted 6.0 to 7.5 lbs. In addition to ureteritis and

cystitis fibronecrotic membranes were present.

Foss (l9hu) isolated.§, renalis from the kidneys and bladder mucosa
of a horse. The organism was a deatroe fermenter, indole non-producer,
occurred as a rod in groups, showing typical bars and metachromatic granules

and was gram positive.

Beck g£_gl (1945) reported on the penicillin treatment of six

cows affected with pyelonephritis and cystitis with encouraging results.

Feenstra gt gl_(l9k5) artificially produced pyelonephritis and cystitis
in a Jersey steer by intra-urethral inoculation. At autopsy nine months
later gross and microscopic changes were identical with the lesions seen

syyadbd

in naturally<animals. 2, renale was isolated from the kidneys.

Feenstra 35 El (19H5L) studied morphological, cultural, biochemical
and serological properties of 19 diphtheroids isolated from cases of pyelo-
nephritis. These findings suggested that there were two distinct species

of corynebacteria - fine growers and rapid growers - involved in the

etiology of pyelonephritis. Of the 19 strains studied 6 were slow growers,

xylose fermenters, hemolysis and H28 producers.

1n
Eggnst£§_:t.gl (19kb) described the invasiveness of the diptheroids

of pyelonephritis. In the acute fulminating type the lesions were more
severe and were scattered throughout the kidney. In the mild and chronic

type the lesions involved mostly the calyces, ureters and bladder.

Lovell (lahb) in a morphological, cultural, biochenical and sero-
logical study of 36 strains of diphtheroids found 26 to be 9, renale.
All the 26 strains were gram positive, occurred in groups and showed a

beaded appearance.

These grew well in plain broth and on agar. Bacterial deposit
occurred in the broth leaving the supernatant fluid clear. No markediim-
provement in growth was seen in blood agar or serum agar. Neither hemolygis

of blood nor liquefaction of gelatin was produced by any of the strains.

Catalase was produced by all. ChromOgenesis was marked on blood agar,

6’

solid serum and egg medium. A distinct "halo” around the colonie‘ was
produced when grown on 10% sterilized milk agar. High alkalinity in.
litmus milk, splitting of urea in urea broth were produced by most of these
26 strains. Glucose was attacked by all the 26 strains. Some of the

strains wele V. P. and M. R. positive. Most of the strains were serologically

alike when treated with anti serum of three different strains of Q. renale.

Mice when were infected intravenously with 2, renale at a dosage
rate of 20 millionf'living organisms, in most of the cases the organisms
were isolated from the kidneys and urinary tract. In a few cases organisms
were isolated from the blood and spleen. This suggested that 9, renale

was pathogenic to mice when given in adequate dosage.

Lovell and Cotchin (19M6A) gave 20 milliond of 9, renale to 50 mice -

15
25 male, 25 female - intravenously. They were able to isolate the
organisms from the blood, spleen, kidneys, urine of infected animals
when they were killed after an interval of 6 hours to 14 days. More or
less the same picture was noticed in those animals which died from the
3rd to 12th day after infection. In all these cases lesions were marked
in the kidneys. There was papillitis, pyelitis and pyonephrosis with
the invading degenerative processes extending downward in the ureter

and outward to the peri-renal tissue.

In a second study they gave 20 million 9, renale organisms intra-
venously, intraperitoneallyyisubcutaneotaly and vaginally to four groups
of 10 mice. Kidney lesio‘sioccurred in three mice, infected intravenously
and one mouse vaginally infected. The infection was not spread by contact

as 12 healthy mice failed to show lesions in the urinary tract when placed

with 12 infected mice for 56 days.

The renal damage in guinea pigs and rabbits to an intravenous in-

oculation of 60 million of Q, renale was mild as compared to the lesions in

mice.

Thickened edematous condition of the chorioallantoic membrane was
noticed when an inOCulum of E, Eggalg'was injected into a nine-day-old

chick embryo.

Morgan gt al (19ub) identified 18 strains of 9, renale isolated

 

from the sheaths of 5N bulls.

Runnells (l9h6) mentioned that infections pyelonephritis has been

recorded irh swine.

lb

Weitz (l9h7) isolated nu strains of E, renale from the vagina
of 100 normal cows in three herds. Er renale was not found in the
abnormal discharge of 180 cows suffering from endonukritis. E, renale

was recovered from the normal urine. This led to the Opinion that

2, renale possibly might be a normal inhabitant of the posterior part
of the vagina around the urinary meatus of dairy cows. Under certain
favorable conditions they became pathOgenic. Transmission of infection
from animal to animal was not likely to occur because of the fact that

many or most of the animals were potentially infected.

Eggs: (19H8) in a study of M3 strains of Q, renale found that a
granular deposit occurred when they were grown in serum infusion broth.
The cultural growths had a tendency to become mucoid when stored in the
refrigerator for one month. No hemolysis was observed in blood agar plates.
Appreciable luxuriant growth was more pronounced in blood or serum agar

media. Chromogenesis appeared to be present.

Neither serum digestion nor gelatin liquefaction occurred. Dextrose
was attacked by all strains. Urease was produced in urea medium. In.

hibition of growth was noticed in the medium containing 12.5% to 15%
sodium chloride. They were non-motile, Hes, indole, M.B., V.P. negative.

Except for two all the strains did not reduce nitrate to nitrite.

Morse (19MB) made a bacteriological study of eight cases of pyelo-

nephritis treated with penicillin and found the results encouraging.

Feenstra 3£_gg_(19u9) reported that E, renale was pathogenic for

22 rabbits artificially infected by the intravenous route. Very sig-

nificantly altered clinical blood picture was noticed. The hemoglobin

dropped and the heterophil percentage was increased in the infected rabbits,

17
9. renale could be recovered from the lungs, liver. bone marrow, spleen,

kidneys and urinary bladder.

In early cases congestion of the capillaries, and with the chronicity
of the infection necrosis of tne pelvis, leucocytic infiltration and
proliferation of fibroblasts were observed in the kidneys. Uretferitis

and cystitis were marked.

Eaten gt_gl (19M9) isolated eight cultures of E, renale from the

cervix and vagina of infertile dairy cows.

@3333 (19u9) in a study of 51 strains of Q, renale described
the organisms as gram positive pfipmorphic rods occurring in clumps with
palisade arrangements. They fermented dextrose. All the strains produced
alkalinity in litmus milk medium. Indole and hydrogen sulfide were not

produced. Urea was hydrolyzed.

@2533-(1950) studied six strains of C. renale in plain-extract-

broth in which the pH became 8.2 after 28 days against tne initial pH

6.8.

#8 strains of 9, renale produced acid in dextrose medium with the

pH 6.1 against the initial 7.6.

,4 high alkalinity of pH 9.0 was observed in urea broth against the
initial pH 7.5 by 51 strains. Increase in pH was noticed in litmus milk
by H} strains. Of the 50 strains studied in 10% milk agar media 7 did
not produce a "halo". 9, renale showed very poor growth in cnocolate

blood agar medium containing 5% of 0.9% of sodium tellurite solution.

Morse (1950A) made a survey of the incidence of 2, renale in 523

13
animals. Of these, 215 animals were in herds where clinical cases
were absent. The author was able to recover g. renale from the vaginal
and penile swabs‘ of clinically affected and non-affected cases, but
failed to isolate the organisms from the urinary bladder of 108 animals.
In an infected herd periodic examinations brought out latent infections.
It appeared that E, renale was disseminated from the apparently healthy

carriers to the neighboring healthy animals.

Morse (19503) reviewed the various medicaments employed in the

treatment of bovine pyelonephritis and found that penicillin was the

effective therapy.

MATnhlALS AKD METHODS

l. Bacteriological Procedure:
For studying tne morphology of these cultures Gram’s stain
(Husker modification, l9&{) and Wright's stain were used.
Motility in broth culture was cnecked after 18-2“ hours incuba-
tion at 37 C.

The media used in the stuo of tne cultural characteristics are

shown under results.

2. Pathological Procedure:

A. figugg inoculation

 

1. Selection and 9355 of Mice

“- -.~*-.

 

Apoarently healthy waits mice of two to three months of
age, weighing an average of 20 to 25 gms each, were se-
lected. They were housed in galvanized wire mesh cages
having raised bottoms. Before use cages, trays, pans and
all ieceptacles for food and water were thoroughly washed
and were placed in flowing steam for an hour. The diet
was a commercial rabbit-pellet. supplemented with tankage.
During a preliminary period of at least a week animals not
showing signs of illness were selected for use.

II. Preggrgtion 2i Bacterial Suspension

 

The micro organisms were inoculated onto t:3ptose agar

slants and incubated at 37 C for three to four days. The

 

*Aanady Sunkist Rabbit Pellets Prepared by Acrady Farms Milling Co.,
Chicago 6, Illinois.

