TY - JOUR
AU - Royal,, Angela
AB - Abstract This report describes the phenotypic characteristics of a novel Penicillium species, Penicillium labradorum, isolated from a 3-year-old male, castrated, Labrador retriever with disseminated fungal disease. The dog's presenting clinical signs included lethargy, lymphadenopathy, tachypnea, moderate pitting edema, and nonweight bearing lameness associated with the right hind limb. Fine-needle aspirate biopsies from the sublumbar and prescapular lymph nodes were initially examined. The cytologic findings were consistent with pyogranulomatous inflammation with abundant extracellular and phagocytized fungal fragments and hyphae. Based on the morphology of the organisms and lack of endogenous pigment, hyalohyphomycosis was considered most likely, with Fusarium, Penicillium, and Paecilomyces species being considerations. Fungal isolates were obtained via culture of samples from the lymph nodes, and molecular identification testing originally identified an undescribed Penicillium species belonging to the Penicillium section Exilicaulis. BLAST searches and phylogenetic analyses performed approximately 1 year and 9 months after the isolation date revealed an isolate within the Penicillium parvum clade in the Penicillium section Exilicaulis but phylogenetically distant from the other species in the section, thus representing a new species, Penicillium labradorum. Antifungal susceptibility testing was also performed on the isolate and low minimum inhibitory concentrations were observed with terbinafine, voriconazole, and posaconazole, while in vitro resistance was observed with fluconazole. The dog had been previously treated with fluconazole, itraconazole, amphotericin B lipid complex, voriconazole, and terbinafine. Approximately 587 days after the initial diagnosis, the dog was euthanized due to worsening of clinical signs and concerns for quality of life. fungi, Penicillium, dog, lymphadenopathy Introduction Penicillium is a ubiquitous fungus present in the general environment with >300 known species.1 Most species of Penicillium are considered to be nonpathogenic (opportunistic) in humans and animals.2 Climatic conditions, genetically predisposed defects in immunity, and/or immunosuppressive therapies (steroids, azothiaprine, cyclosporine, etc.) have been suggested to be predisposing factors in dogs.1-5 German Shepherd dogs, particularly young females, are susceptible to systemic fungal infections, presumably due to a hereditary immunologic defect in this breed in which IgA immunoglobulins are decreased.1-5 Other genetically predisposed defects in immunity are generally not recognized in most cases described in the veterinary literature.2,3 According to one review paper in which 157 cases of disseminated mould infections in dogs were identified, 30 cases (19.1%) reported previous implementation of immunosuppressive therapy, 55 cases (35%) specifically stated that no predisposing factors were recorded, and 72 cases (45.9%) provided no data on the immune status. In human medicine, most cases of disseminated fungal disease have been attributed to a lack of suitable macrophage and/or neutrophil reaction in immunocompromised patients.5 More specific predisposing factors recognized in humans include acquired human immunodeficiency virus (HIV) infection, those on immunosuppressant therapy or cancer chemotherapy, neutropenic patients, or patients with poorly controlled diabetes mellitus. Some fungi reported to cause disseminated disease in immunocompromised humans include Aspergillus sp., Fusarium sp., Mucor sp., Cryptococcus sp., Penicillium sp., and Candida sp.6 Additionally, localized infections in otherwise healthy humans (usually following penetrating trauma) and infections of the nasal passages and lower airways of dogs have also been described.7,8 In cases of disseminated disease, discospondylitis, osteomyelitis, and diffuse lymphadenopathy in dogs have been observed.1,3,9 Overall, opportunistic filamentous fungal infections in dogs are infrequently reported, with Aspergillus species being the most commonly identified in cases of disseminated fungal disease.1,3,5 The infrequent nature of Penicillium infections (with the exception of Talaromyces marneffei, formerly known as Penicillium marneffei) has been attributed to the inability of most Penicillium species to grow at 37°C.9 Many of the previously reported Penicillium species have recently been identified as Talaromyces species infections. This includes: disseminated infection clinically resembling multicentric malignant lymphoma due to P. radicum (now identified as T. radicus),9 disseminated mycosis in a German Shepherd due to P. purpurogenum (now identified as T. purpurogenus),3 intra-abdominal eumycotic mycetoma caused by P. duponti (now identified as Thermomyces dupontii),10 pulmonary infection with P. commune,11 and two cases of osteomyelitis in which P. verruculosum (now identified as T. verruculosus) and P.canis were isolated.1,2 A review of opportunistic fungal infections