Isolation, enumeration and survival methods for the study of Leptospira in the environment
by Thomas Dean Roberts
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Microbiology
Montana State University
© Copyright by Thomas Dean Roberts (1977)
Abstract:
Cultural media and methods for leptospiral growth were reviewed to find a system to promote growth
from a small inoculum size, to obtain high cell densities in a short time period, and to detect leptospiral
growth early in culture. Ellinghausen and McCullough's albumin-tween medium was found to be the
best media for culturing and growth.
A 70% isolation success from natural water was achieved using natural leptospiral motility across a
0.22 μm pore size membrane into semisolid culture medium. The addition of 100 μg/ml 5-fluorouracil
to the semisolid recovery medium helped control background contaminants.
A purification system which relied on leptospiral motility and drugs, in particular colistin, was used.
This was found to be satisfactory for removing the major problem contaminants, which were small
spiral organisms. Further purification was accomplished by membrane passage and other conventional
methods.
Survival studies were made with membrane side-wall, diffusion chambers. Leptospiral hardjo and L.
pomona were found to survive for periods of a week or more when they were suspended in water
environments. Leptospires were also found to survive and reproduce in algal supernatant.
Leptospiral recovery was possible from moist soils and most natural waters. Areas of high pollution
showed higher leptospiral numbers than those of lower pollution, with "pristine" sites showing no
leptospiral recovery. The ability of leptospires to survive in water and algal supernatant as well as
recovery from water and moist soil reflects a wide distribution. Harboring of leptospires in these
various environments may assure a continuous infectivity of surface waters with leptospires. STATEMENT OF PERMISSION TO COPY
In presenting th is thesis in p a rtia l f u lfillm e n t o f the require­
ments fo r an advanced degree a t Montana State U n iv e rs ity , I agree th a t
the Library shall make i t fre e ly a v a ila b le fo r inspection.
I fu rth e r
agree th a t permission fo r extensive copying o f th is thesis fo r scholarly
purposes may be granted by my major professor, o r, in his absence, by
the D irecto r o f L ib ra rie s .
I t is understood th a t any copying or p u b li­
cation of th is thesis fo r fin a n c ia l gain shall not be allowed without
my w ritte n permission.
Date
r W .,
Cl 3 . / ^ 7 7
ISOLATION, ENUMERATION, AND SURVIVAL METHODS FOR THE STUDY
OF Leptospira IN THE ENVIRONMENT
by
THOMAS DEAN ROBERTS
A thesis submitted in p a r tia l fu lfillm e n t
o f the requirements fo r the degree
of
MASTER OF SCIENCE
in
Microbiology
Approved:
Head,'Major Department
MONTANA STATE UNIVERSITY
Bozeman, Montana
December, 1977
' ^
iii
ACKNOWLEDGEMENTS
The author wishes to thank Dr. David G. S tuart and Mr. John
S c h illin g e r who suggested th is research and le n t th e ir guidance and
assistance throughout the course o f study.
Sincere thanks are also
due to Drs. Gordon A. McFeters, Sam Rogers and Guylyn Warren fo r th e ir
contributions o f advice, a n a ly s is , and ideas.
Thanks are due to Dr. Malcolm H. Smith fo r discussion and
supplies.
Thanks are also due to Mrs. Kay Newman fo r providing e xp eri­
mental organisms and help in serotyping.
I am indebted to the M illip o re
Corporation fo r th e ir provision o f equipment fo r is o la tio n attempts.
Special thanks are due to C harlie C antrell and Dr. Ken Lee fo r
th e ir help in electron micrograph preparations and preparation of other
visual aids.
This project was supported by Grant Number 14-34-001-6207 from
the O ffice o f Water Research and Technology, Department o f In te r io r as
authorized under the Water Resources Research Act of 1964, Public Law
88-379.
I'
TABLE OF CONTENTS
Page
VITA............................................................................................................................
ACKNOWLEDGEMENTS. . . . .
...............................................................................
ii
iii
TABLE OF CONTENTS...............................................................................................
IV
LIST OF. TABLES.....................
vi
LIST OF FIGURES........................
ABSTRACT................................................................................................
v ii
ix
Chapter
1.
INTRODUCTION ...........................................................................................
Statement o f Purpose .................
I
2
2.
LITERATURE REVIEW...................................................................................
3.
MATERIALS AND METHODS....................................................................................17
Organisms U tiliz e d .....................................
Media......................................................................
C u ltiv a tio n and Observation.........................................
Equipment...............................................................................................
Is o la tio n ...............................................................................................
P u r i f i c a t i o n .............................................................................. .... •
Use of Drugs ..........................................................................
E n u m e ra tio n ..................................
Serotyping . ■.............................................
Survival S tu d ie s '. ...........................
4.
RESULTS. .
....................................................................................
Media. . ................................... : .................................... ....................
Is o la tio n ...............................................................................................
P u r i f i c a t i o n ..............................................' ......................... .. . .
Electron Microscopy.......................................................... . . . . .
S e ro ty p in g ..................... . . '..................... ..................................
Enumeration.........................................................
3
17
17
22
22
30
32
34
41
41
42
45
.4 5
47
52
59
59
59
V
Chapter
4.
Page
RESULTS (Continued)
Chamber D iffusion Experiments..................................................
Survival ,....................................................................................................
MPN EnumerationFromNatural W a t e r ..........................................
64
64
68
5.
DISCUSSION ....................................................................... . . . . . .
73
6.
SUMMARY............................. .......................................................................... '
94
LITERATURE CITED. ..........................................................................................
98
.
vi
LIST OF TABLES
Table
1.
Page
Leptospira] Growth Media Used in These Studies
and the Major Components Other Than Basal S alts .....................
19
2.
Drugs Used AndTheir A c t i v i t y ....................................................
3.
Results o f Is o la tio n Attempts From Natural Water
and Recovery o f Leptospires From Water Samples
Spiked With L.pomona, Using Various Methods....................................... 49
4.
Results o f 40 Is o la tio n Attempts Using the Is o la to r
Vial and Some o f the Water Conditions At The
Sample S ite s ...........................................................................
5. - Comparison o f Membranes and Membrane Combinations
For Use In Is o la tio n and P u r if i c a t i o n ................................. .... .
6.
The Results o f the Drug Study on the Mixed Culture
Al in Semi-Solid EM Medium. . . ...................................................... 55
7.
Results of a Most Probable Number (MPN) Enumeration
o f Leptospires a t Four Natural Water S ite s ......................... .
35
50
53
71
LIST OF FIGURES
Figure
1.
Page
Swinnex M odification I Used fo r Is o la tio n
Attempts and P u r ific a tio n ......................
25
Polycarbonate Cell Used fo r Is o la tio n
Attempts and P u r ific a tio n .........................................
27
Is o la to r Vial Used fo r Is o la tio n
Attempts and P u r ific a tio n .......................................................... .
29
Drug Plate Configurations 1-6 For Study of
Drug E fficacy in P u r ific a tio n ......................................................
38
Drug Plate Configurations 7-12 fo r Study of
Drug E fficacy in P u r ific a tio n ......................................................
39
Culture Tubes o f EM Semisolid Medium Showing
the Dinger Ring Growth Phenomenon............................. .
48
7.
Examples o f Drug Plates Used fo r P u rific a tio n .
57
8.
Electron Micrograph of Water Is o la te C ..................................
9.
Comparison o f Nephelometry and D irect
Cell Count as Measures of Cell Densities
in a Survival Study of L. pomona . . . . . . . . . . . .
2.
3.
4.
5.
6.
10.
11.
12.
13.
. : . . . .
Comparison o f D irect Cell Count and Absorbence
a t 420 nm as Measures of Cell Densities
in a Survival S t u d y ....................................................................
.
61
62
•
63
Comparative D iffusio n o f Carbon-Containing
Compounds Across a 0.45 ym Membrane, a D ialysis
Tubing Sac and a 0 .1 -0 .4 5 ym Double Membrane
Layer. Ton D iffusion Across a 0 .1 -0 .4 5 ym Double
Membrane Layer as Measured by Conductivity ........................
65
Comparison o f Glucose D iffusion Across a 0.45 ym
Membrane and a 0 .1 -0 .4 5 ym Double Membrane Layer . . . .
66
Survival of L. pomona and L. handjo in Continuous
Flowing Well Water and a Laboratory Water Environment
As Measured by D irect Cell C o u n t..................................... '- •
67
viii
F igure
Page
14.
Survival o f L. pomona in a Laboratory Water
Environment With a Maximum and Minimum V ia b ilit y
Range Based on D irect Cell Counts and the Percentage
o f M otile C ells in the C o u n t.......................................................... 69
15.
Comparison of L. hardjo and L. pomona survival to
Salmonella Iyphl3 Salmonella entevlt-idls ser.
paratyphi B and D, and Vibrio oholerae survival
in continuous flowing well w ater. Maximum and
Minimum V ia b ilit y Ranges Based on D irect Cell
Count and M otile Cell Percentage of the
D ire ct Cell Count are Shown fo r L. hardjo and L. pomona. .
70
ix
ABSTRACT
Cultural media and methods fo r le p to sp ira l growth were reviewed
to fin d a system to promote growth from a small inoculum s iz e , to
obtain high c e ll densities in a short time period, and to detect le p ­
to s p ira l growth e a rly in c u ltu re . Ellinghausen and McCullough's
albumin-tween medium was found to be the best media fo r c u ltu rin g and
growth.
A 70% is o la tio n success from natural water was achieved using
natural le p to s p ira l m o tility across a 0.22 ym pore size membrane into
semisolid c u ltu re medium. The addition o f 100 yg/ml 5 - f l uorouracil to
the semisolid recovery medium helped control background contaminants.
A p u rific a tio n system which r e lie d on le p to s p ira l m o tility and
drugs, in p a rtic u la r c o lis t in , was used. This was found to be s a tis ­
facto ry fo r removing the major problem contaminants, which were small
s p iral organisms. Further p u rific a tio n was accomplished by membrane
passage and other conventional methods.
Survival studies were made with membrane s id e -w a ll, d iffu s io n
chambers. Leptospiral hardjo and L. pomona were found to survive fo r
periods o f a week or more when they were suspended in water environ­
ments. Leptospires were also found to survive and reproduce in algal
supernatant.
Leptospiral recovery was possible from moist s o ils and most
natural waters. Areas o f high p o llu tio n showed higher le p to sp ira l
numbers than those o f lower p o llu tio n , with "p ris tin e " s ite s showing
no le p to s p ira l recovery. The a b il it y o f leptospires to survive in
water and algal supernatant as well as recovery from water and moist
soil re fle c ts a wide d is trib u tio n . Harboring o f leptospires in these
various environments may assure a continuous in f e c t iv it y of surface
waters with le p to sp ires.
CHAPTER I
INTRODUCTION
The increased use and reuse o f the water resource has in te n s i­
fie d in te re s t in water q u a lity and in methods fo r evaluating water
q u a lity .
The search fo r a bio lo g ical in d ic a to r which can be e a s ily
assayed and which gives a c le a r c o rre la tio n with s p e c ific types o f p o l­
lu tio n has long been sought.
The fecal coliforms are widely used
organisms fo r detecting animal and human fecal p o llu tio n , which could
contain water-borne pathogens.
The in te r r e la tio n o f the fecal c o li­
forms and other fecal p o llu tio n indicators with water-borne pathogens
remains obscure, although probable pathogenic contamination can be
suspected when fecal c o li form counts are high.
D if f ic u lt ie s in obtaining d e fin itiv e information concerning the
po ten tial health hazards of pathogenic bacteria through the use of
in d ic a to r systems has increased in te re s t in the survival and virulence
of pathogenic bacteria in the environment.
Of the hundreds of diseases
involving animals there are more than 150 which are zoonotic (1 6 ).
Some
of the b e tte r known ones are salm onellosis, brucellosis and le p to ­
s p iro s is.
The complex epidemiology, the increasing recreatio n al exposure
(80) and the improved diagnosis o f previously unrecognized mild forms
o f lep to sp iro sis (47.) have made lepto sp iro sis a disease of public s ig ­
n ific a n c e .
Leptospires have been shown to survive in water (6 6 ,8 ,4 ) ,
2
soil
(59,37) and animal wastes (1 6 ,1 7 ), however l i t t l e
is known of th e ir
actual a b ilit ie s to endure h o s tile environments and to maintain th e ir
virulence apart from th e ir host animals.
e
The inform ation gleaned about Iepto sp iraT behavior in the envi­
ronment has la rg e ly been gathered through laboratory experiments,
through u t iliz a t io n o f host animals, and by follow ing lepto sp irosis
cases.
Methodology enabling in vitro and in situ observation and
experimentation needs to be expanded to f u lly explore the in teractions
and impact o f pathogenic leptospires in the environment.
Statement o f Purpose
The aim o f th is research is to develop methods and to u t iliz e
these methods fo r in situ and in vitro examination o f le p to sp ires.
In
p a r tic u la r , the goals are to look a t the survival o f leptospires in
water as compared to in d ic a to r organisms and to recover leptospires from
the natural water systems.
CHAPTER 2
LITERATURE REVIEW
■
■
The f i r s t observance o f Iepto sp ira was in 1905 by Stimson who,
/
■■
'
in examining tissue from a "yellow fever" case, noted long thin s p iral
organisms.
I t was re a liz e d la t e r th a t these thin s p ira l organisms were
ic teric-p ro d u cin g leptospires and were themselves the cause o f the
"yellow fe v e r."
(7 4 ).
Stimson c a lle d these organisms Spiroahaeta interrogans
In 1913, S. B. Wolbach and C. A. L. Binger described the same
type o f organisms from fresh water sources.
tiv a te these organisms (8 4 ).
They were unable to c u l­
Later researchers, working with a
pathogenic s p iral organism, were able to c u ltiv a te them in seracontainihg media.
They named th e ir is o la te s Leptospira iaterohaemor-
rhagiae, and id e n tifie d i t as the etp'ological agent o f W eil's disease
described in 1886 (4 0 ,5 7 ).
,
Increasing is o la tio n o f these th in s p ira l organisms gave
evidence th a t the epidemiology o f le p to s p ira l in fectio n s is complex.
i
There are now over 190 ^erovars described and o f these 150 are named
and recognized (6 8 ,8 2 ).
These serovars are placed in 18 serogroups
based on cross-agglutination and in two complexes based on th e ir
p a ra s itic or saprophytic character.
The "Interrogans Complex" includes
the serovars which have been is o lated from sick animals or c a rrie rs .
They are pathogenic to some.and may be commensal to other animals.
The
" B ifIexa Complex" includes about 70 nonpathogenic, f r e e - liv in g serovars
4
which have been is o la te d from surface waters and moist s o ils .
Epidemiology
Leptospirosis is cosmopolitan in nature.
F ifte e n o f the sero-
groups have been confirmed or suspected as in fe c tiv e agents by sero­
lo g ical means (4 7 ).
Before 1948, L. ioterohaemovvhagiae was recognized
as the major in fe c tiv e serovar, with L. aanicola recognized to a lesser
e x ten t.
A fte r 1948, with improved is o la tio n , c u ltu rin g and maintenance,
the complexity o f le p to s p ira l epidemiology became apparent.
The r a t
lepto sp ire L. icterohaemorvhagiae was displaced by L. can-Loola as the
most frequent e tio lo g ic a l agent.
In 1971 and 1972 the fiv e major common
source outbreaks occurred in Minnesota, Texas, M issouri, New York and
Oregon, and were a ttrib u te d to the serogroups Autumnal is , Canicola,
Interohaemorrhagiae, Pomona and Autumnal is , re sp ec tiv e ly .
The outbreak
in Minnesota was a ttrib u te d to rid in g go-carts in a farm yard a fte r .
seasonal ra in s .
In Texas the outbreak was among children playing in
pools o f water with in fected dogs.
The outbreak in Missouri was
associated with infected dogs th a t were shedding leptospires in th e ir
urin e.
The outbreak in New York was a ttrib u te d to contact with urine
o f infected c a t t le , and th a t in Oregon with an infected dog and a moist
environment (3 1 )..
The mild a n ic te ric forms o f lep to sp iro sis are recog­
nized as more prevalent than the severe ic t e r ic forms.
Pathogenic leptospires are found as parasites o f both w ild and
5
domestic animals.
One or more host species w ill act as th e /re s e rv o ir
fo r a s p e c if ic .Ie p to s p ira l in fe c tio n with other animals, including man,
being in fected in lo c a liz e d outbreaks.
The Norway r a t is a c a rr ie r and
shedder o f L. 'Lotevohaemorrhagiae. Counts o f 10
9
organisms per m i l l i ­
l i t e r have been shown in the urine o f the asymptomatic Norway rats (3 ) .
Leptospira pomona is usually associated with swine, and to a lesser
e x te n t, with c a ttle and sheep.
■canicola.
Dogs are the major c a rrie rs o f L.
