Cultural characteristics of certain pathogenic anaerobes isolated from sheep
by Theodore Thomas Chaddock
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
Degree of Master of Science in Bacteriology at Montana State College
Montana State University
© Copyright by Theodore Thomas Chaddock (1935)
Abstract:
1. Three anaerobes isolated from sheep suspected of having died of a disease resembling "black
disease" were studied© These were com-pared with a New Zealand strain 7 which had been isolated
from a known ease of "black disease".
2. A brain-liver broth medium with a pH of 7.2- 7.4, consisting of minced beef brain, liver broth plus
0.1 per cent dextrose, an iron Wire, and .5 cc. laked sheep blood, was found to produce optimum
growth© 3. Optimum growth conditions were determined by direct counts of bacteria in definite
quantities of culture media.
4. A medium composed of a sugar free broth, laked sheep blood and an iron wire was found to favor
optimum growth in carbohydrate test cultures.
5. Two strains (572 and 591) proved to be Clostridium sporogenes, while strain 590 was Clostridium
oedematiens and identical with strain 7. 6. It could not be demonstrated that immunity could be
produced by bacterins prepared from strain 590 of Clostridium oedemations. CULTURAL CHARACTERISTICS OF CERTAIN PATHOGENIC ANAEROBES
ISOLATED FROM SHEEP
by
Theodore T 0 Chaddoek
A THESIS
Submitted to the Graduate Committee in
partial fulfillment of the requirements
for the Degree of Master of Science
in Bacteriology a t Montana
State College
Bqkeman8 Montana
June8 19S5
a**
•2**
TABLE OP CONTENTS
Ie I n t r o d u c t i o n .......... .
. ......... ................ .
IIe Methods of procedure............................ .............
A. Morphology and staining ................... .
1. Size .....................
6
2. Shape .............
6
3. Motility ............................................
6
4. Staining .................................
6
5. Sporulation ...........
7
a. Factors favoring s p e c u l a t i o n ...........
7
b. Position of the spore .
7
......... .
^vft^o-r
B. Physiology ......... .............. .
'rr
6
6
6. Colony characteristics in deep agar .................
I 9 '35
Page
5
7
7
1. Nutritional requirements .........................
7
a. Description of medium for optimum growth .........
8
b* Methods of determining best medium .••••••........
8
2. Bioohemic r
e
a
c
t
i
o
n
s
.
.
.
.
.
.........
9
.. .....
9
a. Oxidation and reduction ..................
I 1. Carbohydrates, Table No. I .......... .
10
a*. Carbon dioxide formation ................. 11
b *. Acid formation ............................... 10
2*. Proteolysis ....................................
H
a ** Hydrogen sulphide formation .............. 11
I*. Blackening of brain
5 0 4 8 3
H
=4529
Page
GLOtIOH
T) %
o o oo o o o o o » o o o o o o o e e 6 o o
O^o
^KotiOH OH. Xli^nms imXlC
d%
Coagulated serum liquefaction
© o ooooooeoooooeeoedo
11
12
©eeooooeoeeo
12'
Oxygen requirements oooooooeoeoooooeoooeoeaooo
12
Co Table Ho© IIj? Morphology and cultural reactions of
organisms isolated from sheep dead of suspected "black
Do
d i s e a s e " o © & o © o o o o o « * o o « 6 o o * i> o o t > o © e o o o o o o & o o <i o o © o & o © o o o © o
13
Pathogenic ity
14
0 0 0 © 0 0 0 0 ©-*0 0 0 0 © 0 © 0 0 © 0 0 0 0 0 o o o o o 0 9 0 0 0 0 0 0 0 0 1 0 0
I
X e
- E x p © I * X lT lS I ltE L X
S V X G L S H lC S
o o o o o o o o o o o o o o d o o o o o o o o o o o o o o o o
.14
Z q L o s s of pathogenicity on artificial media osooooooot . ® 14
3 o .Maintenance of pathogenicity oeo&ooooooeoo-eeeoeoooooe 14
Toxin production
X Q -Preparation
o o o ie-e
O'OvO o o
o e o o e o o o e e o o e- e o o o o e a o o e o o o o
@ o @ 9 0 0 0 o o o o o o p o @ @ o o @ o o o o o o c g o *c * o » & o » o»oo
a o Kind of toxin
o o o o e o o e e o e o o e o e o e e d o o . e e . e o o e o ’o o o e o o o
