Evaluation of Microbial Culture Techniques for the Isolation of Pythium Insidiosum from Equine Tissues

Abstract

Abstract. The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.

Diagnosis and Treatment of Pythium Insidiosum Corneal Ulcer in a Chinese Child: A Case Report and Literature Review

“…P. insidiosum is an oomycota with filaments resembling fungi. In accordance with previous reports [19,24], the hyphae of P. insidiosum stain positive for acridine orange hydrochloride and lactophenol blue, showing a thick cell wall, sparse septate, and numerous vehicles inside. At the end of some filaments, an “end-plate”- like structure was discovered in this case, and was assumed to be associated with sporangias release (Figure 2B).…”

“…In retrospect, P. insidiosum can be cultured in various types of agar, including potato dextrose agar, Sabouraud dextrose agar, and chocolate agar [5,24]. In this case, the pathogen was cultured successfully on potato dextrose agar at 37°C (Figure 3).…”

Development of an Anti-Elicitin Antibody-Based Immunohistochemical Assay for Diagnosis of Pythiosis

“…The current diagnostic modalities, including culture identification (9)(10)(11), serodiagnosis (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), and molecular-based detection (22)(23)(24)(25)(26)(27), are fraught with problems. For example, culture identification is time-consuming and often fails to grow and to identify the organism.…”

Micosis por Pythium Insidiosum primer caso con diagnóstico definitivo en Colombia

“…Desde el punto de vista anatomopatológico el diagnostico presuntivo de Pitiosis en la especie equina se correlaciona con la presencia de masas muy endurecidas dentro de las lesiones tegumentarias, como incrustadas en ellas, Figura 1c, que al extraerse representa hallazgos similares a masas de aspecto "coralino" llamados "kunkers" descritas en Pitiosis equinas (16,(29)(30)(31)(32)(33)(34)(48)(49)(51)(52), Figura 1d.…”

“…Las hifas halladas morfometricamente fueron similares a las descritas en equinos y sugestivas de P. insidiosum, esto es; con ramificaciones en ángulo recto, Figura 3a, (29,30,34,43,49,52), raramente septadas (29,31,34,43,52) y con diámetro hifa oscilando entre 2,5 a 6,2 micras, Figura 3b, ó de 2 a 6 micras (14,29,31).…”