o I-nci...‘ . ‘ ' vc'w “e"(:"" d“. "’ .‘ " ‘ "' ‘ ‘ A cOO-tugt a ’ . .to. . . _ ‘ -.v .. ..‘ u. ,. ‘ ,V,” , _ .. . . u .“ ‘ ~‘.‘,':'.’|‘f‘5 \‘,“‘,'.. .' '1‘. 0“ -'.'.V}. ,' ,‘,. 'J. O ‘ v. ," a, 't " ' ' . K" ‘ " . u :. .. I. .l.“ " _'v|. t.. .. , , ,, ...o..' 4‘. '.' ‘.. . h. ' ... ‘ “. .I . ‘ 1‘ M” < ' . | .1 "g _ '2. , .'.". ' I . I , l . n .‘.| a "' . a v.“ . e. H. . J: renal-0.. .'*..b.-.'.‘.|‘.. .‘I | ...‘..,'. o... | .‘ ‘.IV I .. STAPHYLOCOCCALACID PHOSPHATASE: A - . ANTIGENICITY,ASSAY-OF-ITS Amman, a r_- AND PROTECTION STUDIES- Thesis for the Degree of, M. S. - MICHIGAN STATE UNIVERSITY BARBARA K. BODNER 1972 . LIBRA RY Michigan L are University swims av " HUAG a SDNS‘ BUDK BINDERY INC. LIBRARY BIN DEBS . ABSTRACT STAPHYLOCOCCAL ACID PHOSPHATASE: ANTIGENICITY, ASSAY OF ITS ANTIBODY, AND PROTECTION STUDIES BY Barbara K. Bodner Purified staphylococcal acid phosphatase was pre- pared from Staphylococcus aureus (PS 55) according to Malveaux and San Clemente (1969). In a series of ten injections given every other day, three New Zealand White rabbits were inoculated with the purified enzyme, mixed 1:1 with Freund's complete adjuvant. The first five in- jections were given in the foot pad and the last five subcutaneously in the cervical region. One rabbit received a total of 25 units phosphatase, the second, 50 units, and the third, 100 units. One unit of acid phosphatase is the amount necessary to liberate from p-nitrophenylphosphate, disodium salt, one uM of p-nitrophenol per minute at 37 C. Antibody was first detected on day 12 following the first inoculation in the two rabbits given the largest amount of phosphatase. Eventually all three rabbits produced an 1 Barbara K. Bodner antibody titer of 1:16. Titers were determined by an im- munodiffusion test in which serial dilutions of antisera were permitted to diffuse against purified staphylococcal acid phosphatase. Development of precipitin lines and identification of the antibody was made with a specific stain. A colorimetric procedure for antiphosphatase quantitation was also deve10ped; however, it is not as sensitive as the immunodiffusion method. As determined by mercaptoethanol treatment of an antiphosphatase serum sample, antibodies to staphylococcal acid phosphatase were apparently localized in the 196 serum fraction. Serum samples from normal and staphylococcus in- fected individuals were tested for staphylococcal anti- phosphatase. The antibody was not found in sera from normal individuals or those with acute staphylococcal disease. It was detected in most serum samples from individuals with chronic staphylococcal skin disease and those in which deep tissues were infected. Its presence, therefore, seems to indicate a chronic or severe staphy- lococcal infection. Three rabbits previously immunized with the pur— ified staphylococcal acid phosphatase, and one nonimmunized 2 Barbara K. Bodner control rabbit, were challenged with subcutaneous injec- tions of Staphylococcus aureus (PS 55) and talc. The talc served as a foreign body irritant. The degree of erythema and necrosis, as well as the total and differential leu- kocyte counts were determined at 24, 48, 72, 96 hr and then at frequent intervals until healing occurred. Data obtained the first week following inoculation suggested that the antiphosphatase was providing some protection. Later the immunized rabbits' lesions increased in severity and took longer to heal than the control rabbit, probably due to the organisms' ability eventually to overcome the antiphosphatase or to delayed hypersensitivity. STAPHYLOCOCCAL ACID PHOSPHATASE: ANTIGENICITY, ASSAY OF ITS ANTIBODY, AND PROTECTION STUDIES BY Barbara K. Bodner A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1972 A ACKNOWLEDGEMENTS The author wishes to express her thanks to Dr. C. L. San Clemente for his interest and enthusiasm during the course of this study, to Dr. C. C. Morrill for his guidance, to Steven Glenn for his helpful discussions and mechanical assistance and to her husband, Dick, for his constant help and encouragement. ii "f - .L ”m I\'.‘ A“? U. “v.1. Maw n.~‘ ‘ TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . LIST OF FIGURES O O O C O O O O C C O O O O O O 0 INTRODUCTION 0 O O O O O O O O O O O O O O O O O 0 REVIEW OF LITERATURE. . . . . . . . . . . . . . . Acid Phosphatase as an Indicator of Patho- genicity. . . . . . . . . . . . . . . . . . Acid Phosphatase Preparation. . . . . . . . . Determination of Staphylococcal Acid Phospha- tase ActiVity O O O O O O O O O O O O O O O Immunological Aspects . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . Organism Cultivation and Maintenance. . . . . Production of Acid Phosphatase. . . . . . . . Media . . . . . . . . . . . . . . . . . . Cultural Conditions . . . . . . . . . . . Purification of Acid Phosphatase. . . . . . . Quantitative and Qualitative Methods. . . . . Assays on the Purified Enzyme . . . . . . iii Page vi vii 12 13 24 24 24 24 25 26 28 28 TABLE OF CONTENTS (cont.) Page Antiphosphatase Detection and Semi- quantitative Assay. . . . . . . . . . . . 30 Colorimetric Antiphosphatase Assay. . . . . 32 Total White Blood Count and Differential. . 34 Immunology. . . . . . . . . . . . . . . . . . . 35 Experimental Animals. . . . . . . . . . . . 35 Immunization Schedule . . . . . . . . . . . 35 Serum Collection and Preparation. . . . . . 36 Pilot Protection Studies on Immunized Rabbits . . . . . . . . . . . . . . . . . 37 Antisera Absorption . . . . . . . . . . . . 38 Mercaptoethanol Treatment of Antisera . . . 40 RESULTS . . . . . . . . . . . . . . . . . . . . . . 42 Preparation of Staphylococcal Acid Phosphatase. 42 Cultural Modifications. . . . . . . . . . . 42 Acid Phosphatase Purification . . . . . . . 43 Colorimetric Antiphosphatase Assay. . . . . . . 50 Immunodiffusion Antiphosphatase Assay . . . . . 51 Parameters of the Assay . . . . . . . . . . 51 Staining Immunodiffusion Assays . . . . . . 54 Antiphosphatase Titers. . . . . . . . . . . . . 55 Immunization of Rabbits . . . . . . . . . . 55 iv TABLE OF CONTENTS (Cont.) Page Antiphosphatase Titers in Normal and Infected Subjects . . . . . . . . . . . . 59 Cross Reaction of Staphylococcal Antiphospha- tase with Nonhomologous Phosphatase . . . . . 61 Antiphosphatase Immunoglobulin. . . . . . . . . 61 Pilot Protection Studies on Rabbits Immunized with Acid Phosphatase . . . . . . . . . . . . 63 DISCUSSION. . . . . . . . . . . . . . . . . . . . . 70 LITEMTURE CITED 0 O O O O O O O I O O 0 O O O I O O 78 r—il C" V ABLE A: Table LIST OF TABLES Page Assay of staphylococcal antiphosphatase in sera of immunized and control rabbits using immunodiffusion and colorimetric methods . . . . . . . . . . . . . . . . . . 52 Staphylococcal antiphosphatase titers in concentrated and unconcentrated sera of normal and infected subjects using immunodiffusion and colorimetric methods. . 53 Course of staphylococcal.antiphosphatase titers in rabbits immunized with purified staphylococcal acid phosphatase . . . . . . 58 Absorption of rabbit staphylococcal anti- phosphatase serum . . . . . . . . . . . . . 62 Effect of 2-mercaptoethanol on the titer of rabbit antiphosphatase. . . . . . . . . . . 63 Normal leukocyte counts of experimental rabbits prior to immunization with dif- ferent amounts of acid phosphatase and counts taken approximately 2 months later following establishment, by subcutaneous inoculation, of a staphylococcal skin infection . . . . . . . . . . . . . . . . . 65 Normal differential leukocyte counts of experimental rabbits prior to immunization with different amounts of acid phosphatase and counts taken approximately 2 months later following establishment, by subcu- taneous inoculation, of a staphylococcal skin infection. . . . . . . . . . . . . . . 66 vi LIST OF I? IGURES Figure Page 1. Rate of whole culture acid phosphatase production and cell growth (O.D. at 625 nm) measured in shake cultures (Trypticase Soy Broth) of S. aureus (PS 55) at 37 C, pH 7.5, Dow B anti- foam 0.05 ml/liter. . . . . . . . . . . . . 44 2. Rate of whole culture acid phosphatase production and cell growth (O.D. at 625 nm) measured in shake cultures (CCY medium) of S. aureus PS 55 at 37 C, pH 7.5, Dow B antifoam 0.05 ml/liter . . . . . 46 3. Rate of whole culture S. aureus (PS 55) acid phosphatase production measured in shake cultures of Trypticase Soy Broth and CCY medium at 37 C, pH 7.5, and without antifoam. . . . . . . . . . . . . . . . . . 