Detection and identification of probable endemic fungal pathogen, Cryptococcus gattii, and worldwide pathogen, Cryptococcus neoformans, by real-time PCR

A real-time PCR method for detection and identification of Cryptococcus neoformans and Cryptococcus gattii was developed and evaluated using DNA from single-colony or koala nasal smears. Two TaqMan minor groove binder probes that distinguished between these species were designed corresponding to the internal sequences of the CAP59 gene for both species. The real-time PCR assay had 100% specificity, as assessed using 13 reference strains and 300 environmental strains. Twelve smear samples from healthy koalas were analyzed by direct real-time PCR. This method successfully detected C. gattii and C. neoformans in one and three koalas, respectively.     

Cryptococcosis and pets

This paper is a review of cryptococcosis in domestic animals. Cryptococcosis is an uncommon mycosis in domestic animals and its occurrence is sporadic. The disease is caused by Cryptococcus neoformans, although Cryptococcus gattii has been also isolated from different animal species. Although cryptococcosis has been reported in several animal species, the most frequently affected domestic animal is the cat. The present paper deals with feline and canine cryptococcosis, its common clinical signs and the different clinical forms of the disease in these species. The diagnosis and treatment of cryptococcosis is also discussed. Diagnosis usually includes cytologic examination, capsular antigen detection and culture and identification of the Cryptococcus species. The management of criptococcosis will review the most common therapeutic agents and their role in therapy. Finally, we will address the situation of cryptococcosis in domestic animals in Spain and the role of cryptococcosis as a zoonotic disease and its public health importance.     

Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States

Cryptococcus gattii has recently emerged as a primary pathogen of humans and wild and domesticated animals in British Columbia, particularly on Vancouver Island. C. gattii infections are typically infections of the pulmonary and/or the central nervous system, and the incidence of infection in British Columbia is currently the highest reported globally. Prior to this emergence, the environmental distribution of and the extent of colonization by C. gattii in British Columbia were unknown. We characterized the environmental sources and potential determinants of colonization in British Columbia. C. gattii was isolated from tree surfaces, soil, air, freshwater, and seawater, and no seasonal prevalence was observed. The C. gattii concentrations in air samples were significantly higher during the warm, dry summer months, although potentially infectious propagules (<3.3 microm in diameter) were present throughout the year. Positive samples were obtained from many different areas of British Columbia, and some locations were colonization "hot spots." C. gattii was generally isolated from acidic soil, and geographic differences in soil pH may influence the extent of colonization. C. gattii soil colonization also was associated with low moisture and low organic carbon contents. Most of the C. gattii isolates recovered belonged to the VGIIa genetic subtype; however, sympatric colonization by the VGIIb strain was observed at most locations. At one sampling site, VGIIa, VGIIb, VGI, and the Cryptococcus neoformans serotype AD hybrid all were coisolated. Our findings indicate extensive colonization by C. gattii within British Columbia and highlight an expansion of the ecological niche of this pathogen.     

中国广西地区格特隐球菌菌种复合体的基因型分析

目的探讨中国广西地区格特隐球菌菌种复合体的基因型特点、种群结构特征和全球菌株的进化关系。方法收集2014—2018年间分离自临床确诊为隐球菌病患者的隐球菌临床株,利用CGB培养基初步筛选格特隐球菌菌种复合体。采用多位点序列分型方法(MLST)确定基因型。通过MEGA7软件构建系统发育树,利用R语言进行主成分分析。使用微量肉汤稀释法M27-A3方案行体外抗真菌药物敏感性检验。结果120株临床隐球菌中,分离出11株格特隐球菌菌种复合体,6株属于C.deuterogattii(AFLP6/VGII),5株属于C.gattii sensus stricto(AFLP4/VGI)。分离自广西的AFLP6/VGII呈遗传多样性,主要起源进化自南美巴西格特隐球菌菌种复合体。11株分离菌株均对常用抗真菌药物敏感。结论中国广西可能出现高致病性AFLP6/VGII,对格特隐球菌菌种复合体进行有效的全国性监测是必要的。     

Laccase activity in Cryptococcus gattii strains isolated from goats

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.     

