JOURNAL OF BACTERIOLOGYVol. 87, No. 5, pp. 1216-1220 May, 1964Copyright X 1964 by the American Society for Microbiology

Printed in U.S.A.

ANTIBIOTIC CONTROL OF TISSUE REACTIONS IN DOGS VACCINATED WITHVIABLE CELLS OF COCCIDIOIDES IMMITIS'

MERIDA W. CASTLEBERRY,2 JOHN L. CONVERSE, AND PETER J. SOTO, JR.'

U.S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland

Received for publication 18 December 1963

ABSTRACT

CASTLEBERRY, MERIDA W. (U.S. Army Biologi-cal Laboratories, Fort Detrick, Frederick, Md.),JOHN L. CONVERSE, AND PETER J. SOTO, JR. Anti-biotic control of tissue reactions in dogs vaccin-ated with viable cells of Coccidioides immitis. J.Bacteriol. 87:1216-1220. 1964.-A total of 12 dogs(15 to 25 lb each), vaccinated with viable Coccid-ioides immitis (subcutaneous injection of 260viable arthrospores in the medial surface of thehind leg), resisted a respiratory challenge (aerosol)with the same organism (13,000 viable arthro-spores) administered (aerosol) 2 months aftervaccination. Oral amphotericin B therapy (150mg of Fungizone per day for 21 days) of 6 of the 12dogs, initiated immediately after vaccination,eliminated the undesirable side reactions of theviable vaccine (ulcerated vaccination site andinguinal lymphadenopathy exhibited by the 6untreated dogs) without affecting the immuno-genicity of the vaccine. Clinical observation(blood-urea nitrogen levels) during and aftertherapy and histological examination approxi-mately 3 months after respiratory challenge failedto disclose any evidence of nephrotoxicity or renaldamage due to the oral antibiotic therapy (totaldoses of more than 3 g of amphotericin B).

The immunogenesis of various antigeniccomponents of the saprophytic and parasiticstages of Coccidioides immitis was studied byNegroni, Vivoli, and Bonfiglioli (1949), Friedmanand Smith (1956), Pappagianis et al. (1961),Levine, Cobb, and Smith (1960), Converse et al.(1962b), Kong, Levine, and Smith (1962), and

I Animals were maintained in compliance withthe "Principles of Laboratory Animal Care" aspromulgated by the National Society for MedicalResearch.

2 Present address: U.S. Army Medical Researchand Nutrition Laboratory, Fitzsimons GeneralHospital, Denver, Colo.

3Present address: Department of Pathology,St. Louis University School of Medicine, St. Louis,Mo.

others. A fair degree of immunity was developedby several of these preparations, if death wasused as the unit of measure. Absolute preventionof coccidioidomycosis lesions after respiratory orintraperitoneal challenge, however, has neverbeen attained with a killed vaccine.

Pappagianis et al. (1960) and Converse,Castleberry, and Snyder (1961) reported theresistance of monkeys to a second infection(respiratory) with C. immitis after the sub-cutaneous administration of viable C. immitisarthrospores. The investigations of Converse etal. (1961) demonstrated that, even in very lowdoses (ten arthrospores), the viable vaccineprotected against subsequent, extremely heavyaerosol challenge. However, ulceration at the siteof vaccination or regional lymphadenopathyor at both sites was occasionally encountered.

Several methods to circumvent these unde-sirable tissue reactions to the viable vaccine areunder study. One of these studies was based onthe hope that concomitant oral administration ofamphotericin B (Fungizone; E. R. Squibb &Sons, New York, N.Y.) at the time of the vac-cination would alleviate the adverse reaction tothe vaccination. Campbell and Hill (1959) andCastleberry et al. (1963) described the use oforally administered amphotericin B in C. immitis-exposed animals. These investigators ascribed noadverse physiological reaction to its use in thismanner.The present report concerns an evaluation of

the effects of amphotericin B on the untowardlocal reactions of a viable vaccine against coc-cidioidomycosis, and a determination of theeffectiveness of such a vaccine in dogs.