48 4. Stained precipitin lines obtained during the titration of antiphosphatase by immunodif- fusion of two different rabbit serum samples. Each has a titer of 1:8 . . . . . 56 5. Lesions in rabbit 1, rabbit 2 and rabbit 3 occurring at 1 week post inoculation. . . . 68 vii locccc Kaila for vi V man t q A. c 5. x .g‘ - . iuq~ao‘ f). INTRODUCTION Most studies concerned with the nature of staphy- lococcal disease and its prevention have focused on cer— tain bacterial products toxic to the infected individual. While it is now obvious that no one factor is responsible for virulence, it is also apparent that products other than toxic ones should be investigated. Staphylococcal acid phosphatase, which produces no known toxic effect, has been considered a reliable indi- cator of pathogenicity in staphylococci (Barber, Brooks- bank and Kuper, 1951; White and Pickett, 1953; Gupta and Charavarti, 1954). The enzyme has been purified, charac- terized and localized (Malveaux and San Clemente, 1969), and there is some evidence that it is antigenic in rabbits and cows (Arai and San Clemente, 1971). This investigation was undertaken to confirm the antigenicity of staphylococcal acid phosphatase. If there should be an antibody response we would proceed to develop a rapid, easy method of antibody assay, and subsequently use this assay to screen sera of staphylococcal infected l and noninfected individuals for antiphosphatase. Further- more, we would determine the possible protective effect of an acid phosphatase immunization program to intracutan- eously injected, virulent Staphylococcus aureus. REVIEW OF LITERATURE Acid Phosphatase as an Indicator of Pathogenicity The majority of the publications on staphylococcal acid phosphatase involve its correlation with pathogeni- city. Since coagulase production is the classic indicator 0f pathogenicity, many workers have correlated the occur- rence of phosphatase with coagulase. Barber, Brooksbank and Kuper (1951) tested strains of staphylococci for phos- Phatase, glucuronidase and sulfatase but only phosphatase correlated with coagulase production. All 160 coagulase- poSitive organisms tested were phosphatase-positive, and of 75 coagulase-negative strains only one was phosphatase- positive. Similar results were reported by White and Pi(Ikett (1953) , despite their use of a different phospha- tase test system. In another study 400 strains of Sfighylococcus aureus were screened for phosphatase ac- tiVity and approximately 90% were both coagulase- and phosphatase-positive (Gupta and Charavarti, 1954). Cor- relation between these two enzymes was further 3 sudostantiated by Adolfo and Gallardo (1959) who reported 98 . 7% agreement. After consideration of mannitol fermen- tation, hyaluronidase, coagulase and phosphatase produc- tion, Angyal (1960) concluded that coagulase was the most reliable indicator of potential pathogenicity. However, he recommended that a coagulase-negative strain be re- garded as pathogenic if it was positive for phosphatase or hyaluronidase . Several investigators have tried to relate the enzyme production of staphylococci to phage pattern. When comparing phage type, coagulase, hyaluronidase and phos- Phatase activity Fodor, Rizgonyi and Csepke (1962) noted that Staphylococcus aureus, phage type 80/81, had low coa-gulase and hyaluronidase and high phosphatase activity. They explained, hoWever, that phosphatase could not be regarded as the sole indicator of virulence since virulent 8“trains from other phage types had low phosphatase activity. cannon and Hawn (1963) reported that the relationship be- t“Wee-3n phosphatase production and penicillin resistance was inconsistent. However they observed a close relationship between phosphatase production and phage type. They also E011nd the greatest percentage of high phosphatase producing Staphylococci in the 80/81 and Group I strains, regardless of source. Other reports substantiating these results were published by Solomon and San Clemente (1963) and Pan and Blumenthal (1961) . In the strains tested by Solomon and San Clemente coagulase and phosphatase activity and mannitol fermentation all occurred together or not at all. The quantitative results of Pan and Blumenthal (1961) supported previous qualitative evidence that acid phos- phatase activity was higher in coagulase-positive strains than in coagulase-negative ones; coagulase-positive Strains of S. aureus produced an average of five times more acid phosphatase than coagulase-negative strains. But individual coagulase-negative and -positive cultures Could not be separated on the basis of their acid phos- Phatase content since some coagulase-negative strains pro- CInk-Ted more phosphatase than coagulase-positive strains. In a later study Kedzia gt a_]_._. (1966) found a quantitative difference in the phOSphatase production of organisms isolated from various types of infection. They demon- strated that strains isolated from healthy carriers pro- duced considerably less phoSphatase than those from cases of septicemia and pyemia as well as furunculosis. More— QVer, the activity of strains from septicemia and pyemia was generally greater than that of the strains from furuncles. These authors suggested that organisms iso- lated from patients had a higher acid phosphatase activity because of adaptation to the acid pH within the lesion. There has been some disagreement with the observation that phage Group I contained the majority of high phosphatase producers. Choudhuri and Chakrabarty (1970) reported that phage Group II had maximal correlation with a and 8 hemo- 1ys in, lipase and phosphatase production. Earlier work by Thatcher and Simon (1956) showed isolates from phage Group IV to be more consistent in the production of coagu- lase, phosphatase and of potent amounts of a and B l"leltuolysins and enterotoxins. Since some of their strains Were isolated from dairy foods, they were interested in finding a good correlation between coagulase or phospha- tase and toxin production. However, neither of these eIlzymes consistently indicated toxigenic strains. Acid phosphatase has been demonstrated in a vari- ety of microorganisms and yet little is known about the thsiological role of this enzyme and how it contributes to pathogenicity (Tirunarayanan and Lundbeck, 1968) . BElsed on work done with Escherichia coli, Torriani (1960) s"uggested that phosphate, a product of phosphatase action, controlled the level of enzyme synthesis via a negative feed back system. Such a phosphate-phosphatase regulatory system could play an important part in the phosphorous economy of the cell. However Kuo and Blumenthal (1961) reported that such a phosphate repressible system could not be found in Staphylococcus aureus. Ivler (1965) the- orized that some staphylococcal enzymes might contribute to virulence by maintaining the cell at a high rate of metabolic activity. Kedzia _e_t_ E}.- (1966) presented exten- sive evidence that phosphatase activity is closely related to some mechanism for regulating the concentration of the inorganic phosphate pool and consequent metabolic pro- cesses within the bacterial cell. They cited the study by Volk (1954) who found that bacterial cells grown in the presence of sodium fluoride were unable to ferment phos- phorylated compounds. It was theorized that phosphorylated c"31npounds did not penetrate the membrane freely, and pre- viOus growth in fluoride must have prevented formation of the phosphatase necessary for dephosphorylation and subse- quent penetration of these compounds. Moreover they sug- geEkted that the high phosphatase activity in clinical s“lirains might play an important role in the intensity of penetration of some compounds into the cell and thus in the regulation of the bacteria's phosphate pool. A clue to the action of phosphatase in the infected host was re- ported by Mukherjee 2E El° (1965) who found an increase in host inorganic phosphate associated with staphylococcal infection. Staphylococcal acid phosphatase could contri- bute to the fall of organic phosphate and the rise of in— organic phosphate in the host, although the substrate involved 113 Hg is not known. Although the concept of phosphatase as an indi- cator of virulence is well supported, several conflicting studies have been published. Yoshida and Takeuchi (1970) found that diffuse variants exhibited lower coagulase, deoxyribonuclease, phosphatase and hemolysin activity than the compact strain they were derived from. The diffuse Strain was encapsulated and these authors suggested that