Genetic differentiation, recombination and clonal expansion in environmental populations of Cryptococcus gattii in India

Cryptococcus gattii is a ubiquitous eukaryotic pathogen capable of causing life-threatening infections in a wide variety of hosts, including both immunocompromised and immunocompetent humans. Since infections by C. gattii are predominantly obtained from environmental exposures, understanding environmental populations of this pathogen is critical, especially in countries like India with a large population and with environmental conditions conducive for the growth of C. gattii. In this study, we analysed 109 isolates of C. gattii obtained from hollows of nine tree species from eight geographic locations in India. Multilocus sequence typing was conducted for all isolates using nine gene fragments. All 109 isolates belonged to the VGI group and were mating type α. Population genetic analyses revealed limited evidence of recombination but unambiguous evidence for clonal reproduction and expansion. However, the observed clonal expansion has not obscured the significant genetic differentiation among populations from either different geographic areas or different host tree species. A positive correlation was observed between genetic distance and geographic distance. The results obtained here for environmental populations of C. gattii showed both similarities and differences with those of the closely related Cryptococcus neoformans var. grubii from similar locations and host tree species in India.     

Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.     

Genetic Diversity of the Cryptococcus gattii Species Complex in Mato Grosso State,Brazil

Mycopathologia - Cryptococcosis is caused by fungi of the genus Cryptococcus. Owing to its importance, this study aimed to analyze the genetic diversity of C. gattii isolates from animals, humans,...     

利用红外相机网络调查古田山自然保护区的兽类及雉类多样性

2014年5月至2015年4月,采用红外相机技术系统地调查了浙江省古田山国家级自然保护区内大中型地栖兽类和鸟的多样性组成。调查共获得244个有效位点的数据,其中兽类和雉类的照片视频共67 086份,有效探测数16 129次,包括分属6目15科的野生兽类21种、野生雉类5种、家畜及家禽4种。记录到雉类以外的其它鸟类52种,分属6目15科。在野生兽类和雉类中,国家一级重点保护野生动物2种,分别为黑麂（Muntiacus crinifrons）、白颈长尾雉（Syrmaticus ellioti）,国家二级重点保护野生动物7种,合计占物种总数的35%。IUCN物种红色名录评估为“易危VU”的物种2种,“近危NT”的物种5种,合计占物种总数的27%。中国生物多样性红色名录评估为“濒危”的动物1种,“易危”7种,“近危”5种,合计占物种总数的50%。独立探测数最高的兽类为小麂（Muntiacus reevesi）,雉类为白鹇。调查显示古田山保护区内受威胁的野生兽类和雉类的比例较高。本次调查将红外相机均匀布设于整个保护区,持续时长一年,全面监测保护区内兽类和雉类的物种组成与相对多度,为古田山保护区后续的科研项目和科学管理提供了重要依据。     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

四川省老河沟自然保护区兽类多样性红外相机调查

自然保护区的生物物种编目是区域性和全国性生物多样性研究与监测的基础,而红外相机调查技术已成为兽类物种编目的重要手段之一。老河沟自然保护区位于四川省岷山北部,位于大熊猫岷山种群分布核心地区,是2012年新建保护区。从2011-2014年,我们把保护区划分为1km×1km的方格,采用红外相机技术,对区内的兽类多样性进行了系统全面的调查。经过9,188个相机日的调查,共记录到分属7目18科的兽类。其中,有81,709份记录可以鉴定出具体物种,包括野生兽类24种,家畜1种（家狗）,分属5目14科,独立探测数总计1,766次。其余未能鉴定出具体物种的兽类记录为小型翼手目、食虫目和啮齿目动物。在24种野生兽类中,食肉目物种数最多,共4科9种;其次为偶蹄目（4科7种）与啮齿目（3科6种）。就独立探测数而言,偶蹄目兽类是记录到次数最多的类群（占总探测数43.97%）,其次是啮齿目（25.61%）和食肉目（22.44%）。被IUCN红色名录评估为“濒危EN”、“易危VU”和“近危NT”级别的物种各3种,被列为国家I级和II级重点保护野生动物的分别为4种和5种。本次调查对老河沟自然保护区内的大中型兽类进行了首次系统性编目,了解了区内兽类群落的物种组成、空间分布与相对多度,为后续的科研项目和保护管理提供了本底数据和基础信息。     

Whole-Genome Sequencing of an Uncommon Cryptococcus neoformans MLST43 Genotype Isolated in Nigeria

Mycopathologia - Cryptococcosis is a human infection caused mainly by two species of the Cryptococcus genus, Cryptococcus neoformans and Cryptococcus gattii, whose populations contain several...     