in 10 dogs also noted one case of nonspecified penicilliosis.4 The prognoses for these cases are poor, with cures unlikely despite aggressive anti-fungal therapy. Favorable outcomes with treatment, however, have been infrequently reported and was initially seen in the present case following multimodal medical therapy with amphotericin B lipid complex (Abelcet; 2 mg/kg, IV slow over 2 hours, 2–3 treatments per week to total dose of 26 mg/kg) followed by long-term oral voriconazole (3.3 mg/kg PO q12hr) and terbinafine (29.2 mg/kg PO q24hr). The dog reported to have infection caused by P. canis also notably had a favorable outcome, with no clinical signs apparent 250 days after diagnosis following multimodal medical therapy with amphotericin B lipid complex (Abelcet; 75 mg IV over 1 hour, three treatments per week for 4 weeks) and long-term oral antifungal therapy with ketoconazole (1 g/day) and terbinafine (1 g/day).1 In the presented case, a previously undescribed Penicillium species belonging to the Penicillium section Exilicaulis was isolated. This report describes the phenotypic characteristics of a new species, P. labradorum, as another agent of disseminated fungal disease in a dog. Methods Dog examined A 3-year-old male castrated Labrador retriever was evaluated by a primary care veterinarian for a 3-week history of lameness, lethargy, and lymphadenopathy. The dog recently moved to Kansas but had previously resided in Florida and California, USA. Pertinent physical examination findings at the time of initial evaluation included mild tachypnea and moderately enlarged and firm prescapular, inguinal, and popliteal lymph nodes. Ultrasound guided fine-needle aspirate biopsies of an abdominal lymph node were obtained and examined by the primary care veterinarian with cytologic recognition of suspected fungal hyphae and pyogranulomatous inflammation. Treatment with fluconazole (10 mg/kg PO q12hr) and doxycycline (5 mg/kg PO q12hr) were initiated at that time. Within 2 days of treatment, the dog developed a depressed mentation and became increasingly more tachypneic and dyspneic. The dog was evaluated again by the primary care veterinarian and in addition to increased respiratory abnormalities, moderate pitting edema and mild weight bearing lameness associated with the right hindlimb were noted. There was also mild accumulation of edema along the ventral thorax and abdomen. Antifungal therapy was switched from fluconazole to itraconazole and the patient was additionally prescribed carprofen (2.4 mg/kg PO q24hr) and tramadol (3.2 mg/kg PO q6–8hr) for pain management. The dog became anorectic and mildly ataxic in the following 2 days, thus prompting referral to the University of Missouri Veterinary Health Center for further evaluation (day 1). Initial diagnostics during this visit included a plasma biochemical profile and urinalysis which were both unremarkable. A complete blood count (CBC) revealed mild leukocytosis (19.17 K/µl; RI: 4.53–14.99 K/µl) characterized by a mild mature neutrophilia (15.14 K/µl; RI: 2.270–10.140 K/µl). Thoracic radiographs revealed mild perihilar lymphadenopathy. Abdominal radiographs and ultrasonography showed sublumbar lymphadenopathy and multiple enlarged intra-abdominal lymph nodes that measured up to 3.5 cm in diameter. Bony proliferations or other abnormalities associated with the right hind limb were not observed on radiographs; therefore, it was concluded that the pitting edema present was most likely due to impingement of the lymphatic system or occlusion of venous return by the enlarged sublumbar lymph nodes, or less likely due to thrombus formation. Sampling, cytology, immunological testing Fine-needle aspirate biopsies of the sublumbar and prescapular lymph nodes were submitted for evaluation and to confirm fungal infection. All samples were collected using a 22-gauge needle, and slide preparations were stained with a Wright-Giemsa stain. Intraabdominal lymph node fine-needle aspirate biopsies were repeated approximately 38 days later after finding multiple enlarged intrabdominal lymph nodes on ultrasound examination, with some being more enlarged than previously seen at the initial referral. Immunological testing was pursued to screen the host's immune response to the pathogen. An immunoglobulin assay for IgA, IgM, and IgG was performed through the Animal Health Diagnostic Center of Cornell University. Other tests to evaluate innate and adaptive immune functions included stimulated leukocyte cytokine production capacity (Sigma-Aldrich, St. Louis, MO, USA), leukocyte MHC-II surface expression (MHC-II antibody from Biorad, Hercules, CA, USA), lymphocyte blastogenesis (CD5 and ki67 antibodies from ThermoFisher, Waltham, MA, USA), and granulocyte/monocyte phagocytic capacity (Phagotest®, Orpegen Pharma, Heidelberg, Germany), all of which were compared to at least one healthy, age-matched control dog evaluated concomitantly with the dog reported here. Evaluation of granulocyte respiratory burst was performed using a commercially available