its virulence in mice was not related to any extracellular products but rather to the capsule and its size. In a study of over 500 clinical isolates (Morton and Cohn, 1972) 200 of 224 coagulase-positive organisms produced Phosphatase as also did 19 of 96 coagulase-negative or— ganisms. The authors concluded that phosphatase produc- tion did not correlate well with coagulase production. Strains of staphylococci from a great variety of clinical Sources that did not produce either coagulase or “9.11”- pf .1. deoxyribonuclease were isolated as frequently as strains which produced these substances. Therefore they concluded that the production of coagulase and deoxyribonuclease were not necessarily indicative of potential pathogenicity of these organisms for man. Acid Phosphatase Preparation A number of methods, yielding products of dif- ferent degrees of purity, have been used to extract acid Phosphatase from microorganisms. Sevag e_t_ 313 (1954) used Water extraction to remove acid phosphatase from Brewers Yeast. A partially purified phosphatase preparation was Obtained when extraction was followed by ammonium sulfate fractionation and dialysis against water. An "osmotic ShOck procedure" has been used to release a group of en- zl’l'nes from S. <_:_<_3_1_i_ (Dvorak, Brockman and Heppel, 1967) . This technique was used as the first step in preparation of acid phosphatase followed by chromatography on Sephadex ion exchangers, zone electrophoresis and gel filtration (Hofsten and Porath, 1962) . A highly purified acid phos— phatase was obtained from Neurospora crassa by using 10 ethanol fractionation, precipitation with cetyltrimethyl- ammonium bromide (CTAB) and ammonium sulfate and finally fractionation on a diethylaminoethyl (DEAE)-cellulose column (Kuo and Blumenthal, 1961) . Precipitation with ammonium sulfate has been the standard procedure for preparation of various staphylococcal enzymes and toxins. Using this method, deoxyribonuclease has been precipitated by Anfinsen, Rumley and Taniuchi (1963), leukocidin by Woodin (1959) , hemolysin by Bernheimer and Schwartz (1963) and staphylokinase by Glanville (1963) . Vesterberg a; _a_];. (1967) using isoelectric focusing were able to separate all these proteins with less loss of biological activity tl'lan occurred with ammonium sulfate precipitation. Mal- Veaux and San Clemente (1969) developed a method of ex- traction and purification of staphylococcal acid phospha- tase by eluting it from the surface of S. aureus cells with 1.0 M KCl (pH 8.5) by gentle agitation and then pur— ifrying it 44-fold by two cycles of dialysis and gel fil- 1:Il’l‘ation. Some investigators have attempted to increase acid Phosphatase production by varying culture conditions. Torriani (1960) reported that S. coli alkaline phosphatase Was only formed when inorganic phosphorus became limiting 11 in the medium. In contrast Hofsten (1961) had shown that the nature and concentration of the phosphate source for growth had no marked influence on the formation of S. 92;}; acid phosphatase. He also reported that acid phosphatase . activity varied considerably with the composition of the Q‘- growth medium. For example, succinate or glycerol as the carbon source supported synthesis of high levels of acid ‘. V-«r. .- w. in“- phosphatase, whereas carbohydrates, such as glucose, had a repressive effect. A biphasic growth medium was used by Pan and Blumenthal (1961) for production of staphylococcal acid phosphatase. The highest yields were obtained when Brain-Heart-Infusion medium was used and the flasks were Shaken. Arvidson, Holme and Wadstrom (1971) compared staphylococcal enzyme and toxin production using membrane c(DVered solid medium and liquid media in shake flasks and $1T—irred, aerated fermentors. For all enzymes, cultivation in liquid media was superior to membrane plates, and yeast extract was required as a medium component for high yield of cells and extracellular proteins. Maximal bacterial yields were obtained in aerated cultures, but it was necessary to control foaming to avoid inactivation of e1lzymes. Another paper by these authors (Arvidson, Holme a11d Wadstrom, 1971) described the importance of optimal 12 pH of the culture medium. The maximal yield of acid phos- phatase was obtained at pH 6.5-8.0. Determination of Staphylococcal Acid I Phosphatase Activity A method for the detection of staphylococcal acid ‘VI: in: w _ phosphatase was first reported by Barber and Kuper (1951) . They incorporated the substrate, phenolphthalein diphos- phate, into either nutrient broth or agar. If the sub- 531:J:ate was cleaved by growing colonies the surrounding Dneaciium turned pink when exposed to ammonia fumes. A more Iratp>id phosphatase test was described by White and Pickett (JLSBSB). By using phenyl disodium phosphate as the sub- Estzlrate results were available in four hours instead of the 'theelve to eighteen hours required for the first procedure. 1¥El .indicator, 2,6, dibromo-N-chloro-p-quinoneimine was “Sad to detect substrate cleavage. Others (Lovell, 1958) have incorporated this same substrate into nutrient agar allél thereby lengthened the procedure to overnight incuba- 'tj43r1. Phosphatase liberates free p-nitrophenol, a dye whOSe yellow color is intensified at an alkaline pH. A 13 recent report applied the indigogenic principal used to demonstrate tissue enzymes to detection of staphylococcal ac id phosphatase (Von der Muehll, Ludwick and Wolf, 1972) . The substrate, 5-bromo-4-chloro-3-indolyl phosphate, offered the advantage of precise enzyme location with *1 very little or no diffusion. They stated that only path- ogenic staphylococci produced a blue green precipitate In _J addition they listed a number of the Enterobacteriaceae [E after thirty minutes incubation with the substrate. in which acid phosphatase was detected by this method. Barnes and Morris (1957) published a quantitative method uSing the phenyl disodium phosphate substrate used by White and Pickett (1953) in their screening test. The resulting colored product is quantitated by spectrophoto— metry after thirty minutes incubation. Cannon and Hawn (1963) modified this procedure by substituting phenolph- t113.1ein diphosphate as the substrate which required ej-.D. at 625 nm and whole culture acid phosphatase were determined. Trypticase Soy Broth was the medium of choice aJid was used for the remaining acid phosphatase produc- IZjuon. Sflgltural Conditions Cultivation was performed in a MP 214 Microferm unit (New Brunswick Scientific Co., New Brunswick, N.J.) 26 in 10 liters of Trypticase Soy Broth at 37 C. A propeller speed of 200 rpm was maintained. The air flow rate was 1.5-2.0 liters per min at 1 atmosphere pressure. The pH of the medium was adjusted to 7.5 before sterilization and did not decrease more than one pH unit during the 24 hr cultivation period. To control foaming Dow B Antifoam was added before sterilization to a concentration of 0.05 ml per liter of media and additional aliquots were added as needed during cultivation. Overnight shake flask cultures in 100 ml Trypticase Soy Broth served as inocula for the 10 liter cultures. Cells were collected by continuous- flow centrifugation using a Serval Model SS-l centrifuge taquipped with the Szent-Gyorgi and Blum continuous—flow System (Ivan Sorval, Inc., Norwalk, Conn.). Purification of Acid Phosphatase Purified staphylococcal acid phosphatase was ob- tained from cells (PS 55) using a slightly modified pro- cedure of Malveaux and San Clemente (1969) . The whole (33118 were washed once with 500 ml of a 0.1 M KCl/0.05 M TIris-chloride solution (pH 8.5), centrifuged, and 27 resuspended in the same volume of 1.0 M KCl/0.5 M Tris- chloride (pH 8.5). The wash solution was discarded. Elution was effected by gentle agitation of the washed cells on a reciprocal shaker at 4 C for 60 minutes. The cells were separated from the eluted material by centri- fugation and the resulting supernatant fluid was dialyzed against 10 volumes of 0.01 M Tris-chloride (pH 8.5) for 12 hours at 4 C. The precipitate formed during dialysis was sedimented in a Serval Model RC-2 refrigerated cen- trifuge (Ivan Sorval, Inc., Norwalk, Conn.) at 4 C, was redissolved in 1.0 M KC1/0.5 M Tris-chloride (pH 8.5), and was redialyzed as described above. The precipitate :formed after the second dialysis was dissolved in several Inilliliters of 1.0 M KCl/0.5 M Tris-chloride. A Sephadex CPharmacia Fine Chemicals, Piscataway, N.J.) G-lOO column (2.5 by 38 cm) was