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Identification of fungi by the fluorescent antibody technique Johnson, Gary Clifford 1972

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IDENTIFICATION OF FUNGI BY THE FLUORESCENT ANTIBODY TECHNIQUE BY GARY CLIFFORD JOHNSON B.S.F., The U n i v e r s i t y of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE MASTER OF FORESTRY DEGREE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1972 In p re sen t ing t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, 1 agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and s tudy. I f u r t he r agree tha t pe rmiss ion for ex t ens ive copying o f t h i s t h e s i s fo r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada 7 i i ABSTRACT The fluorescent antibody technique was investigated as a means of f a c i l i t a t i n g the recognition and identification of the fungal components of a western red cedar (Thuja plicata Donn) heartwood flora i n s i t u . Fungi isolated from the heartwood were grown i n bulk and prepared for two different injection t r i a l s . In one t r i a l the antigen was the particulate matter of the c e l l that could be centrifuged into a pellet after the hyphae were destroyed by a tissue grinder. In the second t r i a l the hyphae were ground up and ultrasonically disintegrated. Only the cytoplasm and small wall fragments were retained for injection. After antisera collection the indirect staining method was employed. Unlabeled specific antiserum was layered over the antigen, allowed to incubate and washed off before fluorescent sheep anti-rabbit globulin was applied to form the f i n a l layer. A l l attempts to detect specific antibodies to the fungal antigens failed. This was probably due to not using antigens rich enough i n protein. Successful production of precipitating antibodies to fungal antigens has been shown by other workers to be more l i k e l y when the antigen contains greater than 10 milligrams of protein per m i l l i l i t e r of antigen solution. It has also been found that in some cases fresh antigen must be prepared for each diffusion test and injection as i t can't be preserved even at -20°C. It i s hoped that i f fresh, high protein antigen were to be used this study could be successfully completed. i i i CONTENTS Page ABSTRACT 1 1 CONTENTS 1 1 1 FIGURES i v ACKNOWLEDGEMENTS v Introduction 1 L i t e r a t u r e Review 5 H i s t o r i c a l Perspective 5 F.A.T. Uses 8 Problems encountered with the F.A.T 12 Methods and Materials 14 Growth of Fungi i n Bulk 14 Antigen Preparation 15 In j e c t i o n 17 Bleeding and Antiserum 17 Antibody Detection 18 Indi r e c t Staining Method 20 Microscopy 23 Results 23 Discussion 23 Glossary 28 References 29 i v FIGURES Figure Page 1 Thuja plicata Heartwood Zones and Fungi Commonly Present 2 2 Direct Fluorescent Antibody Methods 6 3 Indirect Fluorescent Antibody Methods 7 4 Typical Ring Precipitin Test With Controls 19 5 Diffusion Plate Pattern 20 6 Hypothetical Explanation of Extra Sensitivity of Indirect Staining 21 7 Comparison of Direct and Indirect Methods 22 8 Typical Staining- T r i a l With Controls for One Fungus 23 V ACKNOWLEDGEMENTS I wish to thank Dr. Bart van der Kamp, Assistant Professor of Forestry, for his helpful criticism and encouragement during the course of this project and in the preparation of this thesis. Votes of thanks are also due to Dr. J.J. Stock, Associate Professor of Microbiology, for his worthwhile suggestions for the second t r i a l and to Dr. R.J. Hudson, Assistant Professor of Animal Science, for helping me to understand and use serological techniques. To my wife, Noni, I extend my deepest appreciation for her constant interest and encouragement. 1 IDENTIFICATION OF FUNGI BY THE FLUORESCENT ANTIBODY TECHNIQUE Introduction Cross-sections of butts of o l d (approximately 200 years old) western red cedars (Thuj a p l i c a t a Donn) commonly e x h i b i t a c e n t r a l column of decayed m a t e r i a l surrounded by several zones of v a r i o u s l y stained heartwood (see Figure 1). The decay column i s enclosed by a zone of brown stained, but sound wood. Outside of t h i s l i e s a b e l t of red-brown or pink heartwood which i n turn i s surrounded by a straw colored zone. Repeated i s o l a t i o n of fungi from the heartwood of Thuja p l i c a t a has demonstrated that the outer straw coloured heartwood may be considered s t e r i l e but that the red-brown and brown stained heartwood zones are colonized by c h a r a c t e r i s t i c fungi (van der Kamp, unpublished). Such i s o l a t i o n work i s based on the assumption that the l i v i n g organisms present i n the heartwood w i l l grow on the media used f o r i s o l a t i o n . Furthermore, i n cases where two or more organisms occur together i n the wood i t i s not uncommon for one of them to grow much f a s t e r on the i s o l a t i o n medium and i n fact eliminate the other. Thus, the r e l a t i v e frequencies of two or more organisms i n the heartwood cannot 2 red-brown zone decay colui brown zone straw zone heartwood zone VI QQ st e r i l e Figure 1 Thuja plicata Heartwood Zones and Fungi Commonly Present be determined by the frequency of isolation. Furthermore, in the case of western red cedar heartwood i t has proven impossible to separate the various fungal isolates by microscopic examination of variously stained sections of heartwood. Fluorescent antibody staining offered the best po s s i b i l i t y to overcome both these problems. The as yet unidentified fungi isolated from the stained heartwood of Thuja plicata may be viewed as forming stages in a succession of fungi leading to decay. Tests have shown that a l l the fungi commonly isolated from the red-brown and brown stained heartwood zones are able to break down thujaplicin,a potent fungicide