MATERIALS AND METHODS

A total of 16 healthy mixed-breed dogs of bothsexes weighing between 15 and 25 lb were em-ployed in this study.The vaccine was prepared by suspending viable

arthrospores of C. immitis, strain D-76 (highly1216

on February 12, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

COCCIDIOIDOMYCOSIS VACCINE

virulent for dogs), in normal saline (260 spores perml). The high dose and virulence of this straininsured somatic reaction to the subcutaneousdeposition of the vaccine.

Amphotericin B was dissolved in distilled water(15 mg/ml). Each dog was fed twice daily with asplit dose of 5 ml of the amphotericin B solutionmixed in his food. It was accepted readily by alldogs.Of the 16 dogs, 14 were vaccinated subcu-

taneously in the medial surface of the right thighwith 1 ml of the vaccine. Administration ofamphotericin B (150 mg per day) to six of thesevaccinees was initiated immediately and con-tinued for 21 days. The remaining eight dogs wereuntreated. At 54 days after vaccination, 12 ofthe 14 vaccinated dogs and 2 nonvaccinated,nontreated controls were exposed via the respira-tory route in the manner described by Converse

et al. (1962a). These dogs received an averageinhaled dose of approximately 13,000 viablearthrospores of the Cash strain of C. immitis.Two vaccinated, untreated control dogs werekilled at this time, rather than exposed, to eval-uate the gross and histopathological responses ofthe tissue to the vaccine. The remaining 12vaccinated and the 2 unvaccinated, untreatedcontrol animals were also killed 77 days afteraerosol challenge. Intravenously administeredpentobarbital was used for this purpose. The dogswere necropsied, and the tissues were fixed in10% buffered formalin, embedded in paraffin, sec-tioned, and stained. Hemotoxylin-eosin and theGomori methenamine silver stains were usedroutinely. Lung material from all animals wascultured on GPY (2% glucose, 1% peptone,0.1% yeast autolysate) agar slants.

F1G. 1. Ulcerated vaccination sites developing by the 17th day postvaccination in dogs not receiving oralamphotericin B therapy at the time of vaccination (a and b). Dissection, showing enlarged inguinal lymphnodes (c). Dissection, showing involvement of the subcutis (d). Note penetration of the gracilis muscle.

VOJ.. 87, 1964 1217

on February 12, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

CASTLEBERRY, CONVERSE, AND SOTO

RESULTS

By the 17th day, the vaccinated dogs that didnot receive the amphotericin B had developed an

induration at the vaccination site. Withoutexception, these eventually ulcerated (Fig. laand b). The lesions had healed, however, atsacrifice 130 days after vaccination. Histo-pathological examination of these areas showed a

fibroblastic response extending rather deeply intothe subcutis. An increased number of lympho-cytes and plasms cells accompanied the scarring,but C. immitis was not seen. A mild reactivehyperplasia of the regional (right inguinal) lymphnodes was noted in several of the untreatedvaccinees. These changes were attributed to thelesion produced by the subcutaneous depositionof viable C. immitis arthrospores; however, no

sl)herules of C. iinmitis were noted here.Visible reaction to the vaccination did not

develop in any of the dogs that received am-

TABLE 1. Response of dogs to subcutaneous vaccinimmitis a

photericin B. Subsequent histological examina-tion of the skin and subcutis in the area of theoriginal vaccination site failed to reveal anychanges. Histological examination of the rightinguinal lymph glands of these dogs revealed no

significant changes, except for minimal reactivehyperplasia.

