Discovery of a Unique Set of Dog-Seroreactive Coccidioides Proteins Using Nucleic Acid Programmable Protein Array.

Valley Fever (VF), caused by fungi in the genus Coccidioides, is a prevalent disease in southwestern and western parts of the United States that affects both humans and animals, such as dogs. Although the immune responses to infection with Coccidioides spp. are not fully characterized, antibody-detection assays are used in conjunction with clinical presentation and radiologic findings to aid in the diagnosis of VF. These assays often use Complement Fixation (CF) and Tube Precipitin (TP) antigens as the main targets of IgG and IgM reactivity, respectively. Our group previously reported evidence of over 800 genes expressed at the protein level in C. posadasii. However, antibody reactivity to the majority of these proteins has never been explored. Using a new, high-throughput screening technology, the Nucleic Acid Programmable Protein Array (NAPPA), we screened serum specimens from dogs against 708 of these previously identified proteins for IgG reactivity. Serum from three separate groups of dogs was analyzed and revealed a small panel of proteins to be further characterized for immuno-reactivity. In addition to CF/CTS1 antigen, sera from most infected dogs showed antibody reactivity to endo-1,3-betaglucanase, peroxisomal matrix protein, and another novel reactive protein, CPSG_05795. These antigens may provide additional targets to aid in antibody-based diagnostics.

Vaccines to Prevent Coccidioidomycosis: A Gene-Deletion Mutant of Coccidioides Posadasii as a Viable Candidate for Human Trials.

Coccidioidomycosis is an endemic fungal infection that is reported in up to 20,000 persons per year and has an economic impact close to $1.5 billion. Natural infection virtually always confers protection from future exposure, and this suggests that a preventative vaccine strategy is likely to succeed. We here review progress toward that objective. There has been ongoing research to discover a coccidioidal vaccine over the past seven decades, including one phase III clinical trial, but for reasons of either efficacy or feasibility, a safe and effective vaccine has not yet been developed. This review first summarizes the past research to develop a coccidioidal vaccine. It then details the evidence that supports a live, gene-deletion vaccine candidate as suitable for further development as both a veterinary and a human clinical product. Finally, a plausible vaccine development plan is described which would be applicable to this vaccine candidate and also useful to other future candidates. The public health and economic impact of coccidioidomycosis fully justifies a public private partnership for vaccine development, and the development of a vaccine for this orphan disease will likely require some degree of public funding.

The WOPR family protein Ryp1 is a key regulator of gene expression, development, and virulence in the thermally dimorphic fungal pathogen Coccidioides posadasii.

Coccidioides spp. are mammalian fungal pathogens endemic to the Southwestern US and other desert regions of Mexico, Central and South America, with the bulk of US infections occurring in California and Arizona. In the soil, Coccidioides grows in a hyphal form that differentiates into 3-5 micron asexual spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo a unique developmental transition from polar hyphal growth to isotropic expansion with multiple rounds of nuclear division, prior to segmentation, forming large spherules filled with endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides development. We show that Coccidioides Δryp1 mutants have altered colony morphology under hypha-promoting conditions and are unable to form mature spherules under spherule-promoting conditions. We analyze the transcriptional profile of wild-type and Δryp1 mutant cells under hypha- and spherule-promoting conditions, thereby defining a set of hypha- or spherule-enriched transcripts ("morphology-regulated" genes) that are dependent on Ryp1 for their expression. Forty percent of morphology-regulated expression is Ryp1-dependent, indicating that Ryp1 plays a dual role in both hyphal and spherule development. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. Concordant with its role in spherule development, we find that the Δryp1 mutant is completely avirulent in the mouse model of coccidioidomycosis, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live Δryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the Δryp1 mutant fails to make mature spherules and likely does not express key antigens required for effective vaccination. Taken together, this work identifies the first transcription factor that drives mature spherulation and virulence in Coccidioides.

Mouse Model of a Human STAT4 Point Mutation That Predisposes to Disseminated Coccidiomycosis.

STAT4 plays a critical role in the generation of both innate and adaptive immune responses. In the absence of STAT4, Th1 responses, critical for resistance to fungal disease, do not occur. Infection with the dimorphic fungus, Coccidioides, is a major cause of community-acquired pneumonia in the endemic regions of Arizona and California. In some people and often for unknown reasons, coccidioidal infection results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis, prompting genetic investigation. All affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4E626G/+ ). A knockin mouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-type mice. Stat4E626G/+ T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells from Stat4E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4E626G/+ mice failed to produce IFN-γ and related cytokines in the lung and to accumulate activated adaptive immune cells in mediastinal lymph nodes. Therefore, defective early induction of IFN-γ and adaptive responses by STAT4 prevents normal control of coccidioidomycosis in both mice and humans.

