孢子丝菌相关复合种需加强生物安全检控

近年来,随着分子生物学及真菌分类研究的快速发展,包括检疫性真菌在内的物种分类及命名已发生翻天覆地的变化。孢子丝菌病的病原菌长期被认为有且仅有申克孢子丝菌1种,现经多基因系统发育学分析发现孢子丝菌病的病原菌并非仅为申克孢子丝菌,而由6个种构成。我国孢子丝菌菌株的分子生物学鉴定,目前只发现了球形孢子丝菌和申克孢子丝菌,其他4种孢子丝菌尚未发现传入我国。由于这些物种之间形态差异小,但致病力、致病反应、药物反应各不形同,应加强生物安全管控和监测,防止这些有害真菌传入我国。     

中国南北方地区临床孢子丝菌菌种鉴定

目的对分离自我国南、北方地区50株临床孢子丝菌进行菌种鉴定。方法分离菌株分别进行25℃恒温培养和玻片小培养,肉眼和镜下观察形态特征;同时提取菌丝相基因组DNA,用PCR分别扩增部分微管蛋白(β-tubulin)基因和核糖体内部转录间隔区(Ribosomal Internal Transcribed Spacer,ITS),扩增产物进行测序,并采用最大似然法(maximum likelihood,ML)和邻接法(neighbor-joining,NJ)构建联合β-tubulin基因和ITS区域的系统发育树。结果结合形态学及系统发育分析,50株菌均鉴定为球形孢子丝菌(Sporothrix globosa,S.globosa)。结论球形孢子丝菌是目前我国南、北方地区孢子丝菌病的主要致病菌种,为进一步明确我国孢子丝菌病致病菌种的分布提供了依据。     

实时荧光PCR检测巴西孢子丝菌的实验方法的建立

目的建立一种快速、特异、灵敏的荧光PCR方法检测巴西孢子丝菌。方法比对NCBI数据库中所有巴西孢子丝菌内转录间隔区(internal transcribed spacer, ITS)序列,在保守区域设计并合成特异性引物和探针,建立并优化荧光PCR检测方法。对优化后的方法使用标准浓度核酸进行扩增效率、灵敏度及特异度评价。通过巴西孢子丝菌小鼠感染模型,与组织培养比较,对本研究中的方法进行评价。结果建立的实时荧光PCR方法对巴西孢子丝菌的检测限为100fg。该方法对申克孢子丝菌、球形孢子丝菌、其他常见致病真菌28种、常见细菌3种以及人类基因组和小鼠基因组扩增结果均为阴性,特异度为100%。对巴西孢子丝菌感染小鼠脑、肝、肺、脾、肾及淋巴结检测与培养结果相一致。结论本研究建立的荧光PCR方法可快速、灵敏、特异地鉴定巴西孢子丝菌,并能够有效的对感染小鼠模型标本进行检测,有助于孢子丝菌病的早期特异性病原学诊断。     

申克孢子丝菌体外药敏试验研究近况

申克孢子丝菌是孢子丝菌病的致病菌,如何选择高效敏感的抗真菌药物对于有效治疗孢子丝菌病具有重要意义,抗真菌药物体外药敏试验是评价抗真菌药物抗真菌活性的重要手段。综述了申克孢子丝菌体外药敏试验的研究近况,展望了研究及应用前景。     

球形孢子丝菌致婴儿固定型孢子丝菌病1例及相关实验研究

报道1例由球形孢子丝菌所致的婴儿固定型孢子丝菌病。患儿女,3个月,因左眼下内侧皮损2个月就诊,皮损脓液标本进行真菌培养,对培养获得菌株进行形态学、生理学和分子生物学鉴定,并进行药物敏感性检测。真菌培养阳性,镜下可见典型的套袖样菌丝。钙调蛋白基因序列分析鉴定为球形孢子丝菌。药敏试验显示特比萘芬和伊曲康唑对该菌株的菌丝相最低抑菌浓度（minimal inhibitorycon centration,MIC）分别为0．5μg／mL和0．5μg／mL;对该菌株的酵母相的MIC值分别为0．25μg／mL和0．5μg／mL。给予患者口服特比萘芬32．5mg／d治疗10周后皮损消退呈瘢痕化修复。依据临床及实验室检查确诊该病例为球形孢子丝菌所致固定型孢子丝菌病,特比萘芬治疗本病例显示较好疗效。     