naturally occurring i n the heartwood of western red cedar. Naturally dark stained pieces of heartwood are much more susceptible to decay than light colored heartwood. Test blocks of straw colored heartwood inoculated with fungi isolated from the stained heartwood zone show a rapid decrease 3 in thujaplicin concentration. In this instance, however, there i s no parallel decrease i n decay resistance. This suggests that the breakdown product of thujaplicin may be as toxic as thujaplicin i t s e l f . This in turn could mean that the further steps i n the breakdown of thujaplicin are mediated by organisms not commonly isolated. One or more of the occasional isolates could actually be common i n the wood and essential for the loss of decay resistance. Fluorescent antibody staining, a technique used mainly in medicine, appeared to offer an effective means of approaching this problem. According to Nairn (1962), the fluorescent antibody technique (hereafter known as F.A.T.) " . . . i s perhaps at i t s most useful when employed to determine the numerical or spatial distribution of microorganisms among a mixed population."^" Thus,the F.A.T. was investigated as a means of overcoming two specific problems. The F.A.T. i s based on the principle that specific antiserum can be precipitated when i t contacts i t s homologous antigen. This fluorescent complex could be readily viewed under ultraviolet stimulation. It i s important to note that the antigen(s) must be unique to one fungus i f a specific stain i s to be produced. This technique has the advantages of high spe c i f i c i t y , sensitivity and rapidity (once developed). The high degree of specificity i s inherent in the antibody which i s produced i n response to the entry of a foreign, high molecular weight compound into an animals Ideally this antibody globulin w i l l complex only with the antigen (usually protein or polysaccharide) to which i t has been prepared. Tagging ^ Nairn, R.C. 1962, Fluorescent protein tracing. S. Livingstone, London, p. 231. 4 antibody with some fluorochromes doesn't significantly affect i t s biological activity. In addition to being specific the F.A.T. i s also sensitive. It is possible by this technique to detect as l i t t l e as -18 1 x 10 grams of dye (Eren and Pramer, 1966) and to identify a single bacterial c e l l that contains 5 x 10 ^  milligrams of nitrogen (Coons, 1956). Thomason et a l ; (1956) report no d i f f i c u l t y i n locating specifically stained bacteria i n mixtures containing ratios of contaminants to specific cells as high as 10^:1. Once the technique has been developed i t can be used quickly to screen a large number of samples. Staining and microscopic examination can be completed in as l i t t l e as one hour. Also, the presence of contaminants i s of no concern i f proper controls are maintained and the technique i s equally useful for viable and nonviable organisms (Cherry et,al.,1965). 5 L i t e r a t u r e Review H i s t o r i c a l Perspective In the period from 1930-42 several attempts were made to tag antibodies with azo-dyes (Reiner, 1930) and l a t e r with fluorochromes (Creech and Jones, 1941). The former res u l t e d i n low s e n s i t i v i t y while the l a t t e r complexes were deleterious to the antibody. U n t i l the e a r l y 1940's most workers had been preoccupied with studying the e f f e c t of various r a d i c a l s on the immunological a c t i v i t y of antibodies. Coons, Creech and Jones (1941) and Coons, Creech, Jones and B e r l i n e r (1942) were the f i r s t to study the tagged antibody from the point of view of using i t as a t r a c e r . They used f l u o r e s c e i n isocyanate-conjugated antibody to trace pneumococcus soluble polysaccharide antigen i n t i s s u e sections of mice i n f e c t e d with pneumococcus. In the period from 1950-51 a series of papers on the F.A.T.'s t e c h n i c a l aspects were published by Coons and co-workers. The f i r s t (Coons and Kaplan, 1950) d e t a i l e d the synthesis of the fluorochrome f l u o r e s c e i n isocyanate, and i t s conjugation to the antiserum along with i n s t r u c t i o n s f o r microscope f i l t e r i n g systems, for t i s s u e powder removal of n o n s p e c i f i c s t a i n i n g and for Immunological proofs of s p e c i f i c i t y of s t a i n i n g . This p u b l i c a t i o n along with four others (Coons, Leduc and Kaplan, 1951; Coons, Snyder, Cheever and Murray, 1950; H i l l , Deane and Coons, 1950; Kaplan, Coons and Deane, 1950) es t a b l i s h e d the technique's p r i n c i p l e s , b a s i c mechanics and f e a s i b i l i t y . 6 Until the mid-1950's F.A. work was being done exclusively by the direct staining method. In this case the fluorochrome i s conjugated directly to the antibody (see Figure 2). This conjugate i s able to I A n t i b o d y F l u o r o c h r o m e L a b e l e d y . j , a n t i b o d v * P? - tt - - £ 5 3 * X X A n t i g e n L a b e l e d S p e c i f i c a n t i b o d y f k i o r e s c e n c e ( + ) c o * Y* — R P X Y:K A n t i g e n H e t e r o l o g o u s S p e c i f i c a n t i b o d y f l u o r e s c e n c e ( — ) f l u o r e s c e n c e ( — ) X A n t i g e n A n t i b o d y A n t i g e n - a n t i b o d y L a b e l e d c o m p l e x a n t i b o d y Figure 2. Direct Fluorescent Antibody Methods (Kawamura, 1969) 7 adsorb onto the homologous antigen to form a v i s i b l e s p e c i f i c s t a i n when excited by u l t r a v i o l e t i r r a d i a t i o n . Experience has shown the d i r e c t technique to be more s p e c i f i c than the i n d i r e c t , though the reason for t h i s i s not known. Deacon et a l . (1957) varied the procedure to i n d i r e c t l y detect antibodies i n unlabeled test sera. In the i n d i r e c t (also known as sandwich or an t i g l o b u l i n ) method the homologous