Effect of various antibiotics on gastrointestinal colonization and dissemination by Candida albicans

Mice were treated orally with various antibiotics to determine which members of the indigenous intestinal microflora normally suppress Candida albicans colonization and dissemination from the gastrointestinal (GI) tract. The mice were given penicillin, clindamycin, vancomycin, erythromycin, or gentamicin for 3 days, and then challenged orally with C. albicans. Penicillin, clindamycin, and vancomycin, but not gentamicin or erythromycin, decreased the total anaerobic bacterial populations in the animals ceca, and increased the enteric bacilli population levels. All three of the former antibiotics allowed C. albicans to proliferate in the gut and, subsequently, disseminate from the GI tract to visceral organs. The ability of C. albicans to associate with intestinal mucosal surfaces was also tested. It was found that antibiotics which reduced anaerobic population levels, but not enteric bacilli or aerobes, also predisposed animals to mucosal association by C. albicans. It is suggested that the strictly anaerobic bacterial populations which predominate in the gut ecosystem are responsible for the inhibition of C. albicans adhesion, colonization and dissemination from the GI tract.     

Production of anti-Candida antibodies in mice with gut colonization of Candida albicans

BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined. RESULTS: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group. CONCLUSION: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.     

Immunosensing during colonization by Candida albicans: does it take a village to colonize the intestine?

Candida albicans, an opportunistic fungal pathogen and a component of the normal flora of the gastrointestinal tract, is a frequent colonizer of humans. Is C. albicans capable of sensing the immune status of its host, a process we term immunosensing, and, if so, how? C. albicans causes serious disease only in immunocompromised hosts and therefore the ability to immunosense would be advantageous to an organism. We propose a speculative model whereby, during colonization, C. albicans produces phenotypic variants that vary in relative concentration depending on host status. One variant is optimized for persistence as a commensal, whereas the other variant has higher capacity to initiate pathogenic interactions. When the ratio of the two variants changes, the pathogenic potential of the population changes. The critical element of this model is that the C. albicans colonizing population is not uniform but is composed of subpopulations of phenotypic variants that are advantageous under different host conditions.     

An anaerobic continuous-flow culture model of interactions between intestinal microflora and Candida albicans

Summary The finding by earlier workers that Escherichia coli suppressed the growth of Candida albicans in vitro or in gnotobiotic mice has led to numerous, erroneous conclusions regarding the identity of the organisms and mechanisms responsible for the suppression of Candida in the gut. This is due, in part, to the fact that nearly all studies to date have not reflected interactions as they occur in the intestinal tract. This paper describes a series of experiments that establish that an anaerobic continuous-flow (CF) culture model, of the ecology of the large intestinal flora reproduces interactions between bacteria and Candida as they occur in the large intestine. This was determined in the following ways. (i) Bacterial counts in CF cultures of conventional mouse cecal flora or human fecal flora closely resembled that found in the mouse intestine and human feces. (ii) Dense layers of bacterial growth that formed on the glass walls of the CF culture vessels resembled bacterial populations that colonize intestinal mucosa. (iii) Total and individual levels of certain metabolic end-products of the predominant anaerobic bacterial flora present in CF cultures coincided with those found in the large intestine of conventional mice or human feces used to establish the CF cultures. (iv) C. albicans was eliminated from CF cultures of mouse cecal flora at a rate similar to that of untreated experimental animals. (v) Contents of CF cultures fed to antibiotic-treated mice redressed several cecal abnormalities, and suppressed Candida populations to levels found in conventional animals. Thus, a number of complex ecological mechanisms were maintained in CF cultures which normally control Candida populations in the large intestine. It is suggested, therefore, that the CF culture model should help to further define the mechanisms which control C. albicans and other fungi in the intestinal tract, as well as define which components of the indigenous microflora are responsible for suppression of Candida in the gut.     