20

cultures were washed and suspended in sterile distilled
w

water. A uniform. well mixed bacterial suspension was
made by shaking the suspension in bottles containing
glass beads in a Shaking Machine‘ for half an hour. The
suspension was then kept at room temp rature for five to
ten minutes allowing the foam to decrease.
The bacterial suspension according to the required dosage
of organisms (15 million, 20 million, 40 million) was then
standardized by the photelometeru using the standard
opacity tubes of a McFarland Nephelometer (Kolmer and
Boerner, 19MB).

III. Technig for Injection

 

The device used for restraining the mice is shown in

Fig. 1, page 21. After the mouse was placed into the filter
mantle, its tail was wiped off with a plfidget of cotton

and immersed in warm water (approximate h5—5O C) just for

a moment to make the vein more prominent. Holding the

tail of the mouse with one hand the needle was inserted

into the coccygeal vein.

B. Rabbit_ipoculation

 

I. Selectigp_gn§_gfirg of Rabbits
Young healthy rabbits weighing an average of 2000 to 3000 gms

were selected. They were housed in strong galvanized cages

having wire-mesh bottoms.

A L .

‘Miller Paint Mixer Edd by Miller Mfg. Co. 3238 Brynhawr Ave.,
Chicago, Illinois.

I*"'CenconShearda-Sanford--Photelome.ter Central Scientific Company, Chicago

 

.03.—
4.03

 

 

.23... not ET

fl

 

wadm mafia

oofls mnaqaaaumwa we venue:

 

 

 

 

 

 

 

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wig—L «up...» 395

3.

22

The diet was the same as given to the mice but supplemented
with cabbage leaves and hay. Fresh water was given daily.

Animals were observed for a preliminary period of five to

seven days befoie inoculation.

II. Preparation 0f Bactsrial Suspension

 

 

The procedure was tne same as followed for the mice. The
dose was h billion organisms for each rabbit.

III- 3551:1919. 2.0.: English
The rabbit was restrained in a rabbit box. Bacterial
suspension of specific quantity was given in the marginal
ear vein after the site was clipped and 70% alcohol applied.

C. Hematological study

 

Blood smears for differential leucocyte counts (100 cells)

stained with Wright's stain were prepared from both mice and
rabbits. Hemoglobin determinations (Cenco-Sheard-Sanford
photelometer, Hoffman method, 19Ml) were made on the rabbits only.
For white cell and red cell counts, a Bright-line-improved—
Neubauer-Counting figmber was used. The diluting fluid for
erythrocyte count was that of Guy and Leaks (Todd and Sanford,
1911(8) and for leucocyte count, 0.1fl HCL (Coffin, 1945) plus 1%
aqueous solution of 1% gentian violet. Thoma blood cell pipettes

,were used. Hematocrit reading and sedimentation rate were de—

termined by the Wintrobe method (Kolmer and Boerner, 19H5).

Egghpic of Isolation and Tissue Preservation.

Unless otherwise stated heart blood, lungs, liver, spleen, kidneys and
urine of necropsied animals were cultured on litmus milk agar and blood
agar plates. Tissue smears from heart, lungs, liver, spleen, kidneys

and urine sediment were stained with Gram's stain.

23

For histoPathologic study, tissues of heart, lungs, liver, spleen,
kidneys, ureter and urinary bladder were fixed in Zenker's fluid
(Mallory, 1938).

With mice tissue the usual procedure of decreasing the time for fixa-
tion, processing and embedding was followed.

Sections were stained with Hematoxylin and Basin (Mallory, 1938). A
few of the sections were stained by the Goodpasture's Method (MacCallum,
1919) for identifying bacteria in the tissues.

Some of the tissues (liver and kidney) of four rabbits were fixed in

10% neutral-formol-saline and the sections were cut on a Spencer

Freezing Microtome and stained for fat with Sudan IV (Mallory, 1938).

RESULTS

I. Morphologic
All the strains studied were non-motile, non-spore forming gram
positive rods usually arranged in a palisade, especially when isolated
from animal tissue. Pleomorphism was tne chief characteristic. Often
deeper staining granules appeared to be present in the body of the
organism which gave a banded or beaded appearance. Smears made from
old cultures showed coccoid forms with a striking tendency toward gram

negative appearance. This was noticed in all the strains.

II. Cultural Characteristics

Nutrient_broth. After #8 to 72 hr. at 3] C the growth of these

 

organisms in nutrient broth (M.M.P.C.S.B., 19U8)* was discernible with
the formation of granular sediments at the bottom of the inoculated tubes.
The supernatant fluid was clear with not much clouding of the broth. ho
surface pellicle was produCed by any of tne cultures. In one there was
growth on the side of the tube. The following table indicates the nature

of growth produced by each culture.

 

t1
Manual of Methods for Pure Culture Study of Bacteria.

TABLE 1

Cultural characteristics in nutrient broth.

 

 

 

 

No. of ___ Culture number A- ng
Days 1 2 }_ u 5 b J 8 9 10 Control
1 - - - - gus. - - - - - -
2 g.s. g.s. g.s. g.s. g.s. g.s. g.s. g.s. g.s. f.g.s. -
(slight c)
3 g.s. g.s. g.s. g.s. ' g.s. g.s. g.s. g.s. f.g.s. -
34 g.s. g.s. g.s. g.s. "t‘ g.s. g.s. g.s. g.s. f.g.s. ;..
7 g.s. g.s. g.s. g.s. " g.s. g.s. g.s. g.s. f.g.s. -
l2 g.s. g.s. g.s. g.s. " g. s. g.s. g.s. g.s. f.g.s. -
14 5:5. 5:3. gas. ~_A_g.s. ” g.s. g._s. g.s._3g.s. his. -
g.s. - granular sediment with supernatant fluid clear

f.g.s. - fine granular sediment with supernatant fluid clear
g.s.c. - granular sediment with clouding of the liquid
_§.s.t. - granular sediment and growth on the side of the tube.

---_-_L_

25

Tr tose gggg plates. On tryptose agar plates (Difco, pH 7.“)
incubated at 37 C for MS to 72 hr. there deve10ped small, raised greyish
to creamy-white colored colonies, wnicn microsc0pically appeared granular
with slightly erose edges. he colonies were not brittle. On ageig they
became more dry, appeared larger in size and snowed a tendency tohecome
Chromogenic, i. e. from white-grey to paleuyellowish. The plateszwere

kept 30 days for observation.

TABLE 2

Cultural characteristics on tryptose agar plates.

 

 

 

Do. of Days Cultural characteristics Control
1 Very minute greyish white -
3 Very minute greyish white but larger -
b Tendency to become dry, lacy, larger -
11 Dry white yellowish colonies -
13 More tendency towards yellow -
2b Dry yellow -
29 Dry yellow -
Blood ggar plates. Difco tryptose agar plates to which 10% defibrinated

sheep blood was added were incubated at 3] C for N8-72 hr. The colonies
initially were small dew-drop-like. Zones of alpha hemolysis were not
large. The zones were better discernible after 72 to 9b hr. when viewed
under the microscope. As these organisms were fine, slow growing and

fastidious in nature, the cultural characteristics always were noted on

the 3rd or nth day.

TABLE 3

Cultural characteristics on blood agar plates.

 

 

 

 

No. of Culture numben ii:
Days 1 2 3_ 'h _5_ b 1_ 8 _9 lo ngggpi
1 - n a n o n - a - n u
3 + + + e + + + + + + -
h + + + + + + + + + e -
_1:» + + + + tn + + +‘ A + + - A;
- a no hemolysis
¥ = slight hemolysis
;'
4 = complete hemolysis
Potassium_telluritg'ggag. On potassium tellurite agar (M.M.P.C.S;B,19M8)

 

black small raised colonies appeared in and along the streaks of inoculum

made for seven days at 37 C.

TABLE h

Cultural characteristics on potassium tellurite agar.

w-b-——— -

 

 

 

 

No. of Culture number #
Days 1 2 5 u ‘5 b 7 8 _9 10 Control

1 - - - - - - - - - - -
2 + + + + + - + + e - -
I + + + 3: + v. + + + l -
h + + + v T ?~ + + + r -

- = no growth

1 = poor growth

v = growth

Litmus milk a‘ar. This medium was prepared by adding 10% of sterile

litmus milk to tryptose agar (Difco) pH 7.“. Streaked cultures were

made evenly on the plates and incubated at 37 C for 9b hr. The "halo"

or zone of clearing (Lovell, 19Lb) around the colonies were observed as

indicated in table 5.

27
TABLE 5

Occurrence of "halo" on litmus milk agar plates.