The rodent population has been found to be the major source-
re se rv o ir in fe ra l populations.
Leptospires have been is o lated from
nonmammalian hosts such as frogs (22) and tu r tle s (3 6 ).
Insects do not
appear to play a ro le as c a rrie rs or vectors o f in fe c tiv e leptospires
(6 2 ).
Transmission
Transmission occurs through contact with in fected blood, u rin e ,
tissues and organs.
Leptospires are in d ir e c tly transm itted through
contaminated natural fresh water systems, s o ils , muds, vegetation and
food s tu ffs .
Mucosal membranes o f the nose, muzzle or the mouth, skin
abrasions, and conjunctiva are the s ite s of entry.
A study on guinea
pigs, whose abdominal h a ir was shaved and the abdomen then exposed to
slowly flowing water containing L. pomona, indicates th a t,g iv e n tim e,
the leptospires can penetrate in ta c t skin (3 3 ).
D ire ct contact occurs
when handling in fected hosts and th e ir fresh carcasses and organs.
6
Other important routes o f transmission are coitus and transplacental
in fe c tio n o f the fetus (6 2 ,8 0 ).
Leptospira! in fe c tio n in man is often dependent on occupational
hazards.
V e te rin a ria n s , farmers, hunters, slaughter house workers and
miners are examples o f the types o f workers developing le p to sp ira l in ­
fe c tio n s .
While occupational disease incidence has been decreasing,
except th a t o f the farm er, recreational a c t iv it ie s are accounting fo r
increased numbers o f le p to s p ira l cases.
Swimming, wading and contact
with infected pets (in p a r tic u la r , dogs) have become the major sources
o f lepto sp irosis in man.
r
Reported cases o f lep to sp iro sis have been increasing in f r e ­
quency in the la s t fiv e decades.
Sixteen cases in 1925-1934, 230 in
1935-1944, 267 in 1945-1954, 705 in 1955-1964 and 791 in 1965-1974
have been reported (4 7 ).
The increase in numbers of cases is probably
due to improved recognition o f mild cases o f lep to sp irosis and to
increased recreational exposure e s p e cia lly swimming and wading in
contaminated areas.
— ■j) The erad ication o f lep to sp iro sis appears to be unachievable due
to the com plexities o f host reservoirs and the po ten tial survival of
leptospires in w ater.
Some control has been accomplished through vac­
cinatio n o f domestic herds (such as c a ttle and swine) using m u ltiva len t
antigens o f the types most prevalent in th a t area.
However, the e f f i ­
cacy o f vaccines and cross reactions among the pathogenic leptospires
7
are not f u lly understood.
Vaccination o f human populations does not
seem p ra c tic a l because o f the low incidence o f lep to sp iro sis in man.
Some high ris k vocations may warrant vaccination (7 5 ).
Pathogenicity
Leptospirosis is protean in nature, dependent upon the host and
the in fe c tin g serovar as well as other poorly defined c h a ra c te ris tic s .
The c la s s ic al W eil's lepto sp iro sis is usually considered to follow
three separate stages.
A fte r penetration through mucosal membranes or
abraided s k in , the leptospires quickly enter the blood stream.
They
then spread throughout the visceral tissue with the kidneys and liv e r
bearing the brunt o f the in fe c tio n .
A fte r .th is primary leptospirem ia ,
the populations increase in size and a fever w ill occur usually 6 to 8
days a ft e r in fe c tio n .
The septicemic stage is characterized by gener­
a liz e d muscular aches and pains, and exq u isite tenderness o f muscles,
p a r tic u la r ly the gastrocnemisis muscles.
r e fle c t meningeal i r r i t a t i o n .
Headache, nausea and vomiting
Occasional intense conjunctival conges­
tio n and hemorrhage may occur as well as petechial and ecchymotic
hemorrhages appearing on the skin.
. In the th ird to seventh day, the toxic or ic t e r ic stage begins.
Jaundice appears in about 2/3 o f the cases, with an enlarged and tender
li v e r .
The jaundic condition la s ts fo r a few days and gradually de­
creases over the remaining course o f the disease.
At the end o f the
8
f i r s t week, o lig u ria may appear and progress to anuria leading to
uremic death.
The convalescent stage shows the fever slowly f a llin g and urine
output returning to normal.
The jaundice gradually fades and the
p a tie n t generally returns to normal (3 5 ,6 4 ).
Maintenance
Media fo r c u ltiv a tin g and m aintaining le p to s p ira l cultures are
of three types.
(4 0 ).
The o rig in a l successful growth media contained sera
D iffe re n t sera were used with varying success, with ra b b it
becoming the serum o f choice.
A pool o f ra b b it serum made up of sera
from a t le a s t 20 le p to s p ira l negative rabbits has become the most suc­
cessful medium a d d itiv e .
Sera-containing media consist o f a basal
s a lts buffered portion with serum added to form 8 to 10% o f the volume.
Some o f the serovars may require as much as 20% serum fo r growth to
occur.
Examples o f th is type o f media are Fletch er's 1928 (2 9 ),
K o rth o fs 1932 (4 9 ), S tu a rt's 1946 (76) and m odifications o f these (8 1 ).
Leptospiral media in wide use a t present include those con­
ta in in g albumin frac tio n s and polysorbates.
E a rlie r attempts to
u t i l i z e albumin frac tio n s of sera proved unsuccessful, because in these
media form ulations, the importance o f only one vitam in was stressed and
the requirements fo r lip id s was not known.
With the a d d itio n .o f B I2 and
and lip id s in the form o f polysorbates, the albumin media became sue-
9
cessful in c u ltiv a tin g leptospires (2 1 ).
The most widely used albumin-
tween media are those o f Ellinghausen and McCullough and a m odification
by Johnson and Harris (2 5 ,4 5 ).
The th ird type o f medium is the chemically defined serum-free
synthetic medium.
type.
Several researchers have described media o f th is
Shenberg (1967) has had the most success and was able to sub­
cultu re 52 strain s belonging to 12 serogroups (6 8 ).
Work is s t i l l
incomplete in form ulating chem ically-defined media capable of sustain­
ing a ll the serovars.
Thus, most media used in routine cu ltu rin g
consists of the f i r s t two types.
Three forms o f media are used fo r various c u ltu rin g needs.
Liquid medium is essential fo r growth o f antigens fo r serum te s tin g as
flakes o f agar in te rfe re with te s t resu lts (8 1 ).
Semisolid agar media
are the media o f choice fo r maintenance of stock c u ltu re s .
Growth o f
leptospires occurs fa s te r , and with greater success in semisolid media.
Some o f the serovars, e s p e cia lly those of the Hebdomadis serogroup, are
d i f f i c u l t to c u ltu re without the use o f semisolid media.
The n u trie n t
value or other b e n e fic ial properties o f agar are not understood in
c u ltu rin g leptospires (2 1 ).
Solid agar media are used to study colony
morphology and as aids in is o la tio n and p u rific a tio n .
The saprophytic
leptospires w ill usually become v is ib le w ithin 3 to 4 days on s o lid 1%
agar media.
The p a ra s itic leptospires may require 14 to 20 days with
some o f the serovars being very d i f f i c u l t to observe on s o lid agar (7 3 ).
10
Incubation o f leptospires is generally done in the dark with a
temperature o f 2 7 -3 0 °,C. . Some researchers claim primary incubation a t
37° C fo r a 2-3 day period enhances le p to sp ira l growth, but other
workers disagree (8 1 ).
Lower temperatures tend to decrease the growth
o f pathogenic le p to s p ire s , while 56° C fo r a few minutes w ill k i l l
leptospires (8 1 ).
Johnson and H arris have suggested the use o f growth
a t lower temperature as a means to d iffe r e n tia te saprophytic and
p a ra s itic leptospires (4 5 ).
Culturing is also carried on a t room tem­
perature.
Survival of le p to s p ira l cultures usually depends upon tra n s fe r
to fresh media a t 3-4 week in te rv a ls .
Cultures have remained viable
fo r over a year in Ellinghausen and McCullough semisolid medium.
Freeze
drying may be the method o f choice fo r maintaining cultures over long
time periods since subculturing often resu lts in loss o f virulence and
may re s u lt in antigenic a lte ra tio n s giving fa ls e inform ation in sero­
lo g ic a l examination (8 1 ).
Morphology
Leptospires are Gram-negative, long, th in , tig h tly -s p ir a le d
organisms.
They are 4-20 pm long and approximately 0.1 pm in diameter.
Under c e rta in conditions, they may obtain a length o f 30-40 pm.
The
c o ils have 0 .2 -0 .3 pm overall diameter and a pitch of 0 .3 -0 .5 pm.
There are two axial fila m e n ts , one inserted a t each end in terminal
disks w ith the fre e filam en t ends meeting a t the center o f the organism.
/
Tl
A sheath or envelope encloses both the a x ia l filam ents and the cyto­
plasmic body (4 2 ,5 5 ).
Three forms o f movement can be seen.
A to-and-
fro shunting with a short re st a t the change in d ire c tio n and rapid
spinning o f the hooked ends about the axis can be seen in liq u id medium
In semisolid agar, leptospires move in a serpentine fashion, boring
>
through the medium (5 8 ).
Observation
D a rk -fie ld microscopy is the method of preference fo r viewing
le p to sp ire s.
The high, dry ob jective
fo r most observations.
tio n .
o f 43X is generally acceptable
Oil immersion can be used fo r b e tte r resolu­
Phase contrast microscopy can be used, although, due to the
le p to s p ire 's long th in shape, they are not very phase dark and do not
show up without a great deal of focal plane adjustment.
Electron
micrbscopy has been used fo r gross morphology and viewing transverse
and lo ngitu din al sections (7 0 ).
L ig h t-fie ld microscopy is of lim ite d
value fo r observing le p to s p ire s , however, leptospires may be rendered
v is ib le through the use o f s ilv e r impregnation and a n ilin e dyes (5 ,6 9 ).
Macroscopic observation depends on the type o f media used fo r
c u ltu rin g .
Growth in semisolid media generally appears as th in disks
3-5 mm below the surface.
Several disks often appear with the number
o f discrete disks dependent upon the age of the c u ltu re .
In some types
of media or in c e rta in batches o f media, these th in disks do not appear
although growth o f m otile leptospires can s t i l l be observed upon micro­
12
scopic examination.
in 1932;
Dinger f i r s t wrote about these th in disks o f growth
thus, th is growth phenomenon has come to be c a lle d the Dinger
ring (1 5 ).
Leptospira! growth in liq u id media' is recognized by increasing
tu r b id ity and a shot s ilk appearance upon a g ita tio n .
agar was described by Cox and Larson in 1956 (1 2 ).
Growth on solid
Appearance on s o lid
agar depends upon several parameters ou tlined by Cox in 1966 (1 1 ).
Since the leptospires have such a long generation time and incubation
must be c arried out over an extensive period of tim e, i t is necessary
to pour plates of s u ffic ie n t depth to prevent drying.
taken in streaking to allow formation of colonies.
Care must be
Plates should be
incubated fo r a t le a s t 3-4 weeks before they are considered negative.
Some s tr a in s , due to th e ir fastidiousness, may not grow on the solid
agar used.
The concentration o f agar is very im portant;
1% agar •
appears to be the best concentration, although some leptospires form
th in v e ils of growth a t th is agar concentration and may need I . 3-1.4%
agar.
■
While immunofluorescence has been used to detect le p to sp ire s,
th is technique a t present has lim itatio n s , due to cross-reaction
problems.
When demonstration of leptospires is the only goal, flu o re s ­
cent antibody techniques may be the methods of choice (3 7 ).
13
Enumeration
In Chang's e a rly work on the survival of Leptospira ioterohaemorrhagiae counts were made by placing a drop of a le p to sp ira l
suspension upon a s lid e from a c a lib ra te d c a p illa ry .
A standard cover
s lip was then placed over the drop and i t was examined with the high
dry ob jective of a d a rk -fie ld microscope.
fo r each preparation.
Twenty fie ld s were examined
Chang had to re so rt to th is type o f count be­
cause no counting chamber was a v a ila b le which was th in enough to use
on a d a r k -fie ld app licatio n (8 ) .
With the advent of the P e tro ff-
Hausser b a c te ria l counting chamber, researchers began making d ire c t
counts o f le p to s p ires.
P late counts have also been obtained using
spread p la te techniques (5 1 ).
Later Schiemann had success generating
survival curves using most probable number procedures (6 7 ).
Measure­
ment o f tu r b id ity has also been used to monitor the growth of le p to ­
spi res .
Greene used a Klett-Summerson ph otoelectric colorim eter (34)
and Ellinghausen used nephelometry as a measure o f growth (2 7 ).
Since
nephelometry measures only scattered lig h t the color o f the medium does
not in te rfe re with measurements, thus tu r b id ity measured via nephelo­
metry correlates well with d ire c t microscopic counts.
Nephelometry has
become the tu r b id ity measurement o f choice in enumeration o f le p to ­
spi res .
Is o la tio n
Leptospires can be is o lated from the tissue of an infected host
14
Infected tissue is removed a s e p tic a lly , ground, d ilu te d in b u ffe r and
then placed in re p lic a te tubes o f semisolid media (7 7 ).
" I f care is
taken, pure cultures can be obtained in th is manner (6 0 ).
Surface
waters can also be sampled fo r leptospires by inoculating an appropriate
host, such as guinea pigs and hamsters in the weanling stage, in tr a peri tioneal Iy with the sample.
A fte r a period o f incubation, tissue
is then harvested and placed in the appropriate medium (1 9 ,3 2 ).
Is o la ­
tio n through the use o f host animals has been very successful, but cost,
expertise and time are a ll high when using in vivo is o la tio n techniques.
In vitro is o la tio n has met with some success.
Researchers such
as Cox were able to c u ltiv a te leptospires from surface waters by placing
a drop o f f ilt e r e d water on s o lid agar medium and then watching fo r
Teptospiral growth on the advancing edge o f b a cterial growth (1 1 ).
tra tio n has also been used as a method o f is o la tio n (6 ,6 3 ,7 9 ).
F il­
These •
methods usually consist o f f i l t r a t i o n through decreasing pore size mem­
brane f i l t e r s u n til the fin a l f i l t r a t e has passed a 0.45 or 0.22 ym
pore size membrane.
The f i l t r a t e is then inoculated in to cultu re media.
A method described by Smibert to decontaminate a le p to s p ira l culture
has also been used to is o la te leptospires (1 7 ,7 0 ).
This method consists
of placing a s t e r ile membrane of 0.22 or 0.45 ym pore size on a solid
agar surface and sealing a s t e r ile ring to the top of the membrane.
The sample is then placed inside the rin g .
A fte r several days of in ­
cubation, the membrane and ring are removed and the agar checked fo r
15
le p to s p ira l growth.
Leptospira canioola was isolated from the urine
o f an in fected dog using th is method (P. Thomson, personal communica­
tio n ).
P u rific a tio n
A problem in is o la tio n and c u ltiv a tio n o f leptospires is
contamination.by other b a c te ria .
Since leptospires are capable of
passing a 0.22 pm pore size f i l t e r , the methods o f Smibert, Rittenberg
and Braun can be u t iliz e d to elim inate some contaminating bacteria (6 ,
6 3 ,7 1 ).
The addition of drugs such as neomycin (5 7 ), 5 - f l uorouracil
(4 6 ), furazolidone (5 6 ), s u lfa th iazole (1 0 ), acidione (1 0 ), singly and
in combination has also been used fo r decontamination (1 0 ,5 6 ).
Streak plates and d ilu tio n have also been used fo r p u rific a tio n ,
however, due to slow growth and the f a ilu r e of some leptospires to grow
on agar media, these methods may be lim ite d in value.
P u rificatio n ,
may also be accomplished through animal passage, which maintains v ir u ­
lence or may revive lo s t virulence (6 9 ).
Survival
Survival of leptospires in natural systems plays an important
part in transmission to host animals.
Some of the e a rly work by Chang
indicated th a t survival times may be qu ite long (8 ).
At 25-27° C and
pH 7 .0 , L..- icteroharmorrhagiae survived fo r 30-32 days in s t e r ile tap
water and 98-102 days in s t e r ile tap water plus 1% serum.
/
Diesch showed
16
th a t m otile leptospires could be found a fte r six days in selas candles
suspended in a ditch o f manure (1 6 ).
Other studies show survival times
o f a few hours to several weeks and in some cases several months depend
ing on the temperature, pH and type of suspending f lu id (5 9 ,6 5 ).
Sur­
vival in fresh water systems is dependent on a pH range o f 6 .8 -8 .0 and
a temperature range o f 7-30° C (1 3 ).
O
C a ttle have been shown to shed as many as 10
ml in th e ir urine (3 2 ).
leptospires per
Periods of heavy rain and flooding have also
been shown to increase the concentration of leptospires in water (4 ).