equilibrated with this same solution ade.the void volume determined with Blue Dextran 2000 irudicator (Pharmacia Fine Chemicals, Piscataway, N.J.) at 4 <2. The sample (2 ml), containing sucrose at a concen- tration of 10%, was layered on the column and was eluted ‘stth.the same solution while 4 m1 fractions were collected 0:1 a Fractomat fraction collector (Buchler Instruments, F'OrtLee, N.J.). Each fraction was scanned at 260 nm and 28 280 nm with a Beckman DU spectrophotometer (Beckman In- struments, Inc., Fullerton, Calif.). Those fractions with the highest protein content were assayed for acid phos- phatase activity and those with the maximum were combined. This purified product was used in the remainder of the study. Quantitative and Qualitative Methods Agsays on the Purified Enzyme Acid phosphatase activity was measured by a method Inodified from Barnes and Morris (1957) using 0.08% p- IlitrophenylphOSphate, disodium salt (Calbiochem, Los Fungeles, Calif.) as the substrate buffered at pH 5.2 with SCMdium acetate. When a blank was included, acidification with HCl was not necessary to eliminate color due to free £>-rlitrophenol. Crystalline p-nitrophenol (Matheson, Coleman and Bell, Norwood, Ohio.) was used to prepare a Standard curve . Protein determinations were made according to the method of Lowry _e_E ai. (1951) , using crystallized bovine 29 Fraction V albumin (Sigma Chemical Co., St. Louis, M0.) for protein standards. A number of tests were made on the purified acid phosphatase to determine the presence or absence of asso- ciated staphylococcal products. Coagulase was measured by the tube method (Bailey and Scott, 1970) at 37 C using rabbit plasma (Difco Laboratories, Detroit, Mich.). De- oxyribonuclease activity was analyzed by placing 0.2 ml of the purified fraction into wells cut in Deoxyribonu- clease Test Agar (Baltimore Biological Laboratories, Baltimore, Md.). The plates were incubated overnight at 37 C and flooded with l N HCl. A clear zone around the Ivell indicated deoxyribonuclease activity. Lipase ac- ‘tivity was determined according to the method of Nachlas éind.Seligman (1949), using p-nitrophenylpalmitate (Cal- iafornia Corporation for Biochemical Research, Los Angeles, Chalif.) as the substrate. The reaction occurred in ver- <31La1 buffer (pH 7.4) at 40 C. The presence of a yellow cOlor following incubation for one hour indicated lipase aC2tivity. Fibrinolysin activity was analyzed by streaking tine sample on fibrin plates made by adding citrate bovine fibrinogen (Sigma Chemical Co., St. Louis, Mo.) to warm 30 nutrient agar. The plates were read after incubation overnight at 37 C. Antiphosphatase Detection and Semiquantitative Assay Qualitatively, antibodies were detected by immu- nodiffusion using Ion Agar No. 2 (Colab Laboratories, Glenwood, 111.), 0.85% in 5% NaCl at pH 8.1. Two m1 of agar were layered on a microscope slide and a symmetrical five well pattern was out. Each well had a diameter of 1.5 mm and the distance between the central and outside wells was 4.5 mm. Antigen (48.6 units/ml) was added to the center well. One unit of acid phosphatase is the amount necessary to liberate one uM of p—nitrophenol per minute at 37 C. Development was allowed to proceed over— night at room temperature. Quantitatively, antiphospha- tase titers were determined on agar slides with a 14 well pattern in 3 rows. Antiserum was diluted with a commer- cial microtiter loop and plastic plates (Cooke Engineer- ing Co., Alexandria, Va.). Antigen was placed in the middle row of four wells. Concentration of sera was sometimes necessary to detect antibody and was accomplished using two different 31 procedures. Sera were first concentrated by addition of approximately 0.2 gm Lyphogel (Gelman Instrument Co., Ann Arbor, Mich.) to 1 ml serum. The mixture was allowed to stand at room temperature for five hours for maximal con- centration. Sera negative for antiphosphatase following this procedure were treated with ammonium sulfate to pre- cipitate gamma globulin. One-third saturation was ob- tained by dropwise addition of the proper volume of satur- ated ammonium sulfate. The mixture was adjusted to pH 7.8 with 0.5 N NaOH and continually stirred for 2 hr. The resulting precipitate was removed by centrifugation and resuspended in saline to the original volume. This pro- cedure was repeated two additional times and the final precipitate was resuspended in borate buffered saline (pH 8.2) to one half its original volume. Staining of the precipitin bands was carried out by adapting the Gomori histochemical stain for acid phos- phatase as described by Shulman 95 El: (1964). This utilized the following reagents: a) Buffer: 0.05 M acetic acid-sodium acetate; pH 5.0.' Lead nitrate was incorporated into the buffer at a concentration of 1,33 9/ liter. b) Substrate: 3% sodium 8 glycerophosphate 32 prepared in water. This substrate was newly prepared prior to use. c) Rinse: 1% acetic acid. d) Stain: 2% yellow ammonium sulfide. Slides were incubated in the buffer-substrate mixture (10:1) at 37 C for 15 min, rinsed in distilled water, then 1% acetic acid, and then immersed in 2% ammonium sulfide for l to 2 min. Precipitin lines containing acid phosphatase stained a deep brown. Colorimetric Antiphosphatase 9.9221: Antiphosphatase was also quantitated by a colori- metric procedure based on the acid phosphatase determina- tion of Barnes and Morris (1957). Optimal proportion of antigen and antibody to be used in the colorimetric pro- cedure was initially determined by a simplified quantita- tive precipitin test using agar gel diffusion (Nowotny, 1969). Serial dilutions of antigen were made using a microtiter plate and loop. A constant amount of antibody was added to each well and the mixture was incubated for 1 hr at 37 C. A microscope slide previously covered with 2 ml Ion Agar No. 2 (0.85% in 5% NaCl) was prepared by cutting a row of seven wells (1.5 mm diameter) in the center of the slide and a trough above and below the row 33 of wells. Following incubation, each well on the slide was filled with some of the mixture from the corresponding microtiter well. The top trough was filled with antiserum and the bottom well with antigen. The slides were read after overnight incubation at 37 C. Prior to performing the colorimetric assay the sera were inactivated at 56 C for 30 min and diluted 1:10 if they were known to contain high levels of the antibody such as were found in rabbits immunized with large amounts of purified acid phosphatase. Equal amounts (0.2 ml) of serum and purified acid phosphatase (2 units) were mixed and incubated for 1 hr at 25 C. Following incubation the mixture was centrifuged to sediment the fine precipitate present. Four tubes were labeled: test, control, total acid phosphatase, and blank. The following additions were made: 0.1 m1 supernatant from the reaction mixture to the tube labeled test; 0.1 ml serum diluted 1:1 with saline to the control tube; 0.1 m1 acid phosphatase diluted 1:1 with saline to the total acid phosphatase tube; and 0.1 ml saline to the blank. Next 0.3 ml of 0.08% p-nitrophenyl- phosphate, disodium was added to each tube followed by 2.6 m1 sodium acetate buffer (pH 5.2). The tubes were incubated at 37 C for 30 min after which the reaction was 34 stopped by addition of 1.0 ml 1 M NaOH. The O.D. of each solution was read at 400 nm against the blank set at zero O.D. in a Bausch and Lomb Spectronic 20 spectrophotometer (Bausch and Lomb, Rochester, N.Y.). The O.D. of the con- trol, which indicated the amount of serum acid phospha- tase, was subtracted from the O.D. of the test. The ratio of the O.D. of the corrected test to the O.D. of the total acid phosphatase gave the percentage of acid phosphatase that remained in solution. We preferred to take the dif- ference between this value and 100% and expressed our answer as percentage of acid phosphatase bound. Total White Blood Count and Differential Blood was withdrawn from the central ear artery using a 22 gauge needle (Becton, Dickinson and Co., Co- lumbus, Neb.) and 5 m1 glass syringe. It was immediately transferred to a Vacutainer tube (Becton, Dickinson and Co., Columbus, Neb.) containing ethylenediaminetetraacetic aCid (EDTA) anticoagulant. The whole blood was diluted 1:500 in Isoton (Coulter Electronics, Hialeah, Fla.) using a Dade automatic dilutor model 2D (Dade Reagents, Inc., Miandq Fla.). Duplicate diluted samples were each counted 35 twice on a Coulter Model B counter (Coulter Electronics, Hialeah, Fla.) and an average value was calculated. A thin smear of blood was made on a microscope slide and stained using Wright-Giemsa stain (Harleco, Philadelphia, Pa.). A differential count of 100 white blood cells (WBC) was made twice and an average taken. Normal values were determined by performing total and differential WBC counts on four rabbits on the four days prior to their inoculation. Immunology Experimental Animals Adult male New Zealand White rabbits weighing approximately 10 kg were caged individually and supplied with a diet of Triumph Rabbit Pellets (John K. Van Den Bosch Co., Zeeland, Mich.). Immunization Schedule In a series of 10 injections given every other day, three rabbits were inoculated with purified 36 staphylococcal acid phosphatase, mixed 1:1 with Freund's complete adjuvant (Difco, Detroit, Mich.). The first five injections, using 25-gauge needles, were given in the foot pad and the last five subcutaneously in the cervical re- gion. One rabbit received a total of 25 units phosphatase, the second 50 units, and the third 100 units. One unit of acid phosphatase is the amount necessary to liberate from p-nitrophenyl disodium salt, one uM of p-nitrophenol per minute at 37 C. The control rabbit was given only Freund's complete adjuvant. Serum Collection and Preparation After rubbing the ear with xylene, serum samples (about 0.5 to 1.0 ml) were collected using a 22 gauge needle and 5 m1 glass syringe from the central ear artery. The blood was allowed to coagulate at room temperature and the serum was then separated by centrifugation. The serum was transferred to a tube by Pasteur pipette and stored at -20 C. “L D. n“ 0 to “It“ by 1 sea} pare .‘l 37 Pilot Protection Studies on Immunized Rabbits Since rabbits are known to be relatively resistant to skin infection with Staphylococcus aureus, a prelimi- r nary experiment was conducted to determine the number of organisms necessary to establish infection in a normal rabbit; also the feasibility of using a foreign body irri- IF -' a" .'. 1? ‘ tant to enhance infection was explored. Two types of irritants were investigated. Following a method described by Elek (1956) sterile cotton sutures of uniform size were soaked in dilutions of Staphylococcus aureus (PS 55) pre- pared from an overnight Trypticase Soy Broth culture. Each dilution differed logarithmically from 108 organ- isms/m1 to 103 organisms/m1. The concentrations were determined by plate counts done in triplicate (Manual of Microbiological Methods, 1957). The second method (Dajani and Wannamaker, 1970) involved injecting inocula contain— ing talcum powder (Mallinkrodt Chemical Works, St. Louis, Mo.) at a concentration of 1 mg/ml. Again concentrations of organisms ranged from 108 organisms/m1 to 103 organ- isms/ml. The backs of two nonimmunized rabbits were shaved and depilated. Along one side of each rabbit sutures 38 treated as described above were inserted using one stitch which was secured with a double knot leaving some slack to accommodate swelling. A suture soaked in sterile saline was the control. Along the other side 0.1 m1 subcutaneous injections of the organism and talc suspensions described above were given by means of a 25 gauge needle and tuber- culin syringe. A suspension of sterile saline and talc I?" A was injected for the control. Based on the results of this experiment the talcum powder irritant was chosen in the remaining protection studies. The plan was slightly modified by increasing the number of organisms injected. The dilutions ranged from 4.4 x 1010 organisms/ml to 4.4 x 105 organisms/ml. In all other aspects the procedure described above was followed. The degree of erythema and necrosis, as well as the total and differential leukocyte counts were determined at 24, 48, 72, 96 hr and then at frequent intervals until healing occurred. Antisera Absorption The following organisms were used in the absorp- tion experiment: Klebsiella pneumoniae; Enterobacter the susP. trea' Sec) ing a Shape Organ the C EQual immune and 0‘ UntrEG 39 aerogenes; Escherichia coli; Streptococcus faecalis; Proteus vulgaris; Serratia marcescens; Staphylococcus aureus (PS 55). In addition purchased alkaline phospha— tase (hog and calf intestinal mucosa) and acid phosphatase (potato and wheat germ) were used (Sigma Chemical Co., St. Louis, Mo.). The organisms were inoculated into a tube of Trypticase Soy Broth and grown overnight at 37 C. They were separated from the culture medium by centrifugation and resuspended in 0.05 M Tris-chloride buffer (pH 8.2) at a concentration of 0.5 g (wet wt) per ml. To release the firmly and loosely bound acid phosphatase each cell suspension was subjected to sonic oscillation (sonic treatment for 15 sec was alternated with cooling for 30 sec) in a MSE 100 watt ultrasonic disintegrator (Measur- ing and Scientific Equipment Ltd., London, England). Rod- shaped organisms were sonicated for 2 min while coccal organisms were sonicated 4 min. Equal parts (0.2 ml) of the cell lysate and serum containing antiphosphatase and equal parts of the purchased enzyme (30 units/ml) and immune serum were mixed and incubated for 1 hr at 25 C and overnight at 4 C. A negative control consisted of untreated sera incubated in the same manner; for a M _‘ u 40 positive control, purified staphylococcal acid phospha- tase (30 units/ml) was added to the serum. Following incubation the suspensions were centrifuged and the anti- phosphatase in the supernatant fluid titered by the im- munodiffusion method previously described. In addition the suspensions were tested for acid phosphatase using the quantitative procedure of Barnes and Morris (1957). Results indicated that at least 30 units/m1 of acid phosphatase were necessary before absorption of antibody took place. Therefore the procedure was re- peated using 24 hr shake flask (100 ml) cultures of Staphylococcus aureus (PS 55) and Klebsiella pneumoniae. The cells were centrifuged and treated as described above. Mercaptoethanol Treatment of Antisera Serum with a high titer of staphylococcal anti- phosphatase was treated with mercaptoethanol according to Yoshida and Ekstedt (1968). Serum (1 ml) was mixed with an equal volume of 0.2 M 2-mercaptoethanol (Eastman Or- ganic Chemicals, Rochester, N.Y.) and allowed to react at 4 C for 18 hr. The mixture was then dialyzed against 0.02 M iodoacetic acid (Matheson, Coleman and Bell, 41 Norwood, Ohio.) in 0.02 M phosphate-buffered saline for 2 hr and in fresh phosphate-buffered saline (0.02 M) for 24 hr at 4 C. The control serum was treated in the same manner, except phosphate-buffered saline (0.02 M) was used instead of mercaptoethanol. Cult tase modi fOr fiVe 20 h COnc Sequ SuCc grOw 0bSe: Was rela1 repea RESULTS Preparation of Staphylococcal Acid Phosphatase Cultural Modifications To determine the medium for maximal acid phospha- tase production Staphylococcus aureus (PS 55) was culti- vated in Trypticase Soy Broth, casein hydrolysate, a modified casein hydrolysate with succinate substituted for glucose, CCY, and a biphasic growth medium. In all five media growth increased most rapidly between 8 and 20 hr and slowly thereafter. The peak acid phosphatase concentration was reached in approximately 22 hr and sub- sequently decreased sharply, presumably due to denatura— tion. The original casein hydrolysate medium and the succinate substituted medium were eliminated because both growth and acid phosphatase production were far below that observed in the other media. The biphasic growth medium was also excluded because it was confined to the use of relatively small volumes of media. The experiment was repeated using both Trypticase Soy Broth and CCY medium 42 43 in which the pH and foaming were controlled to reduce enzyme denaturation. The rate of acid phosphatase pro- duction in whole culture paralleled cell density in Tryp- ticase Soy Broth shake culture (Fig. 1). In the CCY medium the rate of whole culture acid phosphatase pro- duction also increased with cell number (Fig. 2), but the amount of growth was about one half of that obtained in Trypticase Soy Broth and consequently the total amount of 1r acid phosphatase elaborated was much less. In both cases the control of pH and foaming seemed to inhibit the enzyme denaturation that otherwise occurred by 22 hr (Fig. 3). Also, slightly higher levels of acid phosphatase were reached prior to 22 hr in the controlled media. Based on these results, Trypticase Soy Broth (pH 7.5, antifoam con- centration 0.05 ml/liter) was chosen for acid phosphatase production. Acid Phosphatase Purification Loosely bound acid phosphatase was extracted and purified (Malveaux and San Clemente, 1969) from Staphy- lococcus aureus cells grown in 20 liters of culture. The final product contained 0.63 mg of protein/m1 and an acid 44 .umuwa\as mo.o snowflucm m 30o .m.» an .o am pm Amm may msmusm .w mo Anuoum mom mmmoflu Imwuav mousuaso mxmnm cw pmHSmmmE AEc mmw um .a.ov £u3oum Hamo was cofluosooum wmmumsmmonm pflom mnsuado maon3 mo mummul.a .mHm 45 WU 93969 'CI'O on N o I I P-N V N t l l T T I I o 00 V O '0 N 00 V’ O m N N N I— u— o: x (mm/Iw/‘IONBHdoalIN-d WY!) 8— TIME (hr) 46 .Hmuwa\ae mo.o smomwucm m 309 .m.a mm .o um um mm mm msmusm .w «0 Aesfloms woov mousuaso oxmnm :H consumma “Ed mmm um .o.ov.