Genetic Diversity of Aspergillus flavus Associated with Chili in Nigeria and Identification of Haplotypes with Potential in Aflatoxin Mitigation.

Dried red chili (Capsicum spp.), a widely produced and consumed spice in Nigeria, is often contaminated by aflatoxins. Aflatoxins are potent mycotoxins of severe health and economic concern worldwide. Aspergillus flavus often contaminates crops with aflatoxins in warm regions; however, not all isolates are aflatoxin producers. Nonaflatoxigenic isolates have potential as biocontrol agents for aflatoxin mitigation. The current study examined the genetic diversity of A. flavus (n = 325) associated with chilies in Nigeria and identified 123 nonaflatoxigenic isolates. The Nigerian A. flavus isolates from chili were diverse at 17 microsatellite loci, with 5 to 36 alleles per locus, and included 152 haplotypes. The isolates that are active ingredients in Aflasafe, registered for aflatoxin biocontrol on maize and groundnuts in Nigeria, did not share haplotypes with the chili isolates. Of the 152 haplotypes, 65% produced aflatoxins in autoclaved maize, some of which (17%) produced >100,000 µg/kg of aflatoxins. Aflatoxins were not detected in 35% of the haplotypes. Cluster amplification pattern assay detected large deletions in the aflatoxin biosynthetic clusters of some (32%) of the nonaflatoxigenic haplotypes. Coinfection of chili with nonaflatoxigenic isolates from chilies (n = 7) and A. aflatoxiformans resulted in a significantly greater average reduction in total aflatoxins compared with that achieved by Aflasafe active ingredient isolates (P < 0.01). These nonaflatoxigenic isolates are a genetic resource for the development of biological control products for aflatoxin mitigation in chilies in Nigeria and should be evaluated under field conditions.

Δcps1 vaccine protects dogs against experimentally induced coccidioidomycosis.

Coccidioidomycosis is a significant health problem of dogs and humans in endemic regions, especially California and Arizona in the U.S. Both species would greatly benefit from a vaccine to prevent this disease. A live avirulent vaccine candidate, Δcps1, was tested for tolerability and efficacy to prevent pulmonary coccidioidomycosis in a canine challenge model. Vaccine injection-site reactions were transient and there were no systemic effects observed. Six of seven vaccine sites tested and all draining lymph nodes were sterile post-vaccination. Following infection with Coccidioides posadasii, strain Silveira, arthroconidia into the lungs, dogs given primary and booster vaccinations had significantly reduced lung fungal burdens (P = 0.0003) and composite disease scores (P = 0.0002) compared to unvaccinated dogs. Dogs vaccinated once had fungal burdens intermediate between those given two doses or none, but disease scores were not significantly different from unvaccinated (P = 0.675). Δcps1 was well-tolerated in the dogs and it afforded a high level of protection when given as prime and boost. These results drive the Δcps1 vaccine toward a licensed veterinary vaccine and support continued development of this vaccine to prevent coccidioidomycosis in humans.

Degradation of Aflatoxins B1 by Atoxigenic Aspergillus flavus Biocontrol Agents.

Aflatoxins are potent Aspergillus mycotoxins that contaminate food and feed, thereby impacting health and trade. Biopesticides with atoxigenic Aspergillus flavus isolates as active ingredients are used to reduce aflatoxin contamination in crops. The mechanism of aflatoxin biocontrol is primarily attributed to competitive exclusion but, sometimes, aflatoxin is reduced by greater amounts than can be explained by displacement of aflatoxin-producing fungi on the crop. Objectives of this study were to (i) evaluate the ability of atoxigenic A. flavus genotypes to degrade aflatoxin B1 (AFB1) and (ii) characterize impacts of temperature, time, and nutrient availability on AFB1 degradation by atoxigenic A. flavus. Aflatoxin-contaminated maize was inoculated with atoxigenic isolates in three separate experiments that included different atoxigenic genotypes, temperature, and time as variables. Atoxigenic genotypes varied in aflatoxin degradation but all degraded AFB1 >44% after 7 days at 30°C. The optimum temperature for AFB1 degradation was 25 to 30°C, which is similar to the optimum range for AFB1 production. In a time-course experiment, atoxigenics degraded 40% of AFB1 within 3 days, and 80% of aflatoxin was degraded by day 21. Atoxigenic isolates were able to degrade and utilize AFB1 as a sole carbon source in a chemically defined medium but quantities of AFB1 degraded declined as glucose concentrations increased. Degradation may be an additional mechanism through which atoxigenic A. flavus biocontrol products reduce aflatoxin contamination pre- or postharvest. Thus, selection of optimal atoxigenic active ingredients can include assessment of both competitive ability in agricultural fields and their ability to degrade aflatoxins.

