Influence of Gonadectomy on Candida albicans Urinary Tract Infection in CFW Mice

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1 INFECTION AND IMMUNITY, Mar 1972, p Copyright 1972 American Society for Microbiology Vol 5, No 3 Printed in USA Influence of Gonadectomy on Candida albicans Urinary Tract Infection in CFW Mice DAVID RIFKIND AND JANET A FREY Departments of Microbiology and Medicine, University of Arizona, College of Medicine, Tucson, Arizona Received for publication 22 October 1971 Candida albicans appeared in the urine of approximately 8%,, of CFW mice of both sexes within 2 weeks after intraperitoneal inoculation During a follow-up period of 1 weeks, candiduria tended to clear, but persisted in 37% of males as compared to only 16% in females Autopsy kidney cultures yielded candida in 53% of males as compared to 25 % of females Urine cultures in both sexes when positive contained 13-5 to 14-5 candida/ml regardless of the outcome of the infection Preinoculation gonadectomy in both sexes increased resistance to the infection but did not alter the quantitative aspects of candiduria It is a documented clinical observation that males tend to be more severely affected than females by infectious diseases; however, the basic mechanisms involved in this sex difference remain undefined (13, 16) Insight into this phenomenon is of potential importance as it could provide new information on the development and regulation of the antimicrobial defense systems In an attempt to define an animal model system of this phenomenon, experimental Candida albicans infection in CFW mice was studied After intraperitoneal injection of the yeast-like fungus, the majority of mice of both sexes demonstrated candiduria, following which the infection tended to heal spontaneously However, in the female, the rate of healing was more rapid and the incidence of renal infection at autopsy was lower Furthermore, it was found that preinoculation gonadectomy in both sexes increased the resistance of mice to the infection Some possible mechanisms contributing to the results of these experiments and their relationship to the general phenomenon of greater anti-infective resistance in the female are discussed MATERIALS AND METHODS 1NIice Eight- to 1-week-old CFW mice of both sexes (Carworth Farms, New City, NY) were maintained on antibiotic-free mouse pellets (Purina Mouse Chow, Ralston Purina Co, St Louis, Mo) and water ad lib In most cases the mice were inoculated within 2 weeks of receipt Mice for use in studies on the influence of gonadectomy were obtained at 3 weeks of age and were operated upon within 1 week of receipt The mice were then inoculated at age 8 to 1 weeks Gonadectomy In this procedure the abdomen was 332 epillated with a lotion (Nair, Carter-Wallace Inc, New York, NY), and the animal was anaesthetized with sodium pentobarbital A midline suprapubic incision was made and the gonads were excised The abdomen was closed with a continuous through-andthrough 5- silk suture The sutures were removed 5 days postoperatively The procedure was identical in males and females, as at age three weeks the gonads are intraabdominal in both sexes As controls, animals were subjected to surgery and both gonads were exposed but only one was removed At the termination of the experiments, the accuracy of the surgical procedure was verified by gross autopsy examination Candida C albicans serotype A was isolated from a patient with pyelonephritis and septicemia The culture was grown on Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich) and stored at 4 C For preparation of candida inocula for mice, a veal infusion agar (Difco) Roux bottle was inoculated and incubated at 35 C for 2 days The organisms were harvested in 85%, saline and adjusted to approximately the desired concentration by hemocytometer count Mice were inoculated intraperitoneally with the organisms in a volume of 2 ml of saline The viable cells in the inoculum were then enumerated on veal infusion agar plates Quantitative urine cultures A urine specimen was obtained from each mouse once weekly This was accomplished by holding the mouse over a 12 by 75 mm sterile plastic tube (Falcon Plastics, Oxnard, Calif) and applying suprapubic pressure with the edge of the tube until a urine specimen was collected Using a calibrated 2-mm platinum loop, 2 ml of the urine specimen was transferred to 5 ml of cold veal infusion broth The resulting 5 ml of a 1:25 dilution was poured onto the surface of a Sabouraud dextrose agar plate, spread, and incubated at 35 C for 2 days Colonies morphologically compatible with candida were enumerated Questionable-appearing colonies were checked by means of a Gram stain Downloaded from on October 31, 218 by guest