£u3oum Hamo can coauodcoum wmmumnmmonm pans muduaso ma0£3 mo mummll.~ .mflm 47 um sz9o'a'o ~O '0 V m N :— O l 1 I 1 V N _O N _O 'E 3 2 0 0! Pl— I a m _o m -V 1 1' I T I 0 V o ~O N 00 V O N N '— 8_OlX(U!w/lw/ 'IONSHdOHllN-d w") I . """II- .\W ‘ ‘— _V *7 + 7— — __¥ 7 TIME (hr) 48 .Emom lavas psonuflz was .m.h mm .0 um um Eswpme $00 was nuoum wow mmmoflpmmna mo mmugpaso mxmsm Cw pmusmwmfi coauoscoum mmmumsmmocm cans Amm mm madman .mq wusuaso maoa3 mo mummll.m .me 49 H/‘t/ TSB CCY i r I l l l 29 16 20 I 12 hm \! ‘1 V O ~O (\l 00 fo 0 N N '- "" crow (usw/Iw/10N3HdoalIN—d wfi) TIME (hr) 50 phosphatase activity of 48.6 units. One unit of acid phosphatase is the amount necessary to liberate 1 uM of p-nitrophenol/min at 37 C. The 280/260 ratio of the product was 1.4 or approximately 0.75% nucleic acid (Man- ual of Microbiological Methods, 1957). Tests for coagu- lase, fibrinolysin and deoxyribonuclease were negative; however, a qualitative test indicated that a small amount of lipase was present. Colorimetric Antiphosphatase Assay- Quantitative precipitin tests were done to deter- mine the optimal antigen-antibody ratio for the test. Precipitin lines between the center wells and the upper trough indicated that an antigen excess was present in the antigen-antibody mixture. Precipitin lines between the center wells and the lower trough indicated antibody ex- cess. At the equivalence point there were no precipitin lines. The results of these tests indicated that 2 units of antigen provided a margin of slight excess when incu- bated with the antiphosphatase levels present in most sera. However, when high antibody levels were present, as in the ta ra. wi ph ti Ve Se. (:11 am W315 Bah iti 51 rabbits immunized with large amounts of pure acid phospha- tase, it was necessary to dilute the sera 1:10. Table 1 lists the amount of antiphosphatase in rabbit sera determined by the colorimetric test compared with the immunodiffusion titer. Sera negative for anti- phosphatase by the immunodiffusion method were also nega- tive using the colorimetric procedure as shown in Table 2. Very low levels of the antibody, detectable in horse serum by immunodiffusion, were not detected by the less sensi- tive colorimetric procedure. Immunodiffusion Antiphosphatase Assay Parameters of the Assay Initially, immunodiffusion of immunized rabbit serum versus purified acid phosphatase was attempted using 0.85% Ion Agar No. 2 in 0.85% saline (pH 8.1). No pre- cipitin lines formed, but a broad, diffuse ring around the antigen well indicated impeded mobility of the antigen. When the salt (NaCl) concentration was increased, the enhanced solubility of the antigen produced sharp precip- itin lines. No further enhancement of the pattern was TA} An Ra Ra Ra Cc U, _r) [3.1 52 TABLE l.--Assay of staphylococcal antiphosphatase in sera of immunized and control rabbits using immunodiffusion and colorimetric methods. Colorimetric test- Animals Immunodiffu51on % of acid phosphatase titer bound . a Rabbit 1 1:4 49.6 Rabbit 2b 1:4 49.0 Rabbit 3C 1:8 74.5 Control Rabbitd 0 0 aReceived 25 units staphylococcal acid phosphatase 90 days prior to these determinations. bReceived 50 units staphylococcal acid phosphatase 90 days prior to these determinations. cReceived 100 units staphylococcal acid phosphatase 90 days prior to these determinations. dDid not receive any staphylococcal acid phosphatase. W TAB CI 53 TABLE 2.--Staphylococcal antiphosphatase titer in concentrated and unconcentrated sera of normal and infected subjects using immunodiffusion and colorimetric methods. Source of Number of Immunodiffusion Colorimetric sera samples unconc. conc. Human 29 normal - - infected: Patient Da - + Patient Sb - - - Patient Fb - - - Patient lC + + Patient 2C - t Patient 3c - - Patient 4C - - Cow 5 normal — - 1 infectedd - - Horse 1 infectede - + _ post vaccinef + + - Dog 1 infected - - a I I I I I I Chronic staphylococcal skin infection; received autogenous vacc1ne. b . . . Acute staphylococcal skin infection. c I 0 Staphylococcal endocarditis. d I I Streptococcal mastitis. e Chronic cellulitis from which a Staphylococcus aureus and a Streptococcus were cultured. Sera of same horse following administration of an autogenous vaccine containing both organisms. obseJ stit chlo Prec bat? rat pat C38! I (n r-r (D 'tJ 54 observed when the saline concentration exceeded 5%. Sub- stitution of the sodium salt with 1.0 M KCl/0.5 M Tris- chloride did not increase the sensitivity of the test. Precipitin bands generally occurred after 4 to 6 hr incu- bation at 25 C. Incubation at 37 C did not increase the #3 rate of diffusion or appear to have any effect on the pattern. The concentration of agar (0.85%) or the pH i (8.1) were not critical but were within the parameters generally suggested for immunodiffusion. Staining Immunodiffusion Assays Application of a staining procedure by Shulman £2 31. (1954) specifically stained phosphatase- antiphosphatase precipitin bands dark brown. The phos- phate was split from the B-glycerophosphate substrate by acid phosphatase at pH 5.0. Lead in the substrate solu- tion was precipitated as lead phosphate and was visualized as brown lead sulfide when incubated with ammonium sulfide. Due to this specificity, bands formed by other antigen- antibody complexes remained unstained. When 8- glycerophosphate was absent, no staining occurred. Al- though enzymes are usually completely inhibited by their anti sta cog: OI 55 antibodies, the action of acid phosphatase during the staining procedure indicated that it was active despite coprecipitation with its antibody. This had previously been observed only in systems where the substrate had a low molecular weight (Cinander, 1963). However, when 3 p-nitrophenolphosphate (M.W. 263) or phenolphthalein diphosphate (M.W. 562) were substituted for B- glycerophosphate (M.W. 216) staining was not inhibited. Figure 4 shows a stained antiphosphatase assay on two rabbit sera; one in the top row of wells, the second in the bottom row. Both sera formed precipitin lines as far as the third well which was a 1:8 dilution. Antiphosphatase Titers Immunization of Rabbits Three rabbits were immunized over a 20-day period with increasing amounts of acid phosphatase. Their sera were tested for antibody at frequent intervals (Table 3). On Day 12, following the first inoculation, antibodies were detected in Rabbits two and three by immunodiffusion. 56 .mua mo “mafia m mm: 30mm .mmamamm Esnom panama ucmumMMHU ozu mo aoflmDHMHpossfififi mp ommumnmmonmwucm Mo coaumuuHu was august tmcampno mmcaa cflpflmflomum omcamumuu.a .mam 57 58 TABLE 3.--Course of staphylococcal antiphosphatase titers in rabbits immunized with purified staphylococcal acid phosphatase. gziiimmunization Rabbit 1a Rabbit 2b Rabbit 3C 3 o o o o 3 12 0 + + J 20d 1:2 1:8 1:4 L. 1 26 1:2 1:8 1:8 33 1:8 1:16 1:8 41 1:16 1:16 1:16 55 1:16 1:16 1:16 e 65 1:16 1:16 1:16 70 1:16 1:16 1:16 90 1:4 1:4 1:8 aReceived 25 units staphylococcal acid phosphatase. bReceived 50 units staphylococcal acid phosphatase. cReceived 100 units staphylococcal acid phosphatase. d Date of tenth and final injection of acid phosphatase. eStaphylococcal skin infection established in all rabbits. 