Temperature Influences on Interactions Among Aflatoxigenic Species of Aspergillus Section Flavi During Maize Colonization.

Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

A Chronic Murine Disease Model of Coccidioidomycosis Using Coccidioides posadasii, Strain 1038.

Murine infections with most Coccidioides spp. strains are lethal by 3 weeks, limiting the study of immune responses. Coccidioides posadasii, strain 1038 (Cp1038), while slowly lethal, resulted in protracted survival of C57BL/6 (B6) mice. In resistant (B6D2)F1/J mice, lung fungal burdens stabilized by week 4 without progression through week 16, better modeling human coccidioidal infections after their immunologic control. Immunodeficient tumor necrosis factor (Tnf) α knockout (KO) and interferon (Ifn) γ receptor 1 (Ifn-γr1) KO mice survived a median of 22.5 and 34 days, compared with 70 days in B6 mice (P = .001 and P < .01, respectively), though 14-day lung fungal burden studies showed little difference between Ifn-γr1 KO and B6 mice. B6 mice showed peak concentrations of key inflammatory lung cytokines, including interleukin 6, 23, and 17A, Tnf-α, and Ifn-γ, only after 4 weeks of infection. The slower progression in B6 and the acquired fungal burden stability in B6D2 mice after Cp1038 infection greatly increases the array of possible immunologic studies.

Differential Thermotolerance Adaptation between Species of Coccidioides.

Coccidioidomycosis, or Valley fever, is caused by two species of dimorphic fungi. Based on molecular phylogenetic evidence, the genus Coccidioides contains two reciprocally monophyletic species: C. immitis and C. posadasii. However, phenotypic variation between species has not been deeply investigated. We therefore explored differences in growth rate under various conditions. A collection of 39 C. posadasii and 46 C. immitis isolates, representing the full geographical range of the two species, was screened for mycelial growth rate at 37 °C and 28 °C on solid media. The radial growth rate was measured for 16 days on yeast extract agar. A linear mixed effect model was used to compare the growth rate of C. posadasii and C. immitis at 37 °C and 28 °C, respectively. C. posadasii grew significantly faster at 37 °C, when compared to C. immitis; whereas both species had similar growth rates at 28 °C. These results indicate thermotolerance differs between these two species. As the ecological niche has not been well-described for Coccidioides spp., and disease variability between species has not been shown, the evolutionary pressure underlying the adaptation is unclear. However, this research reveals the first significant phenotypic difference between the two species that directly applies to ecological research.

Phenotypic Differentiation of Two Morphologically Similar Aflatoxin-Producing Fungi from West Africa.

Aflatoxins (AF) are hepatocarcinogenic metabolites produced by several Aspergillus species. Crop infection by these species results in aflatoxin contamination of cereals, nuts, and spices. Etiology of aflatoxin contamination is complicated by mixed infections of multiple species with similar morphology and aflatoxin profiles. The current study investigates variation in aflatoxin production between two morphologically similar species that co-exist in West Africa, A. aflatoxiformans and A. minisclerotigenes. Consistent distinctions in aflatoxin production during liquid fermentation were discovered between these species. The two species produced similar concentrations of AFB1 in defined media with either urea or ammonium as the sole nitrogen source. However, production of both AFB1 and AFG1 were inhibited (p < 0.001) for A. aflatoxiformans in a yeast extract medium with sucrose. Although production of AFG1 by both species was similar in urea, A. minisclerotigenes produced greater concentrations of AFG1 in ammonium (p = 0.039). Based on these differences, a reliable and convenient assay for differentiating the two species was designed. This assay will be useful for identifying specific etiologic agents of aflatoxin contamination episodes in West Africa and other regions where the two species are sympatric, especially when phylogenetic analyses based on multiple gene segments are not practical.

Molecular Analysis of S-morphology Aflatoxin Producers From the United States Reveals Previously Unknown Diversity and Two New Taxa.