 

 

 

 

ho. of Culture number
Days 1 2 _3_ N 5_ b [ u 9 10 Control

1 +* - - 3' ++ #- + ++ + +++ -
2 +++ ++++ ++ ‘# *+*T ++ +++ ++ ++ ++++ -
3 ++++ ++++ ++ ++ +++e ++++ ++++ ++++ +f ++++ -
h ++++ ++++ ++ +7 ++++ ++++ ++++ ++++ ++ ++++ -

- = no "218.10"

3 = slight ”halo"

+ to ++++ = indicates size of "halo"

Starch agar. Cultures were streaked onto the freshly made starch

agar plates (M7B.C.S.B., 1938) preferably on the day prepared. The in-
oculated plates were incubated 7 days at 37 C. A few drops of Lugol's
solution was poured over each plate. There was a bluish appearance in
all the plates except strain No. 10. This showed that starch was not

hydrolysed by the remaining nine strains.

TABLE 6

Test for starch hydrolysis with Lugol's iodine.

__

Culture number

 

 

 

Date 1 2 43 u 5 b 7 8 _9 16¢ conpggi
S/b/hg - - - - - - - - - + -

- = no starch hydrolysis

+ = starch hydrolysis

When seven day old Cultures were treated with a saturated solution
of iodine in 50% alcohol, hydrolysis of starch by cultures 6 and 10 was

noticed.

For this test a saturated solution of iodine in 50% alcohol appeared

to be more sensitive.

TABLE 7

Test for starch hydrolysis with 50% alcoholic solution of iodine.

 

Cultureppumber
Date 1 2 3 h 5 b 7 8 9 10 Control

 

 

8/b/u9-----+---+ -

 

I
|!

no starch hydrolysis

+ = starch hydrolysis
Liguefggpion 3£_gglgglg. The gelatin was prepared as outlined in

M.M.P.C.S.B., 1948. The inoculated tubes were incubated at 37 C. After

intervals of l,2.3,h,7,12, and 1M days they were placed in the refrigerator
for evidence of liquefication. In no instance did the gelatin remain

fluid except for culture 10.

TABLE 8

Liquefaction of gelatin.

 

 

 

 

No. of Culture number
Days 1 2 ‘3, 7h 5 b #7 8 _9 10 Control
1 - - - - - ‘. - - - - -
2 - - - - - - - - - - -
7 n - - - a a. n a - + u
12 u o n u a - n - a + -
- = no liquefaction of gelatin
+ = liquefaction of gelatin
Litmus milk. Litmus milk (Difco) was sterilized before use by

 

heating to 100 C for 15 min. in an Arnold sterilizer for three conseCutive

days. The final pH was 6.h-b.5. In all cases the medium showed a tendency

towards alkalinity. characterized by the bluish coloration of the super-

natant fluid, with total or partial digestion of casein.

29

TkBLE 9

Reaction of litmus milk.

 

 

 

Culture Number of days h
number 1 2 h 7 12 14
l - b b b pd b md b md
2 - b b c b c b md b md
3 - b pd b pd b c md b md b md
N - b pd b pd b c md b md b md
5 b b pd b md b c md lbmd b md
b b c pd b pd b md b c md b md b md
7 bcpd bpd bmd med bmd bmd
8 b c pd b pd b md b c md b md b md
9 b c pd b pd b 1nd b c md b md b md
10 b c pd b md b md b c md b md b md
Control - - - - - -

b = alkalinity

coagulation

O
I!

pd = partial digestion

md marked digestion

Two sets of litmus milk tubes from a recently prepared batch were
inoculated at different times. The pH of the medium after in days incuba-

tion was determined by the Beckman pH meter as noted in table 10.

TABLE 10
hangs of pH in litmus milk after lh days.incubation.

Culture number __ Control
1 2 3 h 5* b 7 8 Q 10 l 2 3

1st set b.9 7.1 7.2 7.0 7.2 7.0 7.b 7.2 7.4 7.1 b.u 6.2 6.3

2nd set 7.2 7.0 7.2 7.2 7.2 b.8 7.7 7.b 7.3 7.0 b.u 6.H b.2

 

pH of litmus milk before inoculation, b.5.

Hydrolysig_g£ urea. Difco urea broth tubes were heavily inoculated

 

with a 3 mm. loopful from a 72 hr. blood agar culture. Good urease production

 

 

_._-__. ___.——

was noticed after 2h hr. incubation at 37 C in some of tne tubes.

Tubes not showing urease activity in 2” hr. were incubated for an

additional N8 hr. to 72 hr. Urease was produced by all tne cultures

except b and 10. Table 11 indicates the pH before and after 120 hr. i-p

 

 

 

 

cubation and the degree of change in the color of the medium.
TABLE 11
Hydrolysis of urea.
Nm fi' mflhuenmwn' A ”
Qays 1 2 3 h 5 b 7__ s 4_9 10 Control._4

0 fl - O O - C O C - Q ‘
l - ++ ++ + ++"’ - ++ :- ++fi - a
2 - 1'+ H'f' + fi++ - 7‘” + ”7+ - n
3 ++ +++ ++++ + ++++ - +++v +++t ++++ - -
u +++ +e. ++w+ e ++++ - ++++ ++++ ++++ - -
5 +++ +++ ++++ t ++++ - ++++ ++++ ++++ - -
5 par 7.9 7.3 7.8 6.0 8.2 b.5 7.9 8.5 ..2 o.7 6.7

I = yellow (normal color of medium)

+ to ++++ = varying degrees of color cnange from yelLOw :; violet.

’ original pH 6.7

Digestion of serum. Loeffler's serum medium was made in accordance

with M.M.P.C.S.B., 19M8. A slisht modification was made in that the tubes
contained 3-h ml of medium and were laid in a tray containing water. The
tempersznre was slowly raised to 75 C and maintained for 6 hr., during

which time the serum coagulated to a yellowish wnite solid. These tubes

were then sterilized at 90 C (in the t0p of the Arnold steam cabinet) for
After eacn day's heating the tubes

20 minute: on 3 consecutive days.

were placed in tne incubator. The medium was inoculated and incubated

at 37 C for 14 days. The colonies apgeared as small discrete yellow-cream

discs. The serum was not liquefied by any of tie cultures except 6 and 10

as noted in table 12.

fir;

 

TABLE 12

Liquefaction of Loeffler's serum.

 

No. of Culture number

Days _ 1 2 3 TL 5 s j 8 ”Control

F4
O

:TR3~J$rnJF‘
I
a
I
I
I
+I414I+Ii
U
n
I
4 4 + 4|!
I

9...:

 

- = no digestion
1 = slight digestion

+ = complete digestion

Proteolysls of alkaline egg medium. The alkaline egg medium was

 

made as outlined in the M.M.P.C.S.B., 19MB. Mixing was done in a Waring
Blendor.‘ The medium was sterilized in the same manner as for the Loeffler's
serum medium. After each heating the medium was kept in the incubator at

37 C. Proteolysis was indicated by progressive clearing of the medium.

The pH change after in days incubation was as noted in table 13.

mats 13

Proteolysis of the alkaline egg medium.

#A4# L

 

 

 

No. of _“-«_ Culture number ___ :)
=Daygs l 2 j N i b 7 8 9__ 10 Control
1 :- - u u a a a u u- u- u
2 - - - - - ‘- - - - _ -
u n o o o n a. - - o o o
7 - c - u- n + u. - - + -
12 o n u - - fl .- - - ++ -
1’4 '- 0 II '- II ++ - s- - +1- .
pH. 8.3 8.0 5.1 8.1 8.2 7.1+ 8.3 8.0 8.0 7;; 7.7- w

 

 

- = no proteolysis

+ = proteolysis

‘ original pH 7.b

 

. I I ' ‘fi ‘
Waring Corporation, 1097 Droadway. New York City, N. Y.

._.._.—_.___._‘ "v

 

 

 

gtiliggtigp g: gitrgtg. The citrate medium was prepared as

cited by Mackie and.McCartney (19th). The pH was adjusted to 6.8, by
the addition of NaOH. The final appearance of the medium was clear.

The organisms were inoculated into the medium and incubated for in days
at 37 C. Citrate utilization was determined by adding an equal volume
of a saturated aqueous solution of lead acetate to the culture tubes.

A flocculent white precipitate was noted in the uninoculated controlthbe

and in those culture tubes exhibiting inhibition of growth. There was

a slight variation :in the pH of the medium after 1N days incubation.

TABLE 1M

Reaction and pH change in citrate broth.

 

 

 

No. of Culture number k AL ..
Days 1 2 437 4 5 6 7 8 EL 10 Control
1 - - - - - :- - - - - -
2 n n - D n - c a Q - o
1+ - :- .- 1. - - o - u a. -
7 - - - - ‘- - - - - - -
12 n - n O n u n - n u -
1n n o :- - - a n o s u .9

PH‘ 7.H 7.3 7.” 7-3 7.3 7.2 7-3 7-3 7-3 7-1 7-0

Treatment
with lead
acetate P P P P P P

'd
’d
to
n
’d

 

- = no growth

p precipitate

* original pH 6.8

Fermentable media. The basic medium was prepared as described by

 

Therp (193s), modified by the addition of 2% Bacto-tryptose, 0.7% beef
extract and the indicator was brom cresol purple. The fermentable media

attacked are shown in table 15. There was no action on arabinose. dulcitgl

glycerol, inositgl. inulin. mannitol, raffinose,

trlhalose.

salicin, sorbitol nor

 

TABLE 1 5

The summary of the fermentation reactions of the C. renale-dike organisms.

