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CYTOPLASMIC ANTIGEN RELATIONSHIPS AMONG STRKPTOMYCES SPECIES

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF ARTS

By

Kenneth W. Brunson, B. A.

Denton, Texas

August, 1966

TABLE OF CONTENT'S

Page

LIST OF TABLES iv

Chapter

I. INTRODUCTION 1

II. MATERIALS AND METHODS 8 Organisms Antigen Preparation Antiserum Preparation Serological Methods

III. RESULTS AND DISCUSSION 15

IV. SUMMARY 25

BIBLIOGRAPHY 27

i n

LIST OF SABLES

Table Pa.£,j

I. Species Tested 9

II. Cytoplasmic Antigen Reactions With Streptomyces Antiserum Recorded in XJunber of Precipitant Bands 17

III. Cytoplasmic Antigen Reactions With Streptomyces Antiserum Recorded in Number of Precipitant Bands 19

17. Cytoplasmic Antigen Reactions With Stroptomyces Antiserum Recorded in Number o"f Precipitant Bands 21

xv

CHAPTER I

INTRODUCTION

Antigenic properties of many bacterial organisms have

long been used as taxonoaic tools. Only somewhat recently,

however, have such tools been utilized in regard to the

order -1c t i n c my c e t a 1 e s» mere especially the genus

StreptoTsyces. Most work with actinomycetes has been con-

cerned with the genera Nocardia and Actinomyces of the

family Aotinonycetaceae and the genus Myco bac terium of the

family Mycobacteriaceae» due to the importance of some

members of these groups as pathogens for mammals. Organisms

of the genus Strcpto-nyces later attained a position of in-

terest primarily due to their ability to produce antibiotics,

Aoki (1) met with some success when he used aggluti-

nation techniques to group aerobic and anaerobic aetino-

mycete isolates; not all of the designated groups were

easily demonstrated, however.

Erickson (7) showed antigenic relationships of

Actinomyces bo vis and A. israelii, but no cross reaction

was observed with saprophytic or parasitic aero 1, o isolates.

Ludwig and Hutchison (15) and Slack jet al. (15) have shown

the presence of common antigens in microaerophilic forms

and have divided then into two serological groups.

Cummins and Harris (5) used antigens obtained from

coll walls to group No car die, with Myc obg.c t erium rather

'Jh&n with Actinomyces. However, studies conducted later

by Xwapincki and Snyder (12), using cytoplasmic antigens,

depicted Myco 1"acter ium and Actinomyces to be antigenically

related. Slack et al. (16), employing the fluorescent

antibody technique and reciprocal adsorption, established

four serological groups of Actinomyces» Corynebacterium,

and anaerobic diphtheroid bacilli.

Cummins (4) reported common antigenic components in

all strains of Mycobacteriura, Nftcardia and Corynebacterium

which had arabinose and galactose as principle cell wall

sugars.

Kwapinski (10), using chemically obtained fractions

of Actinomyces and Nocardia as antigenic material, found

the cytoplasmic materials to be serologically related;

genus-spccific antigens apparently resided in the cell

walls.

The above studies serve to offer presumptive con-

firmation of an early proposal by 7/aksman (17) that thei ~

is a gradual linkage of species among the actinomycetes.

In more particular regard to the genus Stx^rrtomyces,

much of the early work was hampered by weak antigenicity

of these organisms, thus dictating burdensome, lengthy

immunization schedules. Also, once antiserum ;;f satis-

factory titer was obtained, apparent lack of specificity

resulted in numerous cross reactions. These cross reactions

were among the most serious problems of serological studies.

Ehrlieh crt al. (6) checked S. venezuelae and

S. lavendulao strains morphologically and biochemically,

then sent the cultures to a collaborator, A. B. Hillegas.

Hiilegas administered weekly injections of spore suspensions

to rabbits for three and one half months, then performed

agglutination tests. Strong reactions occurred between

antisera and homologous antigens in each strain; cross

reactions were also observed.

Okani (14), in a serological study of S. lavendulae

and other forms, found some species specificity but also

found frequent cross reactions among apparently unrelated

species.

V/odehouse and 3acku3 (18) reported antiserum to

S. auroofaciens which produced nine precipitant bands when

tested against homologous antigens in an agar gel system.

These workers suggested that the method is well adapted as

an aid in differentiating species and strains within the

genus. •

Bunch and Earth (2), during a study on streams pol-

luted by fermentation-wastes, found that the presence of

Streptomyccs could be detected immunologically? at least

five antigens could be detected in undisrupted ayceliuci

of S. lavendulae.

A major advance in methodology cams when Guthrie,

4

Rq c.ch and Ferguson (8) reported an improved shortened

immunisation schedule by the -use of adjuvants together

with a. sonically disrupted antigen preparation. Improved

specificity of in vitro reactions could be demonstrated

in an agar gel system. However, multiple precipitant

"bands occurred in "both homologous and heterologous

reactions.