test kit validated for use in canine blood (Phagoburst®, Orpegen Pharma, Heidelberg, Germany)12 and compared to one healthy, age-matched, control dog. All testings were done after additional antifungal treatment while the patient was clinically well with no peripheral lymphadenopathy noted. Fungal culture, morphology, and susceptibility testing Prior to the implementation of additional antifungal treatment, pre-scapular lymph node aspirates were placed in a BD-BACTEC PLUS™ antimicrobial removal device (Becton Dickinson Diagnostics, Sparks, MD, USA) and a standard blood culture bottle and submitted for aerobic and anaerobic bacterial culture and fungal culture through the University of Missouri Veterinary Medical Diagnostic Laboratory. The prescapular lymph node aspirates inoculated into the BD bottle produced a slow growing fungus and that was then subcultured to Sabouraud dextrose agar (SDA) (Remel™, Lenexa, KS, USA). Two fungal isolates were sent to the Fungus Testing Laboratory (FTL) at the University of Texas Health Science Center at San António, TX, USA for species identification and susceptibility testing. The isolates were accessioned as UTHSCSA DI19-20 and UTHSCSA DI19-21. The specimens received at the FTL were subcultured onto potato flakes agar (PFA) and incubated at 25°C and 37°C. Growth at 10°C, 25°C, 30°C, and 40°C was also assessed on Czapek yeast autolysate agar (CYA), malt extract agar (MEA, Oxoid Limited, Hampshire, UK), yeast extract sucrose (YES), and creatine sucrose agar (CREA) (all prepared in-house). Colonial features were examined after 7 days of incubation and the color recorded following the color descriptions in the Methuen Handbook of Colour.13 Additional DNA sequencing and phylogenetic analysis are described below. Susceptibility testing was performed according to the CLSI M38 reference standard.14 Scanning electron microscopy (SEM) was also used to obtain images of microscopic structures. Phenotypic characterization and molecular analysis Mycelial mass from the isolates were harvested from PFA for DNA extraction, and genomic DNA were extracted following previously described protocols.15 The ITS rDNA region, partial beta tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes were amplified and sequenced to compare with sequences of the same loci in previous studies.16-18 Polymerase chain reaction (PCR) and sequencing were carried out using primer pairs BMBC-R and NL4R (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/) for ITS,19 BT2a and Bt2b for BenA,20 CF1 and CF4 for CaM,21 and 5F and 7CR for RPB2.22 The primer pair BMBC-R1 / NL42 was used since the amplification that is achieved results in a complete sequence spanning both ITS1 and ITS2. This primer pair has been validated by the FTL for species identification of clinical isolates received by this reference mycology laboratory. Since this pair works very well, other primer pairs such as those published by others were not used.16,17 The generated sequences were used to perform BLASTn searches in GenBank.23 BLASTn search results were considered significant with an E-value of 0.0, at 90–100% identity and from 90% query coverage. Phylogenetic analyses were conducted independently with each locus and combined to assess the relationship of isolates UTHSCSA DI19-20 and UTHSCSADI19-21 to members of the Penicillium section Exilicaulis. Sequences were aligned using MUSCLE as implemented in Sequencher ver 5.4.6 build 46289 (Gene Codes Corp. Ann Arbor, MI, USA).24 Substitution models were determined for each locus using the Finding Model program as implemented in Molecular Evolutionary Genetics Analysis ver. 7 software (MEGA 7).25 Phylogenetic analyses using the maximum likelihood method based on the optimal evolutionary models for each locus and combined were conducted. The robustness of the phylogenetic trees was evaluated by 1000 bootstrap resampling26 and Bayesian inference on the combined data set was conducted in MrBayes v3.2.5 using the previously determined optimal substitution model and the Markov chain Monte Carlo algorithm.27 The analysis stopped when the average standard deviation of split frequencies reached 0.01. The sample frequency was set at 100 and the first 25% of trees were removed as burn-in. Results Cytology, immunological testing, case outcome Cytologic preparations consisted primarily of aggregates of extracellular fungal organisms admixed with high numbers of nondegenerate and degenerate neutrophils, lower numbers of heavily vacuolated macrophages, and low numbers of lymphocytes. Macrophages were often seen in aggregates and occasionally contained phagocytized fungal organisms (Fig. 1A). The fungal organisms were basophilic, round to oval, ∼4–5 microns in diameter with irregular borders, and had a 1–2 micron thick, clear halo. Rare septate, parallel walled hyphal structures giving rise to bulbous ends were present (Fig. 1B). The cytologic diagnosis was pyogranulomatous inflammation with abundant intralesional fungal organisms, with hyalohyphomycosis considered most likely. Figure 1. Open in new tabDownload slide A, B: Sublumbar lymph node aspirate biopsies from a dog with disseminated fungal infection caused by a novel Penicillium species, Penicillium labradorumsp.nov. Wright-Giemsa stain. (A) Macrophage containing many phagocytized fragments of fungal organisms. Many degenerate neutrophils are also present in the background. (B) Aggregates of extracellular fungal organisms with rare septate, parallel walled hyphal structures with bulbous ends (arrows). 