Thus, demonstration of survival and transmittance in water systems is ■
dependent upon the number o f leptospires shed in to the system as well
as the a b il it y to detect leptospires through
techniques.
in vivo and in vitro
Another fa c to r confusing le p to sp ira l survival studies is
the saprophytic le p to s p ira l population in te r fe r in g with the recovery
of s p e c ific le p to s p ira l serovars (4 2 ).
CHAPTER 3
MATERIALS AND METHODS
Organisms U tiliz e d
Three serovars representing three serogroups o f leptospires
were u t iliz e d in these s tu d ies .
Leptospira pomona and Leptospira
hardjo. Communicable Disease Center (CDC) serovars, representing the
Pomona serogroup and the Hebdomadis serogroup, re s p e c tiv e ly , were
kindly supplied by Mrs. Kay Newman from the Montana State Department
o f Livestock Diagnostic Laboratory in Bozeman, Montana.
pomona was used fo r most o f the studies.
Leptospira
The saprophytic serovar
Leptospirapatoo (American Type Culture C ollection #235826), repre­
senting the Semaranga serogroup, was used in comparative studies.
Leptospira! is o la te s from the springs and streams surrounding Bozeman
were also used in some o f the studies.
Media
Water U t iliz e d .
The water used fo r media and buffers was e ith e r
double d is t ille d water stored in glass or water.obtained from a M illi-Q
activated carbon, ion-exchange system (M illip o re Corp., Bedford, MA)
follow ing single d i s t i lla t io n .
B u ffe rs .
Sorensen's b u ffe r and peptone-phosphate bu ffer were
used fo r washing, resuspending and d ilu tin g organisms.
Sorensen's
b u ffe r was made by adding 8.33 g NagPO^ (anhydrous) and 1.09 g KHgPO^
(monobasic) to one l i t e r o f d is t ille d water.
The pH was then adjusted
18
to the range o f 7 .4 -7 .6 with 2M NaOH.
Peptone-phosphate b u ffe r was
made by adding 1.25 ml o f phosphate stock solution (2) to one l i t e r o f
d is t ille d water and then adding peptone (D ifco) to 0.1%.
The pH was
then adjusted to 7.4 with 2N NaOH.
S o lid , Semi sol id and Liquid Medial
used fo r these studies.
Three types of media were
SemisoTid medium made with a 0.25% agar con­
centration (BBL, Agar P u rifie d ) was used fo r maintenance, is o la tio n and
enumeration.
Solid medium a t a 1% agar concentration (BBL, Agar P u ri­
fie d ) was used fo r is o la tio n , p u rific a tio n and enumeration.
medium was used to grow leptospires fo r survival s tu d ies .
Liquid
The semi­
s o lid and liq u id media were stored a t room temperature in 100 ml
. •
•
amounts and dispensed as needed. The s o lid media were plated and stored
a t 4° C.
Al I media were checked fo r s t e r i l i t y before use by streaking
on T ry p tic Soy Agar (D ifco ) and by visual inspection.
The various
media u tiliz e d are shown in Table I .
Bovine Serum Albumin Media.
ElTinghausen and McCullough medium*5
(EM) and the Johnson and Harrison m odification (EMJH) thereof were pre­
pared. as described (2 5 ,4 5 ).
A m odification in preparation of E llin g -
hausen and McCullough medium was made as follow s:
to 879 ml of w ater,
40 ml o f 25X b u ffe r, 50 ml o f 20X s a lts , I ml CuSO4BH2O, 10 ml ZnSO4 7H20 and 20 ml FeSO4 -7H20 were added.
This was s tirre d fo r 5 min and
then 200 mg L-cystiene was added and s t ir r in g continued fo r another
5 min.
This basal mixture was then f ilt e r e d through a 0 .8 ym pore size
19
Table I .
Leptospira! Growth Media Used in These Studies and the Major
Components Other Than Basal Sal ts
Media
BSAa
Serum
Tween-80
Ellinghausen
McCullough (EM)
X
X
Johnson Harrison
M odification o f EM (EMJH)
X
X
Minimum essential
medium o le ic (MEM-BOH)
X
Minimum essential
medium tween-80 (MEM-BT)
X
X
Fletcher
X
Stuart
X
Cox (SM I )
X
Cox (SM 2)
X
aBovine Serum Albumin Fraction V.
^Minimum essential medium, Dulbecco1s m odification.
cTryptose phosphate.
MEMb
Oleic
Acid
X
X
Trypc
PO^
X
X
20
membrane f i l t e r and the pH adjusted to 7.4 with ZN NaOH.
This solution
was e ith e r autoclaved fo r 15 min a t 15 lb or f i l t e r s t e r iliz e d through
a 0.22 ym pore size membrane.
Agar was also added to the basal mixture
i f semisolid or s o lid medium was desired.
The albumin solution was made by adding 12.5 g bovine serum
albumin fra c tio n V (Pentex) to 250 ml o f d is t ille d water.
While s t i r ­
ring 1.2 ml o f tween 80 (B aker), 0.1 ml thiamine stock and 0.2 mg B I2
were added.
The pH was adjusted to 7.4 with ZN NaOH.
The medium was
then f i l t e r s te r iliz e d by seq u en tially passing through 0.8 ym, 0.45 ym
and 0.2 ym pore size membranes.
The albumin solution was then added
to the basal s o lu tio n .
Minimum Essential Media.
Two types of media u t iliz in g minimum
essential tissue c u ltu rin g media were used.
Both contained Dulbecco's
modified Eagle medium (Cat #0714, In tern a tio n a l S c ie n tific In d .)
The
minimum essential medium containing bovine serum albumin oleate was
prepared as described by Finn and Jones (MEM-BOH) (2 8 ).
The other
minimum essential medium with bovine serum albumin and tween 80 (MEM-BT)
was prepared by adding 13.43 g Dulbecco's modified Eagle medium, 5 g
bovine serum albumin, 1.2 ml of tween 80, 1.1 ml glycerol and 0.2 mg
BI2 to 500 ml o f water.
ZN NaOH.
The mixture was adjusted to a pH of 7.4 with
This mixture was then f i l t e r s te r iliz e d with a 0.22 ym pore
size membrane f i l t e r (Mi 111 p o re ).
To another 500 ml o f d is t ille d water
0.5 g Na2HPO4 , 0.2 g KH2PO4 and 0.25 g NH4CL were added.
This portion
21
was e ith e r autoclaved or f i l t e r s t e r iliz e d . . Agar was added fo r solid
or semi sol id media.
The pH was adjusted to 7 .4 .with 2N NaOH.
The two
portions were then added to give a to ta l o f one l i t e r .
Serum-Containing Media.
F letc h e r's and S tu a rt's media formula­
tions were used fo r serum-containing media.
They were prepared as
described in Leptospirosis Methods in Laboratory Diagnosis (7 7 ).
S te r ile serum was added to form 8-10% o f the volume.
Fetal c a lf serum
was supplied by Dr. Malcolm H. Smith a t the Montana State Veterinary
Science Research Laboratory.
Rabbit serum was obtained from research
rabbits used fo r the production o f a n tiy .
obtained from Dr. Smith.
Some ra b b it serum was also
The F letc h e r's and S tu a rt's media were ad­
justed to a pH range o f 7 .4 -7 .6 and agar was added fo r semisolid or
solid media.
Serum-Free Defined Media.
Cox described simple serum-free media
fo r is o la tio n and c u ltiv a tio n o f "w a te r-le p to s p ire s ."
Cox's SMl and SM2
media were made as described (1 1 ).
Stock solutions were made up fo r
rapid preparation of SMl and SM2.
A IOX solution of the s a lts of SMl
was made in a l i t e r volume.
medium.
The f i r s t
Two stock solutions were made fo r SM2
IOX solution included NH^Cl, KCl, CaClg and MgSO^.
The second stock solution included the remaining s a lts in a IOOX solu­
tio n .
to 7 .4 .
SMl and SM2 were made up in one l i t e r volumes and the pH adjusted
Agar was added i f desired.
autoclaving a t 15 lb fo r 15 min.
Cox's media were s te r iliz e d by
22
C u ltiv a tio n and Observation
Leptospire cultures were incubated a t 27° C.
On some occasions
the cultures were incubated fo r a period of 3-4 days u n til macroscopic
growth could be seen.
They were then removed and maintained a t room
temperature in the dark.
Macroscopic observations were made by noting
the Dinger ring phenomenon in semi sol id media.
Liquid cultures showed
macroscopic growth in the form o f tu r b id ity and the so-called shot s ilk
appearance upon a g ita tio n o f the c u ltu re tube.
Macroscopic colonial
growth in s o lid agar appeared as spreading translucent growth descending
throughout the depth o f the agar.
D iscrete colonies were round and
usually subsurface with a p u ff b all appearance.
The type o f macroscopic
growth in s o lid media depended upon the type and concentration of agar
used.
Al I cu ltu re manipulations were done in a b acterio lo g ical hood
to prevent cultu re contamination and spread of le p to s p ires.
Al I
laboratory surfaces were d isin fected with amphyl (National Laboratories
Lehn and Fink In d u s tria l Products D iv is io n ).
Equipment
A ll glassware and f i l t e r i n g equipment were taken from general
laboratory stocks.
water and a ir d rie d .
They were machine washed, rinsed with d is t ille d
A ll acid washing was done by soaking in 10% HCl
fo r a minimum of 30 min, rinsin g s ix times with tap water and then
rin sin g with double d is t ille d water or reagent grade M illi- Q water.
23
Any equipment treated as fo r tissue c u ltu rin g was prepared by b o ilin g
in 7X (LINBRO Chemical C o., In c . ) , three rinses o f tap water were f o l ­
lowed by b o ilin g in double d is t ille d water and rinsin g in double d is ­
t i l l e d water.
D ark-Field Microscope.
Microscopic observations were made with
a Bausch and Lomb microscope with a dark f ie ld condenser.
Wet mounts
were prepared fo r observation by placing a drop of the c u ltu re on a
s lid e and covering with a #1 cover s lip .
Care was taken to prevent
any bubbles from forming because o f the lig h t s catterin g a ffe c tin g
observations.
Most o f the observations were made using the 43X objec­
t iv e , while o il emersion was used when needed.
Phase Microscope.
Occasional observations were made with a
L e itz phase microscope using a IOOX ob jective to obtain greater resolu­
tio n .
This was done on isolated cultures to help determine whether or
not they were leptospires based on morphology.
Blendon and Goldberg -
s ilv e r impregnation s ta in (5) was used with some success fo r observa­
tio n o f leptospires on a lig h t f ie ld microscope.
Dark f ie ld microscopy
was the preferred method fo r observing le p to sp ira l growth.
B acterial Counting Chamber.
A Petroff-Hausser b a cterial count­
ing chamber was used to obtain d ire c t c e ll counts.
This chamber had
been used by other le p to s p ira l researchers and was chosen because i t
could be used with dark f ie ld applications due to the th in glass and
chamber depth o f 0.2 mm.
At le a s t 128 squares were counted, and on
24
occasion, duplicate enumerations were made..
Spectrophotometry.
A Varian Techtron model 635 spectrophotom­
e te r set a t 420 nm with a s l i t width o f 0.2 nm was used fo r a ll spectro­
photometry.
Coleman c e lls
with a 10 nm lig h t path and a volume o f I ml
were used fo r small sample sizes.
Nephelometry.
Nephelometry was done with a Turner Designs
Nephelometer with a 4 . dram v ia l sample chamber.
The Turner nephelo-
meter was adapted to a sample size of 5 ml by placing a fiv e ml v ia l
containing the sample in the Turner v ia l and f i l l i n g the Turner v ia l
with water u n til the surface o f the 5 ml v ia l was covered.
An adaptor
was made from a 50 ml sorvall centrifuge tube to hold 25 x 200 mm te s t
tubes fo r measuring tu r b id ity in continuous c u ltu re .
Swinnex M odification I .
A miI l i pore swinnex 13 mm membrane
f i l t e r holder (Figure I ) was cut on the needle side leaving a hole in
which an 8 x 29 mm fermentation tube would f i t snugly.
The swinnex was
then unscrewed and a 0.22 pm pore size membrane (M illip o r e ) placed over
the top o f the fermentation tube.
autoclaved a t 15 lb. fo r 15 min.
The swinnex was then reassembled and
A fte r the assembly cooled, i t was
reopened and semi sol id Teptospiral medium was placed in the fermenta­
tio n tube.
The swinnex was then reassembled and the syringe side was
inoculated with 0.1 ml o f a sample.
The swinnex assembly was then
incubated a t 27° C and was checked d a ily fo r growth o f le p to s p ires.
Swinnex M odification 2.
A 25 mm M illip o re Swinnex membrane
25
Figure I . Swinnex M odification I Used fo r Is o la tio n Attempts and
P u rific a tio n With an Unaltered Swinnex (R ight) fo r Comparison
26
holder was taken apart and the membrane stage removed.
bored through the needle side to f i t snugly
tube.
around a 10 x 40 mm cultu re
The edge o f the tube was placed even with the membrane s h e lf and
the swinnex reassembled.
min.
A hole was then
This u n it was then autoclaved a t 15 lb fo r 15
A fte r cooling, semisolid lepto sp ira medium was placed in the tube
f i l l i n g to the top.
A 0.22 ym pore size membrane f i l t e r (M illip o re )
was then placed on the top o f the f i l l e d tube and the swinnex screwed
back together.
The needle side was then inoculated with a I ml sample.
T)he u n it was incubated a t 27° C and observed d a ily fo r growth o f Ie p to s p ires.
^
Polycarbonate C e ll.
A 25 mm polycarbonate c e ll
(Figure 2)
(MiIlip o r e Cat #XX42 025 10) was f it t e d with a 25 mm swinnex membrane
f i l t e r holder (M illip o r e ) .
swinnex was removed.
The bottom o f the needle side o f the
A 0.22 ym pore size membrane f i l t e r was set in
place a t the needle side and the u n it assembled.
The syringe side
was plugged with a p la s tic syringe t ip which had been cut o f f and
f i l l e d with p la s tic cement.
a t 15 lb .
This u n it was then autoclaved fo r 15 min
A fte r cooling the c e ll was f i l l e d with semisolid Tepto-
s p iral medium through the syringe side o f the swinnex.
A board was
d r ille d with 5 /1 6 -inch holes to hold the u n it. The plugged syringe
"
t
side was inserted in the board. A 10 ml sample was then placed in the
s k ir t above the membrane.
The u n it was then incubated a t 27° C and
checked d a ily fo r macroscopic growth in the semisolid medium.
V
27
Figure 2. Polycarbonate Cell Used fo r Is o la tio n Attempts and
P u rific a tio n
28
Is o la tio n V ia l.
le p to sp ira l studies.
the cap.
A four dram v ia l (Figure 3) was used fo r
A 1 /2-in ch hole was d r ille d through the center o f
Polycarbonate tubing 5 cm long o f I / 2 - inch outside diameter
was inserted in the hole and brought flush with the top side of the
cap.
Epoxy (Duro) was used to cement the tube in place.
The end of
the tube was smoothed by sanding with #100 sand paper and a 13 mm 0.22
ym pore size membrane f i l t e r glued in place, with MF cement (M illip o re )
The cap w ith the tube in place was then screwed back on the v ia l and
autoclaved a t 15 lb fo r 15 mins.
A fte r cooling, semi sol id le p to sp iral
medium was placed in the v ia l u n til i t touched the membrane f i l t e r on
the end o f the tube.
Two ml water samples were then placed in the
polycarbonate tubing through the hole in the outside.
The water was
evaporated by blowing a ir across a lamp and then across the v ia ls .
This procedure allowed fa s t evaporation while m aintaining the v ia ls
a t 27° C.
A fte r evaporation to about 1 / 3 . the o rig in a l volume, the
v ia ls were incubated a t 27° C u n til macroscopic growth was observed.
At th is tim e, the cap was replaced with an unaltered cap arid micro­
scopic observations made o f the medium.
Isolates were then maintained
in these v ia ls .
D iffusion Chambers.
Chambers with membrane f i l t e r side walls
as described by McFeters and S tuart (54) were used in some o f the sur­
vival studies.
Double membrane layers were used on each side of the
chambers with te a r^ re s is ta n t microweb membranes o f 0.45 ympore size
29
Figure 3. Is o la to r Vial Used fo r Is o la tio n Attempts and P u rific a tio n .
A 0.22 yin pore size membrane is glued to the bottom edge of the in t e r ­
tube (c e n te r).
30
(HAWP 304FO, M illip o re Corp.) on the outside' and 0.1 ym pore size
membranes on the inside (E442A6, Mi 111 pore).
These membranes were
obtained in 12-inch by 12 - inch sheets and then cut in to c irc u la r.d is k s
o f 7.5 or 11.5 cm diameter depending on the size o f chamber used.
The
chambers were assembled with the membranes sealed in place with stop­
cock grease (Dow Corning).
They were then autoclaved fo r 15 min a t 15
lb , with the sample ports l e f t open.
