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Conidiobolus lamprauges, a soil and plant entomophthoralean fungus, has been reported only in a horse and, more recently, in sheep with rhinopharyngeal entomophthoramycosis. Thus, little information is available to enable proper identification of this pathogen and its differentiation from other saprotrophic and pathogenic Conidiobolus species. Using classical mycological tools and molecular methodologies, we report for the first time the taxonomic and phylogenetic description of three C. lamprauges isolates recovered from sheep with rhinopharyngeal entomophthoramycosis. The distinctive clinical and pathological features of C. lamprauges are compared with those of other Conidiobolus spp. affecting sheep, as well as with those of the stramenopilan ovine agent Pythium insidiosum. The comparative morphological attributes of Conidiobolus spp. are also diagramed; along with the sequence data generated, they should assist laboratories in the identification of these uncommon species.
Several species of entomophthoralean fungi in the genera Basidiobolus and Conidiobolus have been reported as the etiologic agents of subcutaneous entomophthoramycosis in apparently healthy hosts (13, 21). In contrast, opportunistic members of the Mucorales have rarely been found causing subcutaneous infections in healthy mammalian hosts (20). A difference between pathogenic species of the Mucorales and Entomophthorales is that the latter group of pathogens triggers eosinophilic granulomas with the Splendore-Hoeppli phenomenon around coenocytic hyaline hyphae (13, 21), a unique characteristic shared only with Pythium insidiosum, a fungus-like zoosporic stramenopile pathogen (16, 29). The majority of subcutaneous infections caused by entomophthoralean fungi involve Basidiobolus spp., Conidiobolus coronatus, or Conidiobolus incongruus (13, 21). These fungal pathogens have been studied extensively, and their morphological characteristics are readily available (13). This is not the case for Conidiobolus lamprauges, which has been reported only once previously, from a horse (10). Thus, limited data are available for the proper identification of this newly described Conidiobolus sp. recovered in culture from clinical samples.
Outbreaks of sheep rhinitis involving entomophthoralean fungi are frequently encountered in Brazil (1, 22, 26). The disease occurs in two different clinical forms: (i) a rhinofacial infection affecting the nose vestibule, mucoepithelial junction, and upper lip and (ii) a rhinopharyngeal infection affecting the ethmoidal region, turbinate bones, paranasal sinuses, hard and soft palates, and pharynges (1, 22). Interestingly, Conidiobolus coronatus and other Conidiobolus spp. have been isolated in cases of nasopharyngeal entomophthoramycosis, whereas Pythium insidiosum is usually recovered in cases of rhinofacial pythiosis (22, 24, 26). This study describes in detail, for the first time, the taxonomic and phylogenetic features of three C. lamprauges strains isolated in cases of rhinopharyngeal entomophthoramycosis in three Brazilian sheep.
Tissue samples collected from three sheep with rhinopharyngeal clinical signs of rhinitis were cut into pieces (diameter, 4 to 5 mm) and placed on 2% Sabouraud dextrose agar (SDA) plates. The inoculated plates were then incubated at 37°C or 25°C for a week. The isolates recovered from the infected sheep were macroscopically and microscopically evaluated by placing pieces of their colonies (thickness, 2 mm; diameter, 2 mm) on lactophenol blue (2 ml phenol, 2 ml lactic acid, 4 ml glycerol, 2 ml H2O). The original strains on SDA were also subcultured on 2% potato dextrose agar (PDA) (20 g potatoes, 1.0 g dextrose, 2.0 g agar, and 100 ml H2O), and their macroscopic and microscopic morphological features were evaluated. For the DNA extraction protocol, the three C. lamprauges strains studied—BLDCL-01 (cited in reference 22), BLDCL-02, and BLDCL-03—were maintained at room temperature on SDA, and then pieces of their colonies (thickness, 4 mm; diameter, 4 mm) were subjected to one of two procedures. In the first, the pieces were transferred to 250-ml flasks containing 100 ml of 2% Sabouraud dextrose broth (SDB) and were then incubated at 37°C with rotation at 150 rpm for 5 days. In the second, the pieces were placed on SDA plates, and their hyphae were removed from the surface of the agar after 5 days of incubation at 37°C.
After incubation, the cell mass obtained from SDB and/or SDA cultures was killed with Merthiolate (0.02%, wt/vol). The resulting cell mass was placed in a mortar and ground under liquid nitrogen. The DNA from the hyphae was treated with sodium dodecyl sulfate and proteinase K digestion and was then extracted with phenol, chloroform, and isoamyl alcohol (Sigma, St. Louis, MO). The 18S small-subunit (SSU) ribosomal DNA (rDNA) gene was amplified by hot-start PCR using the universal NS1 5′-GTAGTAATATGCTTGTCTC-3′ and NS8 5′-TCCGCAGGTTCACAACGGA-3′ primers (8). The PCR temperature cycling parameters were as follows: 10 min at 95°C for the hot start and 1 min at 95°C for subsequent cycles, annealing for 1.0 min at 60°C, and elongation at 72°C for 2 min. This procedure was repeated for 40 cycles, followed by a final elongation of 2 min at 72°C. The amplicons were ligated into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA), purified, and then sequenced using BigDye Terminator chemistry in an ABI Prism genetic analyzer, model 310 (Perkin-Elmer, Foster City, CA).