100× objective. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. Open in new tabDownload slide A, B: Sublumbar lymph node aspirate biopsies from a dog with disseminated fungal infection caused by a novel Penicillium species, Penicillium labradorumsp.nov. Wright-Giemsa stain. (A) Macrophage containing many phagocytized fragments of fungal organisms. Many degenerate neutrophils are also present in the background. (B) Aggregates of extracellular fungal organisms with rare septate, parallel walled hyphal structures with bulbous ends (arrows). 100× objective. This Figure is reproduced in color in the online version of Medical Mycology. Repeat intraabdominal lymph node FNA biopsies consisted predominantly of macrophages containing phagocytized fungal organisms with morphologic features similar to those seen from the sublumbar lymph node aspirates submitted ∼1 month prior (Fig. 2). Staining of the organisms, however, varied from the initial aspirates. They instead appeared clear to pale blue with rare sparse internal pink staining, which was presumed to be due to degenerative changes in the organisms as a consequence of the antifungal therapy. The patient was kept in hospital overnight on IV fluid therapy and was discharged with unchanged antifungal therapy while the phenotyping and susceptibility panel was pending. Figure 2. Open in new tabDownload slide Intraabdominal lymph node aspirates from a dog submitted 9 days after the initiation of antifungal therapy with itraconazole. Unlike the initial aspirate preparations, the extracellular fungal organisms are round to oval and are now clear to homogeneously pale blue. Few macrophages, ruptured cells, and scattered erythrocytes are also present. Wright-Giemsa stain, 60 × objective. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. Open in new tabDownload slide Intraabdominal lymph node aspirates from a dog submitted 9 days after the initiation of antifungal therapy with itraconazole. Unlike the initial aspirate preparations, the extracellular fungal organisms are round to oval and are now clear to homogeneously pale blue. Few macrophages, ruptured cells, and scattered erythrocytes are also present. Wright-Giemsa stain, 60 × objective. This Figure is reproduced in color in the online version of Medical Mycology. An immunoglobulin assay for IgA, IgM, and IgG (performed through the Animal Health Diagnostic Center of Cornell University) revealed normal IgM and IgG levels and a slight decrease in IgA (29 mg/dl; RI: 35–270 mg/dl). The slight decrease in IgA was not low enough to be considered clinically significant and was not pursued further. Evaluation of granulocyte respiratory burst, however, revealed that the dog had subjectively decreased Escherichia coli stimulated respiratory burst capacity compared to a healthy age-matched control dog. All other tests evaluating innate and adaptive immune functions were considered unremarkable when compared to at least one healthy, age-matched control dog evaluated concomitantly. Control dogs were determined to be healthy based on a history reported by the owners and the physical exam findings. Low minimum inhibitory concentrations, as measured by the CLSI M38 broth microdilution method, were observed with terbinafine (0.03 μg/ml against both isolates), voriconazole (0.5 and 0.25 μg/ml, respectively), and posaconazole (≤0.03 and 0.06 μg/ml, respectively), while in vitro resistance was observed with fluconazole (>64 μg/ml). An itraconazole blood concentration (MVista®, Itraconazole bioassay, Indianapolis, IN, USA) was also evaluated in the dog to ensure recrudescence of disease was not the result of inadequate therapeutic concentrations of itraconazole. While a specific therapeutic range for itraconazole in dogs is not known, blood concentrations >3.0 ug/ml are recommended for systemic fungal infections when measured by bioassay, and the dog in this report had an itraconazole serum concentration of 10.6 ug/ml. Though the reported concentration represents a combination of both itraconazole and the active metabolite hydroxy-itraconazole (also found in the bloodstream), a switch to amphotericin B lipid complex was recommended given these findings in conjunction with the dog's clinical progression of disease. Treatment with amphotericin B lipid complex began on day 44, and the dog received 2 to 3 treatments per week for a cumulative dosage of 26 mg/kg. During the course of amphotericin treatment, the dog's appetite and attitude improved. The only adverse development was a focal thickening noted