antibody i s not tagged. However, antibody to normal yG-g l o b u l i n (prepared i n a d i f f e r e n t animal) i s labeled and i t i s added to the homologous antigen-antibody complex r e s u l t i n g i n s p e c i f i c fluorescence (see Figure 3). I f there i s s p e c i f i c fluorescence, one e . (J + . i n — - fl* . A n t i b o d y t o F l u o r o c h r o m e L a b e l e d a n t i b o d y t o n o r m a l y G - g l o b u l i n n o r m a l y G - g l o b u l i n • PP + -aJ - [S3 + «« — CS3[ A n t i g e n A n t i b o d y A n t i g e n - a n t i b o d y L a b e l e d a n t i b o d y t o S p e c i f i c c o m p l e x n o r m a l y G - g l o b u l i n f l u o r e s c e n c e ' (+> Figure 3 Indirect Fluorescent Antibody Methods (Kawamura, 1969) can i d e n t i f y the antigen when the nature of the primary antibody (the one that i s not tagged) i s known, or v i c e versa. See methods for d e t a i l s of the i n d i r e c t technique. Numerous workers have investigated the techniques devised by Coons and co-workers and found them to be u s e f u l . Many minor and a few major refinements have been made. One important advancement i n fluorescent antibody a c c e p t a b i l i t y was made by Riggs e_t a l . (1958) who 8 described the synthesis and use of isothiocyanate d e r i v a t i v e s of f l u o r e s c e i n (yellow-green fluorescence) and tetraethylrhodamine B (orange-red). Previously most workers had used unstable f l u o r e s c e i n isocyanate whose preparation required the use of d i f f i c u l t - t o - h a n d l e gaseous phosgene. The isothiocyanate d e r i v a t i v e was prepared with l i q u i d thiophosgene and was a stable product that could be stored f o r months and used to l a b e l proteins with no f u r t h e r chemical manipula-tions (unlike the isocyanate). Thus, with the development of p r o t e i n -l a b e l i n g dyes that were stable enough to be marketed commercially and e a s i l y handled, a major d i f f i c u l t y of F.A. technology was overcome. F.A.T. Uses I n i t i a l l y Coons et a l . (1942) devised the F.A.T. to i n v e s t i g a t e medically-oriented problems. This continues to be the major use, e s p e c i a l l y i n a diagnostic capacity. The technique has been used to i d e n t i f y and study v i r a l , b a c t e r i a l , protozoal, helminthic, fungal, amoebal, mycoplasmal and animal t i s s u e antigens (Nairn, 1962). I t has also been used for the microscopic i d e n t i f i c a t i o n of i n j e c t e d f o r e i g n antigens; i n t r i n s i c antigens, p r o t e i n s , enzymes, and hormones, and s p e c i f i c antibodies (Mellors, 1959). 9 Some F.A.. te s t s have proven so u s e f u l they are conducted r e g u l a r l y i n diagnostic medical labs. Cherry and Moody (1965) have indicated that they consider the following to be the most important diagnostic a p p l i c a t i o n s of the F.A.T. i n bacteriology: i d e n t i f i c a t i o n of Group •A Streptococci, Treponema pallidum (Schaudinn and Hoffmann) Schaudinn, Ne i s s e r i a gonorrhoeae (Zopf) Trevisan, Coasnebacterium diphtheriae (Kruse) Lehmann and Neumann, Bordetel-la. p e r t u s s i s (Pribram) Ldpez, Salmonella typhosus White, serogrouping of eh't'eropathogenic^Echerichia  c o l i (Migula) C a s t e l l a n i and Chalmers and S h i g e l l a . As a d i r e c t r e s u l t of the success of researchers i n medical bacteriology, workers i n other f i e l d s are r e a l i z i n g the F.A.T. 1s p o t e n t i a l and are t r y i n g to adapt i t to t h e i r p a r t i c u l a r needs. Several researchers have used F.A.T. to help overcome problems of working with s o i l microorganisms. F.A. s t a i n i n g aids recognition of the organism d i r e c t l y i n i t s s o i l h a bitat. D i f f e r e n t problems are being attacked with the F.A. as indi c a t e d by the following quotations: Relationships between microorganisms i n s o i l are revealed by i n d i r e c t methods only, and i t i s seldom possible to e a s i l y d i f f e r e n t i a t e organisms of p a r t i c u l a r i n t e r e s t from the myriad of others that comprise the m i c r o b i a l population of s o i l . 2 Even though i t may be shown that a p a r t i c u l a r species e x i s t s i n a s o i l , c u l t u r a l experiments cannot prove whether the organism i s vegetative or dormant, whether i t occurs as sin g l e c e l l s or i n colony form, or whether i t i s associated with any one type of p a r t i c l e , or micro-habitat, i n the s o i l . - * 2 Eren, J . and D. Pramer, 1966. S o i l S c i . 100(1), p. 39. 3 H i l l , I.R. and T.R. Gray, 1967. J . Bact. 93(6), p. 1888. 10 Schmidt et a l . (1968) studied species of Rhizobium i n the s o i l but had d i f f i c u l t y with nonspecific adsorption by s o i l p a r t i c l e s . Paton (1960) showed the r e l a t i o n s h i p between Pseudomonas and the s o i l free root surface of c l o v e r . F.A. studies of fungi i n the s o i l have been able to detect A s p e r g i l l u s flavus Link ex F r . i n mixed cu l t u r e (Schmidt et_ al_., 1962 and 1965). Eren and Pramer (1966) used F.A.T. to i d e n t i f y and measure the abundance i n s o i l of Arthrobotrys coniodes Drechsl. a nematode-trapping fungus. The F.A.T., when applied to fungi, has been mainly f o r the purpose of diagnosing and i d e n t i f y i n g pathogens of man (Kaplan and Kaufman, 1961>. The technique's advantages f o r fungal i d e n t i f i c a t i o n include r a p i d i t y and s e n s i t i v i t y as compared with conventional c u l -t u r a l methods. Immuno-specific s t a i n i n g enabled Kaplan and Gonzales Ochoa (1960) to f i n d fungus elements of Sporotrichum schenckii (. Helet. and Perk.) de Beurm and Goug. i n smears from a human l e s i o n which proved negative by c u l t u r a l techniques. This i s one i n d i c a t i