Identification, genotyping, and pathogenicity of Trichosporon spp. Isolated from Giant pandas (Ailuropoda melanoleuca)

Background Trichosporon is the dominant genus of epidermal fungi in giant pandas (Ailuropoda melanoleuca) and causes local and deep infections. To provide the information needed for the diagnosis and treatment of trichosporosis in giant pandas, the sequence of ITS, D1/D2, and IGS1 loci in 29 isolates of Trichosporon spp. which were isolated from the body surface of giant pandas were combination to investigate interspecies identification and genotype. Morphological development was examined via slide culture. Additionally, mice were infected by skin inunction, intraperitoneal injection, and subcutaneous injection for evaluation of pathogenicity. Results The twenty-nine isolates of Trichosporon spp. were identified as 11 species, and Trichosporon jirovecii and T. asteroides were the commonest species. Four strains of T. laibachii and one strain of T. moniliiforme were found to be of novel genotypes, and T. jirovecii was identified to be genotype 1. T. asteroides had the same genotype which involved in disseminated trichosporosis. The morphological development processes of the Trichosporon spp. were clearly different, especially in the processes of single-spore development. Pathogenicity studies showed that 7 species damaged the liver and skin in mice, and their pathogenicity was stronger than other 4 species. T. asteroides had the strongest pathogenicity and might provoke invasive infection. The pathological characteristics of liver and skin infections caused by different Trichosporon spp. were similar. Conclusions Multiple species of Trichosporon were identified on the skin surface of giant panda, which varied in morphological development and pathogenicity. Combination of ITS, D1/D2, and IGS1 loci analysis, and morphological development process can effectively identify the genotype of Trichosporon spp. Electronic supplementary material The online version of this article (10.1186/s12866-019-1486-7) contains supplementary material, which is available to authorized users.


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Trichosporon is a genus of fungi that belongs to the order Tremellales in the class 45 Tremellomycetes (division Basidiomycota) and is widely distributed in nature(1). 46 Trichosporon spp. can cause superficial fungal infections such as tinea pedis, 47 onychomycosis, and dermoid infections(2). With the increasing prevalence of 48 immunocompromised patients, the incidence of invasive fungal diseases has increased, 49 and Trichosporon has become the second commonest genus of yeast fungus in deep this has become a difficult problem in the study of fungal infections(4). 56 Trichosporon spp. mainly cause skin and organ granuloma, and related 57 pathogenicity studies have focused on Trichosporon spp. that colonize humans, such 58 as Trichosporon asahii, T. asteroides, T. inkin, and T. dermatis. In recent years, reports 59 of trichosporosis in animals have increased. Some cases in animals have been reported 60 in the last decade, such as disseminated trichosporosis in cats(5), canine meningitis (6), 61 and tortoise shell infection(7). A study described a case of systemic infection by T. 62 loubieri in a cat with acute dyspnea, anorexia, and aggressive behavior; a cutaneous 63 biopsy from ulcerated wounds revealed necrogranulomatous dermatitis and 64 panniculitis with numerous intralesional fungal hyphae(5). 65 To enable accurate identification of Trichosporon spp., a number of molecular found no significant differences in colony morphology(13). 82 We collected 29 isolates of Trichosporon spp. from the skin of giant pandas at the All 29 isolates were identified as Trichosporon spp. after molecular identification.

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Microscopic observations of the 29 isolates were made after slide culture on SDA(1).

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A 0.5 ml sample of melted medium was injected into a closed glass Petri dish, which 149 comprised a slide glass, a cover glass, and a copper ring with a hole in the wall, and

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The spores of T. laibachii grew into mycelium after cultivation for 24 h, and no 255 spores divided independently (Fig. 4A). The mycelium spread radially (Fig. 4A) and 256 was folded together (Fig. 4C), and differentiated into spores. Arthrospores were 257 abundant, whereas spores were round and few in number (Fig. 4B, C, and D). The 258 spores and mycelium were unevenly colored: the spores were darker, whereas the 259 mycelium was lighter (Fig. 4B, C, and D). Hyphal folding was typical in structure.

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The structural development of T. guehoae was completed after cultivation for 24 h 261 (Fig. 5A). The mycelium spread radially but was scattered (Fig. 5A, B, C, and D). A 262 large number of round spores were produced as grape-like clusters (Fig. 5A, B, and 263 C). Hyphae and spores were evenly stained (Fig. 5A, B, C, and D). The mycelium 264 grew from a segmented section to form a new mycelium (Fig. 5B), and the new 265 spores were generated by arthrospores (Fig. 5D); this was a typical structure.

