A one year study on the viability of Cryptococcus neoformans in nature

Using four different assay procedures we found a 50 to 86% decrease in viability of Cryptococcus neoformans over a one year period after two towers in the Oklahoma City area were modified to exclude pigeons and weather elements, especially rain. In homogeneous pigeon dropping samples a sharp decrease in viability occurred after 7 to 9 months of storage. The decrease occurred in pigeon dropping samples maintained at the natural sites and at ambient temperature in the laboratory but not in droppings stored at –4 °C. Air sampling studies done at the natural sites before and after tower modifications also showed a decrease in C. neoformans viability. The decrease in viability cannot be explained by seasonal temperature variations that occurred at the natural sites. Our findings suggest that death of C. neoformans occurred because of lack of available intracellular water exacerbated by low humidity, exclusion of precipitation and moisture from fresh pigeon droppings.In light of our experiences, at one site in south central United States, the feasibility of controlling C. neoformans at heavily contaminated sites, other than by the use of noxious or potentially dangerous chemicals, is discussed.     

Gastric colonization withCandida albicans

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.     

A rapid pigmentation test for identification ofCryptococcus neoformans

A rapid pigmentation test for identification ofCryptococcus neoformans is described. The method is based on the formation of a characteristic, mousegrey to violaceous-black pigment when shake cultures ofC. neoformans in a phosphate-buffered,l-DOPA — ferric citrate medium are incubated at 37 C for one hour.This work was carried out in part with financial support from the Council of Scientific and Industrial Research, New Delhi.     

Prevalence of yeast-like fungi and evaluation of several virulence factors from feral pigeons in Seoul, Korea

Aims: Studies of pigeon‐borne yeasts have tended to focus on species, such as Cryptococcus neoformans and Candida albicans, with scant attention to feral pigeons in Korea. We studied the prevalence of yeasts from faecal samples of feral pigeons obtained in various public places in Seoul, Korea, and assessed their potential capacity as human pathogens. Methods and Results: Three hundred and six pigeon faeces samples were collected at city squares and parks in 21 localities in Seoul and Seoul Grand Park and analysed for yeast with conventional methods. Of the 306 samples, 126 (41·2%) were positive for yeast. Seventeen species of yeast were identified. The most frequent species were Candida glabrata (34·1%), Candida famata (12·7%), Cryptococcus albidus (14·3%) and Cryptococcus laurentii (7·9%). The yeast isolates were tested for virulence. Of the 116 isolates (ten isolates missing), 70·7% (n = 82) grew at 37°C. All the Cryptococcus spp. isolates possessed a capsule, 16·4% (n = 19) produced melanin, and 33·6% (n = 39) produced proteinase. Two Ca. glabrata, a Ca. famata and Ca. albicans as well as three C. neoformans, a C. laurentii and Ca. albicans isolates had three virulence factors. Accordingly, 29·3% (n = 34) isolates possessed more than two virulence factors except capsule formation. Conclusions: These results of this study indicate that feral pigeons harbour a variety of yeasts and are a reservoir of human pathogenic fungi. Significance and Impact of the Study: This study is the first time about the microflora (fungi) presents in faecal samples collected from a variety of public areas throughout Seoul, Korea.     

Identification of the perfect state ofCryptococcus neoformans from 195 clinical isolates including 84 from AIDS patients

Filobasidiella neoformans is the teleomorphic state ofCryptococcus neoformans and it is a heterothalic. The purpose of this study was to establish the proportions of each mating types (a, ) from among 195 strains ofC. neoformans isolated from clinical material. The culture medium used was sunflower agar. Cultures were incubated at 20–22 °C for 15 days and observed periodically for one month. Non-reactive strains were mated several times with different reactive strains. Under these conditions 96.8% of the strains were found to be reactors. Among both varieties ofC. neoformans, mating type was found to have the highest frequency of 95% in the varietyneoformans and 84% in the varietygattii. These results showed a higher reactivity in comparison with other investigators. This difference could be due to the medium used or to repeated mating with different reactive tested strains.     

Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

An evaluation of various environmental factors affecting the propagation of Cryptococcus neoformans

Summary Sterilized soil (pH 7.7) seeded withC. neoformans was incubated at different temperatures and under various atmospheric conditions during a summer and winter.Incubated for one year at constant temperatures in atmospheric humidities, the organism survived in greater number and for longer periods of time at 4–6° C. Increased humidity greatly enhanced the survival and proliferation of the organisms incubated at 4–6° C and 20–24° C but had little effect on organisms incubated at 37° C.Summer temperatures in Oklahoma coupled with direct exposure to the sun were lethal to the organism in 100 % humidity. In the winter months, exposure to sunlight had no effect on the viability ofC. neoformans.As incubation time in soil increased the thickness of the capsule decreased.Cryptococcus neoformans probably exists in nature in nearly nonencapsulated state. It survives best in alkaline soils, in areas of high humidity, and where the organisms are protected from high temperatures and direct sunlight.This investigation was partially supported by Public Health Service grants AIO5022 and CC-00081, from the National Institute of Health.This work was done in partial fulfillment for the Ph.D. degree.     

The isolation,characterization and antifungal susceptibilities of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from bird excreta in Klang Valley,Malaysia

The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine–glycine–bromothymol blue agar. All were of α-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.     

Protecting buildings against feral pigeons

Feral pigeons (Columba livia, Gmelin 1789) cause different problems for building owners when using structures for daytime perching, sleeping, and breeding. Problems include fouling of building facades and pavements, transmission of allergens and pathogenic microorganisms, and infestations with ectoparasites emanating from breeding sites. Owners are primarily interested in keeping away unwanted pigeons from their property. Pest control companies offer different deterrent systems, of widely varying efficacy, for proofing buildings against feral pigeons. A better solution is avoiding attractive structures during building design or subsequent alterations of existing structures used by feral pigeons. With our study, we elaborate the relevant structural data to help to maintain a building free of pigeons. We performed experiments with free ranging feral pigeons in a feral pigeon loft in the City of Basel, Switzerland. The maximum outlet width a pigeon is not able to pass through is 4 cm; the respective outlet height is 5 cm and a pigeon-safe square opening is not larger than 6 × 6 cm. The maximum ledge width a pigeon is not able to sit on is 4 cm. The pigeon-safe angle of inclination for smooth construction materials (tinplate, glass, plastics) is 25°, for medium rough materials (wood, plane concrete) 35°, and for rough materials (sandstone, rough concrete) at least 50°. Additionally, we studied the behavioral strategies used by feral pigeons to surmount our experimental constructional restrictions, ledge width, and ledge inclinations. Our data provide the essential data to prevent feral pigeons from using building structures.     

Quantitative complement fixation

Summary In agreement with the investigations ofKent et al. (4), it was found that blood collected in a modifiedAlsevers solution remains constant for a period of at least 14 days.We also succeeded in proving the same with regard to complement, stored according toRichardson (9) and with regard to amboceptor prepared according to the directions of the New York State Department of Health and stored in an equal volume of glycerol at 4° C.The cheap Eel colorimeter proved very suitable for the standardization of the erythrocyte-suspension and the measurement of the percentage of haemolysis.Success was not obtained by means of the method, used in this investigation, to keep the amount of complement, which under the conditions of the test causes haemolysis of 50% of erythrocytes, so constant, as to make daily complement titration unnecessary.     

Kluyveromyces marxianus: a potential source of diacetyl reductase

Kluyveromyces marxianus had a higher specific activity of diacetyl reductase (EC 1.1.1.5) than all other organisms previously reported. The enzyme was NADH-dependent and irreversibly catalysed the conversion of diacetyl to acetoin with an optimum pH of 7.0. It was stable at 40°C but lost 50% of its activity at 50°C in 30 min. The K _m and V _max values for diacetyl were 1.8 mm and 0.053 mm/min, respectively.The authors are with the Department of Food Science and Technology, Comell University, Geneva, New York 14456, USA     

An account of some physiological studies in two species of phytophthora

Summary The paper deals with some physiological studies of two typical strains ofPhytophthora palmivora Butl. andP. parasitica Dast. var.macrospora Ashby causing severe fruit rots ofAchras sapota L. andAnona squamosa L. respectively. The effect of temperature on the germination of sporangia had a marked effect. Maximum percentage of indirect germination (by formation of zoospores) occurred at 20° C, whereas direct germination of sporangia (by formation of germ tube) was at 30° C, which further continued upto 37° C. Maximum indirect germination at 20° C shows the favourable cool temperature for the epidemic of the diseases (Fruit rots) in nature. The best substrate (medium) for germination of sporangia was found to be tap water. Next to this was the host decoction or extract. Glucose solution also accelerated sporangial germination. The effect of two dyes viz., Malachite green and crystal violet was also studied in relation to growth and sporulation ofPhytophthora isolates. Their addition to the medium in various fractional dilutions had a profound influence in the rate of growth and sporulation. An interesting observation noted was that growth of the isolates was inversely porportional to the various concentrations of the dyes, under study. An attempt was also made to study the influence of various vitamins. In all, six vitamins were included in the study. Out of these, thiamine and riboflavin were found to be the best sources promoting good growth and sporulation of both the species ofPhytophthora under study.Forms a part of Senior author's M. Sc. (Agric.) Thesis, University of Poona, Poona (India).Respectively, Ex-Junior Research Fellow, I.C.A.R., New Delhi; Professor of Plant Pathology and Principa, College of Agriculture, Junagad (Gujarat); Plant Pathologist, Wheat Rust Research Station, Mahabaleshwar, Poona, India.     