2 VOL 5, 1972 C ALBICANS URINARY TRACT INFECTION 333 F % =CANDIDURIA 9 =CUMULATIVE DEATHS OZE ~~ 3- MIL F M IM-MALE 8 M F=FEMALE F M 5 F M F 4 mf F * 6 M E F Weeks After Inoculation FIG 1 Comparative inicidenice of canididuria and cumulative deaths in male anid frmale CFW mice after initraperitoneal injectioni of Candida albicanis Autopsy cultures At the completion of the experiment, mice were killed with anaesthetic ether The kidneys were removed aspetically and bisected coronally The cut surface was then used to inoculate a Sabouraud dextrose agar plate After 2 days of incubation at 35 C, the plates were scored as positive or negative for candida RESULTS (i) Candida infection in normal mice Thirty-five mice of each sex were inoculated intraperitoneally with 12 x 17 candida, and urine specimens for quantitative culture were obtained weekly for 1 weeks Candiduria Candiduria occurred within 2 weeks after inoculation in the majority of mice of both sexes (Fig 1) The maximal incidence in the sexes was essentially identical; being 28/35 (8%) in the males and 27/35 (77%) in the females Over the 1-week period of observation, candiduria tended to clear in both sexes, but more frequently in the female Five male and three female mice died during the course of the experiment Of the survivors, 11/3 (37%) of males and 5/32 (16%) of females showed candiduria at week 1 (P = > 5; chi-square method) Serial urine cultures were also obtained from an additional 1 male and 1 female uninoculated CFW mice In no instance was candida recovered from the urine of these mice Autopsy kidney cultures After the 1-week urine specimens were obtained, the surviving 3 males and 32 females were sacrificed, and the kidneys were cultured Autopsy and culture of the eight mice that died before 1 weeks was not possible because of cannabilism by cage mates Kidney cultures yielded candida in 16/3 (53c/%) of the males as compared to 8/32 (25%O) of surviving females (Table 1; P = < 25, chi-square method) A comparison of the 1-week urine culture with the autopsy kidney culture showed that renal infection in certain instances in undetected by urine cultures, The final urine culture was negative for candida in 11/24 (46%-) of mice with positive kidney cultures Conversely, in only one of 38 instances was the final urine specimen positive while the kidney culture was negative Quantitative urine cultures Quantitative cultures of urine for candida showed a mean of approximately 13-5 to 14-5 organisms/ml, and these counts appeared within the first 2 weeks postinoculation (Table 1) The concentrations of candida in the initial positive urine specimens of mice were similarly elevated regardless of the culture status of the kidney subsequently found at autopsy In those mice with positive autopsy kidney cultures, the concentration of candida in the urine remained consistently elevated throughout the 1-week course of observation Among those mice in which negative autopsy kidney cultures were found, the incidence of negative urine cultures increased progressively during the period of observation When positive cultures did occur, however, these urines also contained in in the order of 13 5 to 14-5 organisms/ml The quantitative aspects of candiduria showed no marked sex differences (ii) Candida infection in gonadectomized mice Twelve bilaterally gonadectomized mice of each Downloaded from on October 31, 218 by guest

3 334 ) Z C) C) -C3 Cs ZH CI o CI c Cd r L)4 -O C'E w Cd S:: 2 v) z F73 fjw C) C4-,, - C14 -i4 w 4 "T 4 C) -,- c o o : C) - en _ ' r- _s - s -t mf It o) o o _,4 *~ ¾, e I,C - " -O~ S - o1 a -, * t *-Oen _ O en 6O c r- -OC, OC - o! (L o5 *Cn *ro * ) c *rc ( " It IC 'I fl so so a7 't*r * * It *~*n ) ~ e en * - en # rj ) CJ Tt -C) 5-) c ) *(n ) - 'Cr t _ *C* I - _~~~~~~~~~~ 4) - '- ) > ) > 4 ~ D! I CZ R1FKIND AND FREY INFECT IMMUNITY E o eo co ) C) ) -_ ) _ ) ) O Ḳ C_ C -o ) _ CT )Y C * - )< sex and equal numbers of unilaterally gonadectomized control mice were inoculated intraperitoneally with 2 X 17 viable cells of C albicans Urine specimens for quantitative culture were obtained at weekly intervals for 1 weeks The surviving mice were then sacrificed and the kidneys were cultured Incidence of candiduria The maximal incidence of candiduria was 7/12 in both the bilaterally and unilaterally gonadectomized male mice; and this occurred with the first two weeks postinoculation (Table 2) In the females the maximal incidence was also within the first 2 weeks, but this incidence was 4/12 in the bilaterally gonadectomized group and 