In vitro studies on colonization resistance of the human gut microbiota to Candida albicans and the effects of tetracycline and Lactobacillus plantarum LPK

An anaerobic three-vessel continuous-flow culture system, which models the three major anatomical regions of the human colon, was used to study the persistence of Candida albicans in the presence of a faecal microbiota. During steady state conditions, overgrowth of C. albicans was prevented by commensal bacteria indigenous to the system. However antibiotics, such as tetracycline have the ability to disrupt the bacterial populations within the gut. Thus, colonization resistance can be compromised and overgrowth of undesirable microorganisms like C. albicans can then occur. In this study, growth of C. albicans was not observed in the presence of an established faecal microbiota. However, following the addition of tetracycline to the growth medium, significant growth of C. albicans occurred. A probiotic Lactobacillus plantarum LPK culture was added to the system to investigate whether this organism had any effects upon the Candida populations. Although C. albicans was not completely eradicated in the presence of this bacterium, cell counts were markedly reduced, indicating a compromised physiological function. This study shows that the normal gut flora can exert 'natural' resistance to C. albicans, however this may be diminished during antibiotic intake. The use of probiotics can help fortify natural resistance.     

Protection against Candida albicans gastrointestinal colonization and dissemination by saccharides in experimental animals

Pre- or post-treatment of duodenal discs with mannose, N-acetylglucosamine or chitin soluble extracts (CSE) prevented the adherence of Candida albicans to gastrointestinal tract. CSE was the most effective in blocking the adherence of C. albicans. Treatment of infant mice with saccharides significantly reduced the systemic spread of C. albicans inoculated into the gut. The best protection was obtained when the saccharides were given 2 days prior to the infection and continued over the course of the infection. However, systemic spread was reduced with a single dose of saccharide 30 min before infection. The saccharides may bind to the gastrointestinal mucosa and block the attachment of C. albicans.     

Protection of mice from lethal endogenous Candida albicans infection by immunization with Candida membrane antigen

The protective effects of immunization with Candida membrane antigen (CMA) on a systemic infection originating from intestinally colonized Candida albicans were examined. The colonization of orally inoculated C. albicans in the intestinal tract was established in BALB/c mice that had been concomitantly treated with oral doses of antibacterial drugs. In these animals, a systemic dissemination of C. albicans with fatal outcome was induced by a repeated dosing of prednisolone. In this endogenous infection model, the effects of immunization by CMA on the infection were examined. CMA-immunized mice showed a longer lifespan than unimmunized mice. The protective effect of CMA immunization in immunosuppressed mice was also measured by a decrease in body weight loss after treatment with prednisolone and in the number of viable Candida cells in the target organs, the kidneys and livers. However, the CFU of C. albicans in the intestinal tract was not significantly lowered. These results suggest that CMA immunization inhibited the dissemination of systemic Candida infection from the intestinal tract induced by treatment with prednisolone.     

Horizontal transmission of Candida albicans and evidence of a vaccine response in mice colonized with the fungus

Disseminated candidiasis is the third leading nosocomial blood stream infection in the United States and is often fatal. We previously showed that disseminated candidiasis was preventable in normal mice by immunization with either a glycopeptide or a peptide synthetic vaccine, both of which were Candida albicans cell wall derived. A weakness of these studies is that, unlike humans, mice do not have a C. albicans GI flora and they lack Candida serum antibodies. We examined the influence of C. albicans GI tract colonization and serum antibodies on mouse vaccination responses to the peptide, Fba, derived from fructose bisphosphate aldolase which has cytosolic and cell wall distributions in the fungus. We evaluated the effect of live C. albicans in drinking water and antimicrobial agents on establishment of Candida colonization of the mouse GI tract. Body mass, C. albicans in feces, and fungal-specific serum antibodies were monitored longitudinally. Unexpectedly, C. albicans colonization occurred in mice that received only antibiotics in their drinking water, provided that the mice were housed in the same room as intentionally colonized mice. The fungal strain in unintentionally colonized mice appeared identical to the strain used for intentional GI-tract colonization. This is the first report of horizontal transmission and spontaneous C. albicans colonization in mice. Importantly, many Candida-colonized mice developed serum fungal-specific antibodies. Despite the GI-tract colonization and presence of serum antibodies, the animals made antibodies in response to the Fba immunogen. This mouse model has potential for elucidating C. albicans horizontal transmission and for exploring factors that induce host defense against disseminated candidiasis. Furthermore, a combined protracted GI-tract colonization with Candida and the possibility of serum antibody responses to the presence of the fungus makes this an attractive mouse model for testing the efficacy of vaccines designed to prevent human disseminated candidiasis.     