The DNA sequences of the strains evaluated were aligned with 15 other fungal sequences available in the database using CLUSTAL X (32) analysis (Table (Table1),1), and their alignments were inspected visually. The gaps generated were treated as missing data. The 18 fungal DNA sequences were analyzed phylogenetically by the neighbor-joining (NJ) method, parsimony, and minimum evolution with multiple-hit correction by Kimura's 2-parameter model using MEGA4 software (30). Support for internal branches was assessed by using 1,000 bootstrapped data sets.
The 18S SSU rDNA sequences obtained in this study for C. lamprauges BLDCL-01, BLDCL-02, and BLDCL-03 have been deposited in GenBank under accession numbers GQ478279 to GQ478281.
The three isolates were identified as Conidiobolus species based on their microscopic features (see below). The three strains, BLDCL-01, BLDCL-02, and BLDCL-03, developed fast-growing isotropic, pale colonies in the primary culture on SDA at 25°C and 37°C (Fig. (Fig.1).1). On this medium, within the first 3 days of incubation at either temperature, flat membranous colonies with a smooth, glabrous, and waxy appearance developed (Fig. (Fig.1A,1A, inset). Older colonies (3 days old and older) became powdery in appearance, with short aerial mycelia, and developed radiate folds from the centers of the colonies (Fig. 1A and B). On PDA at 25°C and 37°C, colonies were thin, flat, and smooth, with folds at the center of the colonies (data not shown). The colonies of the three strains studied developed faster on SDA and PDA at 37°C (diameters, 20 mm in 24 h and 55 mm in 72 h) than at 25°C (diameters, 15 mm in 24 h and 45 mm in 72 h).
Microscopically, the three colonies evaluated on SDA showed hyaline, coenocytic, ribbon-type, unbranched hyphae measuring 5.0 to 10.0 (±2 to 4) μm in diameter, with occasional septa, usually on the distal parts of the hyphae (Fig. (Fig.2A).2A). Immature zygospores developed after the encounter of two hyphal segments, producing swelling in the contact area and later becoming globose, with several internal vacuoles (data not shown). Numerous smooth globose to subglobose ripe zygospores, 10.0 to 18.0 μm in diameter, were observed on primary SDA cultures for strain BLDCL-01 (Fig. 2A and B), but not for the other two strains. The internal cell walls of the mature zygospores were 1.0 to 4.0 μm in diameter (±0.5 to 1.0 μm; n = 50). Some of the mature zygospores contained a readily detectable large globule at the center, with some space between the internal globule and the cell wall (Fig. (Fig.2B).2B). The ability of strain BLDCL-01 to develop zygospores on SDA (primary cultures) was lost after the first subculture on the same medium. On PDA, all strains evaluated developed numerous zygospores identical to that described for isolate BLDCL-01 on SDA (Fig. 2A and B). Asexual papillate globose conidia were observed on PDA in the three isolates studied (Fig. 2C to F). The asexual conidia measured 15.0 to 20 (±3.0) μm in diameter and developed in a single hyphal segment from which they were forcibly thrown off (Fig. (Fig.2C,2C, red arrows). The cells wall of the conidia were smooth and lacked the layered features in the inner cell wall characteristic of mature zygospores (Fig. 2C to E). The ejected asexual conidia each possessed a rounded apiculate basal papilla, and some of them developed germ tubes a day or two after discharge (Fig. 2E and F). On PDA medium, migration of cytoplasm from the primary papillate conidia into secondary sporophores was also observed in the three C. lamprauges strains studied (Fig. (Fig.2D2D).
The GenBank accession numbers of the 18S SSU rDNA sequences determined in this study are given in Table Table1.1. Few nucleotide mismatches were found within the three C. lamprauges DNA sequences from sheep. High-stringency BLAST (http://www.ncbi.nlm.nih.gov/) analysis of these three 18S SSU rDNA sequences (1,740 bp) showed strong identity with the DNA sequences of two environmental strains of C. lamprauges, one from a Swiss study (GenBank accession number AF296754) (99% identity) and one, strain CBS 153.56 (AF113420), from Virginia (92% identity), followed by Conidiobolus osmodes (90% identity), Conidiobolus thromboides (90% identity), and other closely related members of the Entomophthorales. By the use of Basidiobolus species as an outgroup, the phylogenetic trees constructed by the three phylogenetic methods in this study (neighbor joining, minimum evolution, and parsimony) showed no significant topological conflicts (Fig. (Fig.3).3). The three C. lamprauges DNA sequences recovered from sheep and the two environmental C. lamprauges DNA sequences available in the database clustered together, forming a strongly supported monophyletic taxon (100% NJ bootstrap and 100% parsimony bootstrap). The C. lamprauges taxon is connected by a long branch to C. osmodes, C. thromboides, Conidiobolus pumilus, and Conidiobolus rhysosporus, which, in turn, are sister taxa to the remaining Conidiobolus DNA sequences in this study (Fig. (Fig.3).3). These remaining sequences cluster into two small, well-supported sister groups (100% NJ bootstrap and 100% parsimony bootstrap), with three C. coronatus strains plus Conidiobolus firmipilleus in one clade and C. incongruus plus Conidiobolus brefeldianus DNA sequences in the other (Fig. (Fig.33).