 

M 3 h 5 b 7 3 ‘ i 9 10 '
observation T51 7 12 Pl TETTflé‘ifi T'émTi'E‘iE T721 7 12 111 1 2‘4 7 12711 T’E’W'Efi' 1-5 H 7 1231 1 2 LL 7'3’117‘51‘7‘51‘17 1 2 7 12 117
__.,_,,, ,, __ M- --_-_l-_.-,_l_ will.---,-,--lll------__.-.-._ -lm.--_..__-_._..-.l._.,__ N...” ._ ._..

 

 

Strains L F.‘

 

 

 

Dextrin -...,.._ ::---u-_~m-»-e»-v-.-w~.c--3».-..—...,,-$+++_.._-___‘,.,_____+++_‘_+++ 7
Fructose -e-r1-<r+.++1-++..-...+++.-....?-1~+ ++++++-+++++—+++++-..+++++..--___..,_+++
GalactOse -...-...-..—~ Cnflhzz‘il'.‘ amta«D-F“s"“'m -"'<H‘-"‘-l--*++v-m-m -m“‘*‘fi¢~~—-m-onn-+++
Glucose ..-+-rw-+.....~.§E..u..+++u-n- 7? ++++++1+++1-+-+++++..+++1-+--++++.....+++-e J
Lactose u~-»~-—-~-~*m*’“""‘"""'”""' ‘"”’""'"“""’++"”'"""’”°'”“ ""'”"""""+
Maltose moan-«---:»mmr-v-m-a--w-=-n--mI-~~ naunnmu—mx-.izw,m_,_,,.n,,1_‘__fl-n_______'_'
Sucrose 'fi'x""W"-M’m1’=fl-Dw&filImus-mun;- ......._...._}3 ""'"""“""{""""”‘3‘3 “2"
tvlose ,....-n--,--’r++-..=m?¢w--w ..- v-r—‘rfl ’”'-§§;—"-m “$“"'€'-‘-'-*-""3 f

A ~‘ *5— ____.-._.l..___..--———~——«-—vo~ V,_“_‘_._~ N»_-_. m-_,_,_-_-l~—~m-—_va—v—s—.n —~ —————— m“ -.——-A-—-—_.< ”'M' w”“

 

 

+ — oositive 1?- : slight ;- : marked 3 = trace No gas was produced in any of the ‘lia. , J

34

HydrOgen sulfide groduction. flydrogen sulfide determination was

 

made as suggested by the M.M.P.C.S.B., 19oz. Readings were made after
the cultures had been incubated for 7 days at 37 C. None of the cultures

produced hydrogen sulfide.

Nitrate reduction. The medium and manner of testing were those described

 

in M.M.P.C.S.B., 19MB. All cultures failed to reduce nitrate to nitrite.

Indole production. Indole production wgs determined according totthe

 

M.H.P.C.S.B., 19h8 with the modification of Tharp (l93b) of using narrow strips
of dry filter paper. Cultures were incubated L days at 37 C. None of

the cultures produced indole.

Methgl red test. The medium was prepared as described by Topley

 

and Wilson (l9hb). The indicator was phenol red. The pH was adjusted
to 7.5. Sterilization was at 100 C for 15 minutes for 3 consecutive days.
After four days incubation at 37 C the cultures were treated with mdhyl red

and were found to be M.R. negative.

Voggs-Proskauer test. The glucose phosphate medium as usedfor the

 

methyl red test was inoculated and incubated for four days at 37 C. One ml
of 10% KOH was added to the medium as suggested by Topley and Wilson (l94b).

All cultures were V.P. negative when observed after 2U hr.

Ammonia test. A peptone water culture (Mackie and McCartney, 13Hb) was

 

grown at 37 C for four days. Addition of a few dr0ps of Nessler's reagent

showed that ammonia was not produced by any of the cultures.

Methylene blue reduction. fine dr0p of 1% aqueous solution of methylene

 

blue was added to a broth culture incubated at 37 C for 72 hr. as recom-

mended by Tapley and Wilson (1936‘. All cultures reduced methylene blue.

 

35

Catalase_test. This test was done an a 72 to 9b hr. tryptose agar

 

slant culture incubated at 37 C by pouring 1 ml of H202 over the growth

(Topley and Wilson, l9hb). Gas bubble production was noticed in each of

the ten cultures.

TABLE lb

Culture number

Culture number

 

 

 

1 2 i I? 3 L j s 9 1:) ‘ Control
++++ ++++ ++++ ++++ + + ++++ ++++ ++++ + -
++++ 2 gas bubble production marked

+
H

gas bubble production slight

no gas bubbles

 

The summary of the cqltura charactAristics usnifseted by tun bEL cultures of C. e

lemlike organismso

 
 

 

 

r‘:k_,11 ;: j: , ." ;~'- *—"‘i"" *‘ ._ a ‘“i“"“ 7"r'—”"7““"‘r—T“‘*”"""‘—‘*'—**~“-~—-—~-—~———
alk'ty otlgare Urgesv }elut1n Blood n.fi.’ Nitrate troteln starch Urea V P
. , -7. g _ r- - . _ _ r [a yr, _ 1. . V v _: , ° °
1H 31;, 1:1 ut1t1rru ulOn o1 linden figs hem3« reduc- m.R.**reduc- hgnrolyss hydiolysis hydrolysi5 test
Qultpres Egg nii§~_ _§3§§;B§f-ifiip§ ~_;:gp&_ah_gj;tlgh-_ lysis Irdole tion test “tion is

1 ~9- w. ~1- - m s a + n d» .- :- - a ‘r

1 Z fositivs reaction *h.3. ~ methylene blue
- ; we roactiot **HDRr ; meth71 rr:

 

'\

RESULTS

In vivo study using mice.

 

§ymptoms and clipical findings. None of the 58 infected mice used

in this study showed clinical signs suggestive of illness. The blood pic—
tnthq

ture of 15 infected miCj‘showed marked variation in the total white cell

and differential counts.' In some, the total wnite cell count was 12 to

20 thousand as compared to the normal 6 to 8 thousand per mgé. Little

importance was attached to these results since they fell within the

normal range (Kolmer and Boerner. 1945). In some of the mice a decrease

in lymphocytes and an increase in neutropnils was noticed 10 to 14 days

after infection. This was not a constant feature in all animals.

Gross;pathology, No gross lesions were observed in any of the inn

 

ternal organs. The weight and size of tne Kidneys. ureters and bladders
of 10 infected mice (table 18) did not show a significant difference when

compared to the same organs of four healthy controls.
Microoathglggy,

Kidney. The pelvis of infected kidneys invariably showed distinct
alterations varying in intensity from slight congestion to exfoliation of

the epithelial lining extending to the papillae of the medullae. In the
progress of pelvic necrosis the epithelia were destroyed and where this
process was intensive the normal architectural design was partially or
completely obliterated (Fig. 2). Frequently blood cells, plasma cells.

polymorphornuclear leucocytes, desquamated epithelial cells and granular

materials were present in the pelvis (Fig. 3). The peripelvic tissue

38
contained a serofibrinous exudate, neutrophils and plasma cells (Fig. 3).
This appeared to be due to the presence of bacteria or their products of
metabolism in the area. The immediate effect was degeneration and death
of the epithelial cells of the collecting tubules (Fig. 2). The amount
of necrosis was so intense and the material stained so deeply that it was
not possible to identify bacterial forms. These changes were not uniformly
observed in all papillae. The presence of g%:::;elar material in the
lumina of the collecting tubules would indicate obstruction and subsequent
dilatation leading to pressure atrophy (Fig. N). In the lumina of the

collecting tubules and ascending limbs of Henele vacuolar material was

often present. which suggested fatty changes.

Leucocytic infiltration occurred around the glomeruli, blood vessels
and between the tubules. Along with the cellular infiltration connective
tissue was proliferating in these areas. The new formed connective tissue
together with cellular infiltration led to pressure atrophy of the con-
voluted tubules. Examination of other sections of the cortex showed that
the distribution of the newly formed connective tissue and cellular in»
filtration was not uniform. Tne distribution of these lesions can be

followed in the photomicrographs (Fig. 5).

The predominant alterations of focal necrosis and intertubular
hemorrhage occurred between and around the convoluted tubules and ”al-
pizfian bodies of the cortex (Fig. b). The convoluted tubules were more
or less dilated and lined by an epithelium of altered shape (Fig. 7).
The individual cells were abnormally distinct and the cytoplasm stained
faintly with eosin (Fig. ?). Connective tissue was seen proliferating

around these dilated tubules leading to compression of the adjacent

tubules. Alterations were ballooning and loosening of the cells of the

39

convoluted tubules.