The autoclave was allowed to cool
without exhausting to prevent membrane rupture from expansion.
A fte r
cooling to room temperature s t e r ile plugs were placed in the sample
ports and the chamber bolts were tightened.
The s t e r ile chamber was
then stored u n til needed.
Is o la tio n
F iltr a tio n .
One l i t e r water samples were taken from ir r ig a tio n
systems, natural streams and c a ttle watering ponds.
These samples were
then f ilt e r e d through Whatman #1 paper f i l t e r s to remove the la rg er
p a rtic u la te m atter.
The samples were then f ilt e r e d through 0.45 ym
pore size membranes ( M illip o r e ) , and one ml aliquots of the f i l t r a t e
placed in 3 tube re p lic a te s of semi sol id le p to sp ira l medium.
The tubes
were then incubated a t 27° C and observed d a ily fo r le p to s p ira l growth.
Some o f these samples were spun a t 5,000 r.p.m . fo r 30 minutes and most
o f the water poured o f f .
The remaining water sample was then remixed
and f ilt e r e d through a 0.45 ym pore size membrane and the supernatant
treated as above.
!
31
Ten ml water samples were forced through 0.45 and 0.22 ym
membranes (M illip o re ) with 25 mm swinnex membrane holders ( M illip o r e ) .
Two fiv e ml washes o f EM medium were passed through these same mem­
branes.
The sample f i l t r a t e was placed in three re p lic a te tubes in
I ml amounts.
A ll the tubes, including the fiv e ml washes, were
incubated a t 27° C and checked p e rio d ic a lly fo r le p to s p ira l growth
and contamination.
This procedure was s im ila r to those described by
Braun e t a l . ( 6 ) , Rittenberg e t a l . (63) and Tripathy e t a l . (2 , 7 9 ).
Is o la tio n by Natural M o t il it y .
S te r ile 0.22 ym membranes were
placed on agar plates containing EM and EMJH media.
S te r ile rubber
washers were then sealed to the membranes with s t e r ile stopcock grease.
Ten ml water samples were then placed in the center o f the ring s.
The
plates were incubated a t 27° C u n til the water sample had evaporated.
The rings and membranes were then removed and the plates inverted and
incubated a t 27° C.
Checks were made a t weekly in te rv a ls fo r le p to ­
s p iral growth in the agar.
This method was patterned a f t e r Smibert's
decontamination method (7 1 ).
The method o f Cox was used to attempt is o la tio n o f leptospires
(T l).
Samples were collected in one l i t e r volumes.
The water was
then f ilt e r e d seq u en tially through Whatman #1 paper f i l t e r s and 0.45 ym
pore size membranes ( M illip o r e ) .
A drop of the f i l t r a t e was then
placed on the center o f the plate and observations made fo r le p to sp ira l
growth a t the periphery of the b a c te ria l growth a fte r incubation
32
a t 27° C.
Is o la tio n s were attempted through the use o f the swinnex modi­
fic a tio n s , the polycarbonate c e lls and the 4 dram is o la to r v ia ls
described under equipment.
Is o la tio n s were attempted by placing a
water sample on the upper surface of the membrane and watching fo r the
appearance o f growth in the semi sol id medium underneath the membrane,
a fte r incubation a t 27° C.
In some cases, the polycarbonate c e lls and
the is o la to r v ia ls were placed in a ir blown over a lamp to evaporate
some o f the sample volume.
P u rific a tio n
M o t ilit y .
Smibert plates were used as described (7 1 ).
medium was e ith e r EM or EMJH.
The
Membrane f i l t e r s o f 0.22 urn pore size
were placed on the agar surface and a rubber washer sealed in place.
The sample was then placed in the washer and incubated a t 27° C.
A fte r
a period o f 3-4 days, the washer and membrane were removed arid the agar
examined fo r le p to s p ira l growth and contamination.
Agar w ells were punched with a #4 cork bore (5 mm diam eter).
A 0.25 ml a liq u o t o f a contaminated le p to sp ira l c u ltu re was then placed
in the w e ll.
The p e tri plates were then sealed and incubated a t 27° .0.
They were then checked d a ily .
Leptospiral growth was seen as trans­
lucent growth through the depth of the agar moving away from the s ite
of in o cu latio n .
Agar plugs were removed a t the leading edge of Tepto-
s p ira l growth and placed in semisolid EM or EMJH media.
The semisolid
33
cultures were then allowed to grow up and were checked under a darkf ie ld microscope fo r m otile leptospires and contamination.
P u rific a tio n was also accomplished using the swinnex m odifica­
tio n s , the polycarbonate c e lls and the is o la to r v ia ls .
They were
f i l l e d with semisolid le p to sp ira l medium and f it t e d with 0.22 ytn pore
size membranes ( M illip o r e ) .
Contaminated le p to sp ira l cultures were
then placed above the membrane.
A fte r incubation, they were checked
fo r macroscopic growth beneath the membrane in the semisolid medium.
Uni-Pore polycarbonate membranes (Bio-Rad Ca. #313-5209) were
used on some o f the is o la to r v ia ls .
o f . 0.2 ym.
They were glued on the smoothed end of the polycarbonate .
tube with MF cement ( M illip o r e ) .
autoclaved.
They had a pore size of a maximum
The is o la to r was reassembled and
A fte r cooling, the is o la to r was f i l l e d with semisolid agar
and a contaminated cu ltu re placed on the upper surface of the membrane.
They were then incubated a t 27° C and observations made fo r growth
beneath the membrane.
Combinations o f double membranes were also used,
including 0.22 ym (M illip o re ) and 0.2 ym (Bio-Rad), 0.45 ym (M illip o re )
and 0.2 ym (B io-Rad), and 0.45 ym (M illip o r e ) and 0.22 ym ( M illip o r e ) .
In a ll cases the la rg e r pore size membrane was glued in place f i r s t and
the sm aller pore size la s t .
The is o la to r v ia ls were then treated as
before and observations were made fo r growth upon inoculation and
incubation.
34
Use o f Drugs
Drugs tested and used in these studies are shown in Table 2.
A ll the drugs, on paper disks were Sensi-Disc (BBL, CockeysvilTe, MD).
Neomycin was an ICN product (ICN L ife Sciences Group, Cleveland, OH).
Furazolidone was obtained from Eaton Laboratories (Eaton Laboratories,
Division Morton-Norwich Products, I n c . ,
Norwich, NY).
The 5-f.luoro-
u racil was from Roche (Roche Laboratories, N utley, Nd).
Based on previous lit e r a t u r e the drugs neomycin (5 7 ), 5 - f luoro-.
u racil
(4 6 ), furazolidone (5 6 ), and combinations of these (10, 56) were
added to le p to s p ira l media to help reduce contamination o f le p to sp iral
cultures as an aid in is o la tio n .
were made up.
Stock solutions of these three drugs
A stock solution of neomycin s u lfa te was made by adding
151.5 mg of a 660 yg/mg assay to 500 ml o f double d is t ille d water,
giving a fin a l concentration o f 200 yg/m l.
This stock solution was
then s t e r iliz e d through a 0.22 ym pore size membrane.
A stock solution
of furazolidone was made by adding 200 mg Furoxone to one l i t e r of
double d is t ille d water giving a fin a l concentration o f 200 yg/m l.
This
furazolidone stock solution was s te r iliz e d by autoclaving a t 15 lb. fo r
15 min.
A stock solution o f 5 -flu o ro u ra c il was made by d ilu tin g a 10 ml
volume containing 500 mg with 90 ml o f s t e r ile double d is t ille d water
giving a fin a l concentration of 5 mg/ml.
s t e r ile .
This solution was considered
Neomycin and furazolidone were used a t a concentration o f 5
yg/ml in c u ltu re media. . Fluorouracil was used a t a concentration of
35
Table 2.
Drugs Used And Their A c tiv ity
t
Drug
Abv.
Cone.
A c tiv ity
N a lid ix ic acid
NA
30 ug/disk
Potent in h ib ito r o f DNA
synthesis
Streptomycin
S
10 ug/disk
In h ib itio n o f polypeptide
synthesis and misreading of
the genetic message
Kanamycin
K
'30 yg/disk
Same as streptomycin
Neomycin
N
200 ug/ml
stock
Same as streptomycin
T h io s u lfil
TH
I .0 mg/disk
Sulfonamides bactero static
acts as an a ritilo g to
paraaminobenzoic acid
T e tra cy c lines
Te
30 ug/disk
B a c te rio s ta tic e ffe c tiv e .
in h ib ito rs o f phosphorylation
and o f protein synthesis
C b lis tin
Cl
10 ug/disk
Disrupt the osmotic properties
of membranes ric h in phos­
phatidyl ethanol ami ne
N itro fu ra n to in
F/M
100. ug/disk
B a c te rio s ta tic and b a c te ricid a l
mechanism not known
Furazolidone
F
200 ug/ml
Same as n itro fu ra n to in
5-Fluorouracil
Fu
5 mg/ml
Acts as an analog of uracil
a ffe c ts nucleic acid synthesis
36
100 M g /m l.
Further studies on drug e ffic a c y in c o n tro llin g contamination
and in is o la tio n o f leptospires were necessary.
is o la te la b e lle d Al was used in th is study.
A contaminated water
Fourteen screw cap tubes
containing 5 ml o f EM semi sol id medium were inoculated with 0.5 ml
o f a contaminated Al c u ltu re .
Drugs were added to the tubes with two
tubes without drugs fo r c o n tro l.
A n itro fu ra n to in paper disk with a
concentration o f 100 ug was added to tube qne.
A sulfam ethizole paper
disk with a concentration o f 1.0 mg was added to tube two.
A c o lis tin
paper disk with a concentration of 10 yg was added to tube three.
N a lid ix ic acid paper disk with a concentration o f 30 yg was added to
tube fo u r.
A te tra c y c lin e paper disk with a concentration of 30 yg was
added to tube f iv e .
A streptomycin paper disk with a concentration of
10 yg was added to tube s ix .
A kanamycin paper disk with a concentra­
tio n o f 30 yg was. added to tube number seven.
to a concentration o f 10 yg/ml furazolidone.
Tube e ig h t was brought
Furazolidone and neomycin
were added to tube nine with a concentration of 5 yg/ml per drug.
Tube
ten contained. 100 yg/ml 5 -flu o ro u r a c il, and 5 yg/ml neomycin and
furazolidone.
10
yg/m l.
Neomycin was added to tube eleven to a concentration of
Tube twelve contained 200 yg/ml 5 -flu o ro u ra c il.
Tubes
th irte e n and fourteen contained EM medium with no drugs added. ■
Drug P la te s .
Solid agar plates were prepared by adding. 7 ml of
EM medium to 47 mm p la s tic p e tri plates ( M illip o r e ) .
Various drugs and
37
le p to sp ira l cultures were then placed on the agar medium as diagrammed
in Figures 4 and 5.
Audries1 River is o la te was streaked on a quarter
of p late I .
A trough was cut in the center of the agar p a ra lle l to
the streak.
This trough was f i l l e d with 0.1 ml o f stock 5 -flu o r o u r a c il.
An F/M disk was then placed on the surface o f the agar opposite the
streak.
Is o la te IR Mun Son was inoculated into a trough on a quarter
o f p late 2.
On the opposite quarter F/M and NA disks were placed.
Plate 3 was inoculated in four slots with Al is o la te .
A disk of TH
was placed a t the narrow end and a disk of F/M was placed a t the large
end o f the s lo ts .
center of p late 4.
Is o la te AS was inoculated into two slots across the
On one side of the slots a K disk was placed with
an S disk on the other side.
inoculated with A2 is o la te .
Plate 5 had two slots through the center
A s lo t on the outside running p a ra lle l to
the other two slots was f i l l e d with 0.1 ml o f stock furazolidone.
Another s lo t on the opposite side of the p late running p a ra lle l was
f i l l e d with 0.1 ml o f stock 5 -flu o ro u r a c il.
inoculated in a s lo t in the agar of p la te 6,
Kakuchska is o la te was
On the opposite side of
the p late a well was f i l l e d with 0.1 ml o f stock 5 -flo u ro u ra c i.l.
Two
slots running across the center o f p la te 7 were inoculated with is o la te
F.
A kanamycin disk was placed on one side of the slots and a Cl disk
placed on the other side.
8.
Is o la te C was inoculated in to slots on p late
On one side of the slots an F/M disk was placed;on the other side
a TH disk was placed.
Is o la te A l2 was inoculated in a s lo t on the edge
38
Pl at e
2
Al
I sol at e
Plate
Pl at e 4
3
KaKuchsko
Isolate
------v.
well
Fu
AZ
Pl at e
5
Isolate
Plate
6
Figure 4. Drug Plate Configurations 1-6 fo r Study o f Drug Efficacy in
P u rific a tio n . S tip lin g indicates surface streaking o f the mixed
c u ltu re . The s tra ig h t lin e s in d icate s lit s in the agar medium. The
small c irc le s represent paper drug disks.
39
,Isolate F
Isolate C
Plate
7
L i oomono
Plate Il
Plate 8
L oomona
Plate 12
Figure 5. Drug Plate Configurations 7-12 fo r Study of Drug E fficacy in
P u rific a tio n . S tio lin g indicates surface streaking of the mixed
c u ltu re . The s tra ig h t lin e s in dicate s lit s in the agar medium. The
small c irc le s represent paper drug disks or wells as in Plate 12.
40
of p late 9.
Three slots running a t angles to each other but converging
a t a d ire c tio n toward the inoculated s lo t were re s p e c tfu lly f i l l e d with
0.1 ml of stock 5 - f l u o ro u ra c iI, furazolidone and neomycin.
Plate 10 had
one center s lo t inoculated with L. pomona and slots on e ith e r side
f i l l e d with 0.1 ml of stock neomycin and furazolidone.
Plate 11 had a
single s lo t across the center f i l l e d with 0.1 ml o f stock furazolidone
and 5 - f l uorouraci,I .
P late 12 contained a center Well inoculated with
L. pomona and three wells spaced evenly around th e ,c e n te r well with 0.1
ml of furazolidone and neomycin in two and a disk of Cl in the other.
These plates were incubated a t 27° C and examined both macroscopicalIy
and m icroscopically fo r le p to s p ira l growth and contamination.
Agar
plugs o f apparently pure le p to s p ira l growth were removed and placed in
tubes o f semisolid EM medium.
These tubes were then incubated and
checked fo r le p to s p ira l growth and contamination.
Streak P la te s .
Streak plates using EM s o lid agar medium in
glass p e tri plates were used to pick isolated le p to s p ira l colonies.
The le p to s p ira l c u ltu re was streaked on s o lid EM medium of s u ffic ie n t
thickness to allow fo r extended incubation.
The plates were then
sealed, inverted and placed in a 27° C incubator.
checked d a ily fo r growth.
They were then
When macroscopic growth was seen, a repre­
sentative colony was observed under d a rk -fie ld and i f i t appeared pure,
the colony was tran sferred to semisolid EM medium.
41
Enumeration
T u rb id ity and D ire ct Count.
Spectrophotometry and nepheVometry
were used to measure le p to sp ira l concentration.
T u rb id ity measure­
ments were compared to d ire c t count done with a Petroff-Hausser
b a c te rial counting chamber.
Serotypinq
Serology was performed on water isolates which were grown to
high densities in liq u id EM medium.
Each is o la te was then added in
0.05 ml amounts to 0.05 ml o f a 1:25 d ilu tio n o f each o f the sera in a
p la s tic -w e lle d serology p la te .
This 1:50 sera d ilu tio n was incubated
a t room temperature fo r I -1 /2 hours.
A fte r incubation low power dark-
f ie ld microscopy was used fo r examination.
The is o la te s Ag22, A l, A2,
AS, Ag23, A ll, Ag21 and Kakuchska were tested with a n tis e ra .
The
iso lates were tested against e ig h t sera s p e c ific against L. canicola,
L. pomonaj L. ictevohaemorvhagiae3 L. bdllum} L. pyvogenes3 L. grippotyphosa, L. bataviae and L. hardjo.
These sera represent eight
d iffe r e n t serogroups with the f i r s t seven being synonymous with the
serogroup and L. Iiardjo representing the Habdomadis serogroup.
An
e a r lie r t r i a l had tested is o la te C against sera s p e c ific fo r L. oanicola3 L. pomona, L. -Icterohaemorrhagiae3 L. grippotyphosa and L.
hardjo.
Enumeration by MPN o f Natural Water Systems.
is o la to r v ia ls were f i l l e d with semi sol id EM medium.
F o rty-eig h t
Fluorouracil was
42
added to a concentration o f 100 ug/m l.
to four s ite s .
These is o la to rs were then taken
A most probable number using three tubes and four
d ilu tio n s was made a t each s it e .
to each o f three is o la to rs .
Two ml of undilute
water was added
A 1:5 d ilu tio n was then made by adding
2 ml o f water to 8 ml o f phosphate b u ffe r.