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After T. gracile was cultured for 24 h, a large amount of hyphae appeared and 267 became segmented, and no spores could be seen (Fig. 6A). It could be seen that the 268 mycelium differentiated into spores (Fig. 6B, C and D). A large number of spores 269 were produced, which were mostly square (Fig. 6C) and became oval after reaching 270 maturity (Fig. 6D). The mycelia and spores were evenly stained (Fig. 6C). Some 271 hyphae did not divide into sections and differentiated into spores at intervals; this was 272 a typical structure (Fig. 6D).

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After culture for 24 h, the spores of T. domesticum swelled to form mycelia and no 274 spores divided independently (Fig. 7A). Hyphae were abundant and parallel to each 275 other and had spindle-type buds (Fig. 7B). A large number of hyphae differentiated 276 into spores ( Fig. 7C and D). The number of spores was small with no arthrospores, 277 and the spores were round (Fig. 7B, C, and D). The mycelia and spores were evenly 278 stained (Fig. 7A, B, C, and D).

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After culture for 24 h, the spores of T. brassicae exhibited no significant changes 280 (Fig. 8A), whereas after culture for 48 h the spores swelled and formed hyphae (Fig.   281 8B). No spores were found to divide independently ( Fig. 8A and B). The mycelia 282 became segmented and were distributed parallel to each other (Fig. 8C). It could be 283 seen that the mycelium differentiated into spindle-shaped spores (Fig. 8D). The 284 mycelium and spores were evenly stained (Fig. 8D), and there were few arthrospores 285 (Fig. 8C).

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After cultivation for 24 h, some spores of T. shinodae swelled (Fig. 9A), whereas 287 other spores divided independently (Fig. 9B). The hyphae were short and sparse and 288 grew very slowly (Fig. 9C). A large number of round arthrospores were produced (Fig.   289   9D). The spores and mycelium were evenly colored ( Fig. 9C and D). Crude short 290 mycelium was produced after culture for 72 h (Fig. 9C); this was a typical structure.

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After culture for 24 h, T. asteroides formed slender hyphae, and no spores divided 292 independently (Fig. 10A). The mycelium was elongated (Fig. 10C) and could 293 differentiate into spores ( Fig. 10B and D). Spores on bifurcated mycelium aggregated 294 into spheres (Fig. 10C); a large number of spores were produced, and the spores were 295 round, oval, or spindle-shaped ( Fig. 10B and D). The spores were not evenly 296 pigmented and some were dark in color (Fig. 10D). The bifurcation of the mycelia 297 and the aggregation of spores into spheres were typical structures (Fig. 10C).

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After T. middelhovenii was cultivated for 24 h, hyphae were generated and no 299 spores were found to divide independently (Fig. 11A). The hyphae were elongated 300 ( Fig. 11A and D), and their segments were small and indistinct ( Fig. 11B and D). No 301 hyphae differentiated into spores. Spores at bifurcations were spindle-shaped ( Fig.   302   11A, B, C, and D). The spores were few in number and darker ( Fig. 11C and D). Shuttle-type articular spores were characteristic structures (Fig. 11A, B, C, and D).

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After cultivation for 24 h, spores of T. cutaneum swelled and budded, and some 309 spores divided independently (Fig. 13A). The mycelium was curved and segmented 310 ( Fig. 13B and C) and differentiated into spores (Fig. 13D). A large number of round 311 arthrospores (Fig. 13C) were produced. The mycelium and spores were evenly stained 312 (Fig. 13D), and curved mycelium was a typical structure ( Fig. 13B and D). middelhovenii all produced spores in the skin infection model (Fig.S1~S10). In 327 particular, T. asteroides gave rise to disseminated infections in the reticular layer of 328 the skin (Fig. 14G1) and budding in the dermis (Fig. 14G2). T. gracile, T. 329 moniliiforme, and T. domesticum caused inconspicuous pathological changes, and 330 hence their pathogenicity was weak.     The morphological development process of Trichosporon spp. was significantly 391 different, and the majority of Trichosporon spp. had a typical structure: for example, 392 septal differentiation of the mycelium in T. gracile (Fig. 8D); short thick mycelium 393 during the development of T. shinodae (Fig. 11C); elongation and bifurcation of the 394 mycelium and the aggregation of spores into spheres in T. asteroides (as shown in Fig.   395 10C); and a spindle-type articular spore structure in T. middelhovenii (Fig. 13A, B, C,   lower than that caused by T. asteroides, and it was inferred that genotype 1 of T. 454 jirovecii was opportunistically pathogenic. Four strains of T. laibachii were identified 455 as having new genotypes by phylogenetic analysis of the IGS1 sequence (Fig. 2).

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Although there have currently been no reported cases of infection involving T. 457 laibachii, T. laibachii (JYZ3252) caused skin ulceration in mice (Fig. 4)