The glomeruli infrequently showed an increased number of poly-
morphonuclear cells in the capille-‘ tufts giving the appearance of over

distension and indicated an acute inflammatory process (Fig. 6).

The lesions occurring in the capsule were not uniform nor con-
sistent. In a number of instances focal leucocytic infiltration in
the subcapsular space was noticed. The capsule appeared to be thickened

owing to the proliferation of connective tissue (Fig. 8).

Spleen. The alterations in the spleen were interesting in that
the infiltration of lymphocytes had gone beyond the splenic corpuscles.
Most of these lymphocytes appeared to be immature. The increased number
of giant cells in some cases was more pronounced in the red pulp and this

probably was the result of the chronicity of the diseased process in

that area (Fig. 9).

Liver. Among the most striking changes found in the liver were
those relating to the disturbances of the hepatic cords. The cords had
undergone varying degenerative processes. The basic changes remainedpthe
same, but in one instance (Fig. 10), there appeared to be the presence
of faintly eosin-staining hyaline droplets or fused masses within the
liver cords and also around the hepatic vessels. The hyaline—like
materials had a tendency to encroach upon the medial layer of hepatic
vessels and actually replaced this layer. This was probably the beginning
of amyloid degeneration. Within the same area round cells were observed

in between the hepatic cords.

Lungs. The striking feature in the lung was the marked inter-

stitial and peribronchiolar type of lesion. The inflammatory reaction

MO
was often severe and was accompanied by extensive desquamation of the
lining epithelium of the bronchioles. Some of the valveoli were filled
with an exudate consisting almost entirely of degenerated polymorpho-
nuclears and red blood cells. In other alveoli, large mononuclears,

19nu

fibrin and blood cellfi‘. At the same time tne alveoli walls were in-
filtrated with inflammatory cells. The peribronchiolar inflammation ex-
tended out into the interstitial lung parenchyma for a variable distance
giving rise to interstitial pneumonitis (Fig. 11). The cellular activity
of the peribronchiolar lymphoid tissue together with the hyperemic condi-
tion of th; vessels (Fig. 11). The bronchioles showed catarrhal inflam-
mation. Desquamated epithelial cells along with invading neutrOphils were

present in the lumina. Some of the polymorphonuclear leucocytes had

undergone necrosis (Fig. 11).

Adrenal. A marked focal lymphocytic infiltration into the sub-

u;
ycapsear space was noticed in one case.

Ln

 

 

 

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#2
TABLE 19
Hematological characteristic of infected and

normal mice.

 

 

 

 

Animal W.B.C. Differential count
count L N M B E ___
Normal Controls blOO 73 25 2 0 O
b500 b9 2b 5 0 0
8000 b9 2b 5 O O
7200 b9 2b 5 0 0
Infected - blag/M9
Dose Interval in
in millions daxg
15 10 11,320 17 77 b O 0
15 10 12,000 22 7b 2 O 0
25 10 10,b00 72 23 u 1 O
20 10 n.8b0 55 MM 1 O 0
25 ll 3,bOO 70 27 3 O 0
25 11 3,b50 72 2M N O 0
25 11 9,500 72 2M M O 0
20 IN lk.250 72 2“ h 0 0
20 in 20,750 72 22 5 1 O
15 1h 1b.OOO 72 22 5 l o
25 lb 8,500 39 b0 1 O 0
25 lb 17,750 77 17 b 0 O
25 1b 7,250 70 25 5 0 0
20 lb 7,000 b8 2b h 2 O
15 1b 3.000 71 22 7 0 0

 

Owing to difficulty in securing blood, the white cell pifiette was

filled only to the 0.1 mark and the éiluting fluid (0.1 HCL) was drawn

to the 11 mark and thus making the dilution 1:100.

In vivo study using rabbits.

 

§Zmotoms and clinical findings. The first six infected rabbits did

 

not show symptoms suggestive of illness. They gained in weight. The ten

rabbits in the second and third groups lost weight and went off-feed.

M3
The blood picture of all the lb rabbits, on the other hand, showed de-
finite change. There was an increase in the percentage of heterOphils
and decrease in the percentage of lymphocytes (table 20). As the per-

oxuullken

centage of polyuevfls increased immature forms appeared. The progressive
drop in hemoglobin values after inoculation was striking (table 20). This
drop in hemoglobin probably could be attributed to the fact that the bone
marrow was concerned primarily with the production of heterophils in order

to combat the infection and thereby interfering with the production of

red blood cells.

QIQEE pathglggz. Mottling appearance of the livers and kidneys
suggestive of fatty metamorphosis was noticed. Some of the livers of

infected rabbits showed lesions of coccidiosis which were not referred

to in the following description. Other organs appeared normal.

MicropathoIng,

 

Kidney. The lesions were similar to those observed in the mice.
The pelvic epithelium showed focal necrosis which was extensive enough

to cause exfoliation of epithelial lining. (Fig. 12)

Infiltrated into the pelvis were pseudoeosinphils, a few mono-
nuclears, faintly stained acidophilic granzles and desquamated epithelial
‘finqmaga.
cells. In the turica‘of tne pelvis edema was evident along with the

presence of acidophilic material (Fig. 13).

A general vacuolization and granular degeneration of the epithelium
of the collecting tubules were evident. In the sections stained with
Sudan IV, fat droplets in the collecting tubules were discernible. The

darker shade in the photomicrograph indicated the degree of fatty degenera-

1V4
tion in the affected tubules (Fig. 1R). In some of tne collecting tubules
the granular and cellular casts had caused no damage at the point at
hicn they were observed (Fig. 15). On the other hand in locations
where tne tubules were damaged and necrotic, the adjacent capillaries
were congested and some a polymorphonuclear leucocytes had migrated into

tne interstitial spaces (Fig. 15)-

we

Changes in the thick limbs of Henele were present in nearly all

A

the kidneys. The epithelium was irregularly swollen and granulur and
snowed mucn variation in staining. Considerable vacuolization of the
cytoplasm was seen, some of which probably was due , hydropic degeneration
or fatty metamorphosis. The tubules contained varying amount of cellular
and granular materials with necrotic Changes in the epithelium. With

fat stain these tubules showed a considerable amount of intracellular

fat (Fig. 1a).

In general the thin limos presented no marked nor consistent
pathological changes. Granular acidOphilic materials were freg-e sly
present in the tubules. ; - epithelium appeared to be in a state of bal-

looning degeneration.

In general, the glomeruli presented a mild cellular infiltration.
The glomerular tufts showed an increased number of leucocytes. In some
instances, there were partial or complete areas of granular degeneration
in the glomeruli. The granular materials coalesced diffusely to form
hyalinized MOSSand took a deep stain (Fig. 1b). This hyalinized M45;
was also seen in some of the perivascular axes“. ‘Aeound the affected

glomeruli newly formed connective tissue led to the thickening of the

capsules. Following this glohsrulonephritis increase of the inter-
stitial connective tissue in the adjoining inoe-rubular spaces vas

seen.

Albuminous degeneration was present in the proximal convoluted
tubules of all kidneys. This was more pronounced in the tubules ad-
jacent to the glomeruli. The epithelial cells were not only swollen
but éompletely filled the lumina of the tubules and also snowed in-
creased granularity of the cytoplasm. (Fig. 17) In some of the cells
there were vacuoles near the nuclei. ce.iular damage was severe in some

of the tubules.

The varying process of necrosis in the cellular structure of the
convoluted tubules was evident (Fig. 17). Many of the cells had ruptured
as a result of severe ballooning degeneration. The nuclei were extfifided
intr the lumina. Pyknotic or caryoclasic changes were seen in some areas.
Faintly eosin stained granules were present in the tubules. The unusual

appearance of intracellular fat in some of the tubules was also noticed.

The distal convoluted tubules presented the same pathological picture.

Bladder. The changes in the bladder were not consistent. Focal

areas of necrosis in the epithelium were evident. The mucosa in the af-

fected areas had undergone partial to complete exfoliation. Characteristic

pyknotic changes were well marked (Fig. 13).

Ureter. Mild focal necrosis of the epithelium was noticed in the

ureter of only one rabbit (Fig. 19).

Liver. The hepatic lesions were widely diverse ranging from

fulminating lesions to a chronic form of hepatitis. In some areas there

 

 

as
was albuminous degeneration of the hepatic cells. This was most marked

in the area around the central veins. The liver cells were markedly
swollen, the cytOplasm was excessively granular and stained lightly with
eosin. In other locations the liver cells showed varying degrees of
necrosis. The architecture of tie hepatic cords was still present. There
was no line of denarcation between the healthy and necrotic liver cells

‘i‘

(it'- 20)-

In some areas there appeared to be a reaction with giant cells
forming a narrow zone of demarcation between the lesion and the livinb
cells (Fig. 20). Within the same field fibroblasts were proliferating
around the necrotic zone in an attempt to wall of: degenerative process

(Fig. 20).