9/12 in the unilaterally operated controls (P = < 5; Fisher exact probability test, one-tailed) The clearance of candiduria in the males was not greatly affected by gonadectomy In the females, however, bilateral gonadectomy increased the rate of clearance as well as decreased the initial incidence of candiduria Mortality Five of the 48 inoculated mice died before termination of the experiment at 1 weeks (Table 2) These deaths (three in the males and two in the females) all occurred in the unilaterally gonadectomized controls Renal infection The mice surviving at 1 weeks were sacrificed, and cultures of the kidneys were obtained for candida Positive cultures were found in three bilaterally and three unilaterally gonadectomized males and in one bilaterally and four unilaterally gonadectomized females (Table 2) Thus, 2/24 (83%,-) bilateral gonadectomized mice of both sexes had survived and were free of renal infection by culture, as compared to 12/24 (5%7^) unilaterally operated controls (P =< 25; chi-square method) DISCUSSION In CFW strain mice injected intraperitoneally with C albicans, candiduria occurs in the majority of mice of both sexes within 1 to 2 weeks During a follow-up period of 1 weeks the incidence of candiduria decreases in both sexes, but this clearing is more rapid in the females At 1 weeks postinjection, kidney cultures show candida in approximately one-half of the males as compared with one-fourth of the females Positive urine specimens usually contained 1 - to 14 5candida/ml regardless of the eventual outcome of the infection In those mice with positive autopsy kidney cultures, almost all of the urine specimens throughout the 1-week followup period contained this concentration range of candida In those mice with negative autopsy kidney cultures, the incidence of positive urine cul- Downloaded from on October 31, 218 by guest

4 VOL 5, 1972 C ALBICANS URINARY TRACT INFECTION 335 TABLE 2 Effect of gonadectomy onl canididuria, mortality, and renial infectiont in CFW mice -No of No of mice with positive urine culture/cumulative deaths Positive Sex Gonadectomy mice ~~~~flocu kidney lated Week 1 'Week 2 Week 3 WN'eek 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 1 ~~~~culture' Male Bilateral 12 7/ 5/ 6/ 6/ 3/ 3/ 3/ 1/ 2/ 2/ 3 Unilateral 12 5/ 7/ 6/1 6/1 5/1 3/2 1/3 3/3 3/3 3/3 3 Female Bilateral 12 4/ 2/ 2/ 3/ 1/ 2/ 1/ 1/ / 1/ 1 Unilateral 12 7/ 9/ 8/ 6/ 5/2 6/2 4/2 4/2 2/2 1/2 4 a Survivors autopsied and cultured at 1 weeks tures progressively decreased with time However, the urines that were positive still showed approximately 13 5 to 145 candida/ml This level probably reflects the population size that bladder urine in the mouse will support These patterns were essentially the same in both sexes In those mice gonadectomized as weanlings and then inoculated as adults, the same general pattern of quantitative candiduria also obtained Gonadectomy, however, did result in a lower initial incidence of infection in the females, but not in the males; and in both sexes clearing of the candiduria was augmented Although the mortality in this system was low, the deaths that did occur were in the unilaterally gonadectomized control mice and not in those bilaterally gonadectomized In rats the incidence of pyelonephritis after intravenous injection of Escherichia coli is higher in males (2) The incidence in both sexes was increased by diethylstilbesterol, and the mechanism was reported to be a hormone-induced squamous metaplasia of the collecting system transitional epithelium Friedlander and Braude noted protection of male rats against ascending Proteus mirabilis urinary tract infection by gonadectomy (7) They interpret this protection as resulting from atrophy of the prostate and thereby removal of a locus of infection that acts as a nidus for continual seeding of the urinary tract Gonadectomy and gonadal hormones have been shown to influence infections not localized to the urinary tract For example, ovarectomy increases survival of guinea pigs and mice with experimental tuberculosis (3, 1) Consistant with these observations are reports of decreased resistance to infection induced by exogenously administered gonadal hormones Estrogens and diethylstilbesterol decrease the resistance of female mice to intravenously or intraperitoneally injected Staphylococcus aureus (14, 15) Estradiol and testosterone decrease the resistance of guinea pigs of both sexes to tuberculosis (3), and estrone decreases resistance of female mice to encephalomyocarditis virus (8) Whether the sex difference in urinary tract infection and the influence of gonadectomy on this difference reflects