at the catheter site after several treatments that eventually became ulcerated. Punch biopsies of the lesion were obtained on day 51 and submitted for histopathology and for bacterial and fungal cultures. Histopathology revealed marked ulcerative dermatitis and extensive vasculitis. No specific etiology was identified, though a systemic process was suspected. Culture results yielded multidrug resistant Staphylococcous pseudintermedius, and no fungal organisms were identified or cultured from the lesion. The dog was treated with chloramphenicol based on the culture and susceptibility results, and skin lesions subsequently resolved entirely. Following successful delivery of a cumulative dosage of 26 mg/kg on day 79, amphotericin B lipid complex was discontinued, and the dog was switched to voriconazole (3.3 mg/kg PO q12hr) and terbinafine (29.2 mg/kg PO q24hr). All clinical signs of illness and all lympadenomegaly resolved completely. The dog continued to do well for many months but moved away from Missouri, and care was transferred to the University of Florida-Small Animal Hospital. After the dog had been in apparent remission for well over a year, antifungal therapy was discontinued. Disease was confirmed to have recurred 535 days after the first amphotericin treatment. Although a plan was developed to restart treatment with amphotericin B, his clinical signs had worsened quickly; he was hyporectic and developed a cough with a retch. Thoracic radiographs showed marked enlargement of the mediastinal lymph nodes and a new nodule in the left lung lobe. Due to the radiographic results and concerns for his quality of life, humane euthanasia was ultimately elected. Macro- and micromorphology The initial fungal culture performed at the University of Missouri yielded a slow growing fungus that formed white fungal colonies on SDA. The hyphae were sterile, septate, and devoid of any identifying characteristics such as metulae, phialides, or conidiophores, thus prompting submission of the isolates to FTL for species identification and susceptibility testing. At the FTL, microscopic features were studied on PFA slide cultures incubated for 3 and 7 days at 25°C and mounted in lactophenol cotton blue (Fig. 3i,j). See Taxonomy below for culture description results. Figure 3. Open in new tabDownload slide Penicillium labradorum (UTHSCSA DI19-20). a–d. Colonies (obverse) at 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. e–h. Colonies (reverse) 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. i–j. Light micrographs of short conidiophores at 3 days (i) and 7 days (j). k–m. Scanning electron micrographs of short conidiophores and conidia. This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. Open in new tabDownload slide Penicillium labradorum (UTHSCSA DI19-20). a–d. Colonies (obverse) at 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. e–h. Colonies (reverse) 30°C, 7 days, left to right: MEA, CYA, YES, and CREA. i–j. Light micrographs of short conidiophores at 3 days (i) and 7 days (j). k–m. Scanning electron micrographs of short conidiophores and conidia. This Figure is reproduced in color in the online version of Medical Mycology. Top BLASTn searches with 0.0 E-values were 99% matched with the following species, P. vinaceum CBS 389.49T, P. pimiteouiense CBS 102479T, P. vinaceum CBS 389.48T, P. guttulosum NRRL 907T, P. parvum CBS 359 .48T, P. rubidurum CBS 609.73T for ITS, 91% matched with P. pimiteouiense CBS 102479T for CaM, 95% matched with P. vinaceum CBS 389.49T, P. parvum CBS 359 .48T and P. rubidurum CBS 609.73T for BenA and 95% matched with P. vinaceum CBS 389.49T for RPB2. Phylogenetic relationships were inferred using the Maximum Likelihood based on the combined datasets of BenA, CaM, and RPB2 sequences and on individual data sets comprising species in the P. parvum clade of the Penicillium section Exilicaulis. The combined data sets had a total length of 2154 characters (individual data sets: CaM = 578; BenA = 509; RPB2 = 1067). Optimal substitution models based on the Akaike information criterion (AIC) were Kimura 2-parameter using a discreet Gamma distribution (K2P + G) for BenA and CaM, Kimura 2-parameter with invariant sites (K2P + I) for RPB2 and Kimura 2-parameter using a discreet Gamma distribution with invariant sites K2P + G + I for the combined datasets of BenA, CaM and RPB2. Results of the BLASTn searches and phylogenetic analyses showed that our isolates are within the P. parvum clade in the Penicillium section Exilicaulis but phylogenetically distant from the other species in the section and represent a new species. The phylogram from the combined data sets showed isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 clustered together at 100% bootstrap support (BS) and 1.00 Bayesian posterior probability value (BPP) and as a distinct clade and sister to P. parvum at 92% (BS) and 1.00 BPP within the P. parvum clade (Fig. 4). Independent analyses of BenA, CaM, and RPB2 confirmed their placement in the P. parvum clade as a distinct