球形孢子丝菌DNA提取方法的比较

目的获得一种高效、快速、简便的提取球形孢子丝菌DNA的方法。方法以不同地区来源的球形孢子丝菌为实验材料,采用CTAB法、异硫氰酸胍法、试剂盒法、碱裂解法4种方法分别提取球形孢子丝菌DNA。通过紫外吸光度法测定DNA的浓度和纯度,琼脂糖凝胶电泳、PCR扩增评价DNA的质量。结果虽然不同方法均可得到球形孢子丝菌的DNA,但碱裂解法获得DNA的浓度和纯度明显优于其他3种方法。利用钙调蛋白基因的引物对DNA进行PCR扩增后,各样本均出现阳性条带,且条带亮度较好。结论碱裂解法是获得高质量球形孢子丝菌DNA的理想方法。     

特比萘芬联合伊曲康唑治疗孢子丝菌病失败1例

伊曲康唑和特比萘芬治疗孢子丝菌病（sporo-trichosis）有效安全,目前已成为孢子丝菌病治疗指南中的推荐用药[1].但少数患者单用伊曲康唑或特比萘芬不能有效控制病情,研究显示两药分别联合碘化钾溶液或两药联合治疗皮肤型孢子丝菌病显示疗效增强2-6].最近,我们诊治1例由球形孢子丝菌引起的皮肤固定型孢子丝菌病患者,采用特比萘芬联合伊曲康唑治疗失败,之后经特比萘芬联合碘化钾溶液治疗才获得痊愈.鉴于该病例治疗过程的特殊性,我们对其分离菌株进行了一些相关的实验研究,现报道如下.     

儿童孢子丝菌病的临床分析及致病菌的基因型检测

目的 对吉林省扶余县35例儿童孢子丝菌病进行临床分析并对部分菌株进行基因测定,了解该地区孢子丝菌病的致病菌菌株基因是否发生变异而致致病力增强及发病率增高.方法 收集2011年1月1日～4月30日来自扶余县就诊于我科并确诊为孢子丝菌病的患儿35例,进行临床分析;从中选取8株菌,对其核糖体保守区及内转录间隔区(ITS)、非转录间隔区(NTS)区进行基因测定,了解基因水平上的异同.结果 8株孢子丝菌菌株中5株NTS区可见较大范围的碱基缺失.结论 吉林省扶余县孢子丝菌菌株可能由于NTS区碱基较大范围缺失而致致病力增强,导致该地区短时间内发病率增高.     

申克孢子丝菌体外药敏试验研究进展

申克孢子丝菌是孢子丝菌病的致病真菌,作为一种双相型真菌,室温培养为菌丝相,37℃则为酵母相。哪一种生长状态的药敏试验结果更准确可靠、并能指导治疗尚有争议。就此对其体外药敏试验的影响因素、试验方案的改良以及试验条件的探索等研究进展作一综述。     

申克孢子丝菌毒力因子及宿主抗感染免疫的研究进展

申克孢子丝菌是一种重要的双相型真菌,其引起的孢子丝菌病是一种常见的侵袭性皮肤感染.研究该菌毒力因子及宿主对其抗感染免疫对于深入了解其致病性及防治该病具有重要意义.该文介绍了申克孢子丝菌毒力因子及宿主对其抗感染免疫方面的研究进展.     