Cryptococcus neoformans mates on pigeon guano: implications for the realized ecological niche and globalization

The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe.     

Viruses isolated from captive and free-ranging wild ruminants in Alberta.

Nasal secretions, leukocytes and preputial or vaginal swabs from a group of 15 captive wild ruminants, comprising six pronghorn antelope (Antilocapra americana), seven fallow deer (Dama dama) and two mule deer (Odocoileus hemionus), and from 50 free-ranging pronghorns in southern Alberta, were examined for viral agents. Captive animals were given injections of dexamethasone daily for 6 days in attempts to reactivate latent infections. Specimens were collected at 2-3 day intervals from days 0 to 18. Free-ranging pronghorns were sampled only once, at the time of capture. Fifteen viral isolates were obtained from the animals: six isolates of parainfluenza 3 (PI3) from nasal swabs from one fallow deer and one mule deer; five isolates of herpesvirus from leukocytes, vaginal, preputial and nasal swabs from three fallow deer; and four isolates of PI3 from nasal secretions of the 50 free-ranging pronghorns.     

Cryptococcus gattii: a resurgent fungal pathogen

Cryptococcus gattii and Cryptococcus neoformans are causal agents of cryptococcosis, which manifests as pneumonia and meningitis. C. gattii has recently received widespread attention owing to outbreaks in British Columbia, Canada and the US Pacific Northwest. The biology of this tree-dwelling yeast is relatively unexplored, and there are few clues about how it causes infections in humans and animals. In this review, we summarize recent discoveries about C. gattii genetics and its ecological niche and highlight areas ripe for future exploration. Increased focus on epidemiology, ecological modeling and host-pathogen interactions is expected to yield a better understanding of this enigmatic yeast, and ultimately lead to better measures for its control.     

Isolation and characterization of Cryptococcus neoformans var. gattii from samples of Eucalyptus camaldulensis in Mexico city.

The habitat of Cryptococcus neoformans var. gattii is not fully known in Mexico. We investigated the relationship of the yeast with Eucalyptus camaldulensis soil in three main Avenues of the city. A total of 135 trees of the species E. camaldulensis, were selected. Samples were taken in duplicate from the ground containing vegetable debris, tree cortex, leaves and flowers. Isolation of the yeast was made on Guizotia abyssinica media, using Staib technique. The identification was accomplished by biochemical and morphologic tests, and caraterization of the variety was made by bromotimol canavanine-glycine-blue (CGB) and D-proline tests. Isolation of 87 strains of Cryptococcus spp. was acomplished and eight of them were identified as C. neoformans var. gattii. These findings confirmed the close relationship of C. neoformans var. gattii and E. camaldulensis. To our knowledge this is the first report concerning the isolation of this variety from E. camaldulensis trees in Mexico.     

The PRP8 inteins in Cryptococcus are a source of phylogenetic and epidemiological information

Only two nuclear encoded inteins have been described. The first, SceVMA, was found in a vacuolar ATPase gene of Saccharomyces cerevisiae and related yeasts. The second, CnePRP8, was found in the PRP8 gene of Cryptococcus neoformans. CnePRP8 contains protein sequences associated with intein splicing but no endonuclease domain. We compared allelic mini-inteins in both varieties of C. neoformans (var. neoformans and var. grubii) and in the related primary pathogen C. gattii to study the evolution of both the mini-intein and the host. We also describe a full-length, endonuclease-containing intein in Cryptococcus laurentii, a moderately distant relation of C. neoformans. We did not detect an intein in the PRP8 gene of other species of Cryptococcus including species closely related to the C. neoformans/C. gattii group. It is therefore probable that the C. neoformans/C. gattii mini-intein was derived from horizontal transfer in which C. laurentii or another intein-containing species was the source.     

格特隐球菌病暴发流行机制的研究进展

目前已证实, 格特隐球菌病可暴发流行。近10 年来, 在加拿大温哥华岛暴发的格特隐球菌病发病率显著高于其他地区, 且大部分感染者免疫力正常, 该致病菌已蔓延至周边地区。本文就格特隐球菌病暴发流行机制, 包括致病菌的分子流行病学分析、可能来源、毒力分析和播散机制进行综述。     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity

Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor approximately 40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MATalpha-specific SXI1alpha gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATalpha strains sequenced, the SXI1alpha gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations.