Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD₅₀ at 28 days post infection ranged from 3.6–7.5×10⁵ cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.     

Pseudomonas aeruginosa,a facultative pathogen of red palm weevil,Rhynchophorus ferrugineus

Pseudomonas aeruginosa was identified as a facultative pathogen of red palm weevil. Intra-haemocoelic injection of the pathogen within larvae and pre-pupae was more effective at killing the insects [with a median lethal dose (LD₅₀) of 9×10² to 2×10³ bacteria/insect] than inoculation by force feeding (LD₅₀ of 10⁵ to 4×10⁵ bacteria/insect) or by wading the insects in a suspension of the pathogen (LD₅₀ of 10⁵ to 2×10⁵ bacteria/insect). Injection of 3×10³ bacteria/insect killed 69% of larvae; small larvae were more susceptible (LD₅₀ of 9×10⁵ bacteria/larva) than either larger larvae (LD₅₀ of 10³ bacteria/larva) or pre-pupa. The median time to death of the small larvae following injection of P. aeruginosa was about 6 days but that following force feeding or wading was about 8 days. A secondary invader, Serratia marcescens, had no effect on the pathogenicity of P. aeruginosa but hastened death of larvae by about 3 days.A. Banerjee and T.K. Dangar were with the Central Plantation Crops Research Institute, Regional Station, Kayangulam 690 533, Kerala, India. They are now with the Central Rice Research Institute. Cuttack 753 006, Orissa, IndiaCPCRI research paper no. 870.     

Clinico-pathological studies on experimental cryptococcal mastitis in goats

Unilateral intramammary inoculation of 10 goats withCryptococcus neoformans (2×10⁶ yeast cells) resulted in the development of mastitis, with gross and microscopic lesions being restricted to the infected udder halves only and there was no dissemination of infection to the opposite uninfected udder halves as well as to other organs of the body. The experiment was continued for 40 days, with 2 animals each from the infected and control groups being killed on 5th, 10th, 20th, 30th and 40th day postinoculation (DPI). Initial enlargement of the infected udder halves was followed by marked decrease in size leading to very small, firm and nodular udder halves. After infection, there was also sharp fall in the milk yield. Cryptococcal organisms were demonstrated in the mastitic milk and udder impression smears with special stains.C. neoformans was reisolated from the milk of only infected udder halves up to 25th DPI. Microsopically, there was initially acute diffuse purulent mastitis which later on became chronic, characterised by marked infiltration of lymphocytes, macrophages, extensive fibrosis and development of multiple granulomas. The cryptococcal organisms could be demonstrated in the udder sections only up to 30th DPI. It is concluded that intramammary inoculation ofCryptococcus neoformans in goats leads to severe mastitis with sharp fall in milk yield.     

Olfactory mucosa response in guinea pigs following intranasal instillation withCryptococcus neoformans

A central nervous system isolate from an acquired immunodeficiency syndrome (AIDS) patient of 10³ Cryptococcus neoformans cells was instilled intranasally into guinea pigs. These were killed to evaluate the fate of the organisms and the response of the olfactory mucosa. Olfactory epithelium prevented the penetration ofCryptococcus neoformans and showed focal hyperplastic responses. The organisms, which manifested an affinity for the olfactory portion of the nasal cavities, were cleared from the olfactory space with no intervention from the immune system cells. By the end of the fifth week almost no organisms could be found and there was no histological evidence of dissemination. In contrast, destruction of the olfactory epithelium with zinc sulfate allowed the invasion of the subepithelial tissues, demonstrating the role of the olfactory mucosa in preventing infection withCryptococcus neoformans through the nasal route. The results and the model described in this report may be useful in clarifying the pathogenic mechanisms of cryptococcosis and the non immune mediated host responses toCryptococcus neoformans.     

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14–28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10⁶ cryptococci, while no mortality was observed in mice exposed to 10³ or 10⁴ cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.     

Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans

Aims: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. Methods and Results: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD₅₀ value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. Conclusions: The silkworm–C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. Significance and Impact of the Study: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.     