Cress and Spooner (3), who used similar methods of

antibody production and immunodiffusion tests, investigated

serological relationships among some species and strains of

Streptomyces used for antibiotic production. Numerous

precipitant bands were observed. However, the situation

was alleviated by recording the number of bands formed "L'.\

each instance? thus certain serological relationships

could be shov.ii.

Further investigation by Guthrie, Reach and Ferguson

(9) showed that standardisation of conditions of growth

medium and culture age carved to increase the accuracy and

specificity of serological grouping. A two-week culture

age was suggested as optimal for a peak titer of antigenic

components present in largest amounts.

Kwapinski (11) has used cytoplasmic antigens obtained

from three strains of one Streptpayees species (S. griseus)

to establish serological relationships of the entire genus

v/ith other members of Actinonycetales. Even with the many

cross reactions observed within the genus S trapt payees.

certain dissimilarities within the group would make the

vising of more members of the group seem desirable in a

study of this nature.

c:The taxonomy of the streptomycetes is thus complex

and presently somewhat confused* Classification "based

essentially on morphology and biochemical characteristics

has "been described; however, no really comprehensive

sc• logical method has gained wide acceptance for iden-

tification of species of Streptorayces. The present study

was conccrned with the testing of antigenic material de-

rived from a number of otreptomyeetes in an effort to

establish an auxiliary taxonomic scheme based upon cyto-

plasmic antigen relationships among these organisms.

CHAPTER BIBLIOGRAPHY

1. Aoici, K., "Agglutinaterische XJntorsuching von Aktinoniyzetin, M Zeitschrift Im-aunitatsforach, LXXXVI (March, 1§£D, 518-524.

2. Bunch, R. 1., and Earth, E. F., "Serological Detection of Fermentation Y/astes," Nature, CLXXXII (August., 1958), 1680-1683.

3. Cress, 1., and Spooner, D. F., "The Serological Identi-fication of Streptonycetes "by Agar Gel Diffusion Techniques,*1 Journal of General Microbiology, XXXIII (February, 19* 3), 2"?5-282.

4. Cunniins, C. S., "Chemical Composition and Antigenic Structure of Cell '//alls of Corynebac terrain, Mycobacterium, Nocardia, Actinomyces and Arthrobacter, " Jeurnal of General Microbiology;, XXVIII (January, 19&2), 35-50.

5. Curamins, C. 3., and Harris, H., "Studies on the Cell Y/all Composition and Taxonomy of Actincraycctales and Related Groups," J ournal of General Microbiology, XVIII (February, 1958"), 173-189.

6. Shrlich, J. and others, "Strcptcmycea venazuelae, n. sp., the Source of Chloromycetin," Journal of Bacteriology, LVI (March, 1948), 467-477.

7. Erickson, D., "On the Nomenclature of the Anaerobic Actinornycetes," International Congress for Microbio-logical Procedures, 3rd Congress, New York (1940), 192-193.

8. Guthrie, R. K., Roach, A. ;h, and Ferguson, J• K., "Serology of Aerobic Aquatic Actinomycetcs. I. Fac-tors Involved in Antiserum Production and In Vitro Antisen-Antibody Reactions," J ournal of Bacteriology, 1XXXIV (August, 1562), 313-317.

9. Guthrie, R. X., Rcach, A. W., and Ferguson, J. X., "Serology of Aerobic Aquatic Actinomycctcs. II. Effects of Growth Medium and Culture Age on Antigen Levels,'1

Journal of Bacteriology, LXXXVI (August, 1963), 173-178.

10. Kwapinski, J. B., "Antigenic Structure of Actinomycetales. VI. Serological Relationships Between Antigenic Fractions of Actinomyces and Nocardia," Journal of Bacteriology, LXXXVI (July, 195377^79-155:

11. Kwapinski, J. B., "Cytoplasmic Antigen Relationships Anion*? the Actinomycetales," Journal of Bacteriology, LXXXVII (May, 1964), 1234-1237.

12. Kwapinski, J. B., and Snyder, M. L., "Antigenic Struc-ture and Serological Relationships of Mycobacterium, Actinomyces, Streptococcus, and Mplococcugt," J carnal o f " Bac t eri o1o gy, LXXXII '(March. 1961), 632-

13. Ludwig, S. H., and Hutchinson, W. G., "A Serological Study of Selected Species of Actinomycetes," Journal of Bacteriology, LVIII (January, 1949)> 89-101.

14. Okami, Y., "A Study for Classification of Streptomyces. On the S. lavendulae Group With Reference to its Immunological Properties," Journal £f General Microbiology, II (January, 1956), 63-75•

15. Slack, J. M. and others, "Studies With Microaerophilic Actinomycetes. II. Serological Groups as Determined by the Reciprocal Agglutinin Adsorption Technique," Journal of Bacteriology, LXX (March, 1955), 400-404.

16. Slack, J. M., Winger, A., and Moore, D. W., "Sero-logical Groupin- of Actinomyces by Means of Fluorescent Ant-todies," Journal of Bacteriology, LXXXII (January, 1961), 54-65.

17. Waksman, S. A., "Species Concept Among the Actinomycetes With Special Reference to the Genus Streptomyces," Bacteriological Reviews, XXI (January, 1957), 1-24.