Colonization of congenitally athymic, gnotobiotic mice by Candida albicans

Colony counts, scanning electron microscopy, and light microscopy were used to assess the capacity of Candida albicans to colonize (naturally) and infect the alimentary tract of adult and neonatal (athymic [nu/nu] or heterozygous [+/nu] littermates) germfree BALB/c mice. When exposed to yeast-phase C. albicans, the alimentary tract of adult germfree mice (nu/nu or +/nu) is quickly (within 24 to 48 h) colonized with yeast cells. Neither morbidity nor mortality was evident in any mice that were colonized with a pure culture of C. albicans for 6 months. Yeast cells of C. albicans predominated on mucosal surfaces in the oral cavities and vaginas of adult athymic and heterozygous mice. In both genotypes, C. albicans hyphae were observed in keratinized tissue on the dorsal posterior tongue surface and in the cardial-atrium section of the stomach. Conversely, neonatal athymic or heterozygous mice, born to germfree or C. albicans-colonized mothers, do not become heavily colonized or infected with C. albicans until 11 to 15 days after birth. Although yeast cells adhered to some mucosal surfaces in vivo, neither widespread mucocutaneous candidiasis, i.e., invasion of mucosal surfaces with C. albicans hyphae, nor overwhelming systemic candidiasis was evident in neonatal (nu/nu or +/nu) mice. Thus, even in the absence of functional T-cells and a viable bacterial flora, athymic and heterozygous littermate mice (adult or neonatal BALB/c) that are colonized with a pure culture of C. albicans manifest resistance to extensive mucocutaneous and systemic candidiasis.     

Colonization of congenitally athymic, gnotobiotic mice by Candida albicans.

Colony counts, scanning electron microscopy, and light microscopy were used to assess the capacity of Candida albicans to colonize (naturally) and infect the alimentary tract of adult and neonatal (athymic [nu/nu] or heterozygous [+/nu] littermates) germfree BALB/c mice. When exposed to yeast-phase C. albicans, the alimentary tract of adult germfree mice (nu/nu or +/nu) is quickly (within 24 to 48 h) colonized with yeast cells. Neither morbidity nor mortality was evident in any mice that were colonized with a pure culture of C. albicans for 6 months. Yeast cells of C. albicans predominated on mucosal surfaces in the oral cavities and vaginas of adult athymic and heterozygous mice. In both genotypes, C. albicans hyphae were observed in keratinized tissue on the dorsal posterior tongue surface and in the cardial-atrium section of the stomach. Conversely, neonatal athymic or heterozygous mice, born to germfree or C. albicans-colonized mothers, do not become heavily colonized or infected with C. albicans until 11 to 15 days after birth. Although yeast cells adhered to some mucosal surfaces in vivo, neither widespread mucocutaneous candidiasis, i.e., invasion of mucosal surfaces with C. albicans hyphae, nor overwhelming systemic candidiasis was evident in neonatal (nu/nu or +/nu) mice. Thus, even in the absence of functional T-cells and a viable bacterial flora, athymic and heterozygous littermate mice (adult or neonatal BALB/c) that are colonized with a pure culture of C. albicans manifest resistance to extensive mucocutaneous and systemic candidiasis.     

The presence of bacteria and yeast like fungi in specimens from surgical patients

The aim of this study was to determine the incidence of Candida spp. strains in specimens obtained from surgically treated patients as well as to analyze the accompanying bacterial flora, both aerobic and anaerobic. The material came from two groups of patients. In the first group consisting of patients operated for colon and rectum carcinoma, the samples included peritoneal fluid, colon or rectum bioptates, pus, blood, and wound swabs. In the other group, biopsy material and smears from post operation wounds were taken from patients who underwent a surgical treatment of larynx carcinoma. Altogether, 282 various clinical specimens from 165 patients were analysed, and 41 Candida spp. strains were isolated: 39 strains of C. albicans and 2 strains of C. tropicalis. In 20 out of 41 specimens infected with Candida spp. (48.8%) the co-infection with bacterial aerobic flora was found. In 10 cases (24.4%), the fungi were isolated together with aerobic and anaerobic bacterial flora, whereas in 2 specimens (4.9%) the anaerobes and Candida albicans were diagnosed. The remaining 9 samples showed only the presence of Candida spp. (21.9%). From among aerobic bacterial flora Enterococcus spp. strains (n = 17) and Gram negative rods from Enterobacteriaceae family (n = 13) were the most frequently isolated. The bacterial strains of Streptococcus spp. (n = 5), Pseudomonas spp. (n = 3), Staphylococcus spp. and Corynebacterium spp. (2 strains, both) were identified more rarely. Bacteroides spp. were the most frequent members of bacterial anaerobic flora (n = 10). Other isolated anaerobic bacteria were classified as Fusobacterium spp. or Peptostreptococcus spp. (1 strain each). E. coli and Enterococcus spp. strains of aerobic bacterial flora were more frequently isolated together with Candida spp. CONCLUSIONS: (i) Mixed bacterial flora was found to predominate in the clinical material from the patients after surgery. (ii) Candida spp. were most frequently found together with aerobic bacterial flora.     