Fat stain showed tne degree of fatty degeneration present in the

hepatic cells, eSpecially around the central vein. This became less

prominent towards the periphery of the lobule (Fig. 21).
No lesions were observed in the bile ducts.

Lungs. The primary lesion in the lungs was a general congestion.
In some it was well marked in others it was Very mild. The alveoli con-
tained a number of pseudoeosinophils. The interalveolar walls were
heavily infiltrated with these cells. The infiltration of inflammatory

cells both in and out of the alveoli was so great and diffuse in some

sections that the lung tissue was hardly recognizable (Fig. 22).

Exudate in the bronchioles was evident. Necrotic bronchiolar
epithelium and polymorphonuclears formed the main constituents of the

exudate. The polymorphon.clears in the exudate had undergone necrosis

(Fig. 23).

Mononuclears, a few pseudoeosinOphils and fine faintly stained

granules were present in the subpleural space (Fig. 2%).

Organisms identical with those inoculated were isolated from the

kidney, urine, liver, lungs and heart blood of omrabbit and from the

urine of two others.

 

 

 

 

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DISCUSSION

Ernst (1905) in a study of the etiolosy of pyelonephxitis in cattle,
found gram positive rod shaped bacteria in the urine of affected animals.
This author classed these organisms with tne Corynebacteria and designated
them "Egrypgbagtprium renalis Efllli"o Later Jones and Little (l92b),
Merchant (1935), Thorp, gt El (19u3), in extensive studies of the morphology
and cultural characteristics of these organisms referred to them as

Qorypebacterium_renale. Brooks and Hucker (19th) described the morpho-

 

logical and chemical properties of two cultures isolated from the urine

of cattle affected with pyelonephritis. These cultures were designated
Corynebacterium pseudodiphthericum. The properties of these cultures com-
pared very closely to those of E, renale. Feenstra. ££_§l‘(l9h5) on the
basis of morphological, biochemical and serological prcperties described
two distinct species of Corvnebagteria.involved in the etiology of pyelo—

nephritis in cattle.

The ten cultures reported in this thesis were similar to 2, renale
in ecology, morphology and in that they fermented glucose and fructose
(culture No. 9 did not attack fructose). Similarly there was no indole,
nor HES production, and no liquefaction of gelatin (culture No. 10
liquefied gelatin). Alkaline reaction in litmus milk, formation of halos

in milk agar and catalase production were shown by all the strains.

They differed from Q, renale in that all 10 cultures produced alpha
nemolysis. Tnis hemolytic preperty was found in six cultures reported by
Feenstra, SE a; (1535). All the cultures grew slowly, produced small

colonies on artificial media, reduced methylene blue, failed to utilize

citrate and to reduce nitrate, were ammonia, methyl red and V0geg-Proqkal'r

\Jl
0

negative. Out of 2b strains of 9, renale studied by Lovell (lfihb) 23
were methyl red positive and 2M were Voges-Proskauer positive. Two of
51 strains of g, renale studied by Morse (1939) reduced nitrate. All
these cultures except Nos. b and 10 Split urea and failed to hydrolyze

starCh and protein, but digested serum.

The possibility that cultures no. 6 and 10 might be similar to
Q, byogeneg-like could be easily suggested, but not substantiated, in
that although they fermented glucose, xylose, lactose, sucrose, hemolysed
blood, digested serum they did not produce acid in litmus milk, and tnere
was no beta hemolysis. Moreover, culture no. b fermented maltose but
did not liquefy gelatin, wnile no. 10 produced an opposite effect on

these two media.

The ten cultures studied in this investigation fell broadly into
two main groups on the basis of cultural and biochemical properties. Cul-

tures no. 6 and 10 appeared to comprise one group although minor dissimilarity

occurred. The remaining eight cultures in spite of minor dissimilarity

between one another were classed as a second group.

In view of these differences it was not possible to classify

these cultures as 9, renale. These groups of Corynebacteria may be a

 

possible etiological factor in bovine pyelonephritis. This was further

substantiated by an animal inoculation study using mice and rabbits.

Enderlen (1691) produced typical renal lesions in two ureter
ligated rabbits. Jones and Little (192b) could not produce the disease in

wnite mice, guinea pigs nor rabbits. Doll (19N2) confirmed the findings

of Enderlen and stated that lesions procuced in rabbits were similar to

those found in the cow. Lovell and Cotchin (19Hba) described experimental

51
pyelonephritis in mice. Jeenstra, gt gl_(1949) produced typical renal
lesions in 22 young rabbits woicn were identical with the lesions found
in naturally affected cattle. These workers, in addition to reporting

changes in the urinary tract, described lesions in the liver, lungs and

spleen.

In this study variation in the lesions was noticed. Hematoloéical
study of the mice did not reveal any significant variation in the infected
animals when compared to the normal. The white cell and differential
counts remained within the normal limits (Kolmer and Boerner, 19L5). Boyd
(1913) observed an increase in size of the kidneys and ureters of cattle
suffering from pyelonephritis. No significant difference in weight be-
tween the kidneys, ureters and bladders of tne iniected mice was noticed.
With the higher infective dose of MO millions, the severity of the lesions
were not uniformly met with, bacteria could not be isolated from the viéEra.
However, it might be stated that these organisms were pathogenic to mice.
They produced the same type of lesions as was described by Lovell and

Cotcnin (l9uba). From the protocols it may be presumed that these organisms

were not as invasive as typical 2, renale.

In addition to the above, lesions in the spleen, liver and lungs

were observed.

The lesions and hematological picture in the rabbits were similar

to those described by Feenstra, et a1 (19”?) with tne exception that

lesions were not seen in the spleen. Recording of giant cells in the
liver was noteworthy in this study. Doll (19oz) mentionrd that giant

cells were invariably found in the livers of rabbits infected with 2, renale.
The reason for the formation of these giant cells in the liVer could not

be eStablished.

CONCLUSION

From the protocols of the results of both 33 vitro and in vivo

 

studies it may be stated that these organisms isolated from cows suf-
fering from pyelonephritis were in many features similar to, but not

identical with typical Corypebacterium renal: as described by various

 

workers. They may be possible etiological factors in bovine pyelonephritis.
SLrt‘u'AARY

The diphtheroids studie‘ were non-motile, non-sporing pleomorphic

gram positive beaded rods occurring in palisade groupings.

The bacteria were slow growers. All produced alkalinity in litmus
milk and "halo" in milk agar, hemolyzed blood, reduced methylene blue,

fermented glucose, and were catalase positive. With tne exception of

culture No. 9 all attacked fructose.

These cultures fell into two groups. Nos. b and 10 formed one

group and the remaining 8 comprised the second group»

In mice, hematological study and weight of kidneys, ureters and

bladders of infected animals did not show significant differences when

compared to the normal.

In rabbits, hematological study revealed a drop in hemoglobin and

an increase in the percentage of heterophils in the infected animals.

When these organisms were given to mice in adeeuate doses they

produced pyelonephritis.

The pathogenesis of experimental pyelonephritis in and pathogenicity

to rabbits were similar to those observed in nice.

J'

0-2

r4
(3

_.LJ

Beck, J. D., Thomas De hott and in. B. Baucler.
.A report of six cows witn inf~ctious cystitis and pyelo
treated with penicillin. U. 01 Penn. Vet. Ext. ouar. E
.1 xi-
lJolS, ljep.
Boyd, W. L.
eloneruritis in cattle. Cornell Vet., h; 1?;nlSl, l;
Boyd W. L., G. P. Fitch add w. A. Bil 1i ngs.
Oviue pyelouepkritis. ".inell Vet. E: 2&1, 1513.
Boyd, W. L.

Pyelonephritis of cattle. Cornell Vet. 17:
Boyd, W. L. and L.iV..Bis'.13p.
Pyelone hritis of cattle 81d horses.
90: lj+~102, 1237.

Joni. Amer. Vet.

Breed, R. 5., E. U. D. hurray and A. P. Hitchins.
Bergey's Manual of Determinative Bacteriolo,.. he Wil
Wilkins Co., Baltimore, e5. t, ljdc.

Brooks, R. F. and G. J. Huc cker.
A study of certain members of tzie genus cor»neoacteriun
Bact.. #8: 293-312. 1~hb.

C)€fin, D. L.
The diagr 10818 of specific cstitis and pveloue;ir1t1s b
office-laboratory ;rocedure. U. of Penn. Vet. Ext. Qua
3.1(, 1943.

Coffin, De Le
Manual of veterinary clinical patilolopy. Comstock Publ
Company, Inc., Ithaca, K. Y. 13L3.

*\
I

Difco Lab31atories Incorporated, Detroit 1, Michigan.
Doll, E. R.
Unpublished data. Dept. of animal Pat L51335y, Michigan

‘16
‘76..

East Lansing, 13
Eberson, F.