Another 2 ml was added to
three more is o la to rs from the 1:5 d ilu tio n .
This was continued u n til
a fin a l d ilu tio n of I C f ^
had been made.
A fte r in oculating the MPN a t
each s it e , pH and temperature were taken.
The is o la to r v ia ls were then
placed in a 27° C incubator and observed d a ily fo r le p to s p ira l growth.
S ite A was a t Hood Creek, a high mountain stream.
S ite B was a t Buck­
skin Creek, a mountain drainage used fo r summer c a ttle range.
S ite C
was in the open G a lla tin V alley in the H ya lite Creek ir r ig a tio n system.
S ite D was also in the G a lla tin V alley along Mystic Creek in a combina­
tio n subu rb /agricultu ral area.
The MPN was done in e a rly September.
An MF fecal coliform enumeration was also done a t each s ite (2 ).
Survival Studies
Well Water Chamber Runs.
grown up in liq u id EMJH medium.
Leptospira hardjo and L. pomona were
They were then centrifuged a t 5,000
rpm fo r 30 thin and washed in autoclaved well water.
They were then
suspended in well water to fin a l concentrations of 8.72 x IO^ L. hardjo
per ml and 7.48 x IO7 L. pomona per ml as counted on a Petroff-Hausser
bacteria counting chamber.
Two s t e r ile membrane d iffu s io n chambers
were then f i l l e d with 20 ml of the respective le p to s p ira l suspensions.
I
43
The chambers were then placed in a 40 l i t e r overflow tank f i l l e d with
unchlorinated well water.
Fresh well water was co n tin u a lly added a t a
rate o f 5 li t e r s per minute.
The experimental apparatus and well water
were the same as described by McFeters e t a l . (5 3 ).
At time zero and
th e re a fte r a t 2-day in te rv a ls , the chambers were sampled.
P e tro ff-
Hausser counts and absorbance readings a t 420 nm were made.
Laboratory Survival Studies.
r e fr ig e ra to r set a t 11° C.
Survival studies were done in a
A p la s tic bucket was f i l l e d with stream
water and placed in the r e fr ig e ra to r .
Aeration was provided through
two spargers supplied with a ir from an aquarium pump.
S te r ile membrane
d iffu s io n chambers were f i l l e d with washed suspensions of L. pomona and
L.
patoc and then placed in the bucket.
been grown in EMdH liq u id medium.
The le p to sp ira l cultures had
They were spun a t 5,000 rpm fo r 30
min and washed free o f medium with s t e r ile water from a small stream.
The chambers were sampled d a ily or a t two-day in te rv a ls and enumera­
tions made with spectrophotometry,
nephelometry and d ire c t c e ll count.
Chamber D iffusion Experiments.
A d iffu s io n chamber with 0.1 ym
and 0.45 ym pore size membranes in place as described in equipment was
used in these d iffu s io n experiments.
The chamber was f i l l e d with 20 ml
of d is t ille d water and suspended in 2 li t e r s of water from Keg Creek.
The suspending water had a conductivity of 475 ymhos as measured on a
Lab Line Lectro MHO meter.
F ifty yg o f dextran 500/ml (molecular weight
500,000) had been added to the 2 l i t e r s of suspending water.
The to ta l
44
carbon in the suspending water was then 87.5 yg/mi as measured on a
Beckman Laboratory
ml as glucose.
Carbonaceous Analyzer c a lib ra te d with 100 yg carbon/
At 15 min in te rv a ls samples were taken from the chamber
and analyzed fo r carbon and cond uctivity.
A separate experiment was done on glucose d iffu s io n .
To 2
li t e r s of d is t ille d w ater, glucose was added to a fin a l concentration of
0.2 mg/ml.
A chamber was then suspended in the glucose water as in
previous experiment.
The glucose concentration was determined by the
Glucostat, glucose-oxidase peroxidase system made by the Worthington
Biochemical Corp.
Samples were taken from the chamber every 30 min
and analyzed fo r glucose concentration.
S im ilar d iffu s io n experiments
had previously been done by. McFeters and S tuart (5 4 ).
.CHAPTER 4
RESULTS
Media
Various media were used in these stu d ies.
They were tested
f i r s t fo r th e ir e ffic a c y then fo r ease o f preparation and s t e r i liz a ­
tio n .
Some consideration was given to cost and a v a ila b ilit y of
in g red ie n ts .
Ellinghausen and McCullough (EM) medium and the Johnson and
Harris (EMJH) m odification thereof were used in most o f the studies.
These media were the most dependable and consistently high le p to s p ira l densities could be obtained.
medium is c le a r and c o lo rle ss .
Ellinghausen and McCullough
The Johnson and H arris m odification
is also c le a r, but i t takes on a yellowish cast.
T u rb id ity can be
measured by nephelometry and to a lesser extent by spectrophotometry
e s p e c ia lly in the case o f the colorless EM medium.
A p re c ip ita n t
formed in EM medium when i t was prepared as described by Ellinghausen
and McCullough (2 5 ).
The p re c ip ita tio n problem was elim inated when
the m odification as described in M aterials and Methods was employed.
This preparation m odification did not appear to change the e ffic a c y
o f the medium.
The major drawbacks to EM and EMJH media are the length
of time required fo r preparation and the f i l t e r s t e r iliz a t io n re q u ire ­
ment o f the albumin po rtion.
F i lt e r s te r iliz a t io n was done using
M illip o re S t e r i f i l l Aseptic systems with 0.22 ym pore size membrane
46
filte r s
( M i l l ipore Cat #GSWP 04700).
I t was found th a t the f i l t e r
s te r iliz e d portions o f the media often suffered contamination which was
reduced by double f i l t r a t i o n through two sets o f 0.22 ym membranes.
The use o f autoclaved water in media preparation also reduced contami­
nation.
F i lt e r s t e r iliz a t io n and media manipulations were performed in
a b a c te rio lo g ic a l hood fu rth e r reducing contamination problems.
The minimal essential media (MEM-BOH) described by Finn and
Jones (45) did not perform as well as hoped.
I n i t i a l growth and
appearance o f the Dinger ring was noted in semi sol id media.
Upon f u r ­
ther incubation the Dinger rin g generally became less d is tin c t or
disappeared a lto g e th e r.
M otile leptospires could be observed upon
d a rk -fie ld observation, however the high densities observed in EM and
EMJH media were never obtained.
The other minimal essential media
(MEM-BT) described in the media section of M aterials and Methods
yielded le p to s p ira l growth and la s tin g Dinger rings in semisolid form.
The serum-containing media both worked w e ll.
Leptospira pomona,
L. patoo and L. hardjo were maintained on these m e d ia .. Due to d i f f i ­
c u ltie s in obtaining serum and s t e r iliz a t io n problems, the serumcontaining media were o f lim ite d use.
Cox's serum-free media had the advantage o f s t e r iliz a t io n by
autoclaving.
High densities o f leptospires could be grown using SMl
and SM2.. These media deterio rated or were depleted ra p id ly leaving a
cu ltu re with c e llu la r debris and weakly m otile c e lls .
Dinger rings
47
formed quite ra p id ly with L. pomona3 L. patoc and L. hardjo in semi­
solid Cox's media, but became less d is tin c t and often disappeared upon
fu rth e r incubation.
Often the Dinger ring descended through the
medium u n til i t reached the bottom o f the c u ltu ra l vessel where i t
dispersed.
M otile leptospires could s t i l l be.observed throughout the
medium upon d a rk -fie ld examination.
Some leptospires were isolated
using SMl semi sol id medium. (See Figure 6 .)
Is o la tio n
Is o la tio n attempts were unsuccessful using the f i l t r a t i o n
techniques as well as Sm ibert1s and Cox's methods as seen in Table 3.
A to ta l o f 129 samples were treated by one or more o f these methods.
A number o f s p ira l organisms were is o la te d , but no le p to s p ira l p o sitive
is o la tio n s were noted.
The swinnex m odifications and the polycarbonate
c e lls were also unsuccessful in showing le p to sp iral is o la te s from the
natural systems.
Water samples spiked with cultures o f L. pomona and
L. patoo were used with the swinnex m odifications and the polycarbonate
c e lls .
Recovery o f leptospires from these spiked water samples was
possible although less than 50% o f the t r ia ls proved successful.
As seen in Table 4 , the use o f the is o la to r v ia ls resulted in an
overall is o la tio n ra te o f 70% from 40 samples with an increasing is o la ­
tio n success in the la t e r is o la tio n attempts.
Increased is o la tio n
success was associated with the use o f 5 -flu o ro u ra c il and EM semisolid
medium.
A 100 yg/ml concentration o f 5 -flu o ro u ra c il reduced background
48
Figure 6. Culture Tubes of EM Semisolid Medium Showing the Dinger Ring
Growth Phenomenon. The tube on the l e f t is Leptospira patoo, a
saprophyte. The center tube contains water isolated s p ira l organisms
which develop growth s im ila r to the le p to sn ira l Dinger ring s. The tube
on the rig h t is Leptospira pomona, a pathogen.
Table 3. Results of Is o la tio n Attempts From Natural Water and Recovery of Leptospires From
Water Samples Spiked With L. pomona, using various methods
Methods
Natural Water
Samples Tested
'■ #
Leptospires Recovered
From Natural Water
%
Spiked Water3
Samples Tested
# '
Spike
Recovery
%
F iltr a t io n
1C
2H
3d
24
. 5
15:
0
0
0
5
3
10
0
0
10
Smibert p late
15
0
15
45
Cox p late
20
0
17
25
Polycarbonate
c e ll
20
0
24
45
Zf
15
15
0
0
8
4
10
. 24
Is o la to r v ia l
40
70
50
100
Swinnex
I!
a
'5
^Spiked water samples gave a concentration o f a minimum o f 10 L. pomona per ml.
0One l i t e r sample volumes f ilt e r e d through seq u en tially decreasing pore size membranes.
jSame as b with concentration done by c e n trifu g a tio n f i r s t .
"Pressure f i l t r a t i o n o f 10 ml sample.
f
eSwinnex m odification I .
Swmnex m odification 2
50
Table 4. Results o f 40 Is o la tio n Attempts Using the Is o la to r Vial And
Some o f the Water Conditions At The Sample Sites
Cond?
umohos
Temp*?
0C
Al k .c
Meq/1
Lepto.
Recovered
18
20
27
15
10
14
2.32
2.32
nd
nd
2.32
nd
no
no
yes
no
yes
yes
no
no
no
no
no
no
nd
nd
nd
nd
340
340
340
nd
nd
184
nd
nd
184
184
15
15
20
20
15
17
22
23
20
15
22
27
17
. 17
nd
nd
nd
. nd
3.7
3.7
3.7
nd
nd
2.32
nd
nd
2.32
2.32
yes
yes
no
yes
yes
no
no
yes
no
no
yes
yes
no
yes
no
no
no
no
no
no
no
no
no
no
no
no
no
no
5 /p
5/p
1/P
1/P
6m/s
6m/s
6m/p
8/p
8/p
2/P
3/p .
■ 3/p
2/P
2/P
EM
EM
EM
EM
354
186
382
537
nd
nd
nd
nd
2.57
1.54
2.38
4.24
yes
yes
yes
yes
yes
yes
no
no
6v/p
6v/p
6v/p
6v/s
8 /5
8/5
8/5
8/5
EM
EM
EM
EM
270
228
112
nd
6 • 3q
3.49
3 .7 9
10
2.93
2.45
1 .04
nd
yes
yes
no
no
yes
yes
no
no
6m/s
6m/s ■
1/P
9
4/25
6/16
EM
EM
184
nd
17
19
2.32
nd
yes
yes
no
yes
2/P
6v/p
6/16
EM
552
16
3.21
yes
yes
7/P
6/16
EM
nd
22
nd
yes
yes
4/p
Sample
Date
Media
A
B
C
D
E
F
4/22
4/22
4/22
4/22
4/22
4/22
EMe
EMJH
SM I
SM I
EM
EMJH
184
184.
ndf
nd
184
nd
Al
A2
AS
A4
AS
A6
A7
AS
A9
AlO
All
Al 2
AlS
Al 4
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
5/18
SM
SM
SM
SM
SM
SM
SM
SM
SM
SM
SM
SM
SM
SM
Ag21
Ag22
Ag23
Ag28
7/16
7/16
7/16
7/16
History
Logging
Langahor
Fountain
Keg
Audries
TRMunson
Kakuch/
ska
I
I
I
I
I
I
I
I
I
I
I
I
I
I
.
Drugc*
S ite *
Used Description
2/p
. 2/p
3/p
4/p
2/P
4/p
51
Sample
Date
Cond.a
Media umohos
8/15
8/15
8/15
8/15
EM
EM
EM
EM
271
271
271
271
13
13
13
13
3.05
3.05
3.05
3.05
yes
yes
yes
yes
9/9
9/9
9/9
9/9
EM
EM
EM
EM
184
271
377
78
13
11
12
5
2.32
3.05
4.02
.62
yes
yes
yes
no
Black
Mystic
Buck.
Hood
I
2
3
4
*S ite description:
I
2
3
4
5
6
r—
Bozeman
Bozeman
Bozeman
Bozeman
Temp*?
0C
U
Continued.
"cT
■5 i : i
Table 4.
Lepto.
Recovered
Springs or seeps
Ir r ig a tio n canals
Drain ditches
C a ttle watering
Ponds
Running streams
m mountain
v v a lle y
.
no
no
no
no
6v/s
6v/s
6v/s.
6v/s
yes
yes
yes
yes
2/P
6m/s
6m/p
6m/pr
7 Rivers
8 Bog
9 Drinking water
p Polluted
pr P ris tin e
s S lig h tly polluted
aConductivity
eSee Table I
^Temperature
^No data
cA lk a lin ity
^Average values
dS-Fluorouracil
Drugd
S ite *
Used Description
52
contamination, thus making i t ea sie r to make a microscopic search fo r
le p to sp ira l p o sitiv e is o la tio n s .
P u rific a tio n
The p u rific a tio n procedures which re lie d on a small membrane
pore size o f 0.22 ym or less were e ffe c tiv e in separating leptospires
from large diameter organisms such as E. ooli. and Bacillus subtilis.,
The is o la tio n s made from natural water systems contained rods, cocci
and s p ira ls capable o f passing through these small pore size membranes,
thus p u rific a tio n by single membranes was only p a r t ia lIy successful.
Bio-Rad membranes with s tra ig h t pore configuration versus the
M illip o re membranes with a compressed fib e r matrix showed very l i t t l e
d iffe re n c e fo r p u rific a tio n purposes.
Leptospires passed through the
Bio-Rad membrane fa s te r than through the M illip o re membrane.
This
c h a ra c te ris tic could possibly be used i f the membrane were removed a t a
time when only the leptospires had passed and the s p ira ls had not com­
p le te ly passed the membrane.
This was not tr ie d but i t appeared to have
p o ten tial as a method o f p u rific a tio n .
Double membrane combinations o f 0.22 ym (M illip o r e ) and 0.2 ym
(Bio-Rad) were very e ffe c tiv e in separating the leptospires from s p ira l
organisms and most o f the rods and cocci as shown in Table 5.
A fte r a
three-month incubation period, cultures of leptospires which were
■
I
recovered from th is double membrane system were free o f sp ira ls and
rods, however, very small vib rio s were seen.
These vib rio s had a diameter
53
Table 5. Comparison o f Membranes and Membrane Combinations For Use In
Is o la tio n and P u rific a tio n
Membranes
Is o la tio n
Estimated P otential
For Natural Samples
P u rific a tio n of Mixed Cultures
Passage Through Membranes
----------------------------------------------------Lepto
Spirals
Rods
Cocci
M illip o re
0.45 ym
0.22 ym
fa ir
good
4a
4
4
4
4
2
4
2
good
4
4
.2
2
P o o rjj
poor
good
good
3
3
4
4
2
0
4
4
0
0
2
2
0
0
2
2
Bio-Rad
0 .2 ym
Combinations
0.22M-0.22Mb
0.22M-0.2BC
0.45M-0.2B
0.45M-0.22M
aNo passage 0-4 free passage apparent.
^ M illip o re compressed fib e r m atrix membrane.
cBio-Rad s tra ig h t pore configuration membrane.
^Prolonged incubation necessary.
54
o f approximately 0.1 to 0.15 urn.
apparent in these c u ltu re s .
They were the only contaminants
These vib rio s could then be removed u t i l i z ­
ing the agar well plates by picking the leading edge of le p to sp ira l
growth.
An extended period o f 15 days or more incubation time was
required fo r the leptospires to pass through th is double membrane
system.
The other double membrane combinations shown in Table 5 gave
the same re su lts as the single membrane systems, thus they were not
completely successful fo r p u rific a tio n .
Agar w ells punched in agar medium and f i l l e d with contaminated
water is o la te s were e ffe c tiv e in separating the leptospires from nonmot i l e and slower moving b a c te ria .