Isolation of saprophytic Cryptococcus neoformans from Puerto Rico: Distribution and variety

Until the present decade, no studies had been conducted in Puerto Rico on the saprophytic distribution and variety of Cryptococcus neoformans. Samples (522) of pigeon droppings from 14 western towns were tested for the presence of C. neoformans. The yeast was recovered from 24.7% (129 isolates) of the samples, representing 10 of the 14 towns studied. All environmental isolates were identified as C. neoformans var. neoformans using canavanine-glycine-bromthymol blue (CGB) agar. The yeast was isolated from 79.4% of the samples in one town, Isabela. The average number of yeast cells isolated from sites within this municipality was 5.1×10⁵ per gram of pigeon droppings. This was 2.6 times the average number of yeast cells of C. neoformans isolated from sites in other towns. In addition, the yeast was isolated from four patients with the acquired immune deficiency syndrome (AIDS), each of whom died of cryptococcal meningitis. Each of these poorly encapsulated isolates was identified as C. neoformans var. neoformans using CGB agar. The results of this investigation demonstrate that C. neoformans var. neoformans is prevalent in Puerto Rico.This paper was presented in part at the X^th Congress of the International Society for Human and Animal Mycology, Barcelona, Spain from June 27 to July 1, 1988.     

Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

The three-dimensional ultrastructure ofCryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method.C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10⁷ cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells ofC. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure ofC. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10–13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions byC. neoformans.     

Different Susceptibility of Three Clinically Isolated Strains of Cryptococcus neoformans to the Fungicidal Effects of Reactive Nitrogen and Oxygen Intermediates: Possible Relationships with Virulence

We investigated the susceptibility of three clinically isolated strains of Cryptococcus neoformans with different virulences to reactive nitrogen and oxygen intermediates (RNI and ROI, respectively), representing two important mediators of macrophage microbicidal activity. All mice infected with the highly virulent strain of C. neoformans, YC-11, died within 3 to 6 weeks because of rapid multiplication of the organism in the lungs and dissemination to the brain. In contrast, a weakly virulent strain, YC-13, was almost completely eradicated from the lungs and did not disseminate to the brain, leading to survival of all infected animals during the period of observation (15 weeks). The virulence of the third strain, YC-5, was intermediate between the other two strains. To examine the susceptibility of C. neoformans to the fungicidal effect of nitric oxide (NO) and superoxide anions (O₂-), the organisms were exposed to these oxidants, which were chemically generated in a cell-free system. Interestingly, the number of live YC-13 yeast cells was markedly reduced after exposure to NO and O₂^?. In contrast, YC-11 was almost completely resistant to the killing effect of these oxidants. YC-5 showed an intermediate susceptibility. Our results demonstrate that the resistance of C. neoformans to the fungicidal effects of RNI and ROI is related to virulence, and suggest that the resistance to nitrogen- and oxygen-derived oxidants may be one of the factors to determine the outcome of infection with C. neoformans.     

A toxic substance produced by Nocardia otitidiscaviarum isolated from cutaneous nocardiosis

During our studies on toxic substances from clinically isolated Nocarida, a new isolate identified as Nocardia otitidiscaviarum from cutaneous nocardiosis was found to produce a toxic substance called HS-6 that had strong in vitro as well as in vivo toxicity. The mouse intraperitoneal LD₅₀ value was 1.25 mg/kg and the ED₅₀ value for L1210 cultured cells was 0.3 ng/ml. The structure of HS-6 was determined and found to belong to the 16-membered macrocyclic group with a molecular formula of C₄₃H₆₈O₁₂. HS-6 also showed activity against pathogenic fungi such as Cryptococcus neoformans.     

Atypical Micromorphology and Uncommon Location of Cryptococcosis: A Histopathologic Study Using Special Histochemical Techniques (One Case Report)

Here we report an unusual case of disseminated cryptococcosis in a patient with AIDS. Although typical Cryptococcus neoformans micromorphology was observed in tongue biopsy, cervical lymph node examination revealed atypical histopathologic findings. These included pseudohyphae, chains of budding yeasts and structures resembling germ tubes. Cryptococcus neoformans infection in supraclavicular lymph nodes was also confirmed by culture. The importance of using special histochemical techniques—Mayer’s mucicarmine stain for mucicarminophilic capsule and Grocott’s silver stain—in the diagnosis of cryptococcosis is reinforced.     