A bacteriologic study

reference to Hodgkin's disease.

of tne diftheroid organisms with

Jour. Inf. Dis. 1

2;; -

Migula.

System der Bacterien, 2, 1900. (cited by Ebersoh, ljlb

Feenstra, E. 5., C. F. Clar< a dF. The , Jr.
Artificially induced case of pyelonephritis
5: lHY-lSO, 1945.

in a bovine

115 tint i tis

:1. 12' C1,
ALO.

q

(0

Med. Assoc.

liens and

Jour.

practical

”I

rt. No. ?2:
isuing
ed. 8. 1335.

State College,

eC

$_l He
\4.) 33

0‘.

P
r‘
L

{Tm

\
[e

O ibij 5.0.

Vet.,

54

Feenstra, E. 5., F. Thorp, Jr. and C. F. Clark.
A study of the diptheroid found in infectious bovine pyelonephritis.

Jour. Bact., 5o: M97—50u, igusa.

Feenstra, EL 3. and F. Thorp, Jr.
BacteriOpatholegy of bovine pyelonephritis. Amer. Jour. Vet. Res.
7: h32-h3b, 19st.

Feenstra, E. 5., F. Thorp, Jr. and M. L. Gray.
Pathogenicity of Corynebacterium renale for rabbits. Amer. Jour.
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Foss, J. 0.
Identification of Corgnebacterium_renalis from the kidney and

bladder of a horse. Jour. Amer. Vet. Med. Assoc. 10M: 2b, l9uL.

 

Gair, G.
Bacterial pyelonephritis in a cow. Vet. Jour. 7M: 56-58, 1918.

Hatch, R. D., E. S. Feenstra and L. F. Jennings.
A bacteriologic survey of the reproductive tract of infertile
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HOLfMRIl, we SO
Phot‘lometric clinical chemistry. The Williams Morrow and 60.,
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Specific infectious cystitis and pyelonephritis of cows. Jour.

Exp. Med. #2: 593-507, 1925.

Joist, E.
Spezielle pathOgische anatomie der haustiere, Berlin, iii, 192M
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Jones, F. S. and R. B. Little.
The organism associated witn specific infectious cystitis andp
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Jones F. S. and R. 5. Little.
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Jour. Amer. Vet. Med. Assoc. 72: th-bb7, 1928.

Jones, F. S. and R. B. Little.
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Kolmer, J. A. and F. Boerner.

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Studies on Corynebacterium renale. I. A systematic study of a
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Lovell, B. and E. Cotchin.
Studies on Corynebacterium renale. II, The experimentfil Path0“eniCit
“f .3 y

for mice. Jour. Comp. Path. and Therap. Sb: 205-2114, 19th

I.

I.

0.

O.

\TI
\J‘l

MacCallum, William G.
Tne path-olog;;¢ of the pneumonia in tne United 8 ates Azny Canps
during the winter of 1917-1919. Mono;,raph No. 10. H.1ckefeller
Institute of Medical Research, New York, #7, 1919.

Mackie, T. J. and J. E. McCartney.
Handbook of Practical Bacteriology. E. and S. Livingstone, Ltd.
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Mallory, F. B.
Pathological Technique. The W. B. Saunders Company, Philadelphia,
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Manual of I'lPtLOLS for Pure Culture Study of Bacteria. Biotecn Publicatiers
G’Xleva, N. Y. ’ lng".

McFadyean, J.
Nephnitis in aninals. Jour. Camp. Path. and Thorn}. U2: 55-71,
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IJJCIILtosJVl’ R. A.
Pyelonephritis. Vet. Med. 33: his-M11, 1 38.

Merchant, I. A.
A study of the corgnebacteria associated with diseases of domestic
animaLS. Jour. Bact. 30: 95.11b, 1935,

Morgan, B. B.
Comparison of pH and
Exp. Biol. and Red.

ation of tricnomanas foetus. Proc. Soc.
8‘

“
I'
C. . a
a 1.382, .1 :“TC.

pu ul
3 3
Mar aan, B. 3., K. R. Johnson and E. Z. Emeison.

Some Corvnebacteria isolated fr 3m tne ger.ital tract of bulls.

11.3.0. Vet. b. 68.75, ljwbc

Morse, E. V.
Bovine pyelonephritis. Cornell Vet. 38: 135-197, 19h8.

Morse, E. V.
Pyelonephritis. IV. Physiological and bacteriological studies
of eight cases jof pyelonephritis. Treated with penicillin.
Cornell Vet. 3: 273-285,19L8b.

Morse, E. V.
Criteria for tne identification of cor; nebactnria isolated from
animals. Cornell Vet. 39: Ebb-275, 19L9.

Morse, E. V.
Furtier studies on t‘e cultural an‘d biochemical Characteristics of

some diphtoeroid bacilli isolated from animals. Cornell Vet. “0:
”9-55. 1950-

Morse, E. V.
An ecological study of Corynetacterium.rgnglg. Cornell Vet. MO:
178-187, 1950a.

 

.0

\

Morse,E. V.
The treatment of bovine :yelonephritis. Vet. Med. “5: 221-22h,
1950b.

Olfson, P.
Pyelonepnritis in a dog due to Corynebacteria_rena1i§. Cornell
Vet. 20: 69-72, 1930.

 

Olney, R.
Pyelonepnritis in a Holstein cow. Vet. Med. 38: 397-338, 13M}.

Palmer, C. C.
Pyelonepnritis in cattle. Vet. bed. 26: le-l77, 1331.

Palmer, C. C.
Pyelonephritis in cattle. Jour. Amer. Vet. Med. Assoc. 93:
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Ritzenthaler, M.

The path310gy of bacillary pyelonephritis in cattle. Jour. Comp.
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The following are citsd by Ritzenthaler, 1910.
Bruckmuller: "Lehrbuch der patnolgi-schen Zootomie", Vienna,

18b9, p. 657-

Siedamgrotzky: "Ber, u.d. Veterinarw. i. Korigr. Sachs”, p. 30,
p- 38. 1675.

Pflug: “Die Kranflh.d.Ur0poetiscnen Systems uns. Haust.", Vienna,
Pp. 38 and 1314, 18(bo

Dammann: "Zeitschr. f. Tiermedizin", 1877.

Guillebeau and Hess: “Scnweiz. Arch. f. Tierfipilk"., TXXX, p. 269;
TXXXII, 1590. p. 22h;

TXXXIII, 1891, p. 157;

TXKXIV, 1892, p. 70.

Bang: "Tidskr. fur. Vet.", 1889, p. 3C9.

Schmidt: WMaanedschr. f. Dyrl.". 1800, p. 1L9.

Frieéburger: "Klin. Diagn.", 1909, 5. 508.

Hcflich: "Monatsn. f. prakt. Tierneilk." T. 11., 1391, 5. 53/.

Enuerlen: "Zeitschr. f. Tiermedizin.", 1891, p. 325.

Bollinger: "Zeitscher. f. Tiermedizin.". I. XVII., 1891, p. 34b.

Lucet: "Journ. de Med. Vet. et (a Zootecnnie", T. XLIII., 1892, p.

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57

Kitt: “Monatsn. f. {1. '. Tierheilk.", T. IV. 1893, pp. M33 and H61;
"Patholog. Anat. der Haust.", T. 11., 190b.

Mollereau and Porcher: "Bull. Soc. Centr. Med. Vet.", T. XLIX,
Bartels: "Deutschet. Wocnenschr.", 1897, p. 303.
Albrecht: "WoCnenscnr. f. l r neilK. u. Vieh7.", 1900, p. tog.

ErnSt: "Centralbl. fo 38th "I TXJLAIX., pp. 5149-1300; To X]... P. 79.

Runnells, R. A.

Animal Pathology. The Iowa State College Press, Axes, Iowa, ed. 4,
1940.

Todd, J. 0. an‘A. H. Sanford.

Thorp,

Thorp,

Udall,

Weitz,

Clinical diagnosis 0; laboratory methods. The W. B. Saunders Company,
Philadelphia, ed. 11, 19mg.

F., Jr.
A bacteriological study of aerobic flora OCCurring in the pneumonic
lungs of swine. Thesis, Graduate School, Univ. of Illinois, 193b.

F., Jr., R. F. Langham, C. F. Clark and E. R. Doll.
The pathology of bovine pyelonephritis. soar. Jour. Vet. Res.
u: etc-2H9, 19L}.

D. H. '
Pyelonephritis, nephritis, cystitis. Cornell Vet. l7: IBM-16“, l92b.

B.
The bacterial flora found in the vagina of dairy cows with special

reference to corynebacteria. Jour. Comp. Path. and Therap. 57:
1918195. 19“?-

Wilsons, G. S. and A. A. Miles.

Topley and Wilson's principles of bact riology and immunity.
The Williams and Wilkins Company, Baltimore. ed. 3. Vol. 1, 19Mb.