18. Wodehouse, R. P., and Backus, E. J., "Gel Diffusion as an Aid to Streptomyces Taxonomy," Bacteriological Proceedings. iVII (1957), 43.

CHAPTER II

MATERIALS AND METHODS

Organisms

A list of identified organisms used is included in

Table I; those marked with an asterisk were used for

antiserum production. The souroe of each strain is

designated by initialss ATCC (American Type Culture

Collection, Rockville, Maryland); FD (From a collection

of Dr. Tom Cross, Bradford, England); KRRL (Northern

Regional Research Laboratory, Peoria, Illinois); MCS

(Midwest Culture Service, Terre Haute, Indiana); NTSU

(Stock Culture Collection, North Texas State University,

Denton, Texas). In addition to these, thirty-six uniden-

tified aerobic marine actinomycetes, designated as num-

bered isolates of an "H" series, were used. All stock

cultures were maintained in tubes containing Special

Noble agar (Difco, Detroit, Michigan) slants and a small

amount of starch broth. This starch broth was essentially

that used by Williams and McCoy (4), and each liter, using

distilled water as the solvent, was made up to contain

10.0 grams (1.0 per cent) soluble potato starch, 5.0 grams

(0.5 per cent) tryptone, 0.5 grams (0.05 per cent) dibasic

potassium phosphate, 0.5 grams (0.05 per cent) sodium

chloride and 0.1 gram (0.01 per cent) ferrous sulfate.

8

TABLE I

SPECIES TESTED

Name Source

Micromonospora chalcea ATCG 12452 locardia vaccinli ATGC 11092 Streptoreyc©s "abikoensis ATCC 12766 Streptomyces aTbo^rTseus FD 705 Streptomyces aXbuJT5 7777 ATCG 3004 Streptomyces aTbtia FD 137 Streptoajoes antibioticus0 ATCG 8663 StrcptbiTiycea aiitibloticxas MCS Streptomyces antibioticus FD 139 Streptogiyces iSrulatua NRRL Streptomyces arftenteolus ATCC 11009 Streptomyces aureofaciens MCS Strep7^omyces cacaoi0 ATCC 3082 Strepxomyoes cinnaaoneus0 ATCC 11874 Streptomyces citrous ATCC 10974 StreptPayees coelicolor0 ATCC 10147 Streptomyces c'oelicolor MCS Streptomyces coellcolor FD 461 Streptomyces erythraeus FD 903 Streptomyces flayeolus ATCG 3319 Streptomyces fradiae0 ATCC 10745 Streptomyces fradiae ATCC 11903 Streptomyces fradiae NRRL Streptomyces fradiae FD 474 Streptomyces /rourecrotii0 ATCC 10975 Streptomyces griseus0 ATCC 11429 Streptomyces griseus ATCC 10137 Streptomyces griseus MCS S t r e p t o n y c e s griseolus NTSU Streptomyces ny^roscopicias NRRL Streptomyces Xavenflulae ATCC 8664 "Streptomyces lavenoulae. MCS Streptornyccs lavendulae. ...... FD 822 Streptomyces oAo'rifer° ' "C'C 6246 S tr e p t omy c e s parvus Ai'OC 12433 Streptomyces parvus0 NRRL Streptomyces purpure o chr omo £cen e s 0 ATCC 3343 Streptomyces rimosus. MCS Streptomyces venezuelae. NRRL Streptomyces venezuelae MCS ' treptorayces viridochromo^enes0 ATCG 3356 Streptomyces viridochromogenes NRRL

10

Each organism was subcultured in the above preparation at

least three times prior to -use in the survey.

Antigen Preparation

Stock cultures were used to inoculate three hundred

milliliters of starch broth contained in one liter cotton-

stoppered Erlenmeyer flasks; one loop taken from the stock

culture was used to inoculate each flask. Inoculated

flasks were placed ©n either reciprocal or rotary type

shakers at room temperature and allowed to remain for six

days. At the end of six days each was removed and placed

on a stationary shelf at room temperature and allowed to

remain for eight days; thus a total culture period of two

weeks elapsed in each instance.

At the end of the two-week growth period, immediately

before harvesting the organisms, samples were taken from

each flask and plated in triplicate on trypticase soy agar,

After a suitable period of incubation, these agar plates

were observed macroscopically for determination of any

possible contamination of flask cultures. The mycelial

mat was collected by suction filtration through sterile *

Whatman No. 6 filter paper discs. The mat thu3 collected

was washed three times with sterile distilled water. The

mass of growth which was retained was then placed in small

sterile beakers. Each beaker was partially submerged in

an ice bath, and a Branson Model S-75 sonifier (Branson

11

Instrumenta, Inc., 37 Brown House Road, Stamford, Connec-

ticut) was U3@d for cell disruption. After a uniform

sludge was obtained, the material was subjected to ecu-

trifugation in an International refrigerated (10° C)

centrifuge for twenty minutes at 12,000 rpm, and the

resultant supernatant material collected in sterile vi&?.s.