Humans (2, 9, 20, 31, 35) and several species of lower animals, including deer (14, 28), dolphins (17), horses (15, 27, 37), llamas (7, 18), primates (23), and sheep (3, 12, 19, 26), have been reported with subcutaneous entomophthoramycoses of the nostrils, facial areas, and orbit caused by Conidiobolus species. Reports of C. coronatus (26), C. incongruus (3, 12, 19), and the stramenopilan fungus-like pathogen P. insidiosum (22, 24, 29) causing similar clinical signs in sheep suggest that in this mammalian species, multiple etiologies can be expected. The fact that C. lamprauges has been isolated in rhinopharyngeal cases and P. insidiosum in rhinofacial cases (22) should be further investigated. We believe that this clinical feature could be used as an aid in the differential diagnosis of entomophthoramycoses caused by these species.
Since the first description of C. lamprauges from environmental samples (5), only two reports of the species, affecting a horse and sheep, respectively, have ever been published (10, 22). Indeed, a review of the literature showed that most cases of Conidiobolus infection in humans and lower animals involved either C. coronatus (7, 13, 27, 37) or C. incongruus (2, 3, 9, 14, 19, 25). However, due to the limited information on the proper identification of C. lamprauges in the laboratory, it is quite possible that by use of classical taxonomic tools only, some Conidiobolus species might have been misidentified. For instance, Morris et al. (19) recently described C. incongruus in sheep. A careful review of the microscopic structures in their culture, including the formation of zygospores, showed that the morphological features of the fungus described in their report are similar to those encountered with C. lamprauges. Likewise, Hernandez et al. (9) and Sharma et al. (25) identified their fungal isolates from human patients as C. incongruus, based only on the production of papillate conidia and the absence of villose conidia. Thus, the lack of a laboratory guide for the taxonomic identification of three pathogenic Conidiobolus species has been problematic. To resolve this drawback, we have constructed a table listing several morphological differences and similarities between the three known pathogenic Conidiobolus species (Fig. (Fig.4),4), which can be used by laboratory workers dealing with clinical isolates of entomophthoramycetous fungi. According to Fig. Fig.4,4, C. coronatus in culture can be distinguished from C. lamprauges and C. incongruus by the production of villose conidia and the absence of zygospores on PDA. Likewise, C. lamprauges and C. incongruus can be properly distinguished from each other by the morphological features of their mature zygospores (Fig. (Fig.4).4). Although human cases of C. lamprauges have yet to be reported, Fig. Fig.44 could also aid in the proper identification of C. lamprauges from putative cases of entomophthoramycosis in humans.
Molecular methodologies have proved to be a valuable diagnostic tool in identifying clinical isolates and a variety of etiologic agents directly from infected tissues (14, 36). Using this approach, Madson et al. (14) reported the presence of C. incongruus in a clinical specimen from an infected white-tailed deer. While the latter approach is important in determining the etiologic agent in the absence of culture, the results have to be interpreted with caution. BLAST analysis of entire or partial rDNA or DNA coding sequences, although informative, could produce misleading data (4). The use of phylogenetics is a better approach for the proper placement of DNA sequences obtained from culture or infected tissue sections in the tree of life. Several studies using phylogenetic analysis of rDNA and protein-coding sequences have validated the classical taxonomic placement of some zygomycetes and have challenged the positions of various members within the Mucorales and Entomophthorales (11, 34, 36). Our phylogenetic study showed two well-supported sister groups in the genus Conidiobolus, data in agreement with those published by others (34, 36). The three DNA sequences from sheep that were evaluated clustered with two C. lamprauges sequences from the database, and together with C. osmodes, C. thromboides, C. pumilus, and C. rhysosporus, they formed a sister taxon to C. coronatus, C. incongruus, and the other two Conidiobolus species used in this study. In addition, the phylogenetic data in our study are in agreement with the phenotypic findings reported for the isolates investigated. The nucleotide differences encountered in the DNA sequences in this study could be used to build specific primers for the future identification of pathogenic Conidiobolus species from clinical samples.
Published ahead of print on 9 December 2009.