59 Approximately one week later antibodies were detected in Rabbit one and titers in the other two rabbits had further increased. By Day 41 all rabbits had reached a maximal titer of 1:16 which remained unchanged despite the estab- lishment of a staphylococcal (Staphylococcus aureus PS 55) skin infection on Day 65. Titers determined on Day 90 showed a significant decrease. Several of these serum samples were tested by the colorimetric technique and Table 2 shows these values compared with immunodiffusion titers on the same sera. Antiphosphatase Titers in Normal and Infected Subjects Human, cow, horse, and dog sera from normal and infected subjects were tested for antiphosphatase (Table 2). Unconcentrated and concentrated human sera from 29 normal individuals were tested for antiphosphatase by immunodif- fusion and found to be negative. Several patients with a history of staphylococcal infection were also tested. Human patient D had a history of chronic staphylococcal skin infections that did not respond to antibiotic treat- ment. He had been injected with one series of an auto- geneous vaccine that produced little, if any, relief. 60 Antibodies were detected in his serum only after concen- tration with Lyphogel or in the gamma globulin fraction after ammonium sulfate precipitation. Human patients S and F had acute staphylococcal skin infections, and anti- bodies could not be detected in their serum using either r! immunodiffusion or colorimetric procedures. Human pa- tients l, 2, 3, and 4 had staphylococcal endocarditis. Antibody was found in one patient's unconcentrated serum ~l V7: and in a second following concentration. In three of the sera an antibody other than antiphosphatase was detected but could not be identified. Sera from five normal cows and one with strepto- coccal mastitis were tested and found to be negative for antiphosphatase. A horse with a chronic staphylococcal— streptococcal cellulitis that involved a large part of one leg was found to have antiphosphatase detectable only by immunodiffusion using concentrated serum. Following administration of an autogeneous vaccine containing both organisms the antibody was detected in unconcentrated serum. Serum from a dog with a staphylococcal skin infec- tion of short duration was negative for antiphosphatase. 61 Cross Reaction of Staphylococcal Antiphosphatase with Nonhomologous Phosphatase ' Table 4 shows the results of absorption of anti- phosphatase serum with lysates of organisms containing various amounts of acid phosphatase and with four commer- cially prepared enzymes. Results of the positive control and the Staphylococcus aureus lysate indicated that a minimum of 30 units of acid phosphatase were necessary for absorption to occur. It was possible to obtain this concentration with only one other organism, Klebsiella pneumoniae, and with the four purchased phosphatases. There was no absorption of antibody by any organism or enzyme other than the homologous one. Antiphosphatase Immunoglobulin To determine which immunoglobulin fraction con- tained antiphosphatase, serum containing antibody was treated with 2—mercaptoethanol. If IgM were responsible for antiphosphatase activity, incubation of the antiserum with mercaptoethanol would result in dissociation of the 62 TABLE 4.--Absorption of rabbit staphylococcal antiphosphatase serum. Acid phosphatase Titer of Source (units) absorbed sera Staphylococcus aureus (PS 55) 17.6 1:16 37.5b 1:8 Klebsiella pneumoniae 15.6a 1:16 43.8b 1:16 Escherichia coli 0.7a 1:16 Enterobacter aerogenes 0.3a 1:16 Serratia marcescens 1.4a 1:16 Proteus vulgaris 0.04a 1:16 Streptococcus faecalis 0.2a 1:16 . b ACid phosphatase (potato) 30.0 1:16 . b ACid phosphatase (Wheat germ) 30.0 1:16 . b Alkaline phosphatase (hog mucosa) 30.0 1:16 Alkaline phosphatase (calf b intestinal mucosa) 30.0 1:16 Positive control (purified staphy- lococcal acid phosphatase) 30.0 1:4 Negative control (unabsorbed serum) - 1:16 a . . All of these sources were tested prior to realizing that 30 or more units of acid phosphatase were required to obtain optimal proportion of antigen and antibody. b The fact that 30 or more units of nonhomologous acid phosphatase did not absorb antibody indicates the specificity of the phosphatase- antiphosphatase system. 63 pentameric IgM fraction into ineffective subunits accom- panied by a decrease in antibody titer. The antiphospha- tase titer of serum treated with mercaptoethanol and an untreated control serum were determined by immunodiffusion (Table 5). Both had a titer of 1:8 indicating that the W3 mercaptoethanol had no effect on the antibody. ‘n_ TABLE 5.--Effect of 2-mercaptoethanol on the titer of rabbit antiphosphatase. Serum Antiphosphatase titer samples Mercaptoethanol No Mercaptoethanol a Normal none none Antiphosphatase 1:8 1:8 aNo antiphosphatase activity. Pilot Protection Studies on Rabbits Immunized with Acid Phosphatase Staphylococcal skin infections were established, by subcutaneous injection of organisms and talc, in three rabbits during the period of maximal antiphosphatase 64 titer (1:16) and in one nonimmunized control rabbit. Total and differential WBC count and lesion severity were deter- mined at 24, 48, 72, 96 hr and at frequent intervals until healing occurred. Table 6 shows normal WBC values for all the rabbits and the WBC values post inoculation. Table 7 lists the differential for the granulocytic and lymphocytic series so that relative shifts may be observed. At 24 hr post inoculation, lesions formed at the sites of the three most concentrated injections (4.4 x 109, 4.4 x 108 and 4.4 x 107 organisms/0.1 ml) were red and slightly swollen in the immunized rabbits. In the control rabbit these lesions were also red and swollen and in addition contained some pus. There was no reaction at any control site where suspensions of sterile saline and talc had been injected. After 48 hr the lesions had reached their maximal size and there occurred no further increase in intensity. The lesion produced by the highest number of organisms (4.4 x 109 organisms/0.1 ml) was approxi- mately 2 cm in diameter, the second lesion was 1.0-1.5 cm in diameter and the third 0.6-l.0 cm in diameter. By 96 hr the lesions of the immunized rabbits appeared to have diminished in intensity while those of the control TAB H 65 TABLE 6.--Normal WBC of experimental rabbits prior to immunization with different amounts of acid phosphatase and counts taken approximately 2 months later following establishment, by subcutaneous inoculation, of a staphylococcal skin infec- tion. Animal Average Average post inoculation WBC normal WBC 24 hr 48 hr 72 hr 96 hr 288 hr Rabbit 1a 15,500 16,500 17,800 13,000 14,000 13,100 Rabbit 2b 11,450 13,900 18,000 17,500 14,600 15,200 Rabbit 3c 10,500 11,750 13,500 13,400 11,400 11,800 d Control 12,650 9,450 9,750 13,000 12,400 10,900 a Received 25 units staphylococcal acid phosphatase 65 days prior to inoculation. b Received 50 units staphylococcal acid phosphatase 65 days prior to inoculation. cReceived 100 units staphylococcal acid phosphatase 65 days prior to inoculation. d Did not receive any staphylococcal acid phosphatase. C01 66 TABLE 7.--Normal differential counts of experimental rabbits prior to immunization with different amounts of acid phosphatase and counts taken approximately 2 months later following estab- lishment, by subcutaneous inoculation, of a staphylococcal skin infection. Differential Count White Animal cell , , t e Normal Hours post inoculation yp 24 hr 48 hr 72 hr 96 hr 288 hr neut.a 24 85 33 29 40 34 . b , Rabbit l stabs O 0 O 0 0 0 lymph.C 73 15 66 67 57 60 neut. 38 84 52 55 61 55 Rabbit 2 stabs 0 4 1 6 0 0 lymph. 55 10 44 35 27 42 neut. 37 77 39 40 52 37 Rabbit 3 stabs 0 l 0 0 0 O lymph. 59 22 57 58 46 58 neut. 30 65 13 22 32 35 Control stabs 0 l 0 0 0 0 lymph. 75 33 86 75 67 63 a neut. 8 neutrophils stabs = immature neutrophils c lymph. B lymphocytes 67 rabbit were redder and contained a large amount of pus. At approximately 1 week post inoculation the immunized rabbit lesions, which had appeared to be healing, became extremely red, raised and fluctuating (Fig. 5). Within the next 5 days many of the lesions developed pus and became necrotic. At first necrosis occurred in lesions produced by the highest number of organisms (4.4 x 109 'organisms/0.1 ml) and eventually it occurred in the other two lesions. Following suppuration healing became evi- dent and was complete by 1 month post inoculation. The control rabbit's lesions which had become necrotic much earlier (96 hr), also healed sooner than the immunized rabbits (approximately 17 days post inoculation). 68 Fig. 5.