Shis method for obtaining cytoplasmic fractions of soni-

cated actinomycete cells was patterned after work done by

Hutcheson (2). Cytoplasmic antigenic material obtained

in this way was used for both antiserum production and

immunodiffusion testing. The antigenic material was either

used immediately or placed in the cold at approximately

-65° C until further use.

Antiserum Preparation

Antigenic material was emulsified in equal portions

of JTreund* s complete adjuvant and the subcutaneous injec-

tion schedule set forth by Guthrie, Roach and Ferguson (1)

was followed. Thus each animal was given three injections

of two milliliters each every other day, rested a week,

then given three additional injections of two milliliters

each. The result was that each animal received si>: milli-

liters or less (the complete schedule was unnecessary in

some instances) of cytoplasmic fraction. A few randomly

choson rabbits were injected according to the above

schedule, but using only Preund*s complete adjuvant as the

injection substance.

12

Either male or female rabbits weighing three to five

pounds were "used for immunization. The subcutaneous in-

jection site was in the inguinal region. Blood samples

were taken immediately prior to the initial injection and

weekly thereafter as necessary from suitable ear veins.

Initial blood samples were used as normal serum controls,

and subsequent samples were used to check antibody titer.

Antigen injection was terminated if serum from an animal

attained a titer of at least 1:32 before the schedule was

completed.

Like antisera from at least two immunized rabbits

were pooled for testing, and only serum having a titer o,.

at least 1:32 was used. Each animal was sacrificed after

its collected serum reached the proper titer; exsanguina-

tion was by cardiac puncture. Pooled antisera were stored

at -65° C until further use.

Serological Methods

The technique used was essentially that of the agar

double diffusion method of Ouchterlony (3). Standard size

glass petri dishes containing twenty milliliters of agar

medium were used. This diffusion medium was made up of

lonagar No. 2 (Consolidated Laboratories, Inc., Chicago

Heights, Illinois) in a one per cent concentration, and

containing one-half per cent phenol. Circular reservoirs

were cut in the agar by employing the Weinberg Agar Gel

13

Cutter (Consolidated laboratories, Inc.)* The pattern of

the reservoirs in each petri dish was a relatively large

central well surrounded by six smaller symmetrically ar-

ranged peripheral wells.

For determination of antibody titer, antiserum was

placed in the central reservoir and doubling dilutions of

the homologous antigen preparation placed in peripheral

wells. For the serological survey, antiserum was placed

in the central reservoir while various cytoplasmic anti-

gen preparations were used undiluted to fill peripheral

wells. All reservoirs were completely filled with the

appropriate substance. Humidity was maintained in each

petri dish by placing a 12.5 centimeter Whatman No. 2

filter paper disc saturated with distilled water over the

dish before closing. Reactions were read as precipitant

bands after four days1 incubation at room temperature.

CHAPTER BIBLIOGRAPHY

1. Guthrie, R. K., Roach, A. W., and Ferguson, R. K., "Serology of Aerobic Aquatic Actinomycetes. I. Factors Involved in Antiserum Production and In Vitro Antigen-Antibody Reactions," Journal of Bacteriology, LXXXIY (August, 1962), 313-3TTT

2o Hutcheson, Josephine, "A Serological Study of the Antigenic Fractions of the Aerobic Actinomycetes," unpublished master's thesis, Department of Biology, North Texas State University, Denton, Texas, 1964.

3« Ouchterlony, 0., "In Vitro Method For Testing the Toxin-Producing Capacity of Diphtheria Bacteria," Acta Pathologica et Microbiologica Scandinavica, , ^ ( ; y 5 E _

4. Williams, A. M., and McCoy, S., "Degeneration and Re-generation of Streptomyces griseus," Applied Micro-biology, I (March, 1953), 307-315.

14

CHAPTER III

RESULTS AND DISCUSSION

Sera obtained from rabbits prior to antigenic stim-

ulation, i. e., normal sera, seldom reacted with the an-

tigenic preparations tested. Any such reaction observed

was seen as a very faint precipitant band, so that for

purposes of the survey, sera could be considered non-

reactive. Sera from rabbits injected with Freund* s ad-

juvant alone gave no detectable reactions with any actino-

mycete fractions tested.

These diffuse indistinct bands observed in reactions

of two or three normal sera, although insignificant in the

present survey, are perhaps of some interest in the light

of past work. Cross and Spooner (2), investigating

generic specificity of streptomycete produced antiserum,

observed one faint band when testing a Mycobacterium

strain; serum from apparently normal rabbits occasionally

also produced this reaction, however. Kwapinski (4),

using cytoplasmic fractions of various actinomycetes,

noted weak cross reactivity between antiserum produced

using a strain of Streptomyoes griseua and cytoplasmic

I* butyricum antigen. Since considerable previous work

has depicted a somewhat weak antigenicity of Streptomyces,

15

16

a possible correlation of the studies of Kwapinski and of

Gross and Spooner with the present observations may exist.