Interrelationship betweenCoprinus lagopus andFusarium udum in soil in relation to competitive saprophytic ability and microbial antagonism

Summary The interrelationship between the growth ofCoprinus lagopus andFusarium udum on pigeon pea substrates in soil was studied at 22-2°C and 30±2°C in relation to their competitive saprophytic ability and antagonism.C. lagopus was observed to be a potent and frequent colonizer of pigeon pea substrates in soil along withF. udum. Saprophytic colonization of pigeon pea substrate byF. udum precolonized byC. lagopus was inhibited in all inoculum soil mixtures. Saprophytic colonization of the substrate byF. udum was better at 22°C than at 30°C and that byC. lagopus better at 30°C than at 22°C. The colonization of substrate by each fungus was suppressed by the other fungus depending upon the temperature used. Hyphal parasitism and colony interactions between these fungi and also with a number of other saprophytic fungi were investigated. No antagonistic reaction was observed betweenF. udum andC. lagopus. However, these fungi were observed to be potent antagonists against other microfungi tested. The formation of fruiting bodies ofC. lagopus was also observed in the laboratory on nutrient media but more frequently on pigeon pea substrates.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

Catabolism of arginine byCarnobacterium spp. isolated from vacuum-packed sugar-salted fish

The ability ofCarnobacterium spp. originally isolated from vacuum-packed, sugar-salted fish to catabolize arginine was examined. All strains were able to produce citrulline, ornithine, and NH₃ from arginine, presumably by the arginine deiminase pathway. The metabolism of arginine was concurrent with acid production from glucose for one strain ofCarnobacterium sp. but delayed for one strain ofCarnobacterium piscicola. The arginine catabolism was not inhibited in the presence of 2% glucose for three strains of carnobacteria during growth in test broth and/or shrimp extract. Growth as well as arginine catabolism was delayed for two strains of carnobacteria by lowering the temperature from 9°C to 4°C. A similar result was obtained by incubating one strain ofC. piscicola in CO₂. None of the compoundsl-citrulline,l-ornithine hydrochloride, and (NH₄)₂SO₄ had any effect on growth or arginine catabolism of this strain. Neither did pH of the medium affect the time for initiation of arginine catabolism.     

Urban Pigeons (<Emphasis Type="Italic">Columba livia</Emphasis>) as a Potential Source of Pathogenic Yeasts: A Focus on Antifungal Susceptibility of <Emphasis Type="Italic">Cryptococcus</Emphasis> Strains in Northeast Brazil

To investigate pigeons as a potential source of pathogenic yeast species, 47 samples of pigeon droppings and 322 samples from pigeon cloacae were evaluated. The samples were also collected from trees located near the pigeon habitats, in the city of Fortaleza, Ceará, Northeast Brazil. In addition, we evaluated the in vitro antifungal susceptibility of these environmental Cryptococcus strains to amphotericin B, azoles and caspofungin. C. neoformans var. neoformans (n = 10), C. laurentii (n = 3), Candida spp. (n = 14), Rhodotorula mucilaginosa (n = 6) and Trichosporon sp. (n = 3) were isolated from pigeon droppings. In contrast, only Candida spp. (n = 4), Trichosporon sp. (n = 3) and R. mucilaginosa (n = 2) were recovered from cloacae specimens. Only Candida glabrata (n = 1) was recovered from plant samples. Azole resistance was detected in only one environmental strain of Cryptococcus, which was resistant to itraconazole (MIC = 1 μg/ml). As expected, all Cryptococcus strains were resistant to caspofungin. In summary, the present study confirms that urban pigeons are a potential source of Cryptococcus spp. and other pathogenic yeasts. Additionally, antifungal resistance was observed in one environmental strain of Cryptococcus neoformans var. neoformans in Northeast Brazil.     