In addition to the vaccination-site scars of theuntreated dogs, necropsy also revealed a fewsmall (1 to 3 mm), grayish-tan nodules scatteredover the pleural and cut surfaces of the lungs ofthree of the treated and one of the nontreatedahimals (Table 1). Histopathological examinationof these pulmonary lesions revealed small isolatedgranulomatous lesions that occasionally containeda spherule (Fig. 2). Similar pulmonary lesionswere encountered in three other animals that haddemonstrated no gross lesions. Interestinglyenough, giant cells, which are generally presentin coccidioidal granulomata, were not noted in

ation and aerosol challenge with viable Coccidioides[rthrospores

Pathology

Viable vaccine Amphotericin B Respiratory challenge Gross Histopathology(spores) therapy (spores) Vaccin- Inguinal Vaccin- Inguinal

ation lymph Lungb ation lymph Lungbsitea nodea sitea nodea

g260 3 13,000 +

_ + - +

260 None,d 13,000 + + - + ++ + + + + ++ + - + + +c+ + - + + +c+ + - + + -+ + - + + +

260 None None (vaccine + + - + +controls) + + - + +

None (disease None 13,000 - - + + - - +++controls) - - +++ - - +++

a Ulcerated vaccination site or inguinal lymphadenopathy indicated by +.Degrees of pathological involvement: -, negative; +, minimal; ++, moderate; +++, severe.

c Histological changes compatible with, but not diagnostic of, coccidioidomycosis. No sphertulesseen.

d Lesions noted at the vaceination site; the inguinal lymph nodes of animals in this group were healedat 130 days postvaccination.

1218 J. BACTERIOL.

on February 12, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

COCCIDIOIDO-MYCOSIS VACCINE

these lesions. The remaining five animals demon-strated no gross or microscopic evidence ofcoccidioidomycosis. All animals with the excep-tion of the two nonvaccinated, challenged, controldogs were negative to culture for C. immitis.The pleural and cut surfaces of the lungs of the

two nonvaccinated, challenged, control dogs wereliberally covered with relatively large (0.2 to 1cm), firm, grayish-yellow nodules. Histopathologi-cal examination of these lesions revealed es-sentially an amalgamation of smaller, early, andwell-developed granulomata. These confluentlesions were in turn surrounded by a restrainingcollar of young connective tissue and lympho-cytes. No histological differences were notedbetween the lesions of the control dogs and thoseof the 12 vaccinees, except for the larger size andgreater number of lesions found in the two con-

trols (Fig. 2). C. immitis was cultured from thelung lesions of each of these two dogs.Except at the vaccination site, no lesions

attributable to coccidioidomycosis were noted inthe two vaccinated, untreated, unchallengedcontrol dogs killed at 54 days. The vaccinationulcer of each dog was still oozing pus at the timeof sacrifice (Fig. la and b). This exudate wasnot cultured; however, a smear was made fromeach dog, stained, and examined. No spheruleswere seen here. The histopathology of the affecteddermal layers was characterized by the replace-ment of the epithelial layer with necrotic debris.Underlying this were proliferative collagenouselements, which were liberally interspersed withlymphocytes and plasma cells. This reaction hadpenetrated, in one dog, to the underlying gracilis

FIG. 2. Comparison of histological lung sections of vaccinated and nonvaccinated dogs 77 days afterrespiratory challenge. Top, nonvaccinated control dogs; bottom, vaccinated dogs (diagnosis: left, negative;right, minimal). Hematoxylin and eosin. 7 X.

VOL. 87, 1964 1219

on February 12, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

CASTLEBERRY, CONVERSE, AND SOTO

muscle (Fig. ld). No spherules of C. immitiswere seen.

DISCUSSIONThree very important observations may be

made from the data presented. First, oral treat-ment with amphotericin B immediately aftervaccination blocked the undesirable side effectsof the viable vaccine. This was evidenced by thelack of ulceration at the site of vaccination, thelack of inguinal lymphodenopathy, and the lackof histological changes in these areas.Secondly, therapy at the time of vaccination

did not interfere with the development of im-munity. This was shown by the fact that theresistance to the subsequent respiratory challengewas essentially the same in the vaccinated, un-treated animals and the vaccinated, treatedanimals.

Thirdly, clinical and histological examinationof all dogs receiving the amphotericin B (totaldoses of more than 3 g) failed to disclose anyevidence of renal damage. Previous study (Castle-berry et al., 1963) showed that the blood-ureanitrogen values in dogs remained well withinnormal limits at this dosage level.

It is also evident that the viable vaccine wasas effective in dogs as it was in monkeys (Con-verse et al., 1961). Of the 12 vaccinated dogs, 5remained free of infection. Of the remaining 7dogs, 4 exhibited only very minimal lung changes;3 were in the doubtful category (few focal gran-ulomata; no spherules seen). This was in contrastto the massive involvement of the vaccinatedcontrol dogs. Moreover, all 12 of the vaccinatedanimals showed negative cultures for C. immitis.The fact that C. immitis could not be seen at thesite of vaccination or in the inguinal lymph nodesindicated that the vaccine strain was probablycleared from the tissues at the time of autopsy.

ACKNOWLEDGMENT

C. immitis strain D-76 was kindly supplied byRaymond E. Reed, The University of Arizona.

LITERATURE CITED

CAMPBELL, C. C., AND G. B. HILL. 1959. Beneficialtherapeutic effects of solubilized amphotericinB following oral administration in experi-

mental coccidioidomycosis, histoplasmosis,and cryptococcosis in mice. Trans. 4th Ann.Veterans Administration-Armed Forces Coc-cidioidomycosis Study Group, Los Angeles,Calif.

CASTLEBERRY, M. W., J. L. CONVERSE, J. T. SIN-SKI, E. P. LOWE, S. P. PAKES, AND J. E. DELFAVERO. 1963. Coccidioidomycosis studies:canine vaccination and therapy. Trans. 8thAnn. Veterans Administration-Armed ForcesCoccidioidomycosis Study Group, Los An-geles, Calif.

CONVERSE, J. L., M. W. CASTLEBERRY, AND E. M.SNYDER. 1961. A viable prophylactic vaccineagainst coccidioidomycosis in monkeys.Trans. 6th Ann. Veterans Administration-Armed Forces Coccidioidomycosis StudyGroup, Los Angeles, Calif.

CONVERSE, J. L., E. P. LOWE, M. W. CASTLE-BERRY, G. P. BLUNDELL, AND A. R. BESEMER.1962a. Pathogenesis of Coccidioides imimitis inmonkeys. J. Bacteriol. 83:871-878.

CONVERSE, J. L., M. W. CASTLEBERRY, A. R.BESEMER, AND E. M. SNYDER. 1962b. Immuni-zation of mice against coccidioidomycosis. J.Bacteriol. 84:46-52.

FRIEDMAN, L., AND C. E. SMITH. 1956. Vaccina-tion of mice against Coccidioides immilis. Am.Rev. Tuberc. Pulmonary Diseases 74:245-248.

KONG, Y. H., H. B. LEVINE, AND C. E. SMITH.1962. Primary locus of immunogens in coccid-ioidal spherules. Trans. 7th Ann. VeteransAdministration-Armed Forces Coccidioido-mycosis Study Group, Los Angeles, Calif.

LEVINE, H. B., J. M. COBB, AND C. E. SMITH.1960. Immunity of coccidioidomycosis in-duced in mice by purified spherule, arthro-spores, and mycelial vaccines. Trans. N.Y.Acad. Sci. 22:436-449.

NEGRONI, P., D. VIVOLI, AND H. BONFIGLIOLI.1949. Estudios sobre el Coccidioides immitisRixford et Gilchrist. VII. Reacciones immu-noalergicas en la infeccion experimental delcobayo. Rev. Inst. Malbran (Buenos Aires)14:273-286.

PAPPAGIANIS, D., R. L. MILLER, C. E. SMITH,AND G. S. KOBAYASHI. 1960. Response of mon-keys to respiratory challenge following sub-cutaneous inoculation with Coccidioides immi-tis. Am. Rev. Respirat. Diseases 82:244-250.

PAPPAGIANIS, D., C. E. S-MITH, G. S. KOBAYASHI,AND M. T. SAITO. 1961. Studies of antigensfrom young mycelia of Coccidioides imntitis.J. Infect. Diseases 108:35-44.

1220 J. BACTER1IOL .

on February 12, 2021 by guest

http://jb.asm.org/

Dow

nloaded from