o n of i t s s e n s i t i -v i t y though a study (Porter ejt a l . , 1965) of t i s s u e s i n f e c t e d with Histoplasma capsulatum Darling and Blastomyces dermatitid-is G i l c h r i s t and Stokes indi c a t e d that h i s t o p a t h o l o g i c a l techniques were more e f f e c -t i v e than F.A.T., and both were bettor than c u l t u r l n g . This l a t t e r paper also points out the great d i v e r s i t y of r e s u l t s from studies i n v o l v i n g d i f f e r e n t methods and organisms. In other cases Gordon (1958) used F.A.T. to d i f f e r e n t i a t e Candida  albicans (Robin) Berkh. from other Candida species and from yeasts of other genera . Al-Doory and co-workers (1963) were able to d i s t i n g u i s h between Cladosporium c a r r o n i i Trejos and C_. bantianum B o r e l l i . Histoplasma capsulatum yeast c e l l s have been s e l e c t i v e l y stained i n the 11 presence of Blastomyces d e r m a t i t i d i s by Gordon (1959). Conjugated an t i s e r a to Cryptococcus neoformans (Sanfelice) V u i l l . has been tested on 96 i s o l a t e s of t h i s organism and on a number (23) of heterologous species (Kase and Marshall, 1960). These workers reported s p e c i f i c s t a i n i n g of 95 i s o l a t e s of C_. neoformans and none of the other species. In 1962 May used F.A.S. to study and i d e n t i f y the s i t e s of c e l l wall extension of Schizosaccharomyces pombe Lindner. Two years l a t e r Goos and Summers used a s i m i l a r technique to study the morphogenesis of two fungi. They found that wall material of the Candida albicans parent c e l l i s incorporated i n t o the wall of daughter c e l l s or hyphal walls. Conidia of Fusarium oxysporum f . cubense (E.F. Smith) Snyder and Hansen stained i n t e n s e l y but germ tubes emerging a f t e r s t a i n i n g d i d not. In 1964 Paton made a s i g n i f i c a n t c o n t r i b u t i o n to a i d plant patholo-g i s t s with use of the F.A.T. He described the preparation of plant t i s s u e f o r examination and included valuable information on c o n t r o l of host autofluorescence and nonspecific s t a i n i n g . In s p i t e of t h i s a i d F.A. studies of plant pathogenic and saprophytic fungi are not common. Beno and A l l e n (1964) used immuno-fluorescent s t a i n i n g to r a p i d l y i d e n t i f y a s p e c i f i c l i n e of germinating Puccinia sorghi (Schw.) uredospores. The presence of POlyporus tomentosus i n mixed c u l t u r e s , s o i l , and in f e c t e d pine roots was detected by F.A. (Kumar and Patton, 1964). In 1965 some fa c t o r s a f f e c t i n g the a n t i g e n i c i t y of the mycelia of 3 species of Phytophthora F r . were reported by B u r r e l l e_t a l . They found the.type and concentration of nitrogen source, the amount of inoculum, and the age of mycelium g r e a t l y a f f e c t e d the antigenic e f f i c a c y of the preparations. Amos and B u r r e l l the following year found the F.A.T. to be the most u s e f u l technique (in comparison with agglutination and g e l d i f f u s i o n ) i n d i f f e r e n t i a t i n g among eight 12 species of Ceratocysis. In another study a fluorescent antibody reagent for Botrytis cinerea Pers ex Fr. was able to distinguish spores and mycelium of that organism from those of three others (Preece and Cooper, 1969). Price (1970) was unable to produce a fluorescent antibody reagent specific for Sphaerothecia pannosa conidia. A large number of his comparative test species fluoresced along with S_. pannosa while other species did not. Problems Encountered with the F.A.T. The problems that can and do arise with the F.A.T. are manifold but most can be controlled to some extent. One problem i s auto-fluorescence of the organism being studied, of other contaminating members and of the organism's substrate or host. Tissue auto-fluorescence i n particular masks the low intensity immune-specific reactions. Autofluorescence can be minimized by prompt use of cut sections and i s partly removable by f i l t e r i n g and counterstaining. Counterstains are useful i f b r i l l i a n t l y fluorescent and i f they have an emission maximum well separated from that of the fluorescent conjugate (Hall and Hansen, 1961). Another problem i s nonspecific fluorescence which may result from the unreacted fluorochrome substance, from antibody with too strong a negative charge, from improper tissue fixation, or from letting the specimen dry out during the staining procedure (Kawamura, 1969). Nonspecific staining may be reduced or controlled by purification and fractionation of antigenic components, dilution of antiserum, improved tissue 13 preparation techniques, removal of unconjugated fluorochrome and adsorption with tissue powders or homogenates. Thirdly, i t i s possible to obtain false positive staining reactions due to the antibody already being present in experimental animals after natural infection. This w i l l be identifiable i f controls are taken unless animals are naturally infected after normal serum i s drawn. Fourthly, the same antigens i n heterologous organisms may result in cross-staining. F i f t h l y , fluorescent artifacts may be produced by manipulat-ing (fixing, etc.) the section to be observed. Once stained, conditions can be aLtered such that the antigen-antibody complex can be broken down. In addition Gooding (1966) points out that in most cases i t i s necessary to work with fungal extracts which consist of multiple antigen systems which are d i f f i c u l t to analyze. He successfully isolated and used a crude DNA fraction from Fomes annosus Fr. i n double diffusion tests. Finally, as Kaplan and Kaufman (1961) point out, there i s a need for standardization of reagents, procedures and equipment so results w i l l be more comparable.