Often several leading edges of le p to ­
s p ira l and contaminant growth could be seen moving away from the s ite
of in o cu latio n .
While the leading edge could be picked, some of the
contaminants, e s p e cia lly the s p ira l organisms, would outdistance the
le p to s p ira l growth, making th is technique un satisfactory fo r p u rific a ­
tio n u n til the s p iral organisms were removed by other means.
The resu lts o f the drug study on is o la te Al are given in Table
6.
Several drugs and drug combinations were shown to in h ib it spiral
growth.
The drugs Ts, S, K, N and the combination o f N, F and Fu
in h ib ite d the growth of these s p ir a ls , however the i n i t i a l le p to sp iral
growth was also r e s tric te d .
C o lis tin appeared not to r e s t r ic t the
growth of the leptospires while in h ib itin g the s p ira l organisms.
The
55
Table 6. The Results o f the Drug Study on the Mixed Culture Al in SemiSolid EM Medium
Leptospires
Tube
Day 3
Drug .
' Day 20
Other
Contaminants
Spirals
Day 3
Day 20
Day 3
Day 20
I
F/Ma
4b
4
4 .
0 .
4
3
2
TH
4
4
4
0
4
3
3
Cl
4
. 4
0
o
4
3
4
NA
2
4
4
+
2
+
5
Ts
2
+—
'0
0
2
4
6
S
2
4
0
0
2
4
7
K
+
4
0
0
2
4
8
2X F
4
4
4
0
4
2
9
N&F
4 .
3
+
0
+
2
10
N,F&Fu
+
3
0
0
3
2
11 .
2X N ..
3
4
0
0
+
2
12
2X Fu
2
+.
0
+-
+
0
13
Control
4
2
4
2
4
2
14
Control
4
2
4 .
2
4
2 .
15 .
Cl &Fu
16
2X N&F
17
C l&2X Fu
'
.
"
2
+-
0
+-
0
+-
2
+
0
aSee Table 2 fo r abbreviations.
bRating scheme:
4
3
2
+
+0
Too numerous to count.
300-400 organisms per f ie ld .
200-300 organisms per f ie ld .
100-200 organisms per f ie ld
less than 100 organisms per f ie ld .
None seen.
56
rods, cocci and other contaminants were very re s is ta n t to most of the
drugs, except fo r Fu which appeared to in h ib it them a t a concentration
o f 200 pg/m l.
At a concentration o f 200 #g Fu/ml, the leptospires also
were s lig h tly re s tric te d as evidenced by reduced lep to sp ire density.
Drug P la te s .
Examples o f the growth and e ffe c t o f the drugs
in these plates are shown in Figure 7.
The resu lts o f the drug
plates set up as diagrammed in Figures 4 and 5 in M aterials and Methods
are given below.
I.
Audries1 River sample grew confluently across plate
Microscopic examination showed contamination by s p ira l organisms
and some rods and c o c c i.
ahead o f the le p to sp ire s.
disk.
Plate 2 showed s p iral organisms growing out
They were seemingly a ttra c te d to the NA
A fte r a longer incubation, both s p ira ls and leptospires grew
up to and under the drug disks.
Is o la te Al as well as s p ira l organisms
grew confluently over p late 3 with no apparent in h ib it io n by the drug
disks.
Growth on p la te 4 was very slow and perpendicular to the lin e
between the drug disks, however with prolonged incubation the is o la te
grew up to the drug d isk s .
S pirals and leptospires grew confluently
on p la t e '5 with no apparent in h ib itio n by the drugs.
The Kakuchska
is o la te was a very slow growing is o la te , but i t did eventually appear
and grow across p late 6 a f t e r a confluent growth of s p ira l organisms
had been established.
Plate 7 showed growth o f s p ira ls and leptospires
up to kanomycin, but only leptospires growing up to C o lis tin .
A zone
o f s p ira l in h ib itio n approximately 10 mm in diameter was seen around
Figure 7. Examples o f Drug Plates Used For P u rific a tio n . Each plate
has a 10 yg c o lis tin drug disk on it s surface. The upper l e f t plate
shows a zone of clearin g around the drug disk and a growth fro n t
approaching the disk from the l e f t . The zone of c le a rin g represents
s p iral organism avoidance o f the drug and the advancing edge is the
slower moving le p to s p ires. The lower rig h t plate shows the lep to sp iral
growth passing under and around the drug disk. The other two plates
show le p to sp ira l growth moving away from the s ite o f in oculatio n.
58
the Cl disk.
No in h ib itio n was noted on p late 8 with the contaminants
and leptospires growing evenly over the p la te .
Plate 9 never did show
le p to s p ira l growth, however s p ira ls were found growing confluently over
the p la te .
Plates 10, 11 and 12 were very slow in growing but eventual
confluent growth was seen on a ll plates with no apparent zone of drug
in h ib itio n .
The le p to sp ira l cultures inoculated in w ells and s lit s in
the agar medium were found to appear macroscopicalIy sooner than those
streaked on the agar surface.
Often areas which were streaked never
showed le p to s p ira l growth, but the streak areas were grown, over by
le p to s p ira l growth through the agar from the s lit s and w e lls .
Streak P la te s .
some is o la te s .
Streak plates were useful fo r p u rific a tio n o f
Individual colonies were picked from the streak p late .
and then placed in a tube o f EM or EMJH semisolid media.
A fte r macro­
scopic . growth was apparent in the semi sol id medium the is o la te was
again streaked and an individual colony picked upon appearance of
colonies on the streak p la te .
This method of p u rific a tio n was lim ite d
by the general observations made by Cox (11) fo r le p to s p ira l growth on
s o lid media.
Other problems in streak plate p u rific a tio n were those
of contamination by fungi and of surface and subsurface contamination by
m otile bacteria before le p to sp ira l colonies could be observed and re ­
moved.
The s p ira l organisms were found to be the main source o f sub­
surface and surface contamination, thus, p u rific a tio n by streak plates
was generally not successful in the presence of s p ira l organisms'.
59
Electron Microscopy
Shadow cast and negative s ta in preparation o f isolate, C were
made by Dr. Ken Lee fo r use on a transmission electron microscope.
An example o f one of the is o la te C organisms with shadow casting is
shown in Figure 8.
The fig u re shows ty p ic a l le p to sp ira l morphology
with the axial filam ents ( f la g e lla ) c le a rly v is ib le .
Serotyping
A ll the is o la te s , with the exception of Ag21 were negative fo r
the eigh t sera.
Is o la te Ag21 showed agglutination with a l l the
sera as well as a negative sera c o n tro l, thus with no fu rth e r testin g
done a nonspecific agglutin ation was the possible cause fo r Ag21 agglu­
tin a tio n of a ll the sera as well as the negative c o n tro l.
Enumeration
T u rb id ity and D irect Count.
T u rb id ity measurements correlated
well with d ire c t counts made on a Petroff-Hausser b a c te ria l counting
chamber.
Figure 9 shows nephelometry vs. d ire c t c e ll count from the
data o f a survival study and Figure 10 shows absorbance.
The lin e a r
regression c o e ffic ie n t is .97 which indicates a closeness o f . f i t to a
lin e a r r e la tio n .
Lim itations are seen a t organism concentrations below
106/m l, with both the tu r b id ity and d ire c t count.
At concentration
below IO6 organisms/ml the tu r b id ity was below the s e n s itiv ity of the
nephelometer which was set with a standard of 20 nephelometry tu r b id ity
60
Figure 8. ' Electron Micrograph o f Water Is o la te C. This shadow case
preparation was done by Dr. Ken Lee and has a to ta l m agnification o f
14,225. The axial filam en t ( a f ) can be seen running along the
cytoplasmic body.
61
v
SI
it;
ii.
i
.
. .V v •
I E
l
y
\
'4■
- '
' * w
. X
f
d
.
-
'.
'
'
;
I
"
:m
62
R= 9.7
I Cr ORGANISMS / ml
DIRECT CELL COUNT
Figure 9. Comparison of Nephelometry and D irect Cell Count as Measures
of Cell Densities in a Survival Study of L. pomona
63
I Cr ORGANISMS/ ml
DIRECT CELL COUNT
Figure 10. Comparison of D irect Cell Count and Absorbance a t 420 nm as
Measures of Cell Densities in a Survival Study of L. pomona
64
u n its .
Ellinghausen also used nephelometry fo r estim ation o f le p to -
s p iral concentration.
His data likew ise show th a t concentrations of
10® organisms/ml or more were required before tu r b id ity could be
measured (2 3 ,2 7 ).
Larson e t a l . and Ellinghausen e t a l . used the
Petroff-Hausser b a c te rial counting chambers to do d ire c t counts.
They
found th a t the chambers were only useful above 10® organisms/ml which
amount to I organisms per counting square (25, 5 1 ).
Th erefore, other
methods o f enumeration must be u t iliz e d fo r organsm concentration below
10®/ml.
Chamber D iffusion Experiments
The d iffu s io n o f glucose, inorganic ions and to ta l carbon
through the double la y e r membranes o f 0.45 ym and 0.1 urn pore size
was s im ila r to th a t shown by McFeters and Stuart with th e ir single
membrane o f 0.45 ym pore s iz e .
Figure 11 and 12 show the. d iffu s io n
rates obtained with the double membranes as compared to the d iffu s io n
rates obtained by McFeters and S tuart (5 4 ).
Survival
The le p to s p ira l response o f exposure to well water and to a
laboratory water environment are shown in Figure 13.
These
survival
curves based on tu r b id ity and d ire c t c e ll counts in d ic a te a minimum
survival time o f 8 days.
I t was f e l t th a t the double membrane system
provided a reasonable exposure to the suspending f lu id w hile containing
Z
65
0 .4 5 p m
TOTAL
O l p m - 0. 45pm
CARBON (pg/m l)
0.1 (irn - 0 . 4 5 pm
HOURS
Figure 11. Comparative Diffussion of Carbon-Containing Compounds Across
a 0.45 pm Membrane (--------------) , a D ialysis Tubing Sac ( - ------ ) (54) and a
0 .1 -0 .4 5 pm Double Membrane Layer ( ------- ) .
Ion d iffu s io n across a 0.1 0.45 pm double membrane layer (-------------- ) as measured by conductivity.
66
0.1 pm - 0 .4 5 pm
HOURS
Figure 12. Comparison o f Glucose D iffusion Across a 0.45 urn Membrane
(54) and a 0 .1 -0 .4 5 pm Double Membrane Layer
67
DAYS
Figure 13. Survival of L. pomona and L. hardjo in Continuous Flowing
Well Water3 and a Laboratory Water Environment^ as Measured by D irect
Cell Count
68
the le p to s p ires.
The d iffu s io n rates o f carbon, inorganic ions and
glucose are shown to have reached equilib rium w ithin approximately 6
hours as seen in Figures 11 and 12.
At the rate of d ie - o f f shown in
these experiments, a h a lf-tim e o f 39 hours fo r L. pomona in the labora­
tory and 35.4 hours fo r L. hardjo in the well water was determined
grap h ically from Figure 13.
Assuming th is d ie -o ff remains stable u n til
d ie -o ff is complete, a th e o re tic a l survival o f 26 days could be expected
O
when the s ta rtin g population is 10 leptospires per ml as in the labo­
rato ry experiments.
D a rk -fie ld observations o f the leptospires during d ire c t c e ll
counts showed a range o f 30 to 60 percent m o tility per counting square.
A maximum v ia b i lit y number is represented by the to ta l number of c e lls
counted and a reasonable minimum v i a b i lit y is represented by 25 percent
of the to ta l count based on 30-60% m o tility which f a l l s below the
lowest m o tility range observed, thus a minimum and maximum v ia b ilit y
range may be noted as indicated by the cross-hatching in Figures 14 and
15.
MPN Enumeration From Natural Water
The resu lts of the enumeration o f leptospires from four natural
water s ite s by MPN procedures is given in Table 7.
These results show
a c o rre la tio n o f le p to s p ira l numbers to fecal c o li forms and temperature.
The f i r s t three s ite s showed fecal c o li form counts in the same range,
thus the f a ilu r e to recover leptospires a t Hood Creek is possibly due
69
— IO
DAYS
Figure 14. Survival of L. pomona in a Laboratory Water Environment With
a Maximum and Minimum V ia b ilit y Range Based on D irect Cell Counts and
the Percentage of M otile Cells in the Count
70
L. pornona
L. h&rd.1o
DAYS
Figure 15. Comparison o f L. hardjo and L. pomona Survival to Salmonella
enteritidis s e r. paratyphi B ( ------), Vibrio cholerae (-------------- ),
Salmonella enteritidis ser. paratyphi D (--------------) , and Salmonella typhi
( --------) Survival (53) in Continuous Flowing Well Water.
The L. pomona
[\ \ \ ) and L. hardjo ( / / / ) areas represent the maximum and minimum
v ia b i lit y range based on d ire c t c e ll count and m otile c e ll percentage
o f the d ire c t count.
71
Table 7. Results of a Most Probable Number (MPN) Enumeration of Leptospires a t Four Natural Water Sites
S ite
Hood
Temp.
C° .
PH
Fecal
Conforms
Per 100 ml
Leptospire MPN Data
0
.2
.04
MPN
- per
.008 100 ml
95%
Conf.
5.0
7.3
172
0/3
0/3
0/3
0/3
<1.8
(0 -3 )
Mystic
11.5
7.2
232
2 /3
0/3
0/3
0/3
5
(1-36)
Buck
12.5
7.0
152
3/3
0/3
0/3
0/3
11
(4-120)
Black
13.0
7.2
2080
3/3
1/3
0/3
0/3
23
(7-210)
/
72
to the cold and p ris tin e w ater.
The high fecal c o li forms a t Black
c o rre la te with the numbers of leptospires recovered.
CHAPTER 5
DISCUSSION
Transmission through water systems is a major route o f le p to ­
spirosis spread.
Thus survival o f leptospires in w ater, e s p e cia lly the pathogenic
serovars, is an important consideration in the epidemiology and control
of le p to s p iro s is .
The e ffe c t of pH, temperature, moisture and types of
suspending flu id s have been examined by various researchers.
Okazaki
and Ringen found th a t survival in a pH range of 6 .0 -8 .4 was possible,
but above or below these ranges the leptospires were ra p id ly rendered
nonmotile.
At freezin g temperatures, pH did not appear to a ffe c t sur­
v iv a l, however, a t temperatures of 7-10° C a lower pH was conducive to
longer survival than a t temperatures of 20-26° C.
The opposite pH
e ffe c t was noted a t a higher pH with a longer survival a t 20-26° C and
decreased survival a t the lower temperatures of 7-10° C.
Moisture was
found to be very important with survival of 30 min in a dry so il and
3 days in a moist s o il.
193 days.
A super saturated soil showed survival up to
Thus Okazaki and Ringen considered a temperature range of
7-26° C and a pH range o f 6 .0 -8 .4 to be necessary fo r survival of
Leptospira ppmona in the environment (5 9 ).
Diesch found th a t Leptospira
pomona survived fo r a period of 6 days when placed in selas candles and
suspended in manure.
Survival o f 61 days was noted in the manure o f an
oxidation d itc h , 5 days in ditch e fflu e n t and 4 days in sludge (1 6 ).
74
Crawford e t a l . found th a t I. pomona survived fo r 13 days but not more ■
than 20 days in natural water under laboratory conditions (1 3 ).
Studies
by Chang e t a l . o f L. ioterohaemorrhagiae showed survival, of 8-9 days a t
5-6° C, fo r 5-6 days a t 25-27° C and fo r 3-4 days a t 31-32° C in r iv e r
water.
Survival in domestic sewage was 12-14 hours but increased to
2-3 days when aerated (8 ) .
Ryu and LIU found th a t le p to s p ires {l .
australis H and L. semavang) lo s t th e ir v ia b i lit y above 40° C and were
able to maintain v ia b i lit y a t 0-30° C (6 5 ).
Survival conditions with a temperature range o f 7-26° C and a
pH range o f 6 .0 -8 .4 are shown to correspond with the incidence of le p ­
tospiro sis (3 0 ,3 1 ,4 7 ).
Thus in la te summer when water temperature is
high and water levels low (re s u ltin g in less d ilu tio n ) an increase in
lepto sp iro sis cases is seen.
These survival studies u t iliz e d four procedures to determine
survival and/or v ia b i lit y of the le p to sp ire s.
Susceptible hosts such
as hamsters, guinea pigs and weanling mice (Webster Swiss) (13 ,3 2,5 9)
were used to recover and to check fo r virulence of leptospires from
various environmental models.
Leptospira! m o tility as seen under dark-
f ie ld was also used as an in d icatio n o f survival in various testing
systems (8 ,1 3 ,1 6 ).
Some attempts were made to enumerate through the
use of d ire c t microscopic counts (8 ) .
Cultural attempts were also
made in le p to s p ira l c u ltu re media to show survival in te s t systems (13,
1 7 ,6 5 ).