Aflatoxins are highly toxic carcinogens that detrimentally influence profitability of agriculture and the health of humans and domestic animals. Several phylogenetically distinct fungi within Aspergillus section Flavi have S-morphology (average sclerotial size < 400 µm), and consistently produce high concentrations of aflatoxins in crops. S-morphology fungi have been implicated as important etiologic agents of aflatoxin contamination in the United States (US), but little is known about the diversity of these fungi. The current study characterized S-morphology fungi (n = 494) collected between 2002 and 2017, from soil and maize samples, in US regions where aflatoxin contamination is a perennial problem. Phylogenetic analyses based on sequences of the calmodulin (1.9 kb) and nitrate reductase (2.1 kb) genes resolved S-morphology isolates from the US into four distinct clades: (1) Aspergillus flavus S-morphotype (89.7%); (2) Aspergillus agricola sp. nov. (2.4%); (3) Aspergillus texensis (2.2%); and (4) Aspergillus toxicus sp. nov. (5.7%). All four S-morphology species produced high concentrations of aflatoxins in maize at 25, 30, and 35°C, but only the A. flavus S-morphotype produced unacceptable aflatoxin concentrations at 40°C. Genetic typing of A. flavus S isolates using 17 simple sequence repeat markers revealed high genetic diversity, with 202 haplotypes from 443 isolates. Knowledge of the occurrence of distinct species and haplotypes of S-morphology fungi that are highly aflatoxigenic under a range of environmental conditions may provide insights into the etiology, epidemiology, and management of aflatoxin contamination in North America.

Coccidioides posadasii in a Dog With Cervical Dissemination Complicated by Esophageal Fistula.

A 5-year-old male, neutered mixed breed dog with a history of a mass with an associated draining tract on the ventral cervical region was diagnosed with an esophageal fistula. The dog exhibited serosanguinous discharge from the draining tract, with enlarged left superficial cervical and mandibular lymph nodes, and was reported to have difficulty with deglutition of solid foods. Computed tomography revealed a communication of the draining tract with the esophagus along with enlargement of the left lateral retropharyngeal, left medial retropharyngeal, and mandibular lymph nodes. This prompted surgical exploration and debridement of the site, with closure of the esophageal fistula. Histopathology of thyroid gland, skeletal muscle, and adipose tissue obtained during surgical exploration showed spherules consistent with Coccidioides spp. infection. Antibody titers performed post-operatively were consistent with an active Coccidioides spp. Infection. By fungal culture and subsequent PCR and DNA sequencing, C. posadasii was identified as the species infecting the dog. Over the course of 85 days of antifungal therapy, discharge from the draining tract, lymphadenomegaly, and cutaneous and subcutaneous nodules resolved. In conclusion, this is the first reported case of disseminated coccidioidomycosis to the cervical region of a dog with involvement of the thyroid gland, skeletal muscle, adipose tissue, connective tissue, and secondary esophageal fistula. Coccidioides spp. infections should be considered a differential diagnosis in unusual cases for dogs that live in or have traveled to endemic areas.

Aspergillus texensis: A Novel Aflatoxin Producer with S Morphology from the United States.

Aflatoxins are carcinogenic metabolites produced primarily by fungi within Aspergillus section Flavi. These fungi infect a wide range of crops in warm regions. Molecular phylogenetic analyses of fungi with S morphology (average sclerotium size < 400 µm) within section Flavi collected from across the United States (US) resulted in the discovery of a novel aflatoxin-producing species, Aspergillus texensis. Aspergillus texensis was isolated from maize grown in Arkansas, Louisiana, and Texas, and from soils cropped to maize in Texas. Aspergillus texensis produces sparse conidia and abundant sclerotia on various culture media, and on maize. Physiological studies have revealed optimal growth on culture media at 35 °C. All isolates of A. texensis produced B and G aflatoxins, cyclopiazonic acid and aspergillic acid. Aspergillus texensis and A. flavus S strain morphotypes produced similar concentrations of total aflatoxins on maize (p > 0.05). Phylogenetic analyses of aflatoxin-producers based on partial gene sequences of the ß-tubulin (0.9 kb), calmodulin (1.2 kb), and nitrate reductase (2.1 kb) genes placed A. texensis in a highly supported monophyletic clade closely related to A. minisclerotigenes and a previously reported unnamed lineage designated Lethal Aflatoxicosis Fungus.

Comparative Genomics of Aspergillus flavus S and L Morphotypes Yield Insights into Niche Adaptation.