- Undoubtedly many anomalies and discrepancies tp date are due to variation in immunizing strain, immunization schedule, the fluorochrome used in labeling, host reactivity, antibody t i t e r s , optical equipment used, etc. 14 Methods and Materials Growth of Fungi in Bulk Three fungi commonly isolated from western red cedar heartwood were chosen to be studied. They were a Kirschsteiniella-like species, a Verticillium-like species and another unidentified species, each respectively known in the U.B.C. Forest Pathology Collection as C4D4, VIQQ and IVTB. These fungi were transferred from storage slants to petri plates containing malt agar where they were allowed to grow u n t i l approximately two-thirds of the plate was covered with an actively growing culture. Then the agar embedded mycelial mat was transferred into a Waring Blendor with 150 m i l l i l i t e r s of sterile water and ground up for 15 seconds. Ten m i l l i l i t e r s of this slurry were added to each of ten 250 m i l l i l i t e r shaker flasks containing 50 m i l l i l i t e r s of culture medium. This was repeated for each of the three fungi. The growth medium was as follows: 15 0.75 g of MgS04*7H 20 0.75 g of KH 2P0 4 10.0 g of yeast extract 20.0 g of glucose 1000.0 ml of water The two filamentous fungi (C4D4 and IVTB) grew for 9 to 16 days u n t i l s u f f i c i e n t mycelium had been obtained. VIQQ, unlike the other two fungi, grew slowly i n a y e a s t - l i k e fashion i n l i q u i d c u l t u r e and as a r e s u l t required 27 days to produce enough fungal m a t e r i a l to harvest. The fungi were then centrifuged ( S o r v a l l SS-1 Angle Centrifuge) and washed three times i n phosphate buffered s a l i n e (here-r a f t e r known as P.B.S.) (Cherry and Moody, 1965). Antigen Preparation Numerous attempts were made to disrupt the fungal c e l l s to expose antigenic m a t e r i a l while at the same time minimizing denaturation. F i r s t a Pyrex t i s s u e grinder with a tolerance of 0.005 to 0.007 inches was used, sometimes i n combination with glass chips. I t d i d not prove s a t i s f a c t o r y as only about 20 percent of the my c e l i a l c e l l s and s i g n i f i c a n t l y l e s s of the y e a s t - l i k e c e l l s were broken up. The second attempt involved f r e e z i n g the organism i n l i q u i d nitrogen and grinding with mortar and p e s t l e . In a s i m i l a r case the fungi were frozen i n l i q u i d nitrogen and struck repeatedly i n a c h i l l e d compression c y l i n d e r . The l a t t e r attempts weren't any more su c c e s s f u l than the f i r s t method and were much more d i f f i c u l t to complete. The fourth time, the hyphal fungi were ground up by the Pyrex t i s s u e grinder and 16 then subjected to ultrasonic disintegration (Bronwill Biosonik III) in short (12 second) bursts f° r a total time of one minute while being cooled by an ice-water bath. Microscopic observation of the treated tissue showed the hyphal fungi to be macerated but such was not the case with VIQQ u n t i l i t had been subjected to a further one minute of treatment. This f i n a l and most successful treatment ruptured at least 50 percent of the c e l l s . In the f i r s t injection sequence ( t r i a l one) the antigen used was basically the particulate matter of the c e l l . Any c e l l component that could be centrifuged into a pellet at a R.C.F. of 6500 i n 20 minutes was used. The fungus was macerated with the tissue grinder only. . Fungal particulates were suspended at five milligrams per m i l l i l i t e r in P.B.S. and frozen (-20°C) i n three m i l l i l i t e r lots. To this suspension an equal amount of Freund's complete adjuvant was added at the time of injection to .enhance antigenicity. In the second t r i a l the antigen was prepared by f i r s t grinding the fungus and then subjecting i t to ultrasonic disintegration. The heavy c e l l wall components were centrifuged and discarded. The supernatant containing cytoplasm and very small wall fragments were diluted to five.milligrams per m i l l i l i t e r . An equal volume of Freund's complete adjuvant was added and mixed vigorously just before injection. Prepared antigen, i n P.B.S., was stored at -20°C in small v i a l s . 17 Injection Young, three to four pound rabbits were injected subcutaneously on the scapular region of the back using st e r i l e disposable syringes. The syringes had a capacity of three m i l l i l i t e r s with one and one-half inch long, 21 gauge needles. Each of the three fungi were injected into two rabbits for the f i r s t t r i a l and into three rabbits for the second t r i a l . On injection day each rabbit received a total of one m i l l i l i t e r of emulsion. For the f i r s t t r i a l each rabbit received a single injection once a week while the rabbits i n the second received half a m i l l i l i t e r i n each of two places every two weeks. Each t r i a l had four injection days. When two injections were given to each rabbit on. one day they were at least two inches apart. The Freund's sometimes caused open wounds which were quickly treated 4 with Cicatrin antibiotic powder and the subsequent injection did not contain the adjuvant. Bleeding and Antiserum Two weeks after the last injection the rabbits were bled from the midvein of the ear. Fifteen to 20 m i l l i l i t e r s of blood were collected from each animal and l e f t overnight i n a cooler 4°C for the clot to form. Antiserum was decanted from the clot and centrifuged to remove any large debris. A small amount (1.5 m i l l i l i t e r s ) of raw antiserum was immediately frozen to -20°C. The remainder of the antiserum was cross-adsorbed with heterologous antigens to remove 4 Cicatrin, Calmic Limited, Toronto 18 n o n s p e c i f i c antibody. Then the remainder was divided i n t o small v i a l s and frozen (-20°C) u n t i l