75
A, system of enumeration remains to be worked out fo r survival
o f leptospires in various environmental conditions.
The results
obtained in our well water and laboratory survival studies were obtained
by d ire c t c e ll counts, with attempts to estimate v ia b i lit y by observa­
tio n o f m o t ilit y .
An assumption o f v ia b i lit y based on m o tility seems
reasonable, however as shown by Okazaki and Ringen (59) v ia b i lit y demon­
s trated by c u ltu rin g and by m o tility did not always correspond.
These
measures o f v i a b i lit y were reasonably close in some cases such as sur­
vival in supersaturated soil being p o sitiv e fo r 193 days based on
m o tility and fo r 183 days based on c u ltu rin g .
However, in simulated
natural conditions, Teptospiral m o tility showed v i a b i lit y a t 14 days,
while c u ltu rin g indicated survival only up to a maximum of 6 days.
Using the assumption of m o tility representing v i a b i l i t y , a survival
curve fo r a period of time can be generated.
The numbers of c e lls must
remain high fo r counts to be made with counting chambers.
A concentra­
tio n o f 10,000-20,000 leptospires per. ml is probably the minimum number
y ie ld in g detection of even one c e ll on high-power d a rk -fie ld (8 1 ), and
a concentration of a t le a s t 10
counts can be made (5 1 ).
6
c e ll s/ml is needed before accurate
The other method used fo r obtaining density
in these survival studies was tu r b id ity as measured by spectrophotometry
and nephelometry.
D irect c e ll counts made with a Petroff-Hausser bacteria counting
chamber correlated well with tu r b id ity by nephelometry and spectrometry
76
as seen in Figures 9 and 10 in Results. As with d ire c t c e ll counts,
tu r b id ity measurements are lim ite d by the need fo r high b a cterial
densities and lack the a b il it y to d iffe r e n tia te between viable and
nonviable
c e lls .
T u rb id ity measurements become useful fo r observing
increasing population growth and can provide accurate densities pro­
vided the organisms do not grow in clumps or chains and do not absorb
lig h t (2 7 ,4 3 ).
With the lim its of d ire c tin g c e ll counts and tu r b id ity in mind,
we can obtain some information on possible survival o f le p to s p ires over
a period of time as compared to water-borne en teric pathogens in the
same water system.
Using data obtained by McFeters e t a l. on en te ric
pathogen survival in well water (53) and comparing i t to le p to sp ira l
data obtained in the same system, i t can be seen th a t the leptospires
remain a t higher densities where the e n te ric pathogen concentrations .
f a l l o f f quite ra p id ly , as shown in Figure 15 in R esults.
The d iffe re n c e in survival between L. hardjo and L. pomona may
be due to the greater s e n s itiv ity of L. hardjo to th is well water
environment re s u ltin g in a decreased survival a fte r four days.
The
Hebdomadis serogroup, which includes L. hardjo, has been known to be
more d i f f i c u l t to c u ltiv a te in vitro ( I ) .
This apparent fastidio us
expression upon in vitro c u ltiv a tio n may also be re fle c te d in decreased
survival as compared to L. pomona, a more e a s ily c u ltiv a ta b le le p to ­
spi re .
77
The survival studies o f the e n te ric pathogens were begun a t
lower concentrations.
The lower concentrations were used to help
prevent c ryp tic growth from high n u trie n t concentration obtained from
d ie -o ff o f a portion o f the s ta rtin g population.
Lower c e ll numbers
of those organisms can be enumerated with plate c u ltu rin g and membrane
f i l t r a t i o n techniques.
Thus the problems encountered with enumeration
o f leptospires with tu r b id ity and d ire c t count are avoided.
A problem which fu rth e r complicated the w ell-w a te r survival
studies was clumping, making d ire c t counting d i f f i c u l t and changing
the lig h t sc atte rin g patterns in the tu r b id ity measurements.
This
clumping phenomenon has been described in certain liq u id media (8 1 ,8 2 ).
I t was suggested th a t th is clumping may be a survival response giving
the leptospires some protection and/or advantage in the h o s tile well
water environment.
This clumping may be the re s u lt o f natural chemical
constituents causing flo c c u la tio n of the le p to sp ires.
Clumping was
not noted in any of the other survival experiments.
The laboratory survival experiments show re su lts s im ila r to
those in the well water as seen in Figure 13 in Results.
The s ta rtin g
concentrations of the laboratory experiments were higher in an attempt
to follow the d ie - o f f in the d iffu s io n chambers over a longer period o f
t i me.
Due to the lim ita tio n s o f tu r b id ity and d ire c t count as enumera
tio n techniques fo r le p to s p ire s , other methods of enumeration were
78
sought.
An enumeration technique which showed accurate Iep to sp irai
v ia b i lit y and which could be used a t low concentrations was needed to
follow the survival over a longer period o f time.
Enumeration by
c u ltu re techniques has as its major problems long incubation times
and d i f f i c u lt y in showing growth with small inoculation s izes.
Long
incubation time leads to problems of media drying and increased con­
tam ination.
Enumeration by an MPN technique appears to be the best
method fo r use on low concentrations and to provide information on
v ia b i lit y .
Most probable number methods re ly on the a b il it y to detect
growth or the lack o f growth in a d ilu tio n series (2 ) .
e a r lie r detection were explored.
Methods of
The MPN fo r fecal c o li forms re lie s
on the production of acid and gas from lactose a t 44.5° C a fte r an
incubation time o f 24 hrs (2 ) .
This reaction is enzyme
mediated,
therefo re i t seemed reasonable to look fo r an enzyme or enzyme product
produced by a c tiv e ly growing le p to s p ires. Two enzymes were studied.
Ellinghausen and Sanvik described trib u ty rin a s e a c t iv it y and a method
fo r demonstrating soluble trib u ty rin a s e (2 4 ).
I t was thought th at
tr ib u ty r in could be added to a semisolid medium which was inoculated as
an. MPN.
Upon growth, the leptospires would produce the lipase t r ib u ty r ­
inase and c le a r the tr ib u ty r in from the media long before the lep ­
tospires themselved could be detected.
T rib u ty rin was added to EMJH
semisolid medium a t a concentration of 2 y l/m l.
appearance.
.Upon addition o f one ml o f a 10
7
This produced.a cloudy
L. pomona/m\ cultu re and
79
incubation a t 27° C, the cloudy appearance began to disappear.
fu rth e r incubation the cloudy appearance disappeared.
spires were noted with d a rk -fie ld microscopy.
Upon
M otile le p to -
Further incubation
resulted in a reappearance of the cloudy m aterial rendering the method
ambiguous.
Some bacteria such as the genus Staphylococcus produce a
nuclease which is an e x tra c e llu la r enzyme capable o f hydrolyzing DNA
and RNA (1 4 ).
(4 1 ).
Methods fo r detecting th is enzyme have been worked out
DNase te s t agar was made up and placed in glass p e tri plates.
Drops o f L. pomonaj L. patoc and L. hardjo were then placed on the
p la te .
Small zones o f clearing could seen a t the periphery of the
drops upon the addition of a cold 25% tric h lo ro a c e tic acid.
upon prolonged incubation the zones did not increase in s iz e .
However
This
enzyme was abandoned as a possible candidate fo r rapid MPM detection.
I t is o f in te re s t th a t a zone of c le arin g was seen.
This may in dicate
the p o s s ib ility of a exocellu la r nuclease being present with a low
a c tiv it y and/or concentration.
A contaminated system may have been
the cause o f the c le a r zone with a nuclease being produced by the
contaminant.
The detection of an enzyme or enzyme product may be a possible
answer fo r e a rly growth detection in le p to sp ira l MPN1s.
However, care
must be taken to e ith e r fin d a unique enzyme system or pure cultu re
work must be done to prevent contamination by enzymes from the con­
80
taminating b a c te ria .
Tributyrinase a c t iv it y was noted with spiral
organisms presumably belonging to the genus Spivilliwn.
DiNase a c t iv it y
has been shown fo r Baoillus3 Stveptomyoes3 Sevvatia3 Pvoteus and
Staphlooocous (4 1 ).
This illu s tr a te s the need fo r a pure culture
system or a unique enzyme or enzyme product as a method o f e a rly MPN
detection.
The detection of a s p e c ific c e ll component may be advantageous
in enumeration.
Living organisms have a constant concentration o f the
energy tra n s fe r molecule adenosine triphosphate (ATP) (3 9 ).
A method
of measuring th is concentration has been developed using a lu c if e r in lu c ife ra s e system with a luminescence Biometer (3 8 ).
This technique
was applied to a c u ltu re of L'. pombna grown in liq u id EMJH medium.
A
s e ria l d ilu tio n was made with an i n i t i a l c e ll concentration obtained
by d ire c t counting with a Petroff-Hausser chamber.
Adenosine trip h o s -
phate was extracted and three re p lic a te s a t d ilu tio n s o f 10
IO '4 were read on a duPont luminescence Biometer.
"I
-3
, 10
and
The resu lts obtained
in th is single experiment did not show c o rre la tio n between d ire c t c e ll
counts, s e ria l d ilu tio n s or ATP concentrations.
At th a t time no e f fo r t was made to repeat th is experiment to
fin d possible problems and to obtain c o rre la tin g data.
This method
remains as a promising p o s s ib ility fo r le p to sp iral enumeration.
The
necessity fo r pure cultures and the high cost involved are possible
lim itin g fa c to rs .
81
Early detection o f an MPN or. Ie p to sp ira l v i a b i lit y in cultu re
may be possible through the use o f microscopic observations.
Postgate
e t a l . has suggested a method fo r observing b a c te rial v ia b i lit y by
s lid e cu ltu re (51)
The use of m icroslides (rectangular glass c a p il­
la r ie s ) as counting chambers and to provide an environment conducive
to fa s te r le p to s p ira l growth may have applications fo r detection and
enumeration o f le p to s p ires (5 0 ).
Macroscopic observations may be in d ic a tiv e o f le p to sp ira l
growth, however in cases where c u ltu re p u rity is in question such
as in
in situ survival studies, care must be taken in in te rp re tin g
apparent le p to s p ira l growth.
Several observation methods may be
employed to f a c i l i t a t e readings o f MPN or viable growth.
Nephelometry
has p o s s ib ilitie s as an e a rly detection method in liq u id c u ltu rin g .
Growth in semisolid may be Shown by Dinger ring form ation, however
other organisms have been shown to produce the Dinger ring growth
phenomenon.
The small s p ira l organisms which are continual contami­
nants in membrane is o la tio n techniques show a ring growth which closely
resembles le p to s p ira l Dinger rings (7 ).
Other researchers have shown
a s im ila r growth phenomenon in semisolid medium with other bacteria
(1 5 ).
Leptospiral growth in s o lid agar medium usually forms opaque
growth descending throughout the depth o f the agar.
Various forms of
le p to s p ira l growth in solid agar have been noted including small
opaque colonies and translucent colonies with a la rg e r diameter and
82
v e i l- l ik e appearance (1 2 ,7 3 ,7 8 ).
Spiral contaminants often produce
v e i l - I ike colonies descending through the depth o f the agar.
C anale-■
Parola described these s p ira l colonies as subsurface, spreading, semi­
transparent, unpigmented colonies which were round or nearly so (7 ).
Various types o f media have been used fo r c u ltu rin g leptospires
Several types were used in th is study (Table I , M aterial and Methods)
with the objectives o f providing a medium capable of promoting rapid
growth from small inoculum sizes and high le p to sp ira l densities in a
short time span.
The f i r s t type o f medium u tiliz e d was a tween-albumin
medium described by Ellinghausen and McCullough (EM) (2 5 ).
This media
had been formulated as a su b stitu te fo r serum-requiring media.
The
f a tty acids found in the tween complex were found to be less toxic y e t
s t i l l a v a ila b le fo r le p to sp ira l u t iliz a t io n as an energy source,
The
previous attempts a t a serum-free media had incorporated free longchain f a t t y acids such as o le ic .
I t was f e l t th a t leptospires grown in
these media with free long-chain f a t t y acids developed a resistance to
the ly t ic a c t iv it y of f a t t y acids.
There was also concern about
possible loss o f virulence in these media employing free long-chain
fa tty acids (1 1 ,2 1 ,6 8 ,7 2 ).
cessful.
The i n i t i a l use of EM medium proved unsuc­
Some le p to s p ira l growth was obtained with
l
. pomona, how­
ever, the cultures never did obtain high densities and they appeared
to die a t.a rapid ra te .
A maintenance medium was sought to prevent
loss of the experimental cultures a f t e r the poor growth results with
83
EM medium.
Rabbit serum and fe ta l c a lf serum was obtained from Dr.
Malcolm H. Smith a t the Veterinary Science Research labs.
U tiliz in g
these sera, F letc h e r's and S tu a rt's semisolid media were made as
described (2 9 ,7 6 ).
These media were successful as maintenance media.
A m odification of EM as described by Johnson and Harris (EMJH) (45)
was then made which showed success a t c u ltiv a tio n o f L. ipomona-, L.
Hardjo and L. patoo.
EMJH media was then used ro u tin e ly fo r le p to -
s p ira l growth with the only major problem being th a t o f contamination.
The need fo r fa s te r cu ltu re growth and to overcome the problem of slow
growth with a small inoculum size prompted the use o f a minimum
essential medium described by Finn and Jones (2 8 ).
They ran an evalu­
ation o f media based on temperature, inoculum and cost and found th a t
a medium (MEM-BOH) incorporating minimum essential medium o le ic acid
and bovine serum albumin fra c tio n fiv e was the le a s t c o s tly per c e ll
concentrations produced and th a t higher c e ll concentrations could be .
obtained in a shorter tim e.
This medium did not produce well when
made as described by Finn and Jones.
Several m odifications were then
tr ie d which included the su b stitu tio n o f tween-80 fo r the sodium oleate
and increasing the albumin supplement.
unsuccessful.
These m odifications also proved
A fu rth e r minimum essential medium was then trie d with
its form ulation based on le p to sp ira l metabolic requirements and com­
parison o f the formulae o f various le p to s p ira l media..
Ammonium chloride
was used as the nitrogen source, tween-80 as the source o f f a tty acids
84
(in p a r tic u la r , bound o le ic ) fo r energy production* bovine serum albumin
(fra c tio n fiv e ) as a serum substitu te and vitamin B I2 as a growth sup­
plement (2 1 ,4 3 ).
Leptospires do not require any amino acids, however
i t was thought th a t the amino acids as well as the other nutrients found
in Dulbecco's m odification o f minimum essential media would promote
growth a t lower c e ll d e n s itie s .
This minimum essential medium formula­
tio n (MEM-BT) did promote the growth o f le p to sp ire s, although no advan­
tage over EMJH medium was noted.
Em medium was again t r ie d .
In fu rth e r attempts to promote growth
A ll the previously made stock solutions
were discarded and fresh ones made.
A m odification in preparation as
described in M aterials and Methods was u tiliz e d to elim in ate the pre­
c ip ita tio n problem which had been encountered.
The e ffic a c y of th is
newly-modified EM medium was found to be very good and EM became the
c u ltu rin g media o f choice.
The i n i t i a l reasons fo r the fa ilu r e of EM
medium to produce expected growth remain unknown, but blame can possibly
be placed on one or a ll o f the stock solutions and perhaps a contaminant
in the preparatory water.
At one time during.these studies, the supply o f bovine serum
albumin was low.
A substitu te medium was needed.
The media described
by Cox (SMl) (SM2) (11) were made and found to be a good growth media,
however upon any prolonged incubation rapid death o f the leptospires
was noted.
The fa ilu r e o f leptospires to grow or the prolonged incubation
85
needed to obtain growth with small inoculum sizes has been a continual
problem in le p to s p ira l c u ltu rin g (4 4 ,8 1 ).
A 1:10 inoculum size is
generally recommended fo r cultu re tra n s fe r and growth fo r experimental
purposes as an attempt to bypass the small inoculum problem (7 7 ,8 1 ).
Attempts to overcome the d i f f i c u lt y o f small., inucolurn sizes were made
in these studies fo r the purpose o f enumeration through MPN techniques
which require small inoculum sizes and demand p o ten tial growth from
each in divid ual c e ll placed in the medium.
present in the c u ltu rin g media.
Toxic substances may be
The water used fo r media preparation
is the most probable source fo r chemical contaminants such as heavy
m etals, pesticides and herbicides.
The use Of d is t ille d water or water
produced from activated carbon,, ion-exchange may prove to be less
to x ic , however the p u rity o f these waters may be in question.
Studies
in these lab o rato ries in dicate th a t water obtained from a M illi-Q
■
activated carbon, ion-exchange system may contain to x ic m aterials and
retard growth o f bacteria when media are prepared u t iliz in g th is water.
Early researchers found th a t attempts to c u ltiv a te leptospires
on s t e r ile human feces fa ile d u n til the feces had f i r s t been inoculated
with Bacillus coli (E. coli). T h e re a fte r, le p to sp ira l growth was noted
(2 0 ).