species clustered at 100% BS and closest to P. parvum but with low BS at <70% bootstrap for BenA, 80% for CaM and 84% with RPB2 (Fig. 5). Figure 4. Open in new tabDownload slide Maximum likelihood tree based on the combined dataset of BenA, CaM, and RPB2 sequences showing the isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 placed within the Penicillium parvum clade of the Penicillium section Exilicaulis. Bootstrap values (left, ≥70%) and Bayesian posterior probabilities (right, ≥0.95 pp) are shown above the branches. Penicillium glabrum CBS 125543T was used as outgroup. CBS = CBS culture collection, housed at the Westerdijk Institute, Utrecht, The Netherlands, NRRL = Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, US Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA, DAOMC = Canadian Collection of Fungal Cultures, IMI = International Mycological institute (CABI = Centre for Agriculture and Bioscience International, UK). T = type species. This Figure is reproduced in color in the online version of Medical Mycology. Figure 4. Open in new tabDownload slide Maximum likelihood tree based on the combined dataset of BenA, CaM, and RPB2 sequences showing the isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 placed within the Penicillium parvum clade of the Penicillium section Exilicaulis. Bootstrap values (left, ≥70%) and Bayesian posterior probabilities (right, ≥0.95 pp) are shown above the branches. Penicillium glabrum CBS 125543T was used as outgroup. CBS = CBS culture collection, housed at the Westerdijk Institute, Utrecht, The Netherlands, NRRL = Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, US Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA, DAOMC = Canadian Collection of Fungal Cultures, IMI = International Mycological institute (CABI = Centre for Agriculture and Bioscience International, UK). T = type species. This Figure is reproduced in color in the online version of Medical Mycology. Figure 5. Open in new tabDownload slide Maximum likelihood trees based on the individual data sets of BenA, CaM, and RPB2 sequences showing the relationship of the isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 with species within the Penicillium parvum clade of the Penicillium section Exilicaulis. The numbers to the left of the species names are GenBank accession numbers. Bayesian posterior probabilities (Left, ≥0.95 pp) and Bootstrap values (Right, ≥70%) are shown above the branches. The trees are rooted with Penicillium glabrum CBS 125543T. This Figure is reproduced in color in the online version of Medical Mycology. Figure 5. Open in new tabDownload slide Maximum likelihood trees based on the individual data sets of BenA, CaM, and RPB2 sequences showing the relationship of the isolates UTHSCSA DI19-20 and UTHSCSA DI19-21 with species within the Penicillium parvum clade of the Penicillium section Exilicaulis. The numbers to the left of the species names are GenBank accession numbers. Bayesian posterior probabilities (Left, ≥0.95 pp) and Bootstrap values (Right, ≥70%) are shown above the branches. The trees are rooted with Penicillium glabrum CBS 125543T. This Figure is reproduced in color in the online version of Medical Mycology. Taxonomy Mycological assessment Isolates UTHSCSA DI19-20 and UTHSCSADI19-21 had identical sequences from all loci and were originally identified as an undescribed Penicillium species in section Exilicaulis based on DNA sequencing of the ITS rDNA region and a part of the beta tubulin gene. Culture description Colony diameter: 7 days, in mm, 25°C: MEA 16–17; CYA 10–11; YES 15–16; CREA 3–4. Colony characteristics: Mycelia white. Reddish brown (9E7) soluble pigments present on PFA but absent on MEA, CYA, and YES. On PFA, colonies were flat and floccose, producing sparse to moderately dense sporulation, with conidia dull greyish green (30E4 obverse) and en masse and reddish brown reverse (8E8). On CYA, colonies grew slowly attaining a diameter of 10–11 mm, erumpent, floccose, folded, wrinkled, and crateriform, appearing dull cream with an olive (3D3) perimeter (Fig. 3b,f). Colonies on MEA were low, with radial folds, centrally raised, displaying greenish gray to olive (3E3) at the periphery and pale orange (6A3) towards the center (Fig. 3a,e), yellowish brown reverse (5E4) with moderate to dense sporulation. Growth on YES was yellowish olive gray (3D2–3D3) on obverse, olivaceous (3D3–3D4) on reverse (Fig. 3c,g) and showed a similar obverse pattern as on CYA. Colonies on CREA were restricted attaining only 3–4 mm in diameter after 7 days, flat and greenish gray obverse (30C3) and grayish brown (6D3) reverse, acid and base production were not observed (Fig. 3d,h). Micromorphology: Conidiophores were monoverticillate with smooth short stipes (7.5–15 um in length); metulae absent, phialides smooth, ampulliform, 2–4 per stipe (3.2–5 um in length). Conidia were finely roughened, globose to subglobose (1.9–2.3um × 1.9–2.3um). No sclerotia or ascomata were observed on all media after >6 months incubation. Growth was good