The very faint reaction of apparently normal serura observed

in this survey might well be due to Mycobacterium induced

antibody, a cross reaction occurring with like antigenic

components of the streptomycete cytoplasm. The M. tuber-

culosis cells contained in the Preund* s complete adjuvant

likely would not have as many antigens in common with the

streptoraycetes as the saprophytic mycobacteria, hence the

absence of cross reactions with that substance.

Numerous cross reactions were observed among the

streptomycete fractions tested. The results of the sur-

vey, recorded as reactions involving two or more precip-

itant bands, are shown in Tables II, III, and IV. The

number of precipitant bands is recorded for each reaction,

except where only one band was observed. All single band

reactions have been omitted in the interest of clarity.

At least one band was observed in almost all heterologous

reactions, and apparently indicated the presence of genus-

specific cytoplasmic antigens.

Antiserum Groups I through IV in Table II are those

which exhibit relatively few multiple group reactions; the

strains listed in Group IV show somewhat weak reactions

even in homologous diffusion patterns. As can be seen

upon inspection of reactions, this is a somewhat aetero-

geneous serological group. The first three strains of

17

TABLE II

CYTOPLASMIC ANTIGEN REACTIONS WITH STREPTOMYCINS ANTISERUM RECORDED II NUMBER OF PRECfPITANT'MSDS

Group I Reactions With. S. parvus Antiserum Only

S. parvus 'MRRJJ S S. parvus ATCC 124-33 3

Group~Tl Reactions With S. griseu3 Antiserum Only

S"7'"jffiaeu"s ATCC 11429 • . . . •• g" 3. griseus ATCC 10137 4 S. argenteolus ATCC 110G9 3 S. flaveolus ATCC 3319 2

Group III Reactions With S. griaeu3 (1). S. parvus (2), and

erotii (3) Antisera {1} {2) (3±

*S» "lavenaulae ATCC 8664 4 5 3 |L lavenduTae MCS 3 3 3 S. rimosus NRRL 0 4 4

Group~IV Weak Reactions With S. odorifer (1), S. albua (2), S. cacaoi (3) * and S. purpureochroiao genes (4) Antisera""

- a) (2) (3) (4) S. g.nreofaciens MCS 0 0 0 3 S, purpureochromogenes 0 0 0 3

ATCC 3543 S. venezuelae MCS 0 0 0 2 "S, venezuelae NRRL 0 2 0 2 S. cacaoi ATCC 3082 0 2 2 2 J>. erythraeus FD 903 2 0 0 0

Group IV (S. aureofaciens MCS, S. pur pur e o c hr o mo en e s

ATCC 3343 and S. venezuelae MCS) show reactions only with

S. purpureochroiaogenes antiserum, while S. erythraeus

PD 903, the last member of the group, reacts only with S.

albus antiserum.

18

In Table III, those strains are listed which show

strong reactions with four or five different antisera.

Group V is perhaps the best defined serological group,

with each of its members showing a good reaction of two

or more precipitant bands with all five of the antisera

involved. Each member of this group is a strain of S.

fra.fl.iac (from various sources), and a remarkably similar

reaction pattern can be noted anong the different isolates.

In Groups VI and VII, it is interesting to note the

similarity of reactions of different strains of the same

species; these reactions apparently could serve to further

set the members of these groups apart into subgroups. An

exception to the rule of recording reactions on the basis

of two or more precipitant bands should be mentioned in

regard, to the two S. albus strains of Group VII. An ap-

parent incongruity occurs here in regard to the reactions

of these strains with homologous antiserum, where only one

precipitant band was noted in each case. However, when

seen in the agar gel medium, these bands appeared quite

broad and diffuse relative to the other bands of the sur-

vey. Bands of this nature have been observed in multiple

antigen-w -"ibody reactions when the antigens were present

in low concentration; this may well be the case here.

The antigens are perhaps in the necessary concentration

to produce antibody in the animal with the help of adju-

vant , but of an insufficient quantity to produce definitive

bands in agar gel.

19

TABLE III

CYTOPLASMIC ANTIGEN REACTIONS WITH STREPTOMYCES ANTISERUM RECORDED IN 1UMBER OF PRECrFlTAI^T^lNDS

Group V Reactions With S. jaxiseus (1), S. parvus (2)? S_. fradiae

C3)» S. odor "ifer (4)~and S. gou^erotii (5 O L

FTlradiae AICO 1074-5 S. "fraciiae ATCC 11903 I. fratfiae NRRL S. fradiae FD 704

:> 3 2 3

2 2 2 3

4 3 3 4

ID (51 2 2 3 4

3 2 3 3

Group VI Reactions With S. ffoufferotii (1)» S, griseus (2), S. parvus (3)» S. coelicoTor (4), " " " " (6) and S. oaorixer (7) Antisera

S. cinnajnoneuaTfTT"S. antTbioticus

(1) (2) (3) (4) (5) (6) (7) S. coelicolor ATCC 10147 3 3 0 5 0 4 0 s. coelicolor MCS 4 3 0 5 0 4 0 s. citreua ATCC 10974 3 0 4 4 3 0 0 s. f.;ou erotii ATCC 10975 3 0 0 0 2 0 0 s. olnnamoneus ATCC 11874 0 3 3 0 3 0 3 s. lay o scop feus KRRL 0 2 2 2 0 0 2 H 35 0 0 0 4 0 0 3 H 46 0 3 0 0 3 0 H 48 0 0 3 0 0 3 0

Group Vll Reactions With S. griaeus (1), S. odorifer (2), S. virido-chrorno^enes (3)7 S. gott^erotii 1*4). S. purpureochronao genes TSlf S. c~Soaoi (67, S. alfrus (7), anl S. cinnarrA^fT8l—

Antisera

S. alfcur. FD 137 3 0 0 0 0 0 1 3 S. 5X5us ATCC 3004 3 3 3 0 0 0 1 0 S*. viri go ehroir.o/rones

ATCC 3356 0 0 2 3 2 2 3 0 S, vir i do cliroreo genes KRRL 0 0 2 2 2 2 3 0 H 9 3 0 3 0 0 3 0 0 H 20 3 0 0 0 0 3 0 0 H 65 3 0 0 0 0 3 0 0 H 66 4 3 3 0 3 3 0 3

20

Table IT (Group VIII) consists of those strains of

Streptonyccs which exhibit cross reactions with a large

number of antisera, plus many numbered raarine isolates

which react similarly. All three strains of S. antibiet-

icus show this characteristic; this wide range of anti-

genic components may then "be typical of this species.

Some of the other strains listed in this group are

apparently variants, however, when compared with reactions

of other strains of the same species (Tables II and III).

S. frrisaus MCS, S. coelicolor I'D 461, and 5. lavendulac

PD 822 show many iaore cross reactions than do other mem-

bers of each species. It is perhaps significant to note

that each of these strains was the last obtained and added

to the stock culture collection of the respective species.

This cross reactivity might then be a manifestation of the

effect of culture medium over long periods of time on anti-

genic components.

It should be pointed out, however, that this was the

exception rather than the rule. In most instances, the

serological characteristics of various strains of one

species from different sources apparently were quite stable.

Flcromonospora chalcea ATCC 12452 gave a rather faint

precipitant band only when tested against antiserum specific

for S. viridoohroTiogenes ATCC 3356. Nocardia vaccinli ATCC

11092 failed to react with any ©f the antisera tested.

21

TABLE IV

CYTOPLASMIC ANTIGEN REACTIONS WITH STREPTCMYCES ANTISERUM RECORDED IN NUMBER OP PRECIPITAMTSAIDS

Group YlTi Reactions With S. odorifer (1), S. vlridoohromogenes (2),

S. f<oii^erotii CD, S* griseus (4)« S» parvus (3)« S« cccxicolor (%), _S. cinnamon en a (7)?""S_* antibioticus 18) ? S. pttrpureochroraogene a Cy)» and S. c.-

f-aoi ( T o j ~ * * Antisera ""

ammiumiLmim) ( (1Q) S.« antibloticiis PD 139 3 3 0 4 4 5 0 4 J 0 S. aHoiMoticns MOS 3 3 0 3 0 5 0 5 4 0 S. g-ntiblotiouB ATCC 8663 2 3 0 0 0 3 0 5 2 0 S. aFikoenais ATCC 12766 3 4 0 3 6 5 0 3 3 0 S. atisens MCS 3 3 3 5 6 5 3 3 3 0 S. afSo/^fsens PD 705 4 3 0 4 4 3 4 4 4 3 3. coolicoior PD 461 3 0 3 4 3 6 3 4 3 4 S. griscolus NTSU 3 0 3 3 4 0 0 0 4 4 S. griscolus NTSU

3 0 3 0 0 0 5 3 3 0 S. TrvvenduTaa PD 822 0 0 3 5 6 0 4 3 4 5 S. odorifer ATCC 6246 2 2 3 3 3 0 2 0 2 0 1 1 3 3 0 5 5 0 4 0 0 0 H 2 3 3 0 5 4 0 4 0 0 0 H 6 0 2 0 5 5 0 0 3 3 0 11 7 0 0 0 3 4 0 0 0 3 3 H 8 0 0 0 0 3 0 0 0 0 3 H 16 4 4 0 6 5 0 3 0 3 0 H 17 4 3 0 3 0 3 3 0 0 0 H 27 4 3 0 0 3 3 5 0 0 0 H 28 5 5 0 3 0 3 0 0 3 0 H 29 3 3 0 3 3 3 3 3 0 0 H 30 0 3 0 4 3 0 4 3 0 0 H 31 3 0 0 0 0 3 0 3 0 0 H 32 3 J 0 0 3 3 0 3 4 0 H 34 5 0 0 3 4 3 3 4 3 0 H 39 0 3 0 0 4 3 0 0 0 0 H 40 0 3 0 4 3 4 0 3 0 0 H 4-2 4 3 0 3 5 3 0 4 3 0 H 49 5 5 0 5 3 5 0 3 5 3 H 53 0 0 0 0 0 3 3 0 0 3 H 54 3 3 0 3 3 0 0 0 0 0 H 55 5 3 0 5 4 L 4 3 4 0 H 56 3 3 0 5 5 0 4 3 4 0 H 57 0 0 0 5 3 0 0 0 0 3 H 61 3 3 0 5 3 3 0 4 4 4