The antagonistic effect of Saccharomyces boulardii on Candida albicans filamentation, adhesion and biofilm formation

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans . We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans , i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.     

Lower filamentation rates of Candida dubliniensis contribute to its lower virulence in comparison with Candida albicans

Candida albicans and C. dubliniensis are very closely related yeast species. In this study, we have conducted a thorough comparison of the ability of the two species to produce hyphae and their virulence in two infection models. Under all induction conditions tested C. albicans consistently produced hyphae more efficiently than C. dubliniensis. In the oral reconstituted human epithelial model, C. dubliniensis isolates grew exclusively in the yeast form, while the C. albicans strains produced abundant hyphae that invaded and caused significant damage to the epithelial tissue. In the oral-intragastric infant mouse infection model, C. dubliniensis strains were more rapidly cleared from the gastrointestinal tract than C. albicans. Immunosuppression of Candida-infected mice caused dissemination to internal organs by both species, but C. albicans was found to be far more effective at dissemination than C. dubliniensis. These data suggest that a major reason for the comparatively low virulence of C. dubliniensis is its lower capacity to produce hyphae.     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

Fungal infection in the oncologic clinic]

Fungal infection in oncologic hospitals is a serious threat for the patients that can undermine the results of the specific treatment of the main disease. Most frequently fungal infection is developing in localities naturally disseminated with microbial flora. The infection is usually mixed: bacterial/fungal and even fungal/fungal. The most frequent members of the associations are the representatives of Candida albicans: 63 per cent in the bacteria + fungi associations and up to 60 per cent in the fungi + fungi associations. The preventive use of azoles can lower the threat of fungal affection of the organs. However, the danger of mixed fungal/bacterial infection remains rather high.     

白假丝酵母与细菌相互作用机制及其与感染的关系

人体内定植着数目庞大、结构复杂的细菌及真菌等微生物群,它们之间存在复杂的相互作用。既往研究主要集中于细菌或真菌的某个单一物种,但最近研究表明,细菌与真菌之间的相互作用对更好地理解体内的微生态系统至关重要。白假丝酵母(又称白念珠菌)是人备体中最常见的机会性致病真菌,通常被认为是人类正常菌群的一部分。白念珠菌与细菌的相互作用近年来备受关注,其协同和拮抗作用有助于维持不同物种间复杂的平衡关系。了解白念珠菌与细菌的相互作用,不仅可加深对微生物致病机制的理解,还可为预防和治疗白念珠菌或细菌感染及新型抗菌药物研发提供新途径。本文就白念珠菌与细菌共存时的相互作用机制及其对人类健康、疾病的影响进行综述,从而为控制念珠菌或细菌感染提供新的策略。     

口服肝素与小鼠肠道菌群的相互作用

口服肝素药物的开发需要系统地理解口服肝素与肠道菌群之间的互作过程。通过荧光体视镜观察荧光素标记的肝素经小鼠口服后在体内的分布情况,利用高效液相色谱法检测肝素在模拟胃肠液中的稳定性和体外培养肠道菌群模拟肠道菌对肝素的降解作用,发现口服肝素主要分布在小鼠胃肠道内,在体外模拟胃肠液条件下肝素结构稳定,但能够被添加肝素的厌氧培养基培养后的肠道菌群降解。为了进一步揭示口服肝素对健康小鼠肠道菌群的影响,利用Illumina MiSeq高通量测序技术测定口服肝素后C57BL/6J小鼠粪便菌群的16S rRNA序列,与口服生理盐水的小鼠粪便菌群进行对比,发现口服肝素的小鼠粪便菌群的生物多样性降低;在门水平上,菌群结构差异不显著;而在属水平上,别样杆菌属Alistipes、副萨特氏菌属Parasutterella和艾克曼菌属Akkermansia相对丰度增高,而嗜胆菌属Bilophila、肠杆菌属Enterorhabdus、瘤胃梭菌属Ruminiclostridium、普雷沃氏菌科Prevotellaceae_UCG_001、瘤胃梭菌属Ruminiclostridium-9、拟杆菌属Bacteroides、Lachnoclostridium、Candidatus_Saccharimonas、Intestinimonas和Dubosiella的相对丰度减少,表明口服肝素能够影响小鼠肠道菌群结构。此外,实验发现口服肝素对小鼠无明显毒副作用,具有较高安全性。研究结果将为开发肝素口服递送策略提供新的思路,为口服肝素类药物的开发提供参考。     