D O Sage
2*o
T im e
3*0
B e s n X ts
P O X m m n n ity
14
15
&q oooooooeooeoooooooeoo
16
o o »-o o o-o o o -o .o-o o o o o e o o e o o o o-o o o e e - o o o e o o o o o o
15
ho Tests for toxin production
1*0
14
o o o o o o o o o o o e o o.e o e b o o o o o o o o o o o o o e o-o
15
o o o o o o o e o o o o o o e o o o o o o o o o o o o o e o o o-o o e-e O o
15
lim its
OO © o o o-o o o o o o e o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
©o
15
o o o o o o o o o o o e ,o o o o 'o o o o o o e o ©
16
X o .Preparation of a formolized whole culture bacteria
2 o Immunization of guinea pigs
15
3« Inoculation of immunized animals o-o o o-oo-ooooooeooooooo 16
Go Identification of the organisms oo-oooo©o.ooooooooooooooo© 16
I 0 According to H a l l ’s sporulating anaerobic key ©ooooeo 17
I(!I-'I'i v:i l
ti-
; .IiUHiI
• . < -I
O
Page
I I I o
D n S O tiS S lO X l
o o o o o o 0-0 O o o o e o o o e O o o ^ o o o o o o o e c o e 0-0 o d o o o o o o o o e<o
18
Ao Relative to the comparison of the organisms oooeoooe.*.* 18
B o Relative to experiments performed *oooooopoooooooooooooo,18
C 0 Relative to variation in reactions different f r o m those
obtained b y other investigators.
Table R o 0 III o*.o.«oo 19
IVo Stuumary and Conclusions oooooooooooo@ooooQoo@@oo*oeoooeoooe 21
V 0 Literature cited
o o o o o e o o o o e o o o o o o o-o © o o o e e o o e o o e e o o © © o o © o o O
x.
xC
I
INTRODUCTION
A disease of sheep occurring in western Montana has been investi­
gated by the Montana Veterinary Research Laboratory,
This disease had
the characteristics of the "black disease" of sheep described b y Turner
(10S I l p 12) and Albiston (l) in Australia®
The data recorded in this paper are derived from a study of the
disease as it occurred in Montana and deals only with the identifieation and cultural characteristics of organisms which were isolated from
sheep that had died of a disease which resembled "black disease"®
METHODS OF PROCEDURE
Strains 572» 590» and 591 were obtained from the livers of two
sheep dying of suspected black disease*.
Strain 7 was obtained from New
Zealand and used as a 'control throughout the worke
Morphology and staining.
These organisms are among the larger anaerobes®
Strain 572 measured 8 to 8®2 microns in length and I micron in
width®
...
Strain 591 measured 5 to 10 microns in length and I to 1®5 microns
in width®
Strain 590 measured 4®5 to 6®7 microns in length and ®8 to le2’
microns in width®
Strain 7 measured I 0S to 5®5 microns in length and ®8 to I micron
in width®
In young cultures» in a modified brain=!iver broth medium (see
page
8 ) the rods usually occurred singly or in filamentous chains of
from 2 to 5 individuals®
In a medium favoring growth the organisms
occurred singly or in pairs, while in an inhibiting medium there was a
tendency for the development of filaments®
In hanging drop preparationss the organism" showed sluggish motil®
ity®
The bacilli in the vegetative state, stained readily with the
'
most of the ordinary staining reagents and were gram positives but showed
a tendency# as the age of the culture increased* to lose the ability to
retain the crystal violet Stain0
Spores formed readily in modified brain-liver broth medium and
were located Subterminally0
tended the vegetative rode
They were more oval than elliptical and dis­
In other kinds of m e d i a 'spore formation was
greatly diminished or entirely Iacking0
The colonies of 572 in deep agar were roughly spherical and clus­
tered*
Those of 591 were small# compact# and roughly spherical$ those of
590 were small# compact and roughly spherical with woolly borders; those
of 7 were small# compact and roughly spherical with woolly borderso
The
above description of the organisms w as based upon the w o r k of Heller (s)
on the.colony formation of anaerobes in deep agar®
Physiology.