22

These phenomena correlate well with the most recent classi-

fication of these organisms in Ber^ey's Manual of Determin-

ative Bacteriology (1). The genus Micromonospora is placed,

according to the manual, together with Streptcmyccs in the

family S t r e p t o my c e t ac e a s. The genus Nocardia, on the other

hand? is included in the family Actinomycetaceae. Thus, of

the two genera, one would expect Micromonospora to be the

more closely related serologically to Streptomyces.

In order to test the facility of serological grouping

of unidentified aerobic actinomycetes, thirty-six marine

isolates were cultured, prepared and tested as above.

These were designated merely as numbered isolates of an "H"

series taken from the Gulf of Mexico. Thirty-one of these

isolates fitted into the scheme of grouping as shown in

Tables III and IV (Groups VI, VII and VIII). Five of the

isolates (H 19, H 21, H 43, H 44 and H 58) did not react

with any of the antisera tested.

This preliminary worlc, using selected strains for

antiserum production, indicates this means of study is a

useful one in identifying streptomycete species and strains,

especially when used as an auxiliary method with more clas-

sical methods. This study also appears to confirm the ob-

servation of Guthrie ejt al. (3) and of Cross and Spooner

(2) of the importance of the effect of certain environ-

mental conditions upon antigen production. The standard-

ization of culture medium, period of growth and other en-

vironmental factors is necessary.

23

The obvious heterogeneity of some of the serological

groups illustrates the need for further antiserua produc-

tion for testing. The testing of additional species and

strains, and perhaps some sophistication of methods for

the separation of antigenic components, may prove desirable

to make a more complete serological scheme possible#

CHAPTER BIBLIOGRAPHY

1. Brood, Robert S. and ©there, Bcrgey*3 Manual of Detorminative Bacteriology» Saltiraore, The "Williams and .vTlkjTns Go., 1*957.

2# Cross, T. p and Spooner, D. P., "The Serological Identi-fication of Streptomycetes by Agar Gel Diffusion Techniques," Journal of General Microbiology, XXXIII (February, 19^JT7~575-28^ ^

5. Guthrie, R. K., Roach, A. Y/., and Ferguson^ J. K., "Serology of Aerobic Aquatic Actinoroyoetes. II. Effects of Growth Medium and Culture Age on Antigen Levels," Journal of Bacteriology, LXXXVI (August, 1963), 173-178.

4. KwapinslriL, J. B., "Cytoplasmic Antigen Relationships Among the Actinonycetales," Journal of Bacteriology, LXXXVI I (May, 19 W7T23~?-12'37.

24

CHAPTER IV

SUMMARY

By employing the methods given of antigen preparation,

antiserum preparation, iamunization schedules and immuno-

diffusion testing, the organisms studied could "be placed

into eight serological groups, as follows. Group I: S.

parvus NRRL and _S. par'/us ATCC 12433- Group lis _S. griseus

ATCC 11429, S. griseus ATCC 10137# S. argentaolus ATCC 11009

and S. flaveolua ATCC 3319. Group Ills S. lavendulae ATCC

8664, S. lavendulae MCS and S. rireosus KRRL. Group IV: S.

aureofacicns MCS, S. purpureochr otr.ogen e3 ATCC 3343, S.

vcnezuelao MCS, S>. venezuelaa NRRL, S. cacaoi ATCC 3032

a:id S. erythraous FD 903* Group V: S. fradiae ATCC 10745,

S. fracliae ATCC 11903, S. fradiae NRRL and S. fradiae FD

704. Group Vis S. coelicolor ATCC 10147? S. coelicolor

MCS, S. citrans ATCC 10974, S, gougerotii ATCC 10975? S.

cinnaijioneus ATCC 11874, S. faygroacopious KRRL, H 35, H 46,

and H 48. Group VII: S. albus FD 137, S. albus ATCC 3004,

S. vir i do chra mo genes ATCC 3356, S. viridochromogenes KRRL,

H 9, H 20, H 65 and H 66. Group VlIIs S. antibioticus FD

139, S. antibioticus MCS, S. antibioticus ATCC 8663, S.

ablkocnsis ATCC 12766, S. griseus MCS, S. aibo/Triseu3 FD

705, S. coelicolor FD 461, S. griseolus NTSU, S. anulatus

KRRL, S. lavendulae FD 822, _S. odorifer ATCC 6246, H I ,

25

26

E 2, H 6, H 7, H 8, H 15, H 17, K 27, H 28, H 29, H 30,

H 51, H 32, H 34, H 39, H 40, H 42, H 49, H 53, H 54, H 55,

IT 56, H 57 and H 61. Although these groups were quite

satisfactorily established for purposes of this survey, the

need is realized for further antibody production and the

testing of more species in a more comprehensive scheme.