used. Antibody Detection V i s i b l e p r e c i p i t a t i o n or clumping of p a r t i c l e s occurs with most antigens as a r e s u l t of the multivalent (many bonding points for antibody) antigen and b i v a l e n t antibody forming aggregates or l a t t i c e s of antigen and antibody. The two basic antigen-antibody reactions are the p r e c i p i t i n i f the antigen i s i n a soluble form and ag g l u t i n a t i o n i f the antigen i s p a r t i c u l a t e . These reactions allow detection and q u a n t i t a t i v e estimation of antigens or antibodies i n s o l u t i o n and t i s s u e s (Kabat, 1968). The a g g l u t i n a t i o n r e a c t i o n was used to test f or antibody prepared to the p a r t i c u l a t e antigen of the f i r s t t r i a l . A p r e r e q u i s i t e fo r the a g g l u t i n a t i o n test i s a homogeneous suspension. Therefore only the small w a l l fragments of the 'hyphal fungi could be used. A f t e r low speed c e n t r i f u g a t i o n to eliminate the l a r g e r w a l l pieces the smaller ones were driven i n t o a p e l l e t and washed s e v e r a l times i n P.B.S. This was the antigen used i n the passive haemagglutination t e s t as described by Campbell et al.(1964). In t h i s procedure f r e s h sheep red blood c e l l s were washed and then tanned with tannic a c i d . Then they were coated with the antigen by mixing together and suspended i n normal serum and s a l i n e . F i n a l l y the coated red blood c e l l s were added to s e r i a l d i l u t i o n s of the serum, shaken and allowed to stand at room temperature f o r three to four hours. A p o s i t i v e r e a c t i o n was i n d i c a t e d by a compact granular a g g l u t i n a t i o n or a d i f f u s e f i l m of agglutinated c e l l s covering the bottom of the tube 19 while a negative one appeared as a heavy ring of cells or discrete smooth button of cells in the center of the tube. The yeast phase of the dimorphic fungus, VlQQ, was studied by the tube agglutination method of Cozad (1958). To se r i a l dilutions of serum (0.5 m i l l i l i t e r s ) in P.B.S. was added 0.5 m i l l i l i t e r s of the antigen suspension and the tubes were shaken vigorously. The tubes were immediately refrigerated (4°C) and l e f t overnight. The degree of agglutination was indicated by the size of c e l l aggregates and turbidity of supernatant. Ring and gel double diffusion tests were used to detect anti-body to the cytoplasmic and fine fragments of fungi used i n the second t r i a l . The ring or int e r f a c i a l test involved carefully over-laying antiserum with a solution of antigen such that a sharp liqu i d interface was formed. Antibody can be detected in amounts as small as one microgram of protein. An example of the test including controls i s given i n Figure 4 . A positive result (as i n Key Tube Number 1 2 3 4 5 B = buffer Top Layer B B Ag B Ag Ab = antibody Bottom Layer Ab Ag N N Ab Ag = antigen Observed Reaction - - - - + N = normal serum Figure 4 Typical Ring Precipitin Test With Controls Figure 4) i s indicated by a fine line or ring of precipitated particles at the interface of the two complementary solutions while a negative one has no ring. The gel diffusion test was also used to detect antibody in the second t r i a l . In this case concentrations of antigen and antibody 20 d i f f u s e toward each other and i f o p t i m a l they form v i s i b l e bands of p r e c i p i t a t i o n i n the s e m i s o l i d medium (Di f c o Noble Agar). Ouchterlony's double d i f f u s i o n method (as described by Campbell et al., 1964) was used. This t e s t r e s o l v e s components i n mixtures. The number of bands i n d i c a t e s the minimal number of antigen-antibody systems present. .Figure 5 shows the p a t t e r n of a d i f f u s i o n p l a t e . i n n e r a n t i g e n w e l l antiserum F i g u r e 5 D i f f u s i o n P l a t e P a t t e r n I n d i r e c t S t a i n i n g Method The i n d i r e c t method was s e l e c t e d r a t h e r than the d i r e c t f o r s e v e r a l reasons. According to Kawamura (1969) the i n d i r e c t i s f i v e to ten times as s e n s i t i v e . One e x p l a n a t i o n f o r the d i f f e r e n c e s i n s e n s i t i v i t y i s the a d d i t i o n a l combining s i t e s o f f e r e d by the antibody molecule sandwiched between the antigen and tagged a n t i - r a b b i t serum when using the i n d i r e c t method ( N a i r n , 1962). In Figure 6 only three 21 Figure 6 Hypothetical Explanation of Extra S e n s i t i v i t y  of Indirect Staining ( N a i r n > i 9 6 2 ) s i t e s i n the antigen are a v a i l a b l e for antibody and these three would be the t o t a l number possible i f using- the d i r e c t method. In the i n d i r e c t method though the second layer i s unconjugated antibody which behaves as an antigen for the conjugated antibody. Thus, i n Nairn's two dimensional model the reactive s i t e s have increased f o u r f o l d . Also, the i n d i r e c t method was chosen as the r e s u l t of a personal communication with Dr. Yasu Hiratsuka who i n d i c a t e d he was having d i f f i c u l t y with the s e n s i t i v i t y of s t a i n i n g from the d i r e c t method and he was switching to the i n d i r e c t F.A.T. i n hopes of improvement. He found that autofluorescence was masking some of his weak immune-specific reactions. Moreover, i t i s possible to buy l a b e l l e d a n t i - r a b b i t g l o b u l i n commercially so i f one uses the i n d i r e c t F.A.T. the conjugation of f l u o r e s c e i n isothiocyanate i s unnecessary. 