Combination Efficacy of Voriconazole and Amphotericin B in the Experimental Disease in Immunodeficient Mice Caused by Fluconazole-resistant <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

The therapeutic efficacy of amphotericin B and voriconazole alone and in combination with one another were evaluated in immunodeficient mice (BALB/c-SCID) infected with a fluconazole-resistant strain of Cryptococcus neoformans var. grubii. The animals were infected intravenously with 3 × 10⁵ cells and intraperitoneally treated with amphotericin B (1.5 mg/kg/day) in combination with voriconazole (40 mg/kg/days). Treatment began 1 day after inoculation and continued for 7 and 15 days post-inoculation. The treatments were evaluated by survival curves and yeast quantification (CFUs) in brain and lung tissues. Treatments for 15 days significantly promoted the survival of the animals compared to the control groups. Our results indicated that amphotericin B was effective in assuring longest-term survival of infected animals, but these animals still harbored the highest CFU of C. neoformans in lungs and brain at the end of the experiment. Voriconazole was not as effective alone, but in combination with amphotericin B, it prolonged survival for the second-longest time period and provided the lowest colonization of target organs by the fungus. None of the treatments were effective in complete eradication of the fungus in mice lungs and brain at the end of the experiment.     

Effect of Some Variables on theIn VivoDetermination of Scorpion and Viper Venom Toxicities

An adequate assessment of scorpion and snake venom LD₅₀is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD₅₀variation of Tunisian scorpion (Androctonus australis garzonii: Aag andButhus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57Bl/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990–1996). Aag venom is 3–4 times more toxic than Bot venom. However for both venoms, the LD₅₀determined in C57Bl/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD₅₀variations (25–50%) were also seen from one electrically prepared batch to another. A good correlation (r=0·982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD₅₀values when assessedin vivo.The LD₅₀variation of Tunisian viper (Cerastes cerastes: Cc andVipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990–1992). No significant LD₅₀variations were observed with the mouse strain, the sex or the season of venom milking. However, LD₅₀varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS–PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD₅₀evaluation or the venom quality used for the development of an efficient antivenom.     

Studies on organ specificity in experimental murine cryptococcosis

The chief histopathological features found in patients with cryptococcosis are both a cystic (gelatinous) lesion and a granulomatous reaction. These two tissue reactions are definitely different from each other, because a cyst is not accompanied with a significant cellular response, while a granuloma is formed as a result of various cell reactions. Therefore, it is very interesting that these two types of lesion can be observed in the same patient or in the same animal infected with Cryptococcus neoformans. From our previous paper (II) the authors reach such a thought that two steps may be required for the granuloma formation against C. neoformans infection: first, of phagocytosis by sessile macrophages of C. neoformans and second is related to T-cell function. This experiment was done to verify that the granulomatous response against C. neoformans infection might occur easily in the organs rich in sessile macrophages as compared with those poor in them and a polysaccharide capsule surrounding cryptococci may have effects to inhibit a migration of polymorphonuclear leucocytes or monocytes toward C. neoformans. C. neoformans strain RIB 12 (serological type A, mating type α) was used in this experiment. After a culture of a brain heart infusion glucose agar slant at 37 C for 3 days, yeast cells of the strain were harvested, and suspended in 1/15 M(p_H7.4) sterile phosphate buffered saline solution. Infective inoculum was prepared by adjusting the number of the yeast cells to 10⁵, 10⁶ or 5×10⁶/0.2 ml in a hemacytometer. Fourty-two male mice strain ddY were divided into 3 groups consisting of 14 each and one group was allotted to one of the cell suspensions. Each mouse was inoculated with 0.2 ml of the cell suspension into a tail vein and one mouse from each group was sacrificed at adequate intervals. At necropsies the brain, thymus, lungs, heart, liver, kidneys, spleen, pancreas, mesenteric lymph nodes, a part of the small intestine, testes and fat tissue were removed. From these organs histopathological sections, stained with HE or by PAS, were prepared. To investigate effects of a polysaccharide capsule to a migration of polymorphonuclear leucocytes or monocytes, double infections with C. neoformans and Aspergillus fumigatus, and an observation by the ‘Agar-Implantation method’ were done. As results, granulomata were formed easily in the organs rich in macrophages or lymphocytes such as the liver, spleen, lymph nodes, thymus, lungs, small intestine and fat tissue. On the contrary, in organs poor in the macrophages such as the brain, heart, pancreas, kidneys, adrenal glands and testes, the chief histopathological feature was a cyst formation containing numerous yeast cells. In the double infection, two types of lesions such as cysts and abscesses were observed in the sections of the brain. The former occurred against C. neoformans infection and the latter, against A. fumigatus infection. Even though a cyst was very close to an abscess, polymorphonuclear leucocytes or monocytes were never induced to C. neoformans. In the observation using the ‘Agar-Implantation method’, a severe cellular infiltration occurred to a perfect (teleomorphic) state of C. neoformans and very weak response, to yeast cells with a polysaccharide capsule. The difference may be due to the existence of the capsule, because a perfect state of C. neoformans is not surrounded by it.     