耐极端环境真菌对犬小孢子菌及孢子丝菌的拮抗研究

目的 检测极端环境分离出的真菌拮抗临床病原真菌的活性.方法 选用极端环境分离出的24株真菌菌株上清液及菌丝体的提取液,来进行临床病原真菌（孢子丝菌和犬小孢子菌）的拮抗试验,采用M38-A2产孢丝状真菌抗真菌药物敏感性试验方案和E-test纸片扩散法.结果 抑菌圈直径≥1.0 cm,具有较强拮抗孢子丝菌活性的极端环境真菌有8株;抑菌圈直径≥2.0 cm,具有极强拮抗犬小孢子菌的活性的极端环境真菌有1株.两种临床菌株的拮抗试验,90％的拮抗结果相一致.结论 70％极端环境真菌菌株上清液及菌丝体提取液具有拮抗临床病原真菌孢子丝菌和犬小孢子菌的活性.     

Molecular identification and phylogenetic analysis of fungal pathogens isolated from diseased fish in Xinjiang,China

The outbreaks of fungal diseases in cultured fish have been severe in recent years, which is harmful to the healthy and sustainable development of fish farming. In this study, an investigation was conducted for significant fungal infections of 12 species of fish in four regions in Xinjiang, China, to understand the distribution of local fish fungal pathogens. Twenty-six fungal strains with pathogenicity were isolated, and the challenge experiment showed that eight strains from Changji area had high infection rate to fish eggs. Based on internal transcribed spacer sequence data and molecular analysis, the 26 strains were classified into nine different species of six fungal genera. Phylogenetic analysis showed that all strains were divided into two clades, namely Cluster 1 (contains only the genus Mucor) and Cluster 2 (consists of five small branches), and the distribution of strains from the same region was scattered in two clusters. There is no strict host selectivity for these fungi to infect fish. Mucor sp. are the main fungal pathogen of fish in these four regions, whereas Hypophthalmichthys molitrix and Carassius auratus are two types of fish that were susceptible to pathogen. In addition, the environmental adaptability experiments showed that eight highly pathogenic strains have different adaptability to the environment, and their optimum temperature and pH were 25°C and 7.0, respectively, whereas the concentration of NaCl was negatively correlated with the growth of strains. Therefore, these results indicated that the coinfection of multiple fungal pathogens in a culture region should be considered in the future study.     

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.     

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.     

Heritability of fumonisin B1 production in Gibberella fujikuroi mating population A.

Fumonisins are mycotoxins produced by strains belonging to several different mating populations of Gibberella fujikuroi (anamorphs, Fusarium section Liseola), a major pathogen of maize and sorghum worldwide. We studied the heritability of fumonisin production in mating population A by crossing fumonisin-producing strains collected from maize and sorghum in the United States with fumonisin-nonproducing strains collected from maize in Nepal. Random ascospore and tetrad progeny from three of these crosses were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography for their ability to produce fumonisins on autoclaved cracked maize. In all three crosses, the ability to produce fumonisins, predominately fumonisin B1, segregated as a single gene or group of closely linked genes. Intercrosses between appropriate progeny and parents were poorly fertile, so we could not determine if the apparent single genes that were segregating in each of these crosses were allelic with one another. Mating type and spore-killer traits were scored in some crosses, and each segregated, as expected, as a single gene that was unlinked to the ability to produce fumonisins. We conclude that G. fujikuroi mating population A provides a powerful genetic system for the study of this important fungal toxin.     

Heritability of fumonisin B1 production in Gibberella fujikuroi mating population A.

Fumonisins are mycotoxins produced by strains belonging to several different mating populations of Gibberella fujikuroi (anamorphs, Fusarium section Liseola), a major pathogen of maize and sorghum worldwide. We studied the heritability of fumonisin production in mating population A by crossing fumonisin-producing strains collected from maize and sorghum in the United States with fumonisin-nonproducing strains collected from maize in Nepal. Random ascospore and tetrad progeny from three of these crosses were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography for their ability to produce fumonisins on autoclaved cracked maize. In all three crosses, the ability to produce fumonisins, predominately fumonisin B1, segregated as a single gene or group of closely linked genes. Intercrosses between appropriate progeny and parents were poorly fertile, so we could not determine if the apparent single genes that were segregating in each of these crosses were allelic with one another. Mating type and spore-killer traits were scored in some crosses, and each segregated, as expected, as a single gene that was unlinked to the ability to produce fumonisins. We conclude that G. fujikuroi mating population A provides a powerful genetic system for the study of this important fungal toxin.     