Based on th is observation, media conditioning was attempted by
growing E. coli and/or leptospires in liq u id EMJH or EM.
A fter a short
period of incubation, the medium was centrifuged and then r e f i l t e r
s t e r iliz e d .
This medium was then reinoculated with leptospires and
86
the ra te of growth was observed as compared to untreated medium.
The
treated medium did support growth of le p to s p ire s , however no medium
conditioning advantage was noted as compared to untreated medium.
Johnson e t a l . found th at the addition o f sodium pyruvate
(WO ug/ml)
was e ffe c tiv e in promoting growth with small inocula and in n u tr i­
tio n a lly fa s tid io u s serotypes (4 4 ).
Leptospires appear to be m icroaerophilic since they form
growth rings a t 3-10 mm below the surface in tubes of semi sol id or
so lid medium.
These rings (see Figure 6) are thought to form
based on n u tritio n a l response to oxygen p a rtia l pressure and as a
response to metabolic waste products d iffu s in g away toward the bottom
of the tube, thus a narrow band of growth forms with a possible upper
lim it being defined by oxygen p a rtia l pressure or COg p a rtia l pressure
and the lower lim it by toxic metabolic or medium substances d iffu s in g
away from the growth mass in the Dinger rin g .
Often m u ltip le rings
may occur with as many as 20 d iscrete rings forming as a function of
incubation tim e.
form ation.
Prolonged incubation resu lts in increased ring
Several explanations fo r these m ultip le rings may be made.
The n u trie n t response may be d iffe r e n t fo r various s tra in s , thus these
in divid ual strain s may seek th e ir own levels based on n u trie n t g ra d i­
ents.
This idea corresponds with the increased ring formation found
a fte r prolonged incubation in d ic a tin g th a t individual strains may show
up as discrete,D inger rings a fte r the appropriate incubation allowing
87
growth dense enough to be observed macroscopicalIy as a rin g .
Another
possible explanation fo r these m ultiple growth rings may be migration
of leptospires to seek new n u trie n t sources.
The prolonged incubation
may allow d iffu sion of gas to an increasing depth pushing the oxygen
sensitive organisms to increasing depth, but leaving behind those lep­
tospires which have developed an. "oxygen tolerance."
Various c u ltu ra l observations were made which may be explained
by one or more of the above discussions.
The use of SMl or SM2 semi -
solici medium was abandoned because the Dinger ring which formed rapidly
began to descend through the medium u n til i t reached the bottom where
i t dispersed.
This may indicate that the leptospires migrated through
the medium in response to n u trien t depletion and, upon reaching the
bottom of the tube, a l l the nutrients were gone, thus the growth ring
dispersed.
The Dinger rings remained v is ib le in a more complete medium
such a EMJH or EM, which may indicate a b e tte r n u trien t source.
In s olid medium, several growth fronts could be seen fo r le p ­
tospires inoculated in wells cut in the agar.
These growth fronts
could possibly be id e n t if ie d as s tra in differences responding to
n u trie n t gradients and displaying varying degrees of m o t il it y .
The apparent microaerophilic nature of leptospires was explored
as a means to decrease the growth time and to promote growth from small
inoculum sizes.
The atmospheres above the leptospiral cultures were
manipulated by evacuation and then replenishing with various gas
88
mixtures with the volumes added controlled by manometric measurements.
Several researchers have explored the e f f e c t of CO2 and oxygen upon
the growth.of leptospires (2 3 ,8 5 ).
Various atmospheres were attempted in these studies including
varying percentages of CO2 , oxygen, nitrogen and decreased 'p a rtia l
pressures through varying evacuation pressures.
These studies did show
response of leptospiral growth to various atmospheres.
However, due
to impure gas mixtures and other experimental problems, no d e f in it iv e
data were obtained.
These lim ited studies indicated th a t changes in
oxygen p a r tia l pressure may be beneficial in growth promotion, although
more research is needed.
The is o la tio n of leptospires was successful in a va riety of
environments.
Most of the isolations were made in free running water
or standing water, however lim ite d is o la tio n attempts in moist soil
also proved positive fo r leptospires.
Other researchers have detected
leptospires in soils and have speculated on the role soil plays in sur­
vival and transmittance (4 ,3 7 ,4 2 ,5 9 ).
Isolation was possible in water
over a wide temperature range ( 3 .5 0-27° C), a wide q u a lity of water
(p r is tin e to polluted) and from stagnant as well as running water, thus
the a b i l i t y to detect leptospires in moist soils as well as these
diverse water conditions indicates a wide adaption and d iv e rs ity in the
environment.
The enumeration study (Table 7, Results) based on most probable
89
numbers a t various s ites ranging from cold (5 .0 ° C), "p ris tin e " waters
to warmer (13° C), "polluted" waters does indicate v a ria tio n in popu­
la tio n densities with v a riatio n in temperature and water q u a lity .
Hood
Creek, a high mountain s it e with cold water (5° C) draining natural and
undisturbed land, f a ile d to show le p to s p ire s .
A high fecal c o li form
count was noted a t this s it e which might indicate p o llu tio n , however,
based on previous fecal c o li form counts and the general appearance of
the sample s i t e , no obvious po llu tion o f pollution source could be
detected.
The high fecal c o li form count may have been the re su lt of
wild animal a c t i v i t y in the area.
The next s i t e . Buckskin Creek, also
a high mountain stream, gave po sitive leptospiral isolations with an
MPN of 11
per 100 ml.
Buckskin drains a larger area, with greater
land use including intensive summer c a t t l e grazing and some human
a c tiv ity .
As noted from Table 7, the temperature was. also higher than
th a t of Hood (12.5° C).
The p o sitive is o la tio n of leptospires a t Buck­
skin may.be expected based on the increase in temperature, the possi­
b i l i t y of av aila b le nutrients and the susceptible host animals providing
a possible re s e rv o ir.
The Mystic s i t e , which, was in the open valley
f lo o r in a m ultiple land-use area, also showed positive leptospiral
is o la tio n with an MPN of 1.8.
Mystic was considered to be only s lig h t ly
polluted and had a la rg e r water volume and fa s te r flow than Buckskin.
These c h a rac te ris tic s would indicate the p o s s ib ility of a leptospiral
population, but because of lesser po llu tion and la rg e r volume and fa s te r
90
flow, fewer leptospires might be expected as compared to Buckskin.
The
fourth s it e was situated in the ir r ig a t io n system branching o f f H ya lite
Creek.
s it e .
There was a herd of c a t t le bedded and grazing a t the sample
C attle were seen standing and drinking in the water a t the
sample s it e and heavy concentrations of. manure were on the bank as well
as in the ir r ig a t io n c anal.
The high fecal coliform counts (2080/100
ml) r e f l e c t the herd p o llu tio n .
The polluted water as well as the
higher temperatures would suggest higher p ro b ab ility o f finding large
lepto sp iral populations.
The lepto sp iral MPN is the same as Buckskin,
however, the additional positive tube a t IO"^'^ d ilu tio n indicates a
la rger number of leptospires a t this s it e .
Based on this study, p o llu tion and temperature seem to correlate
with probable lepto sp iral d ensities.
More work is needed to obtain
d e f in it iv e information in regard to water q u a lity , temperature and
lepto sp iral d ensities.
This type of information w i ll contribute to the
understanding of su rv iv a l.
The leptospires which.have been isolated appear to belong to
the "Biflexa Complex" based on the lim ite d serology and the is ola tion
environments.
' A method using membrane f i l t e r s was chosen fo r isolation
attempts.
Previous researchers were successful in is o la tin g lepto­
spires. by f i l t e r i n g surface water samples through membranes of 0.45 urn
pore size ( 6 ,6 3 ,7 9 ).
Based on attempts a t f i l t e r i n g water samples and
91
water samples spiked with le pto sp ires, i t was f e l t th a t b e tter is o la ­
tion might be achieved i f the leptospires were allowed to pass through
the membranes in what might be described as a chemot a c t ic
response.
Smibert described a technique using membranes and s olid agar medium
fo r separating leptospires from a contaminating bacteria by taking
advantage of chemotaxis and the physical size and shape of the lepto­
spires (7 1 ).
Their extreme m o t il it y and t h e ir thin th re a d -lik e
morphology allowed them to swim through pore sizes too small for
contaminating bacteria to pass, thus p u rific a tio n was effec te d.
Lep­
tospira! growth on semisolid media has been shown to be b e tter than
on s olid media (2 1 ,8 1 ).
u t i l i z a t i o n of Smibert's
Based on th is knowledge, a method permitting
decontamination procedure with movement into
semi sol id instead of solid medium was sought.
The two swirinex modifi­
cations and the polycarbonate c e ll as described in Materials and Methods
served as prototypes fo r the is o la to r v i a l , which gave the best results
i n , is o la tio n attempts.
Is o la tio n using the is o la to r via l was f i r s t accomplished with
EMJH and SMl media, however b e tte r results were seen when EM medium was
used and 5-Fu was added to a 100.pg/ml concentration.
sample was placed in the is o la tio n chamber.
A 2 ml water
The is o la to r via l was then
incubated a t 27° C with evaporation of the water sample being acceler­
ated to help concentrate the leptospires.
evaporation was not necessary.
Later on, i t was f e l t that
The is o la tio n success of these is o la to r
92
v ia ls was probably based on several fa c to rs .
Sampling could be made
a t the sampling s it e with immediate tra n s fe r of the sample to the
is o la tio n chamber where i t was exposed to a nu trien t environment.
The
medium level was kept as low as possible with a miniscus forming at
the medium membrane in te rfa c e .
This was done to promote a natural
gravity flow of sample through the membrane thus concentrating the
organisms in the v ic i n i t y of the membrane.
I t was f e l t th a t the d i f f u ­
sion o f 5 - f l uorouracil upward through the membrane reduced the suscep­
t i b l e bacterial population, thus reducing the problem of antagonistic
interactions among the mixed population which had been noted by other
researchers ( 8 ) .
The mixed population which was capable of passing
the membrane appeared not to influence the leptospiral growth rate or
population s ize .
The immediate exposure to a n u trie n t environment,
reduction in the antagonistic population and the natural concentrating
e ffe c t were f e l t to be positive factors f a c i l i t a t i n g is o la tio n success
with the use of the is o la to r v ia ls .
An observation of high c o li form bacteria counts in an unpolluted
p r is tin e stream prompted laboratory studies on the cause of these high
c o li form counts.
The supernatant from an exenic
Chlovella algal c u l­
ture was used as a growth medium fo r various c o li form bacteria.
Chlovella mats were found to be associated with the sample sites having
high c o li form counts (5 2 ).
with
This same
Chlovella supernatant was tested
L. havdjo, L. pomona and various water is o la to rs .
The leptospires
93
were found to be. surviving a t low densities over a two-month period in
the supernatant a f t e r three passages into fresh algae supernatant.
The
inoculum a f t e r the th ird passage was considered to be free of any Ieptospiral. medium from the i n i t i a l inoculatio n, thus the algae supernatant
was found to be non-toxic and perhaps providing nutrients fo r lim ited
leptospiral growth.
The survival of pathogenic leptospires in soil
(3 7 ,5 9 ), in
water (4 ,8 ,5 6 ) in animal wastes (16,17) and in algae supernatant in d i­
cates an a d a p ta b ility to
in vitro s u rv iv a l.
expands the epidemiological complexity.
This
.in vitro s u r v i v a l
The harboring o f leptospires
in soil and water has been indicated by increased lepto sp iral case
incidence during flooding and heavy rains ( 4 ,4 2 ).
The transmission
of leptospires is probably accomplished through exposure to contami­
nated water, however the leaching o f leptospires from the soil and
wastes as well as the survival in water and continual contamination
by animal reservoirs provide potential continuous lepto sp iral popula­
tion in the water.
CHAPTER 6
SUMMARY
Leptospira! c ultu ral media and methods were examined in an
attempt to promote lepto sp iral growth from a small inoculum size and to
obtain hig h .ce ll densities in a short time period.
Three basic types of
media including serum albumin-tween, minimum essential medium, and
whole serum medium were used.
E llinghausen and McCullough's albumin-
tween medium was found to give the best overall results fo r rapid growth
and culture maintenance, thus, this medium became the medium of choice.
Limited studies were made to find methods fo r early detection
and fa s te r lepto sp iral growth fo r the purpose of enumeration through
the use of most probable numbers techniques.
These methods included
possible enzyme assays, atmosphere manipulations and media conditioning.
None of these methods were used, although they may deserve further
in vestig atio n .
Leptospiral survival was studied a t two sites using membrane
side wall d iffu sion chambers.
Survival of L. hardjo and L. pomona
were studies in a continuous well water flow with a stable temperature
of 11° C.
A minimum survival of 6 days was noted fo r both L. hardjo
and L. pomona.
A d i e - o f f h a lf-tim e was obtained graphically f o r . L.
hardjo and found to be 35.4 hours.
This gives a projected theoretical
survival of 23.6 days with the i n i t i a l experimental concentration of
IO8 c e ll s/ml and with stable d i e - o f f being assumed.
Previous studies
I
95
had been performed with in d icator bacteria and some pathogenic bacteria
in this well water system.
Average d i e - o f f half-tim es fo r coliform and
enterococci in d icator bacteria were found to be 17.0 and 22.0 hours,
respectively.
The pathogenic bacteria Shigella flexneri and Salmonella
enteritidis ser. paratyphi D were found to have d i e - o f f half-times of
26.8 and 19.2 hours, respectively (5 3 ).
Thus, Leptospira hardga
appeared to have.a longer survival potential, than both in dicator and
enteric pathogenic bacteria tested.
Leptospira pomona survival was studied in r e frig e ra te d stream
water held a t 11 ° C in the laboratory.
was noted.
A minimum survival of 8 days
A graphic d i e - o f f h a lf-tim e of 39 hours was calculated
giving a theoretical survival of 26 days with a s ta rtin g concentration
O
of 10
c e ll s/ml and stable d i e - o f f being assumed.
Leptospiral is o la tio n was attempted using various membrane
f i l t e r systems.
Techniques which forced the samples through the mem­
branes were found to be less e ffe c tiv e than a system which allowed the
leptospires to pass through the membrane by natural m o t il it y in a
chemotactic response.
Membranes of 0.22 ym pore size were found to
give the best is o la tio n results based on recovery and fewer contami­
nants.
A device made from a 4 dram vial and 1/2" polycarbonate tubing
was used to allow leptospires in a sample to move across a membrane
into semi sol id culture medium.
I t was found that the addition of 100
yg/ml 5 - f l uorouracil to the semi sol id is o la tio n medium helped control
background contaminants, while not a ffe c tin g the lepto sp iral growth and
recovery.
Seventy percent recovery from natural water was achieved
through the use of this is o la tio n device.
P u rific a tio n of these isolated leptospires was found to be
necessary, because of the large motile population capable of moving
across the membrane.
A p u rific a tio n system which r e lie d on leptospiral
m o t ilit y and drug usage was found to-be sa tis fac to ry fo r removing the
major problem contaminants which were found to be small spiral organisms
presumably belonging to the
Spivillaceae . The drug found to be most
e ffe c tiv e against these spiral organisms was col i s t i n .
Inoculation of
a solid agar plate in a well or s l i t was found to promote fa s te r estab­
lishment of growth.
The drug was placed a distance from the inoculation
s ite and p u rific a tio n effected by the motile leptospires producing a
growth fro n t moving toward the drug with the spirals avoiding the drug.
Further p u rific a tio n was done with streak plate methods and membrane
passage.
Some of the isolated leptospires were tested sero logic ally with
a lim ite d battery of s pecific a n tis e ra .
None of these isolates were .
found to react with any of the sera with the exception of is o la te Ag 21
which appeared to produce a nonspecific agglutination with a ll the. sera
including lepto sp iral negative sera.
Leptospiral recovery.was possible from moist s o ils , rapidly
running streams, deep flowing r iv e r s , ponds, bogs and c a t t le watering
97
troughs.
Enumeration of leptospires a t four natural water sites showed
th a t the r e la t iv e numbers of leptospires varied with po llu tion and
temperature.
Sites with high p o llu tion and temperature (-IS0 C) showed
greater lepto sp iral numbers than those of lower p o llu tion and tempera­
ture (5° C) with " p ris tin e " sites showing no lepto sp iral recovery.
The a b i l i t y to recover leptospires from a v a rie ty o f natural
environments re fle c ts a possible wide d is tr ib u tio n .
Survival in various
natural environments such as s o i l , water and wastes as well as the
a b i l i t y of L. Hardjo3 L. pomona and water isolates to reproduce in the
supernatant of an exenic Chlorella culture in the laboratory gives
credence to this wide d is tr ib u tio n .
The possible harboring of patho­
genic leptospires in these various environments may assure a continuous
i n f e c t i v i t y of surface waters.
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