--Lesions in rabbit 1, rabbit 2 and rabbit 3 occurring at 1 week post inoculation with viable Staphylococcus aureus. \ 0 DISCUSSION The development of an antiphosphatase assay by immunodiffusion has proved to be a useful tool in the .4 immunological study of this enzyme. It is easy and rapid to perform and is more sensitive than the colorimetric test. Concentrated sera are easily tested with this method. Since the number of serum samples available for testing was small, our data are only an indication of the possible usefulness of this assay for diagnosis of staphy- lococcal disease. Antiphosphatase was not detectable in normal human serum. The antibody was present in the sera of individuals with chronic staphylococcal skin infection and with infections of the deep tissues such as staphyj lococcal cellulitis and endocarditis. In addition serum samples of three of the endocarditis patients contained another antibody that we were unable to identify. Since the Klebsiella-Enterobacter-Serratia group of organisms also produces an acid phosphatase in large amounts, we attempted to react this enzyme with staphylococcal anti- phosphatase. The acid phosphatase of these organisms did 70 71 not cross react with antibody to staphylococcal acid phos- phatase; nor was there any cross reaction between this antibody and acid phosphatase preparations of plant origin. Evidently staphylococcal antiphosphatase was a specific indicator of a chronic or severe staphylococcal infection. 1 There are several implications that might be drawn from the staining reaction used in the immunodiffusion Vagina; I test. Since the enzyme had to react with the substrate to produce the staining effect, it was evident that specific aggregation with its antibody does not inhibit reactivity. It is theorized that the enzyme acted on the substrate through catalytic sites not blocked by the antibody. This usually occurs with substrates having a molecular weight less than 200 (Cinander, 1963). The staphylococcal anti- phosphatase system did not comply with this observation since the substrate, B-glycerophosphate, had a molecular weight of 216. Substitution with substrates of higher molecular weight (p-nitrophenylphosphate, M.W. 263; and phenolphthalein diphosphate, M.W. 562) did not inhibit staining. Therefore one can conclude that factors other than the molecular weight of the substrate were involved. Cinander (1959) indicated the complexity of this problem by citing other variables in the inhibition of enzyme by 72 its antibody. The degree of inhibition appears to depend on the quality of the antibody as determined by the con- ditions of immunization, the relative quantity of anti- body, the period of incubation and in some instances, on the concentration of substrates. Sevag, Newcomb and Miller (1954) reported that dissociation occurred in mix- tures of a-glycerophosphate and yeast antiphosphatase in both antigen and antibody excess. There is some evidence for similar dissociation of staphylococcal phosphatase and its antibody. In the colorimetric antiphosphatase determination antigen was still detected in the supernate when antibody was in excess. It was not within the scope of this project to determine which of these factors was responsible for continued reactivity of the precipitated phosphatase; however, this reactivity was notable because of the effect it might have on the protective role of this antibody i3 yiyp. Some investigators (Rogers and Melly, 1965) have concluded that, due to repeated subclinical infections with staphylococci, most adults had developed a maximal titer of antibodies which could not be further increased. Our finding, that rabbits immunized with different doses of staphylococcal acid phosphatase had all achieved the 73 same titer which was not enhanced by subsequent imposition of a staphylococcal skin infection, seemed to support this thesis. However, while it appears that a peak titer had been reached, it should be noted that it was much higher than was found in individuals with chronic staphylococcal “a disease. These same authors claimed that patients with active staphylococcal infection did not demonstrate higher titers of antistaphylococcal antibodies than did normal ‘1’“. adults. We found the opposite to be true; i.e., anti- phosphatase antibodies were not detectable unless chronic or severe staphylococcal disease was present. Therefore from our experience with this particular antibody we con- cluded that increased immunization was worth pursuing. Ekstedt and Yoshida (1968) observed that the anti- body in hyperimmune rabbit antisera specific for Staphy- lococcus aureus teichoic acid was associated with the IgM fraction. The titer persisted for only a short time (2 to 3 weeks) leading them to suspect that there may be a lack of immunological memory in animals undergoing IgM response exclusively. This nonrecognition was suggested as a reason for repeated reinfection of man by staphy- lococci. For this reason we considered determination of the immunoglobulin class of antiphosphatase important to 74 our study. Incubation of the rabbit antiphosphatase serum with mercaptoethanol did not reduce the titer of the serum indicating that the antibody was in the IgG fraction. The high phosphatase activity of pathogenic staphy- lococci as compared to saprophytic nonpathogenic strains (Barber, Brooksbank and Kuper, 1951) prompted us to ex- amine the protective capacity of phosphatase immunized rabbits to subcutaneous injection of Staphylococcus aureus (PS 55). It has been suggested (Kedzia et al., 1966) that phosphatase was involved in regulating the concentration of the inorganic phosphate pool of the organism. Pre- sumably neutralization of acid phosphatase activity would therefore seriously impair metabolic functioning of the staphylococcal organisms. Following challenge of acid phosphatase immunized animals with subcutaneous inocula- tion of Staphylococcus aureus (PS 55), total and differ- ential WBC counts were determined in anticipation of obtaining quantitative evidence of protection in the immunized animals. Dajani and Wannamaker (1972) reported hamster total and differential WBC count values observed during experimental staphylococcal skin infection. Of 12 infected animals the WBC counts were normal in all but one that exhibited leukocytosis, and neutrophilia was marked Tl 75 in all instances. In our experiment a moderate increase in the WBC within 48 hr was observed in the immunized rabbits, but no response in the control rabbit. A neutro- philia and lymphOpenia was observed in the differential of all four rabbits at 24 hr which returned to normal by 48 hr. The rise in the WBC seen in the immunized rabbits might be interpreted as a superior ability to respond to infection; however, for the most part, the results appeared inconclusive. The degree of inflammation and necrosis was also used as a criterion. At the onset lesions in the control rabbit were more severe than in the immunized rabbit. There was pus formation by 24 hr in the control rabbit and necrosis by 96 hr. By comparison the immunized rabbits' lesions were red and swollen with no evidence of pus formation or necrosis. One might conclude at this point that the organisms were able to multiply rapidly and produce enough toxins to cause necrosis in the con- trol rabbit, while in the immunized rabbits multiplica- tion of the organisms was inhibited. This course changed abruptly at 1 week post inoculation when the lesions in the immunized rabbits became intensely red, raised and fluctuating. Eventually these lesions became necrotic and healed two weeks later than the control rabbit lesions. ‘Hs-'.4JF ' ‘ 11: .__ 76 There are several possible explanations for this phenom- enon. Antiphosphatase might have succeeded in inhibiting multiplication of organisms early in infection but this defense was eventually overcome. Similar evidence was __ 1., noted in protective studies following immunization with coagulase (Harrison, 1964). In this study, the number of survivors in the immunized group was significant at 4 days but not at 21 days. The author suggested that anticoag- 5 E r4" | ulase interfered with the pathological processes that lead to death shortly after injection of large numbers of staphylococci, but that it failed to prevent the chronic type of disease that led to death later on. If this were the case, a combination of selected purified staphylococcal products, including acid phosphatase, might elicit a more protective antibody combination. If this sudden lesion intensification was due to the ability of the organism to finally reach a high titer, one would expect a concomi- tant rise in the WBC. Another explanation for this reac- tion might be that it was the result of delayed hyper- sensitivity. 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