The study emphasized the importance of environmental

factors in regard to antigen production.

BIBLIOGRAPHY

Books

Breed, Robert S. and others, Sergey'3 Manual of. Determin-ative bacteriology, Baltimore, The " v / i l T i a m s and Vilkins Co., 1957.

Articles

Aoki, X., "Agglutinatorische Untersuchung von Aktinorayzetin," Z-oitschrift Imraunitatsforsch, LXXXVI (March, 1935)* "5TQ-52T".

Bunch, R. L., and Barth, E. F., "Serological Detection of Fermentation Wastes," Nature, CLXXXII (August, 1958), 1680-1683.

Cross, T«, and Spooner, D. P., "The Serological Identifi- ._/ cation of Streptomycetes by Agar Gel Diffusion Tech-niques, "• Journal of General Microbiology, XXXIII ( F e bruar y"7~l 9 6 3)~ * 27*5- 82.

Cummins, C. S., "Chenical Composition and Antigenic Struc-ture of Cell Walls of Corynebactsriuni, Mycotaot eriuia, Nocardia, Actinomyces and Arthrooacter," Journal "of General Microbiology, XXVIII (January, 19~62), 35-50.

Curaiains, C. S., and Harris, H., "Studies on the Cell Wall Composition and Taxonomy of Actinomycetales and Re-lated Groups, " Journal of General Microbiology, XVIII (February, 1958T,*T73-l59.

Ehrlich, J. and others, "Streptorayces venezuelae, n. sp., the Source of Chloromycetin," Journal of Bacteriology, LVI (March, 1948), 467-477.

Eriokson, D., "On the Nomenclature of the Anaerobic Actino-myeetos," International Congress for Microbiological Procedures, 3rd Congress, New York (1940), 192-193.

Guthrie, R. X., Roach, A. W., and Ferguson, J. K., "Serol-ogy of Aerobic Aquatic Actinomycetes. I. Factors In-volved in Antiserum Production and In Vitro Antigen-Antibody Reactions," Journal of Bacteriology, LXXXIV (August, 1962), 313-3TH

27

28

Gut hi .1 e, R. K., Roach, A. W., and Ferguson, J. K., "Serol-• ' cgy of Aerobic Aquatic Aetinomyeetes. II. Effects of

Growth Medium and Culture Age on Antigen Levels," Journal of Bacteriology, LXXXVI (August, 1963). 173-178. ~ ^ ~

Kwapinski, J. B., "Antigenic Structure of Actinomycetales. VI. Serological Relationships Between Antigenic Frac-tions of Actinomyces and Nocardia," Journal of Bact.criolo jy7^X}QCVI (July, 1963) , 1T5-ISFT

llwaplnski, J. B., "Cytoplasmic Antigen Relationships Among :x-tii 3 Actinomycetales," Journal of Bacteriology* LXXXVI I (May, 1964), 1234-1237.

Kwapinsr:i, J. B., and Snyder, M. L., "Antigenic Structure and Serological Relationships of Mycobacteriurru Actinomvccs, Streptococcus, and Diplococcus,,! Journal of Bacteriology; LXXXII~(March, l9ol), 632-639.

Ludwig, E. H., and Hutchinson, W. G,, "A Serological Study of Selected Species of Aetinomyeetes," Journal of Bacteriology, LVIII (January, 1949)» 89-101.

Okarai, Y., "A Study for Classification of Streptomyces. On the S. lavendulae Group With Reference to its immuno-logical Properties," Journal of General Microbiology, II (January, 1956), 63-75.

Ouchtcrlony, 0., "In Vitro Method For Testing the Toxin-Producing Capacity of Diphtheria Bacteria," Acta Pathologica at Mierobiologioa Scandinavica, XXV "(January, 1948)*, 18^190.

Slack, J. M. and others, "Studies With Microaerophilic Actinomycetes. II. Serological Groups as Determined by the Reciprocal Agglutinin Adsorption Technique," Journal of Bacteriology, LXX (March, 1955)» 400-404.

Slack, J. M., Winger, A., and Moore, D. W., "Serological Grouping of Actinomyces by Means of Fluorescent Antibodies," Journal of Bacteriology, LXXX.II (January, 1961), 54-65.

Wakoman, S. A., "Species Concept Among the Actinomycetes v/ith Special Reference to the Genus Streptomyces," Bacteriological Reviews, XXI (January," 1957), 1-24.

Williams, A. M,, and McCoy, E., "Degeneration and Regen-eration of Streptomyces griseus," Applied Mlcrc'• IqIqat, I (March, 1953), 307-313.

29

Wo&eaouse, R« P., ar.d Backus, E. J., "Gel Diffusion as an Aid to Streptoniycetf Taxonomy," Bacteriological Pro-ceedings, LVII (X957)j 43.

Unpublished Materials

Kutcheson, Josephine, "A Serological Study of the Antigenic fractions of the Aerobic Actinomycetes," unpublished master1s thesis, Department of Biology, North Texas State University, Denton, Texas, 1964.