Fig. 2. l. Pelvic necrosis
2 Necrosis of collecting tubules

3. Hyaline-like materials on the pelvic surface of
the papilla.

The mouse was inoculated with HO million organisms of
culture no. 1. Necropsy after 7 days.

H. & E. stain 100K

58

 

Fig. 2

Fig. 3.

1. Necrosis and exfoliation of the pelvic
epithelium

8. Cellular deposits in the pelvis.

3. Leucocytic infiltration and fibrin in the
peripelvic tissue.

h. Necrosis of the collecting tubules.

The mouse was inoculated with No million
organisms of culture no. 1. Necropsy
after 7 dogs.

H. & E. stain 100x

F1"

,3.

1+.

Dilatation of the collecting tubules.

The mouse was inoculated with 15 million
organisms of culture no. 1. Necropsy

after 55 days.

H. & E. stain 100

b0

 

rig. h

F1

5.

Periglomerular, perivascular and intertubular
leucocytic infiltration with the appearance
of new formed connective tissue.

The mouse was inoculated with 40 million
organisms of Culture no. b. Necropsy

after 30 days.

H. & E. stain 100K

bl

p 9‘;‘::r";? .
L‘ ,. ‘NV“'."‘..&"" 9‘. 5-» ‘

    
  

't‘. v4"

k.

  

 
 

  
 

    
 
 

 

513‘."

4151’”; Q'J‘P" {a a”; “‘3’. a},

    

Fig. 5

Fig. 6.

1. Focal necrosis
2. Intertubular hemorrhage
3. Glomerulitis

The mouse was inoculated with NO million
organisms of culture no. 5. NeCropsy

after 7 days.

H. & E. stain 100x

 

b2

 

Fig.

Fig. 7.

l. Granular degeneration of the proximal
convoluted tubules.
c. Dilatation of proximal convoluted tubules.
3. fewly formed connective tissue around
the dilated tubules.

The mouse was inoculated with 15 million
organisms of culture no. 7. Necropsy
after M7 dajs.

H. & E. stain 100K

 

 

 

b3

z...

. Z..."

'1

van

mm.

b

V

.. .
.—

W.

.1...

s.
J
v I.
new”

.uk

.;.,4,..r\u\.

were.

an

 

Fig. 7

Fig. 8. 1. Leucocytic infiltration in the subcapsular
anace.
2. Thickening of tne renal capsule.

The mouse was inJCulated witn 20 million organisms
of culture as. l. Necropsy after 55 days.

H. & E. stain 1001

 

 

bl),

 
 
   
   

.“._-_5§$.{ .-

qu‘:

.929,{ I"
1; "x ‘
"’ «' i't
c"?..'! ; I >h'
1.. , 3 a].

  

\‘

Q

'5

"A
r w“ a

L.

1. Lymphomtes beyond the Halpig'an
carpuscles of the spleen.
2. Increased number of giant cells.

The mouse was inoculatrd with NO million
organisms of cilture no. 3. Necropsy
after 1“ days.

H. & E. stain bSX

 

 

b5

-._‘

3. , V .J. .a
a .. 325.-,.j_;.. L‘s
V‘- , L J V 9 , ‘ ‘
t'- ‘3‘- ‘ ">‘.'""!-v;"’
' TA 4" t“?.“"l.
‘ , _ ,IQ‘ .la.’.‘n . r '
{filmy-.35"
. ' 5

‘72:;

-.~
.34.!» ._
.

' 3.1:

‘3

.
..
. .
. 4
LI'
,
1‘ ”t;
u .

- ”‘7 i." u. > \
s .é. a 3.":F
43?? .3

V . bu. i

3?
~ 2
.b‘

.g\
4..“
,-

t __‘_x
'Y':

.A I' .A;

."'L:
_'u 1. 2
v
A
‘-

 

1. Fatty metamorphosis in the hepatic cells.
2. Necrosis of tne henatic cells.
3. Amyloid defensration of ne Hepatic cells.

Tue LDUSO was inoculated with #0 million
organisms 91 cilture no. 10. Killed 6 days

after inoculation.

H. & E. stain b5X

 

 

Fig. 10

l. Interstitial pnelmonitis.
2. Peribronchiolitis.
5. Inflanzatory e -nate in tne brancnioles.

The mouse was inoculated with 20 million
organisms of Culture no. 7. Necropsy

after 7 days.

H. & E. stain bRX

I

 

‘27

 

 

 

Us- 1. ....
V"; 1.. .3... .5. a
. L:- L L m.s.x... he h .. O\.:v.l.
. .. .. .3 .19.. ..e...\.
,2. a. ... 3,. a.

 

E ,.
Int J a

.;na sh. my rear.
3

           

l. Exfoliation of tne yelvic agitnelium.
2. Granular and cellular materials with pelvis.

The rabbit vas inoculated with cultLWe no. 13.
NecrOpsy after 10 days.

H. & E. stain o5):

 

b5

.uuyo..e-1\ e . n. a

a ‘11.}! .. ... , . .
I. ‘ o..." un.cp. o. . e, a Q a . .V~.e.\n

   

Fig.

13.

1. Cellular materials in Due pelvis.
2. Edema with acidophilic granules in the tunica’llw.

‘

The rabbit was inoculated witn culture no. .3.
Tecronsy after 10 days.

he & E. Stain 033

L"!

I .l t . .
“PM“... 4». s... . Nwarp... . .

HP?- :95 a.,o.o

.0 .../oe<.e¢e
. .c I
er . r....
. :2.-- .1.
.Ve.e c c .. e...
0 s.
. . .ao

 

"'

cw

Eng. 15

Fig. 1%. Fatty metamorphosis in tne collecting tuoules
and zECending limbs of Hensle.

The rabbit was inoculated with culture no. 6.
Necrogsy after 10 days.

Sudan IV 1001

 

70

.4. r3...

.....« ‘.
. 1.. ~..

 

Fix. 1‘4

Fig.

15.

l. Granular and cellular materials in the
collecting tubules.

2. Necrosis of the epithelium of the
collecting tubules.

3. Hyperemia of the intertubular vessels.

The rabbit was inoculated with culture no. 10.
Necropsy after 10 dRJS.

H. & E. stain lOOX

 

  
   
   

”‘1 a-
O
r e.
«e
._ O
6

§
‘ flit} «I _ .
:__ ‘g; 41%;: 4131‘, I .‘ _
.:4 .‘6‘:,44.. ..o 4444*.i‘

‘ ‘1~‘-._o‘..e‘e'
'3 s
O

71

Fig. lb.

1. Excessive number of leucocytes in the
glomerular tufts.

2. Areas of granular degeneration.

3. Appearance of hyalinized mass.

Rabbit was inoculated with culture no. 10.
NecroPsy after 9 days.

H. & E. stain lOOX

 

Fig. 17. 1. Egalinized bass in the glomerulus.
2. Connective tissue proliferation around the
glomerulus.
3. Granular degeneration of the proximal con-
voluted tubules.

Rabbit was inoculated with culture no. 13.
Necropsy after 9 da;s,

H. & E. stain lOOX

 

 

 

73

 

Fig. 17

Fig. 18.

1. Focal areas of necrosis of the bladder ep

epithelium.
2. Desquamated epithelium in the lumen.

Rabbit was inoculated with culture no. b.
Necropsy after C days.

3. & E. stoin lObX

 

Fig. 18

Fig. 13.

Mild ioosl necrosis of the ureteral epithelium.

The rabbit was inoculated witn culture no. b.
Necropsy after b dogs.

H. & E. stain 100x

 

C 4
., - 4 _ 4,
f_41r I" '
r . '; ’
r.
. ”gt.“ a
' : ‘ ‘ a .
’ [/‘s‘el 4,.4’“

 

Fig. 19

Fig. 20.

l. Hepatic necrosis.
2. Giant cell foxmation.
3 Proliferation of iibroblasts.

The rabbit was inoculated with culture no. 10.
Necropsy after 10 dogs.

H. & E. stain. lOOX

 

7b

 

Fig. 20

Fig. 21. Fatty metamorphosis of the hepatic cells.

The rabbit was inoculated with culture no. 10.
.‘Necropsy after 10 dogs.

m Suduww 100x

 

 

 

1. Cellular infiltration both inside and out-
side of the alveoli. '
2. Inflammatory exudates in tne bronch .

The rabbit was inoculated with culture no. b.
Necropsy after 8 days.

H & E. stain lCOX

 

 

Inflamnatory exudate in the bronchi.
Partial exfoliation of the bronchiolar epithelium.

The rabbit was inoculated with culture no. b.
Necropsy after 0 days.

H. & E. stain 1001

 

 

79

 

Fig. 2“.

Mononuclears and heterophils in tho sub-

pleural space.

The rabbit was inoculated with culture
no. 6. Necropsy after 8 days.

H. & E. stain lOOX

____.,__—-‘—-—-—-

‘-_ __ ——-—.———...._.'. n—n—h. ——_—. _..—— - _-

 

80

 

   

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