on YES at 37°C but slower on PFA, CYA, and MEA and variable at 40°C. Short conidiophores and conidia were also visualized with SEM (Fig. 3k–m). Based on phylogenetic results and their distinct phenotypic characteristics, we describe these two isolates as a new species named Penicillium labradorum sp. nov. Penicillium labradorum Gibas, Wiederhold, Sanders, Rothacker, Rogers et Fales sp. nov. Mycobank: MB831086 In: Penicillium subgenus Aspergilloides section Exilicaulis. Etymology Named after a Labrador retriever dog where it was isolated from. Type and holotype Missouri, United States of America, from prescapular lymph node aspirates from a Labrador retriever dog with disseminated infection. Collected by D.V.M. Erin Rogers (June 11, 2017) and isolated by PhD William Fales (July 17, 2017). Holotype = CBS H-24321. Culture ex-type UTHSCSA DI19-20 = CBS 145775, preserved in a metabolically inactive state at the CBS culture collection, Westerdijk Fungal Biodiversity Institute, The Netherlands. ITS barcode: = MK881918; Alternative markers: BenA = MK887898, CaM = MK887899, RPB2 = MK887900. Other strains UTHSCSA DI19-21 = CBS 145776. ITS barcode: = MK882920 Alternative markers: BenA = MK887901, CaM = MK887902, RPB2 = MK887903. The two strains examined had identical ITS, BenA, CaM and RPB2 sequences. Distribution and ecology Isolated in the United States and distribution in other parts of the world is unknown. Discussion In this report, we describe a new species of Penicillium, P. labradorum sp. nov., within the section Exilicaulis. As described in the case, the morphology of the organism on cytology was most consistent with an organism causing hyalohyphomycosis, with fungal culture and isolation yielding a previously unidentified Penicillium sp. Morphologically, Penicillium is described as having septate hyphae (2–5 μm in width) giving rise to branched or unbranched conidiophores with secondary branches, which give it a brush-like appearance in culture.1 In tissues, septate hyaline hyphae lacking pigment within the walls are a common characteristic, hence categorizing it as one of many organisms which cause “hyalohyphomycosis.” This term is used as a counterpart to the term “phaeohyphomycosis,” in which fungi appear in tissues as septate hyphae containing an endogenous pigment. In the presented case, the preservation of the fungal hyphal morphology was poor on cytology, with only fragments and rare septate, branching, nonpigmented fungal hyphae seen (possibly the result of prior treatment with itraconazole). On the basis of the fungal characteristics that were evident, likely differential diagnoses included infection with Penicillium, Fusarium, or Paecilomyces species. Aspergillus was ruled out based on the lack of characteristic 45° angle branching associated with that genus.2 Penicillium species in the section Exilicaulis are characterized by having mono and biverticillate conidiophores and nonvesiculated stipes.28 Some species in this section have been implicated in several conditions such as allergies from extrolites (e.g., citreoviridin, a mycotoxin extrolite known to cause yellow rice disease or cardiac beri-beri in humans [P. citreonigrum]),29 osteomyelitis in dogs (P. canis),1 and para-vertebral infection and mycetoma (fungus ball) in humans (P. decumbens).30,31Penicillium parvum is the closest relative of P. labradorum. Phenotypically, P. labradorum is similar to P. parvum by its slow growth, production of a reddish-brown diffusing pigment, and short conidiophores. However, P. labradorum differs by its production of finely roughened conidia whereas P. parvum produces smooth conidia.32 Although Penicillium parvum has a sexual state, the sexual state of P. labradorum was not observed in our isolates after >6 months of incubation in all media used; however, mating studies were not performed. In addition, P. labradorum is phylogenetically distant from P. parvum having 24 base pair differences in partial CaM sequences and 45 base pair differences in both partial BenA and RPB2 sequences. Identification of fungal isolates is usually achieved via examination of fruiting structures formed in culture, although evaluation of fungal culture by morphology combined with PCR amplification and sequencing are considered as the criterion standard for fungal identification.2,33 In the present case, molecular and morphological methods were used to identify the organisms. In the beginning, the primary cultures only produced sterile hyphae with no microscopic features to assist with identification. The lack of identifying features may be attributed to the previous treatment with antifungals prior to the samples being submitted for culture. Additionally, the possibility of culture growth due to contamination was considered as Penicillium is a common laboratory contaminant. For the dog in this report, however, this was considered less likely since the morphological features of cultured isolates were similar to those observed on cytology (sterile hyalinized hyphae lacking pigment), and the