Aspergillus flavus, the primary causal agent for aflatoxin contamination on crops, consists of isolates with two distinct morphologies: isolates of the S morphotype produce numerous small sclerotia and lower numbers of conidia while isolates of the L morphotype produce fewer large sclerotia and abundant conidia. The morphotypes also differ in aflatoxin production with S isolates consistently producing high concentrations of aflatoxin, whereas L isolates range from atoxigenic to highly toxigenic. The production of abundant sclerotia by the S morphotype suggests adaptation for long-term survival in the soil, whereas the production of abundant conidia by the L morphotype suggests adaptation for aerial dispersal to the phyllosphere. To identify genomic changes that support differential niche adaption, the sequences of three S and three L morphotype isolates were compared. Differences in genome structure and gene content were identified between the morphotypes. A >530 kb inversion between the morphotypes affect a secondary metabolite gene cluster and a cutinase gene. The morphotypes also differed in proteins predicted to be involved in carbon/nitrogen metabolism, iron acquisition, antimicrobial defense, and evasion of host immunity. The S morphotype genomes contained more intact secondary metabolite clusters indicating there is higher selection pressure to maintain secondary metabolism in the soil and that it is not limited to aflatoxin production. The L morphotype genomes were enriched in amino acid transporters, suggesting efficient nitrogen transport may be critical in the nutrient limited phyllosphere. These findings indicate the genomes of the two morphotypes differ beyond developmental genes and have diverged as they adapted to their respective niches.

Viable spores of Coccidioides posadasii Δcps1 are required for vaccination and provide long lasting immunity.

Coccidioidomycosis is a systemic fungal infection for which a vaccine has been sought for over fifty years. The avirulent Coccidioides posadasii strain, Δcps1, which is missing a 6 kb gene, showed significant protection in mice. These studies explore conditions of protection in mice and elucidate the immune response. Mice were vaccinated with different doses and viability states of Δcps1 spores, challenged with virulent C. posadasii, and sacrificed at various endpoints, dependent on experimental objectives. Tissues from vaccinated mice were harvested for in vitro elucidation of immune response. Vaccination with viable Δcps1 spores was required for protection from lethal challenge. Viable spore vaccination produced durable immunity, lasting at least 6 months, and prolonged survival (≥6 months). The C. posadasii vaccine strain also protected mice against C. immitis (survival ≥ 6 months). Cytokines from infected lungs of vaccinated mice in the first four days after Cp challenge showed significant increases of IFN-γ, as did stimulated CD4+ spleen cells from vaccinated mice. Transfer of CD4+ cells, but not CD8+ or B cells, reduced fungal burdens following challenge. IFN-γ from CD4+ cells in vaccinated mice indicates a Th1 response, which is critical for host control of coccidioidomycosis.

Genome Sequence of Ophidiomyces ophiodiicola, an Emerging Fungal Pathogen of Snakes.

Ophidiomyces ophiodiicola, which belongs to the order Onygenales, is an emerging fungal pathogen of snakes in the United States. This study reports the 21.9-Mb genome sequence of an isolate of this reptilian pathogen obtained from a black racer snake in Pennsylvania.

A Coccidioides posadasii CPS1 Deletion Mutant Is Avirulent and Protects Mice from Lethal Infection.

The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals.

OPHIDIOMYCES OPHIODIICOLA ON A CAPTIVE BLACK RACER (COLUBER CONSTRICTOR) AND A GARTER SNAKE (THAMNOPHIS SIRTALIS) IN PENNSYLVANIA.

Two snakes were presented to the Pennsylvania State University Animal Diagnostic Laboratory with one suffering from external lesions where the scales were raised and discolored, and the other with oral lesions and swelling extending to the left eye, which was opaque. Histopathological analysis revealed multifocal granulomas containing fungal hyphae. Morphological and DNA sequence analyses revealed both suffered from infection by Ophidiomyces ophiodiicola, an emerging pathogen of snakes. This is the first report of this disease in Pennsylvania.

Recent advances in our understanding of the environmental, epidemiological, immunological, and clinical dimensions of coccidioidomycosis.

Coccidioidomycosis is the endemic mycosis caused by the fungal pathogens Coccidioides immitis and C. posadasii. This review is a summary of the recent advances that have been made in the understanding of this pathogen, including its mycology, genetics, and niche in the environment. Updates on the epidemiology of the organism emphasize that it is a continuing, significant problem in areas of endemicity. For a variety of reasons, the number of reported coccidioidal infections has increased dramatically over the past decade. While continual improvements in the fields of organ transplantation and management of autoimmune disorders and patients with HIV have led to dilemmas with concurrent infection with coccidioidomycosis, they have also led to advances in the understanding of the human immune response to infection. There have been some advances in therapeutics with the increased use of newer azoles. Lastly, there is an overview of the ongoing search for a preventative vaccine.