Potential of autochthonous fungal strains isolated from contaminated soils for degradation of polychlorinated biphenyls

Up to now, most studies on polychlorinated biphenyl (PCB) bioremediation have examined the ability of model fungal strains to biodegrade PCBs. Yet, there is limited information concerning the potential of autochthonous filamentous fungal strains in the biodegradation of PCBs and their possible use in the environmental technologies. In this study, we investigated the capacity of autochthonous fungal strains in the biodegradation of PCBs by isolating 24 taxa from former industrial sites highly contaminated by PCBs. Microscopic and molecular analyses using the internal transcribed spacer (ITS) region revealed that the fungal strains belonged to the phyla Ascomycota (19 strains) and Zygomycota (five strains). The chromatography gas analysis revealed evidence of degradation of seven PCB congeners. With the exception of Circinella muscae which presented no degradation potential, the other fungal strains exhibited a rate of biodegradation ranging from 29 to 85 % after 7 d of incubation in liquid medium. Among these strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena, and Thermoascus crustaceus showed remarkable degradation ability (>70 %) regardless of the number of chlorine substituents on the biphenyl nucleus and a high tolerance towards PCBs. To our knowledge, this is the first study that demonstrates the ability of PCB degradation by these species and indicates the potential effectiveness of some autochthonous fungal strains in bioremediation systems.     

Immune Response Against Sporothrix schenckii in TLR-4-Deficient Mice

For many fungal diseases, macrophages are the major cell population implicated in host protection, primarily by their ability to eliminate the invading fungal pathogen through phagocytosis. In sporotrichosis, this remains true, because of macrophages’ ability to recognize Sporothrix schenckii through specific receptors for some of the fungus’ cellular surface constituents. Further confirmation for macrophages’ pivotal role in fungal diseases came with the identification of toll-like receptors, and the subsequent numerous associations found between TLR-4 deficiency and host susceptibility to diverse fungal pathogens. Involvement of TLR-4 in immune response against sporotrichosis has been conducted to investigate how TLR-4 signaling could affect inflammatory response development through evaluation of H2O2 production and IL-1β, IL-6 and TGF-β release during the course of S. schenckii infection on TLR-4-deficient mice. The results showed that macrophages are largely dependent on TLR-4 for inflammatory activation and that in the absence of TLR-4 signaling, increased TGF-β release may be one of the contributing factors for the abrogated inflammatory activation of peritoneal exudate cells during mice sporotrichosis.     

Fungal Biodegradation and Detoxification of Cork Boiling Wastewaters

The ability of several fungal strains to degrade and to detoxify cork boiling wastewaters was investigated. The fungal strains used in this work were Sporothrix sp., Trichoderma koningii, Chrysonilia sitophila and Penicillium glabrum isolated from cork bark as well as Fusarium flocciferum and Phanerochaete chrysosporium. The results obtained in the degradation experiments carried out with each fungus showed that all fungi display similar abilities, with a chemical oxygen demand reduction of 54.2 % (± 4.7 %) attained within five days of incubation. F. flocciferum presented the highest value for the reduction of chemical oxygen demand of 62 %. In addition, a rise in pH values of around 3 units was detected with all the strains, except for Penicillium glabrum. Toxicity tests performed on Vibrio fischeri revealed that fungal treatment of the wastewaters causes the complete loss of toxicity in the cases of Sporothrix sp., T. koningii, P. chrysosporium and F. flocciferum. The other two tested strains were also able to detoxify the raw wastewaters, causing a ten‐fold decrease in toxicity. The results obtained in sequential biodegradation experiments with different pairs of fungi showed that although the increment in the COD reduction did not exceed 10 %, an important reduction in toxicity and a pH rise were attained.