Nutritional requirements.
The bacilli required anaerobic conditions# a rather narrow hydrogen
ion range# and a high concentration of protein in the medium*
In the
preliminary work# H a l l tS brain medium wa s used but growth of the organ­
isms was insufficient for investigational purposes*
After surveying the
literature relative to the conditions favoring the development of the
organism causing "black disease" in sheep# thirty-four different kinds
of media were inoculated, and the results were noted*
F r o m these# a m e d ­
ium was selected which favored the m aximum growth of the organisms and
/
'
■
which later proved to be the best for practical purposes®
This was de­
termined by the variation of BreedtS direct microscopic counts (S) as
stated b y Schaeffer and Fulton (?) in the staining of endosporeso
means of a platinum loop of d
By
ee» capacity9 films from the agitated
culture were spread over an area of I s q 0 cm* on a slide and allowed to
air d r y 0
This was then flooded with a malachite green solution and heat«
ted to steaming three or four times within one half minuteo
stain was washed under the tap for about one half m i n u t e o
The excess
A O 0OS per
cent aqueous, safranine solution was then applied for one half minute
after which the specimen was washed, blotted and dried©
B y taking the
average of bo t h vegetative and spore forms of ten fields, it was possible
to determine which m e d i u m favored sufficient growth for the study of the
organisms and which did not©
It w a s found that the best growth occurred in a modified-brain
liver broth medium©
This consisted of O 0I g m 0 dextrose, 50 gm® of fine=-
Iy minced beef brain and 100 ce0 of liver brotho
The pH of the medium
was adjusted so as to give a pH of 7 02 » 7o4 after forty five minutes
of sterilization at IlS0C 0 at 15 pounds pressure©
The finer the brain was
minced, the more luxuriant the growth of the organisms©
About 5 grams of
the minced brain w a s placed in a culture tube to which 10 ec® of liver
broth w a s added®
A two inch piece of iron wire introduced into the medium
was found to be a factor which affected the growth of the organisms0
Scott and Brandly (.9) recommended the use of reduced iron for the culti­
vation of anaerobic organisms©
Reduced iron was substituted for the iron
wire b u t gave negative results as did also ferric ammonium citrate©
One
half cubic centimeter of sterile laked sheep blood was added aseptically
to each tube after sterilization, followed b y an incubation test of
«9
forty eight hour8 o
This culture medium greatly increased the growth of
the orgatiismsB so it was used throughout the work as the basic culture
mediuuie
Reduction of carbohydrate s<>
A
sugar free broth was prepared b y the inocula tion of a flask of
hormone broth w i t h a culture of Clostridium welchiio
This was incubated
for a period of four days until it was thought that complete reduction
had taken places
It was then clarified b y the addition of a whipped
egg, followed by one hour of sterilization which killed the organisms
and coagulated the. egg»
The reaction wa s adjusted to give a pH of 7®2
after the final sterilization*
After heating again for twenty minutes8
the reduced broth w a s filtered through cotton and filter paper*
Since
the addition of the laked sheep blood,- appeared necessary for active
growths the question arose as to whether the carbohydrates contained in
the blood would lower the pH of the medium*
the case*
This was found not to be
To this medium was added an iron wire* and l/2 cc* of sterile
laked, sheep blood in Dunham's fermentation tubes for the carbohydrate
tests.
The sterile Iaked8 sheep blood and l/2 cc* of a sterile, 15 per
cent solution of each carbohydrate used was added aseptically*
This was
then sterilized for twenty minutes at IlS0 C* at 15 pounds pressure*
The
test for sterility consisted of incubation for 48 hours, after the addi­
tion of the sugars and laked blood®
„
The presence of the laked b l o o d y precluded the use .of an indi­
cator,
So determination of dcid.prociuetion of the carbohydrates was made
-XO
on each test "by use of the L a Motte shell vial hydrogen ion set using
the indicators necessary to cover the various hydrogen ion rangeS0
Table H o 0 Io
I'
Strain
572
%
Dextrose
:
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S
S
4-
8
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%
$
Sucrose
Glycerine
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4-
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8
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%
8
: $
S
$
8
I
S
8
$
Mannose
Xylose
Dulcite
Arabinose
Mannite
8
8
t
$
Raffinose
S
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8
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8
8
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18
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aa
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ess
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Strain
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Maltose
Strain
590
Strain
591
\
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Carbohydrate
Reduction of Carbohydrates
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+ = acid
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Bach test was made in duplicate with the corresponding uninocula­
ted carbohydrate control and the experiment was repeated as a checks
VI
"lire
The pH of each culture was determined every five days for fifteen days6
Ho gas production b y any of the strains in the different carbohydrates
was not e d o
Proteolysis
Hydrogen sulphide formation®
The basic m e d i u m previously described was used®
A small strip of
lead acetate paper, held in place by the cotton plug, was suspended in
the culture tube about one centimeter above the surface of the medium@
Strains 572 and 591 blackened the lead acetate paper within 5=5
days indicating a distinct production of hydrogen sulphide®
Strains 590
and 7 did not blacken the lead acetate paper within that time b ut showed
a partial reaction 10«>14 days latero.
To demonstrate the blackening of brain, a medium consisting of
minced b e e f brain cooked in an Arnold for thirty minutes was used®
The
water content of the med i u m was separated and to it I per cent dextrose
was added®
The brain and the water w e r e then mixed and tubed®
A n iron
wire was placed in each tube and the medium was then sterilized for
forty five minutes at IlS0 C ® at 15 pounds pressure®
Strains 572 and 591
showed distinct blackening of the brain, but strains 590 and 7 did note
A 10 per cent nutrient gelatin medi u m having
a
pH of 7e4 after
sterilization was used to determine gelatin liquefaction®
made at 37°C osiat approximately 26°C. and at IS0C o
Tests were
Strains 572 and 591
liquefied the gelatin at all temperatures and turned the sediment black#
while strains 590 and 7 neither, liquefied the gelatin nor turned the
sediment b Iack0
Action on litmus milk*
Twenty cubic centimeters per liter of a neutral, aqueous solution
of litmus added to skimmed milk was used to determine the action of the
organisms on this medium®
Strains 572 and 591 in two days reduced the litmus, coagulated
the m i l k and showed much proteolytic action b y completely digesting the
curd®
.
Strains 590 and 7 did not reduce the litmus and showed no coagula­
tion or proteolytic effects upon the m i l k within 10=14 days®
To determine
whether growth had taken place, smears made from strains 590 and 7 showed
that moderate growth was present and after two weeks some slight change
to acid in the litmus m i l k was noted®
Coagulation serum liquefaction®
.Sterile horse serum was placed in sterile, plugged tubes, and co­
agulated serum slants were made b y heating in an inspissator®
were inoculated f r o m recent transfers of the organisms®
Wright meth o d for anaerobiosis were made.
cheeks on the first results®
These slants
Cultures by the
Tests were twice repeated as
Strains 572 and 591 showed nearly complete
liquefaction within 7-10 days while strains 590 and 7 did not®
Oxygen requirements®
All four strains were strict anaerobes.
There was no growth on
the surface of solid media in the presence of air®
Table Ho® II summarises the morphologic and cultural characteris­
tics of the organisms®
i'
®i3«j;
Table Ho® IIe
Morphology and cultural reactions of organisms isolated from sheep dead of suspected "black disease",
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Pathogenicityo
Strains 572 and 591 were at all times nonpathogenie for guinea
pigs while strains 590 and 7 were pathogenic®
Pathogenic strains were kept available b y increasing the pathogenicity of the cultures b y guinea pig inoeulationo
The organisms were
recovered after the death of the animals, purified and many cultures
sealed for storage»
Sealed cultures in storage retained their pathogen­
icity 9 b u t those repeatedly transferred on artificial media lost it©
Guinea pigs inoculated intramuscularly with »2 ce© to I ee. of
a twenty-four hour, culture in the modified brain-liver broth medium
usually succumbed in 18 to 24 hours©
of death©
Smaller doses prolonged the time
There was pronounced Swelling9 the edema extending from the
inoculated hind leg to the sternum in some eases©
of gas in the tissues©
There was no evidence
The subcutaneous tissues of the inoculated leg
and the abdomen were infiltrated with a clear jelly-like exudate ©
Some
cases showed a slightly blood tinged exudate hear the site of inoculation®
The abdominal viscera were congested©
The stomach and small intestine®
were inflamed and Congested9 w i th small hemorrhagic areas©
The spleen
and liver appeared normal© but the organism.generally could be recovered
from the liver©
It w a s readily obtained near the site of inoculation®
There was little excess of pleural fluid and the lungs appeared to be
normal w i t h no hemorrhages visible®
Toxin production©
Filtrates were prepared from.cultures of strains 590 and 7 grown
-<=15,M.
in peptic digest brotho
This was prepared by mincing 250 gm© of pig's
stomach, 250 g m 0 of beef, and 250 gm© of beef liver®
A liter of water
plus 2 per cent hydrochloric acid was added to this mi x t u r e ©
This m a ­
terial was thoroughly mixed and held at SS0C 0 for 24 hours, and then
raised to SO0C 0 for fifteen minutes©
vated the enzymes present©
The increased temperature inacti­
The flask of material w as then removed from
the br o t h and allowed to stand for two hours©
poured off and the pH adjusted to 7<,4®
The clear fluid was then
After heating for fifteen minutes
this was filtered through pa p e r 0
Three filtrate cultures were made of each-strain® using for the
toxin producing media, peptic digest broth, modified brain-liver broth
and hormone broth plus O 0I per cent dextrose©
were filtered through Berkefeld V candles©
Forty-eight hour cultures
After sterility had been
proven, six 250 gm® guinea pigs were inoculated intramuscularly with the
filtrates in I e c 0 doses©
Guinea pigs were inoculated w i t h filtrates
from, cultures of b o t h strains 590 and 7 grown in each of the three media®
All tests for toxic effects of the above strains on guinea pigs
were negative©
The experiment was repeated using the same filtrates®
"White mice were inoculated subcutaneously with 1/2 cc© of the filtrates
produced from each strain©
Ho toxic effects were noted and the results
for toxin production were recorded as negative©
Immunity©
Formoliaed whole-culture baeterins were prepared from active
cultures of strains 590 and 7©
A t intervals over a period of three w e e k s #
M n r
=ISira
cultures were made from the bacteria to ascertain if the added O 0S per
cent for mo Iin had killed the organisms©
TOxen the organisms no longer
grew on a suitable medium, and therefore considered deads 250 g m e
guinea pigs w e r e given repeated injections©
The initial dosage of
0e25 oco of the bacterin was increased to I oc0
day intervals were given*
Four injections at five
Following this treatment the two groups of
injected guinea pigs were inoculated w i t h *05 c c 0 and ©025 ce* of viru­
lent culture respectfullyo
Controls were given ©025 CO*, the amount
previously shown to be more than the minimum lethal dose©
All the bae»
terinised guinea pigs and the controls died within 48 hours©
There did
not seem to be any difference between the bac ter ini zed and control groups©
These results led to the conclusion,that the methods of immunizing and
the type of immunizing material used gave no protection to the animals©
Identification of the organisms.
The first step was to separate the proteolytic, from the nonproteolytic strains e
Strains 572 and 591 were proteolytic while strains
590 and 7 were nonproteolytic *
on the followingi
Identification of the organisms was based
morphology, character of the vegetative and spore
forms, motility, cultural reactions, action on proteins, action on the
carbohydrates, and pathogenicity©
Identification was based on the chart
given b e l o w which was taken from H a l l ’s sporul&ting anaerobic key (4)©
M(Spores subterminal rarely central; always when mature swelling the rods
into elostridiumso
Motile rods
»1?«
Coagulated albumin liquefied? brain blackened? gelatin Iiquefied0
Lactose fermented
-
,
e
s
o
e
*
.
B 0 aerofetidus*
Lactose not fermented 0
f
Filtrates toxic for guinea pigs on feeding <===== B^0 botulInus3
Toxic for chickens e»e*e»*oea«os»eaw«<»«»e»weee«ee»«»eee»«»««i«ae»o»m type Ao
Slightly or non toxic for chickens
type B 0
Filtrates non toxic on feedings;
Cultures pathogenic on injection of I
cco
action peculiar to this species ■»»—
—
or less? lytic
«—
B 0 his to Iyticus0
Cultures non pathogenic except in large doses =- B^0 sporogenes0
Coagulated albumin not liquefied? brain not blackened®
Gelatin liquefied? usually pathogenic for guinea pigs (see B 0
novyi)«
Lactose fermented0
Saccharose fermented? salicin not fermented
Saccharose n o t fermented? salicin fermented
Lactose not f ermented
B 0 chauvaei0
Vibrion septIqttec,
B0
^
Strains 572 and 591 may, by the use of this key, be identified as
irogenes while strains 590 and 7 fall under the classifies®
tion of Clostridium n o v y i o
•"3L8*"
DISCUSSION
Sine© strains 572 and 592 have b e e n determined to be Clostridinm
sporogenesg they m a y be considered of no significance in relation to
black disease»
Strains 590 and 7 were considered to be same species
with some slight differences in the fermentation of the carbohydrates0
The cultural characteristics of both organisms were Identical9 and th©
only variation in the fermentation reactions was that strain 590 fer­
mented inulin and 7 did note
Strain 590 was slightly longer than strain 7 0
Otherwise no differ=
ences in morphology were n o t e d o
Both strains were subjected to the different tests at the same
time*
Duplicate tests and:.controls; were made in each © a s e 0
Much of the
cultural w o r k was based on the work of Turner (IO6 Ilfl 12) and Hall (5)o
The 590 strain compared favorably w i t h the strains isolated by Turner
and Hall in respect to cultural reactions* b u t appeared to b e smaller
in Sise0
There were some differences in the fermentation reactions be=
tween strain 590 and the organism isolated b y Turner0
Strain 590 fer»-
mented dextrose* maltose* glycerin* Ievulosefl mannose0 and Inulinfl while
the strain ( B 0 D 0 8) of Turner of Clostridium oedematlens fermented
glucose* maltose, levulose*.and galactose, with;no fermentation of glyeer-x
ine0
Table $To» III shows the comparison of the 590 strain with other
strains of similar organismsc
According to Turner (12) the strains
listed, in this table include one human ( llEeinbergn)? one equine ( nB 0
—19novyl 139” ), and eight strains isolated from ”black disease”, whose re­
actions were identical and are listed as B.D. 8,
Other strains recorded
for comparison are, one strain described by Edgar (B . D . bacillus), two
strains described by Zeissler (^. oodematiens and
g i g a s ), Clostridium
oedematiens described by Bergey (2) and a strain described by McEwen (6)
(B. paludis) isolated from sheep.
Table No. III.
*
® S
O
*o
$
«4
8 «S
S
§\s
£
::::*:*:::::** :$
—*+:-:—:+*—*—
:**::%:::*:*** :*
: :O
;$
N e w Zealand
Strain 7
Montana
Strain 590
’•Weinberg”
!+!-!-!tI-X1*!-,'0!0!0!-!0! 7: 7:O
”B.D. 8 ”
I+:-:":'1-:"-*4-*+':-:0*0*0:**0: O
t : : * :: : : : :: : :*
N e w South Wales
*:0:O
Edgar. B.D. bacillus * + : - * - * + : ° *
: : 7 , 7 7 7 : : :O
ZeIssfer. 6. oeSeina- I : 7 : : 7 7
tiens
:*
Zeissler* B. gigas
!+i-:-:-:-:±i+!-i-;o:o;-i°:"****O
::
B e r g e y 1sManual of D e - I * * : * * * * * * * * * *
ter min ative Bacteriology* +*
-:-*+*-*+*+*-*-*-*-*-*-* * :Clostridium Oedematiens:
::**:::::*:*:
*
: : : :* * * * : t t s : t :
McEwen
t
4
!
4':4':"*"t4-*4-:4-: t t * : * :
B. Paludis
+=definite acidity. - =no acidity. O =not tried.