findings of these organisms were repeated on both the initial and follow-up cytologic examination from the sublumbar and intraabdominal lymph nodes. When the cultures were submitted to the FTL for identification and were subsequently subcultured onto various culture media for identification workup, the isolates grew normally as a typical Penicillium species (i.e., producing structures salient to Penicillium species). Multiple immunologic processes are required to successfully clear fungal diseases including opsonization, phagocytosis, and destruction of fungal organisms utilizing oxidative damage by a respiratory burst.6,34 Therefore, substantial disruptions in immune function at any of these stages could be significant enough to prevent disease clearance, with subsequent dissemination of fungal organisms being more likely. The immune function assessment in this patient suggested that there was suppression of granulocyte respiratory burst capacity. It is suspected that the diminished respiratory burst in this dog increased susceptibility to fungal disease. It is uncertain if the systemic fungal disease further suppressed the dog's immune system, or if the same respiratory burst defect played a permissive role in the secondary bacterial infection with S. pseudintermedius. Iatrogenic infection with this bacteria also could not be entirely ruled out. Aside from prior treatment with steroids,11 other predisposing defects in immunity have not been recognized in previous reports of Penicillium sp. infections in dogs, making this an interesting finding. It is known that some fungal pathogens can exert an ability to mask themselves from the immune system, or dampen immune function to enhance survivability. One such fungal organism is Candida albicans, which actively suppresses the production of reactive oxygen species involved in the respiratory burst.35 Variation of virulence is also known to occur within the genus and species of fungal organisms. For example, Aspergillus fumigatus has been demonstrated to be less pathogenic than A. nidulans in human patients with chronic granulomatous disease (a disease process with features similar to the presented case in which the functionality of the respiratory burst pathway is disrupted).36 In this patient, a novel species of Penicillium resulted in disseminated disease. This leaves the possibility of a more pathogenic species of Penicillium in general, or perhaps a species of Penicillium that is able to exploit this patient's particular immune compromise as an alternative scenario to opportunistic infection. It is uncertain if the long-term favorable response to treatment in this case was a result of the species of fungal organism involved or the aggressive treatment protocol. The in vitro susceptibilities measured against the two P. labradorum isolates in this case are similar to what has previously been reported against P. canis, with potent activity observed with terbinafine, posaconazole, and voriconazole, and in vitro resistance to fluconazole.1 Terbinafine and posaconazole have also been reported by others to have good in vitro activity against Penicillium species.37,38 In contrast, others have reported that the activities of voriconazole and posaconazole may be variable against Pencillium with some species demonstrating reduced susceptibility to these triazoles (e.g., P. citrinum, P. oxalicum, P. rubens) and some reports showing resistance to voriconazole.33,39 This case is unique given the cytologic findings, the novel Penicillium sp. isolated, and the dog's diminished granulocyte respiratory burst functionality compared to a normal dog. Previous cases investigating respiratory burst functionality as a causative factor of S. pseudintermedius infections and disseminated fungal disease in dogs were not found in the literature. Although it is suspected that the diminished granulocyte respiratory burst capacity predisposed this dog to disseminated fungal disease and possibly secondary infection with S. pseudintermedius, additional testing would be necessary to more definitively determine the role of an immune deficiency of this nature as the results were compared to only one healthy, age-matched control dog. Acknowledgments The authors thank Dr. Amy DeClue and Dr. Carol Reinero at the University of Missouri-Veterinary Health Center for providing the assays, lab time, and materials for the immunological testing performed, Hans Rindt for performing most of the immunological testing mentioned in this paper, and Barbara Hunter for performing the scanning electron microscopy. 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Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Novel Penicillium species causing disseminated disease in a Labrador Retriever dog
JF - Journal of Medical and Veterinary Mycology
DO - 10.1093/mmy/myaa016
DA - 2020-11-10
UR - https://www.deepdyve.com/lp/oxford-university-press/novel-penicillium-species-causing-disseminated-disease-in-a-labrador-drmlsEPoms
SP - 1053
EP - 1063
VL - 58
IS - 8
DP - DeepDyve
ER -