unique anatomical features of the sexes or variation in their general antiinfective defense mechanisms is unknown In addition to the gross structural differences in the lower urinary tract, sex-related renal characteristics in the kidney itself are described In outbred Swiss mice, parietal cells of Bowman's capsule in the female are cuboidal as contrasted to a more flattened configuration in the male (6) In addition to differences in susceptibility to various infectious diseases, sex-related differences in the activity of specific systems which contribute to the general phenomenon of resistance are reported The reticuloendothelial phagocytic activity in rabbits for colloidal carbon is greater in the female, and this is stimulated by estrogens (12) Serum complement levels in rabbits and mice are higher in the male, and gonadectomy lowers this level in the male and elevates the titers in females (5) Certain of the lysosomal enzymes within phagocytic cells in the dog are present in a higher concentration in the female (1) How these factors would interact to influence the outcome of any particular infection is at present a matter of speculation Nicol has suggested that the increased resistance of the female to infectious diseases results from the stimulation of the reticuloendothelial system which is produced by estrogens but not by androgens (12) However, it is not immediately apparent how this formulation is to be reconciled with the examples cited above of increased resistance produced by gonadal hormone in both sexes and decreased resistance following gonadectomy It may be that resistance to certain infections is depressed in both sexes by gonadal hormones and that the effect is for some reason more marked in the male Such an effect might be mediated in part through the immune system by both androgens and estrogens which produce lymphoid atrophy (9) Further investigations on the influence of these hormones on the antimicrobial defense mechanism, particularly with regard to cell- Downloaded from on October 31, 218 by guest

5 336 RIFKIND AND FREY INFECT IMMUNITY mediated and humoral immunity, are in progress in this laboratory LITERATURE CITED 1 Abul-FadI, Y, and R B Scott 1969 Effect of estrogen and progesterone on leukocyte glycogen and alkaline phosphatase in dogs Proc Soc Exp Biol Med 132: Andriole, V T, and G L Cohn 1964 The effect of diethylstilbestrol on the susceptibility of rats to hemnatogenous pyelonephritis J Clin Invest 43: Aycock, W L, and G E Foley 1945 Enhancement of tuberculous infection in guinea pigs by steroid hormones J Clin Endocrinol 5: Caren, L D, and L T Rosenberg 1966 Steroids and serum complement in mice: influence of hydrocortisone, diethylstilbestrol and textosterone Science 152: Churchill, W H, Jr, R M Weintraub, T Borsos, and H J Rapp 1967 Mouse complement: the effect of sex hormones and castration on two of the late-acting components J Exp Med 125: Dunn, T B 1967 Renal disease of the mouse, p In E Cotchin and F J C Roe (ed), Pathology of laboratory rats and mice Blackwell Scientific Publications, Oxford 7 Friedlander, A M, and A 1 Braude 1971 Protection against retrograde pyelonephritis by orchiectomy Clin Res 19: Giron, D J, and P T Allen 197 Effect of estrogen and otlher steroids on MM virus infection in mice Infect Immunity 2: Kappas, A, H E Jones, and I M Roitt 1963 Effects of steroid sex hormones on immunological phenomena Nature (London) 198:92 1 Long, E R, and A B Vogt 1941 Relation of sex to the course of experimental tuberculosis in mnice Amer Rev Tuberc 44: Lurie, M B, S Abramson, and M J Allison 1949 Constitutional factors in resistance to infection I Effect of estrogen and chorionic gonadotropin on course of tuberculosis in highly inbred rabbits Amer Rev Tuberc 59: Nicol, T, B Vernon-Roberts, and D C Quantock 1965 The influence of various hormones on the reticulo-endothelial system; endocrine control of body defense J Endocrinol 33: Schlegel, R J, and J A Bellanti 1969 Increased susceptibility of males to infection Lancet 2: Toivanen, P 1967 Enhancement of staphylococcal inifection in mice by estrogens I Effect of the timing, quantity and quality of the hormone Ann Med Exp Fenti 45: Toivanen, P 1967 Enhancement of staphylococcal infection in mice by estrogens 11 Effect of the inoculumii size and the strain variation of bacteria and mice Ann Med Exp Fenn 45: Washburn, T C, D N Medearis, Jr, and B Childs 1965 Sex differences in susceptibility to infections Pediatrics 35: Yotis, W W, and S I Baman 1969 Diethylstilbestrol: a suppressor of induced furunculosis in rabbits Yale J Biol Med 41: Downloaded from on October 31, 218 by guest