Hall nNovyi 139”
wk. = weak acidity production
I
•»20"
F r o m the above table it may be seen that the Montana 590 strain
w a s nearly identical in reaction with some that are listed, b ut differs
in otherSe
According to Bergey (2) Clostridium oedematiens ferments
dextrose, maltose, galactose and levulose*
ment galactose®
The 590 strain did not fer®
In addition to fermenting dextrose, maltose, and l e w ®
lose, it fermented glycerine, mannose and Inulin6
Scott (s) (9) stated that the different media that have been ree®
ommended for the production of toxins b y anaerobes have failed to reveal
the presence of such toxins®
This m ay explain w hy the 590 strain did
not produce a toxin®
F r o m a survey of the literature concerning the cultural and fer®
mentation reactions of Clostridium oedematiens, it is to b e noted that
strain 590 compared favorably with the cultural reactions stated by
other investigators, b u t that there was some difference in the sacchar®
olytic properties®
»21“
SUMMARY AKD COKCLUSIOKS
I0
Three anaerobes isolated from, sheep suspected
of a disease resembling "black disease" were studied*
of
having died
These were com®
pared with a K e w Zealand strain 7 which had b e e n isolated from a known
ease of "black disease"©
2o A brain-liver broth m e dium w i t h a pH of
7a2
- 7*4» consisting
of minced beef brain, liver b r o t h plus O 0I per cent dextrosea an iron
wire* and »5 e o 0 laked sheep blood, was found to produce optimum growth*
3 o Optimum growth conditions were determined b y direct counts
of bacteria in definite quantities, of culture media®
4© A medium composed of a sugar free broth* laked sheep blood
and an iron wire wa s found to favor optimum growth in carbohydrate test
culturesc
5 o Two strains (572 and 591) proved to be Clostridium sporogenes,
while strain 590 w a s Olostridium oedematiens and identical w i t h strain 7«
6 e It could not be demonstrated that immunity could b e produced
b y bacterins prepared from strain 590 of Clostridium oedematiensp
=»22c»
LITERAIURB. CITED
Io AIbiston9 H 0 E 0 Infectious necrotic hepatitis of sheep in Victoria*
A Hraxy-=Iike sheep disease= Australian Jour0 Exper0
Biolo & M e d 0 Sci*, 1927» 4 t 114-125«
2» Bergey9 David H 0 et al*
478-480o
So Bread9 R 0 S0
/
.Man= Deter = B a e t 09 1934» 4th Edition8
Breed (1911-1918) method for direct microscopic counts»
.Jour® B a c t 09 1933» 26$548o
4o H a l l s I 0 C 0 .Differentiation and identification of the sporulating
anaerobeso Jour= Infect* D i s 69 1922» 30$445"504o
i
5* Heller9 H c H 6
The study of colony formation in deep a g a r 0 Studies
on pathogenic anaerobes V I 0 Jour0 Infeet0 D i s 09 1922»
30$.l-18o
6® McEwen9 A 0 D 0
A n e w species of pathogenic anaerobic bacterium. - B 0
p a ludls* Jourc of Cesnp0 P a t h 0 & Therap09 1931» 44$
1- 21»
7 o Schaeffer» A= B 08 and Fultons M 0 A simplified method of staining
endospores=
Scios 1933» 77s:194o
8 0 Scotts J 0 P 0 and Bran d l y 9 C 0 Ao The use of reduced iron for the cul­
tivation of anaerobic organisms0 Jour0 of Bactos 1933#
26$1-7o
9o Scotts J» P 0
The etiology of blackleg and methods of differentiating
C l o s 0 ehauvaei from other anaerobic organisms found in
oases of b l a cklegc Cornell Vet0 » 1928» 18$259-271»
I O 0 T u r n e r » A 0 W 0 and Davesnes J0 Role du B= oedematiens dans I' etiologi© de 1 ’hepatite infectiense neerosante (braxy) du
moutbn Australian© The role of B 0 oedematiens in the
etiology of infectious necrotic hepatitis "(braxy) of
Australian Sheep0 A t m 0 de I 8Inst0 P asteur09 1927»
41$1078-1095a
I l 0 Turner9 A© W 0
Hepatite infectiense nierosante (b r a x y » black disease)
du mouton Australian© Braxy8 black, disease of Austra­
lian sheep= A n n 0 de I 8Inst0 Pasteur09 1928» 42s211-2240
12d T u r n e r s A© Wo
Black disease (infectious necrotic hepatitis) of
sheep in Australia*
Coun* for Sei6 and I n d 0 R e s 0fl
1930» Bulletin 46$5-139®
-- GttItural oharacte
certain pathogenic anaerooes
FE