Pathogenicity of Hansenula anomala in a model of immunocompromised mice

Systemic infections caused by opportunistic fungi have shown an increased frequency in the past 10 years, particularly in immunocompromised patients. Hansenula anomala is an ascosporogenous yeast of the Ascomycetes class found in the skin, throat, and digestive tract transient normal flora. This study was conducted to compare the pathogenicity of H. anomala and Candida albicans in a model of immunocompromised mice. Thirty-eight Swiss mice were divided into two groups as follows: 30 animals received an intraperitoneal (i.p.) injection of cyclophosphamide (200 mg/kg) four days before the induction of infection with H. anomala (1 × 10⁶ yeasts/mL), and 8 animals received 100 mg/kg of cyclophosphamide at 3-day intervals during 3 weeks before inoculation of 1 × 10⁷ yeasts/mL. All animals were treated with amoxicillin/clavulanic acid (40 mg/kg) four days before induction of infection. A group of mice inoculatd with C. albicans (ATCC 64548) served as control. Tissue samples from the lung, spleen, liver, and kidney for histological and mycologic studies were obtained at necropsy. In each animal, the number of viable yeasts per gram of kidney was determined. The organs most frequently infected by H. anomala were the kidneys and the liver (20%), and the lung (10%). However, in conditions of sustained immunosuppression, H. anomala was found in 65.5% of the organs examined. It is concluded that in an experimental model of immunocompromised mice, the pathogenicity of H. anomala was low. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth, morphogenesis, and virulence of Candida albicans after oral inoculation in the germ-free and conventional chick

Balish, Edward (Syracuse University, Syracuse, N.Y.), and A. W. Phillips. Growth, morphogenesis, and virulence of Candida albicans after oral inoculation in the germ-free and conventional chick. J. Bacteriol. 91:1736-1743. 1966.-The effects of intestinal bacteria on the multiplication, morphogenesis, and infectivity of Candida albicans in the alimentary tract were investigated by comparing results obtained in germ-free and conventional chicks after oral inoculation. This challenge resulted in the establishment of large numbers of the pathogen in the alimentary tract of each group of chicks; these numbers were increased in crop contents from challenged bacteria-free chicks wherein hyphae predominated over the yeast form. These animals also had lesions of the crop epithelium containing numerous hyphae and few yeast-like forms. In contrast, challenged conventional chicks receiving an adequate diet displayed no evidence of infection. Their alimentary tract contained the yeast form of C. albicans; no hyphae were seen. Although we found bacterial inhibition of C. albicans multiplication in the alimentary tract, this in itself did not seem to explain the resistance to intestinal candidiasis in our conventional chicks. We argued that this resistance to infection was due chiefly to the prevention of hyphal development in C. albicans by intestinal bacteria. C. albicans in the gut of our conventional chicks resulted in some increase in numbers of enterococci in contents from the crop. Increased pH values in contents from the gut of germ-free chicks were not clearly related to infection after challenge. The E(h) of the above crop contents were only slightly decreased in the germ-free crop. Thus the E(h) did not appear to be involved in susceptibility to infection. Invasion of the blood stream and kidneys of conventional chicks by the yeast form of C. albicans occurred in challenged animals receiving a purified diet which had been radiation-sterilized and stored for 6 months at room temperature (25 C). Their growth rate decreased and they became moribund; no hyphae were observed in tissues or intestine of these animals. Challenged bacteria-free chicks receiving the same diet were resistant to the above invasion, although they had crop lesions containing hyphae as described. The resistance of these chicks to systemic invasion was attributed to absence of intestinal bacteria competing for low levels of vitamins in the stored diet. Germ-free chicks had decreased levels of serum gamma-globulin which increased after challenge, whereas this value was unchanged in conventional birds after challenge.