A proteomic‐based approach for the identification of immunodominant Cryptococcus neoformans proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life‐threatening meningoencephalitis in immune compromised patients. Previous, studies in our laboratory have shown that prior exposure to an IFN‐γ‐producing C. neoformans strain (H99γ) elicits protective immunity against a second pulmonary C. neoformans challenge. Here, we characterized the antibody response produced in mice protected against experimental pulmonary C. neoformans infection compared to nonprotected mice. Moreover, we evaluated the efficacy of using serum antibody from protected mice to detect immunodominant C. neoformans proteins. Protected mice were shown to produce significantly more C. neoformans‐specific antibodies following a second experimental pulmonary cryptococcal challenge compared to nonprotected mice. Immunoblot analysis of C. neoformans proteins resolved by 2‐DE using serum from nonprotected mice failed to show any reactivity. In contrast, serum from protected mice was reactive with several cryptococcal protein spots. Analysis of these spots by capillary HPLC‐ESI‐MS/MS identified several cryptococcal proteins shown to be associated with the pathogenesis of cryptococcosis. Our studies demonstrate that mice immunized with C. neoformans strain H99γ produce antibodies that are immune reactive against specific cryptococcal proteins that may provide a basis for the development of immune based therapies that induce protective anticryptococcal immune responses.     

Comparison of lethalities in mouse versus goldfish for clinical and food isolates ofAeromonas hydrophila

Summary The lethality of 16 clinical or food isolates ofAeromonas hydrophila was assessed by determination of LD₅₀ (i.p.) in mice and goldfish. In mice LD₅₀ values for the variousA. hydrophila strains were similar, ranging from 1.2–21.0×10⁸ cells/animal. A wider range of LD₅₀ values, 0.03–11.8×10⁸ cells/animal, was observed with goldfish. Lethality was not correlated between the two test animals. Further, cytotoxic response in Y-1 adrenal cells did not correlate with lethality in either test animal. It appears that lethality is not a good measure of potential enterotoxigenicity, but may be useful in assessing the invasive character of isolates causing systemic infections in immunocompromised hosts.     

Biological and electron microscopic changes in gamma radiated Cryptococcus neoformans

Samples ofCryptococcus neoformans were irradiated with various doses of gamma radiation. The relatively low doses, 1–5 × 10⁴ rads, resulted in no obvious effect on the fine structure of the cells but did seriously prevent a large proportion of the organisms from reproducing on agar. When subjected to doses of 1 × 10⁶ rads virtually all the organisms lost their ability to reproduce. When such samples were examined in the electron microcsope, almost all the cells were found to have lost their capsules whereas other aspects of their fine structure remained apparently normal.This work was supported by the Sandy Schneider Memorial Fund and by the United Hospital Fund.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Cytoskeletal structures,ultrastructural characteristics and the capsule of the basidiomycetous yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis>

The cytoskeleton, capsule and cell ultrastructure were studied during the cell cycle of Cryptococcus laurentii. In an encapsulated strain, cytoplasmic microtubules and a mitotic spindle were detected. Mitosis was preceded by migration of the nucleus into the bud. F-actin failed to be visualised by rhodamine-phalloidin (RhPh) in encapsulated cells and therefore an acapsular strain was used. The following actin structures were found: actin dots, actin cables and cytokinetic ring. Ultrastructural studies showed the presence of a nucleus in the bud before mitosis. A collar-shaped structure was seen at the base of bud emergence. A lamellar cell wall and a rough outer surface of the cells were detected. Cytoskeletal structures found in C. laurentii are similar to those in Cryptococcus neoformans, which is a serious human pathogen.