22 See Figure 7 f o r a comparison of the d i r e c t and i n d i r e c t methods. Stain i n g was i n i t i a t e d using each fungus fixed to a s l i d e . Fungi f i x e d with ethanol and with heat were dislodged during washing so Haupt's adhesive with phenol was used. Eventually i t was planned to study the fungus i n wood so each fungus, i n pure c u l t u r e , was s t a r t e d growing on western red cedar heartwood. The inoculated samples would allow a more r e a l i s t i c t r i a l of the s t a i n i n g technique, yet with the fungus known. At the same time i t would provide a place where the "bugs" (for example, autofluorescence of wood) could be worked out. Once the tis s u e was f i x e d to the s l i d e the s t a i n i n g procedure of Goldman (1968) was followed as i n d i c a t e d below: Step I (a) A few drops of unlabeled s p e c i f i c (against each fungus) antiserum were layered over the antigen. (b) S l i d e s were incubated i n a moist chamber for 60 minutes at 25°C. (c) Antiserum drops were shaken o f f . (d) Slid e s were immersed i n a s a l i n e , with occasional a g i t a t i o n f o r 10 minutes. (e) S l i d e s were rin s e d i n tap water and dried. Step II (f) Fluorescent sheep a n t i - r a b b i t g l o b u l i n was applied to the antigen f o r 60 minutes i n moist chamber. (g) S l i d e s immersed i n P.B.S. and agitated o c c a s i o n a l l y f o r 10 minutes. (h) S l i d e s were rin s e d with water and mounted with buffered g l y c e r o l (P.B.S.: g l y c e r o l = 1:9). Dir e c t F.A.T. In d i r e c t F.A.T. - simpler procedure - more complex procedure - more s p e c i f i c - more s e n s i t i v e - each antiserum must be - only one antiserum must labeled be labeled - uses greater q u a n t i t i e s - uses less s p e c i f i c of s p e c i f i c antiserum antiserum Figure 7 Comparison of Di r e c t and I n d i r e c t Methods 23 Microscopy Prepared s l i d e s were observed with a L e i t z Ortholux microscope equipped with a high-pressure mercury vapor lamp. The f i l t e r system included a BG12 (4 mm) and UG1 (1 mm) f i l t e r as e x c i t i n g f i l t e r s and a K430 m^ b a r r i e r f i l t e r ( C u l l i n g , 1963). Antigen Homologous Reagent f o r Step I - P.B.S. - normal serum -adsorbed s p e c i f i c antiserum - s p e c i f i c a n t i -serum to C4D4 - s p e c i f i c a n t i -serum to C4D4 Reagent f o r Step II - labeled a n t i g l o b u l i n - labeled " - labeled - labeled - . l a b e l e d normal serum Result Heterologous (VI QQ) s p e c i f i c a n t i -serum to C4D4 - labeled a n t i g l o b u l i n Figure 8 T y p i c a l Staining T r i a l With Controls  For One Fungus Results It was not possible by any of the three methods nor f o r ei t h e r of the t r i a l s to demonstrate the production of antibodies. In addition there was no fluorescent s t a i n i n g when the procedure of Goldman (1968) was c a r r i e d out. Discussion Researchers pioneering the serology of fungi were often f r u s t r a t e d 24 by the extreme variation in the immunizing capacity of fungal antigens. For example, Coons and Strong (1928) and Nelson (1933) were not able to produce antisera, to several species of Fusarium, that was of sufficiently high t i t e r for precipitin tests. However, other Fusarium species produced a high t i t e r easily. Seeliger (1960) reported that no reliable method of immunization had been worked out, as of 1960, for the medically important fungal genus, Cryptococcus. Many experimenters working with fungi have worked with yeast or yeast-like forms or with spore or conidial suspensions which can be treated with techniques similar to those used for bacteria. It i s the hyphal fungi that have proven most d i f f i c u l t to handle. Unfortunately i t was not possible in this study to produce antisera to the hyphal or yeast-like fungi. At the time the f i r s t t r i a l was being attempted the author had only a very cursory understanding of the principles and techniques of serology. As such, two papers (Preece and Cooper, 1969; Eren and Pramer, 1966) successfully applying F.A.T. to fungi were blindly followed with some minor modifications. Cell wall fragments were used as the antigen because i t was the wall of an intact hypha that would be contacted by the conjugated antibody. Gordon stated that "the c e l l wall i s the exclusive site of reactivity in intact cells of most fungus species."^ In spite of not being able to detect antibodies in the f i r s t t r i a l the indirect staining procedure was attempted because of the following statement by Paton:^ 5 Gordon, M.A., 1962. Differentiation and classification of yeasts by the Coon's fluorescent antibody technique, p. 214. 6 Paton, A.M., 1964. J. Appl. Bact. 27(2): 241. 25 On occasion, a serum has been produced which showed no agglutination t i t e r but proved to be of excellent value f o r t h i s technique. The only v a l i d c r i t e r i o n 'of use-fulness i s the s t a i n i n g r e a c t i o n i t s e l f . ^ No s t a i n i n g was observed however so experienced a i d was s o l i c i t e d . Dr. R.J. Bandoni suggested that the c e l l walls of the fungi being studied might be so s i m i l a r that each fungus would cross-adsorb the antibodies f o r the other antigen or, i f not adsorbed, would form a nonspecific s t a i n . Since only the cross-adsorbed a n t i s e r a had been studied i t was p o s s i b l e that antibodies might be present i n the raw a n t i s e r a . This proved negative a l s o . The plan and r e s u l t s of t r i a l one were taken to Dr. J . J . Stock of the U.B.C. Department of Microbiology. He had no experience with F.A.T. but had attempted some fungal serology. He suggested using the cytoplasmic f r a c t i o n as antigen (Hook et a l . , 1967) and i n j e c t i n g i t every two weeks i n two places on the r a b b i t ' s back. Antibodies to the soluble antigen would be easier to detect than they had been i n the f i r s t t r i a l . However, once again no antibodies were detected and no s t a i n i n g observed. Since the