Klebsiella ornithinolytica sp. nov., formerly known as ornithine-positiveKlebsiella oxytoca

The nameKlebsiella ornithinolytica sp. nov. is proposed for a group ofKlebsiella strains referred to previously as NIH Group 12 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, encapsulated, nonmotile rods with the general characteristics of the familyEnterobacteriaceae and of the genusKlebsiella. They give positive results in tests for indole production, Voges-Proskauer, citrate utilization, lysine and ornithine decarboxylases, urease, -galactosidase, malonate utilization, growth in KCN, and esculin hydrolysis, and they produce acid and gas fromd-glucose, and acid froml-arabinose, cellobiose, lactose, melibiose, raffinose, rhamnose, sucrose, trehalose,d-xylose, adonitol,d-arabitol, myo-inositol, sorbitol, arbutin, salicin, -methyl-d-glucoside, and mucate. They give negative drolysis, DNase, pectinase, and acid production fromd-arabinose, melezitose, and dulcitol. They can grow at 4°C and 42°C, and do not produce any pigment. DNA relatedness of eight strains ofKlebsiella ornithinolytica to three strains including the type strain of this species averaged 88% in reaction at 75°C. DNA relatedness to the already recognizedKlebsiella species inEnterobacteriaceae was 1 to 20%. Phenotypic and DNA relatedness data also indicated that a group of organisms referred to as Enteric Group 47 orKlebsiella Group 47 at the Centers for Disease Control (Atlanta, Georgia) was identical withK. ornithinolytica. The type strain ofK. ornithinolytica is NIH 90-72 (JCM 6096).     

First Environmental Isolations of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> in Tunisia and Review of Published Studies on Environmental Isolations in Africa

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts that cause cryptococcosis. These fungi were commonly associated with pigeon droppings and plant materials. The habitat of these pathogens has not been yet studied in Tunisia, although the ecology of these yeasts must be elucidated in order to establish surveillance programs and to prevent infections. The aim of this survey was to recover C. neoformans and C. gattii environmental isolates from pigeon droppings and plant materials in different areas of Sfax region, Tunisia. Nine hundred and fifty samples from leaves, wood, flowers, fruits and soil around trunk bases of 40 almond (Prunus dulcis) and 60 eucalyptus trees were collected as well as 250 pigeon droppings samples from different sites: buildings (n = 150), houses (n = 50) and zoo (n = 50). The identification of Cryptococcus neoformans complex was confirmed using the ID32C auxanogram panel (BioMérieux, Marcy l’Etoile, France); species were determined by multiplex PCR using the CN70 and CN49 primers, and mating type was determined by PCR. C. neoformans was recovered from 26 specimens of pigeon droppings (10.4%). This yeast was obtained more frequently from dry droppings (9.2%) than from moist droppings (1.2%). The mating type was determined. All the 31 environmental strains of C. neoformans and C. gattii were MATα. Out of 700 samples tested from 100 trees, only 5 isolates of Cryptococcus neoformans species complex were recovered (0.6%), two isolates of C. gattii and one isolate of C. neoformans were recovered from the wood of E. camaldulensis trees, and only two isolates of C. gattii were recovered from the wood of almond trees (Prunus dulcis Mill. var. zaaf and var. achek). These two Tunisian almond tree varieties were recorded for the first time in Africa as hosts for C. gattii. These results add new information to the ecology and epidemiology of C. neoformans species complex in Tunisia.     

Effect of inulin on yeast inulinase production in sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K _m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA     

An optimum environment for the culturing of cytospora isolates from stone fruits. I. Temperature

Summary Four isolates ofCytospora cincta Fr. and 2 ofC. leucostoma Fr. were obtained from diseased Italian prune, President plum and Bing cherry trees.The minimum temperature for growth of these fungi was found to be 3° C. Temperatures of 45 °C. were lethal to all cultures. The optimum temperature for theC. cincta isolates on solid and liquid media was found to be 30° C.; for theC. leucostoma isolates, nearly 25° C. OneC. cincta isolate produced greatest radial growth on the solid medium at 35° C., but in the liquid medium produced maximum mycelium at 30° C.All factors considered, the conclusion was reached that the best single temperature for laboratory culture of the fungi was 30° C.Approved by the Director of the Idaho Agricultural Experiment Station as Research Paper No. 493.