Chemically induced disseminated pythiosis in BALB/c mice: A new experimental model for Pythium insidiosum infection

Abstract

Pythiosis is a severe and life-threatening disease that affects humans and various animal species. We report a model of vascular/disseminated pythiosis occurring after subcutaneous inoculation of 2 x 104 Pythium insidiosum zoospores/mL in immunocompromised BALB/c mice. For this model, we carried out two rounds of experiments. First, we evaluated two protocols of immunosuppression before inoculation: cyclophosphamide at 150 mg/kg (CYP group) and cyclophosphamide 200 mg/kg plus hydrocortisone acetate at 250 mg/kg (CYP+HCA group). It was not possible to obtain mortality in the CYP group; however, the combination of CYP+HCA altered disease outcomes, with mortality rates reaching 60%. Second, we used the CYP+HCA immunosuppression protocol to analyze the histological and immunological statuses triggered by disease. When we inoculated immunocompetent mice with P. insidiosum zoospores, self-healing occurred via increased levels of IL-2, IFN-γ and IL-17A, which are characteristic of the Th1/Th17 cytokine response. For infected and immunosuppressed mice, the cytokine profiles showed high levels of IL-10, IL-6 and TNF-α. Increased IL-10 values are related to fungal infection susceptibility and led us to speculate that infection may be established through suppression of the host immune response. In addition, histopathological evaluation of the kidneys and liver demonstrated the presence of hyphae and the cellular findings suggested an acute vascular inflammation that mimics vascular/disseminated pythiosis in humans. This is the first murine model for pythiosis that is useful both for understanding the pathogenesis of this disease and for evaluating new treatment approaches.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection

“…In the experimental model of pythiosis in rabbits, the subcutaneous lesions and histopathological findings resemble those observed in horses and other animals presenting the cutaneous form of pythiosis . Histologically, we observed that the oomycete proliferated by invading blood vessels and forming thrombi; these histological findings resemble those seen in human patients and experimentally infected mice . During the progression of vascular human pythiosis, the pathological findings revealed hyphae invading the arterial wall, with eosinophil infiltration and forming thrombi that affect the circulation and usually lead to limb amputation .…”

“…9 Histologically, we observed that the oomycete proliferated by invading blood vessels and forming thrombi; these histological findings resemble those seen in human patients and experimentally infected mice. 12,30 During the progression of vascular human pythiosis, the pathological findings revealed hyphae invading the arterial wall, with eosinophil infiltration and forming thrombi that affect the circulation and usually lead to limb amputation. 30 Dissemination to internal organs can occur when the infection reaches a main artery (ie the iliac artery, aorta, or renal artery), and the patient usually dies of systemic pythiosis.…”

“…The standard strain of P. insidiosum CBS 101555, which was obtained from a subcutaneous equine pythiosis infection from southern Brazil, was selected for this study. For the zoospore induction, we used the zoosporogenesis technique with the modifications according to Tondolo et al . After counting the number of zoospores in the Neubauer chamber, the final inoculum was prepared by dilution in PBS to achieve the concentrations to be applied to each egg, and the prepared inoculum was grown on corn meal agar to confirm its viability.…”

“…9,10 An alternative model based on nonvertebrate animals was described using fruit flies (Drosophila melanogaster) deficient in Toll-like receptors. 11 Recently, Tondolo et al 12 developed an experimental murine model of pythiosis through the chemical immunosuppression of BALB/c mice. However, the use of both rabbit and murine models is restricted by costs, the requirement of specialised facilities and personnel, legal requirements and ethical considerations.…”

Efficacy of Azithromycin and Miltefosine in Experimental Systemic Pythiosis in Immunosuppressed Mice

“…All mice were rendered neutropenic by the intraperitoneal administration of cyclophosphamide (CYP, 100 mg/kg of body weight; Genuxal, Baxter Hospitalar Ltd., Brazil) on days Ϫ4 and Ϫ1 preinfection (16). On day 0, the mice were anesthetized by an intraperitoneal injection of ketamine-xylazine (Syntec Brasil Ltd., Brazil) administered at doses of 50 and 10 mg/kg, respectively, and were infected by subcutaneous injections of 2 ϫ 10 4 zoospores/mouse (17).…”