second t r i a l an important paper has come to the author's a t t e n t i o n . B u r r e l l , Clayton, G a l l e g l y and L i l l y (1965) studied the f a c t o r s a f f e c t i n g the a n t i g e n i c i t y of the mycelium of three species of Phytophthora. They subcutaneously i n j e c t e d soluble mycelial suspensions of fourteen-day-old cultures i n t o r a b b i t s and f a i l e d to demonstrate the presence of p r e c i p i t a t i n g antibodies. They hypothesized that the preparations were low i n p r o t e i n and therefore antibody production wouldn't be stimulated. B u r r e l l e t - a l . determined the factors that influence the nitrogen content of mycelium and found that the protein 26 content per u n i t weight of mycelium was greatest a f t e r three days of incubation when ammonium s u l f a t e was used at two grams per l i t e r as nitrogen source. Rabbits immunized subcutaneously by t h i s high-protein, soluble antigen y i e l d e d a good p r e c i p i t i n production. I f time had permitted the author would have prepared a cytoplasmic antigen containing ten milligrams per m i l l i l i t e r of p r o t e i n . P r o t e i n concentrations could be measured by the E i u r e t r e a c t i o n or by micro-K j e l d a h l analysis (Campbell et a l . , 1964; Kabat et a l . , 1961). One m i l l i l i t e r of antigen would be subcutaneously i n j e c t e d i n the form of a s o l u t i o n containing 50 percent Freund's adjuvant and 50 percent of the antigen (20 mg of protein/ml) i n P.B.S. Young r a b b i t s would be i n j e c t e d once a week for f i v e weeks (Pepys et al.,1967) with t r i a l bleedings conducted ten days a f t e r the l a s t i n j e c t i o n and weekly thereafter u n t i l a s u f f i c i e n t l y high t i t e r was obtained. The a n t i s e r a would be treated as previously described and s p e c i f i c antibodies detected on g e l double d i f f u s i o n p l a t e s . I f the findings of B u r r e l l et a l . (1965) are generally a p p l i c a b l e i t may be necessary to prepare a f r e s h antigen for each i n j e c t i o n and each d i f f u s i o n t e s t . These workers were not even able to preserve antigens frozen at -20°C. There was a l o s s of p r e c i p i t a t i n g a c t i v i t y w i t h i n a week. I t would be hoped that the use of a soluble high p r o t e i n antigen and fresh antigen f o r each treatment might r e s u l t i n s p e c i f i c antibody production. However, the antibody formed may not be s p e c i f i c i n which case i t would be necessary to f r a c t i o n a t e the antigen complex u n t i l a s p e c i f i c antigen would produce a s p e c i f i c antibody. 27 Though the results of this project were not encouraging i t i s fe l t that new attempts, keeping Burrell et al.'s (1965) studies i n mind, could be successful. Many years of medical mycology research have shown the technique's f e a s i b i l i t y with pathogenic fungi of mammals. The basic methodology i s available and could be extremely useful i n plant pathology. In addition to the uses envisioned in this study the F.A.T. could be used in such identification situations as for spores on spore traps and for mycorrhizae. Other p o s s i b i l i t i e s include using the technique to study disease development, for example i n structures like cankers. 28 GLOSSARY agglutination reaction - a reaction involving the clumping or aggregating of large particulate antigens (e.g., bacterial cells) by specific antisera; in this case the particles are large enough to be seen under the microscope. antibody - a humoral globulin produced in response to the parenteral introduction of an antigen into an animal; this globulin w i l l react with i t s homologous antigen. antigen - any substance which, when introduced parenterally into an animal w i l l cause the production of antibodies by that animal and which w i l l reaction specifically with those antibodies. conjugated - a state i n which the antibody i s coupled to fluorochrome; synonymous with "tagged1,' fluorochrome - a substance which fluoresces under ultraviolet stimulation and can be conjugated to antibody. F.A. - fluorescent antibody abbreviation; indicates antibody conjugated with florochrome. F.A.T., F.A. staining, immuno-specific staining, immuno-fluorescent staining - terms used interchangeably to denote a staining method using fluorochrome-labeled antibody as specific staining agents for antigen. gel diffusion - antigen and antibody are brought together, through diffusion in. a semisolid, where they precipitate to form vi s i b l e bands. parenteral - other than by way of the intestines. precipitin reaction - a reaction occurring between large antigen molecules and antibody molecules resulting i n the formation of an antibody-antigen precipitate. tissue powder - a preparation used to eliminate nonspecific fluorescence. The powder is usually from the tissue to be stained and i s produced by acetone precipitation of the tissue homogenate. t i t e r - the highest dilution of an antiserum that w i l l show detectable agglutination or precipitin reaction; concentration or activity or potency of a serologically active substance. 29 REFERENCES Al-Doory, Y. and M.A. Gordon, 1963. A p p l i c a t i o n of the fluor e s c e n t -antibody procedures to the study of pathogenic dematiaceous fungi I. J . Bact. 86(2): 332-38. Amos, R.E. and R.G. B u r r e l l , 1966. S e r o l o g i c a l d i f f e r e n t i a t i o n i n Ce r a t o c y s t i s . Phytopath. 57: 32-4. Beno, D.W. and O.N. A l l e n , 1964. Immuno-fluorescent s t a i n i n g f o r i d e n t i f i c a t i o n of P u c c i n i a sorghi germinated uredospores. Phytopath. 54: 872-3. B u r r e l l , R.G., C.W. Clayton, M.E. G a l l e g l y and V.G. L i l l y , 1965. 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