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Horse pythiosis is considered an endemic disease in the Brazilian Pantanal region, causing devastating health and economic losses. This study aimed to enhance the understanding of pythiosis epidemiology, map the distribution of horse body lesions, and investigate the correlation between these lesions and warm body surface areas, potentially implicating hematophagous vectors in the disease's transmission. A prospective study was conducted on equids in the Pantanal Mato-grossense and adjacent areas from 2012 to 2022, with 112 horses and three mules diagnosed with pythiosis. Clinical and epidemiological data, lesions' photographic records, and healthy equids' thermal imaging were collected. Most pythiosis cases occurred between January and March, correlating with regional flood cycles. Most lesions were found on limbs and the ventral abdomen, with dark-colored horses exhibiting a higher frequency of lesions. Interestingly, the thermal mapping revealed that warm areas on a healthy horse's body overlapped significantly with lesion distribution - blood-sucking insects also prefer these areas. The results suggest that pythiosis lesions in horses correlate with warmer areas of the animal body, reinforcing the hypothesis of vector involvement in disease transmission. This study underscores the need for further observational research to fully understand the complex epidemiological dynamics of pythiosis in horses.

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We investigated the anti-Pythium insidiosum activity of the antimicrobial peptides (AMPs) MSI-78, LL-37, and magainin-2. To detect the minimum inhibitory concentration (MIC), fourteen clinical strains were incubated with the AMPs following the CLSI M38-A2 protocol. All three AMPs showed antimicrobial activity with an MIC range of 20-80 mg/L against all strains. We concluded that the evaluated AMPs have great potential as anti-Pythium insidiosum agents, and their activity deserves to be more explored in further research. Antimicrobial peptides were tested against Pythium insidiosum, a microorganism that causes a difficult-to-treat disease in animals and humans. These peptides have been shown to be able to kill P. insidiosum and may be candidates for use in the treatment of this infection.

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AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

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This manuscript reports enhanced antimicrobial photoinactivation using tetra-cationic porphyrins with peripheral platinum(II) and palladium(II) complexes against fungal dermatophyte strains. Six different positively charged porphyrins were used and applied in antimicrobial photodynamic therapy experiments (aPDT) against dermatophyte fungi colonies. The microbiological tests were conducted with an adequate concentration of photosensitizer (PS) under white-light irradiation for 120 min and the most effective PS meta isomer 3PtP significantly reduced the concentration of viable fungal colony. In this way, tetra-cationic porphyrins containing platinum(II)-bipyridyl complexes may be promising fungicidal aPDT agents with potential applications in future clinical cases.

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The objective of this study was to determine whether a phytogenic blend (PB), formulated based on organic acids, tannins, curcumin, and essential oils, could replace the antimicrobials commonly used as growth promoters in the poultry industry without compromising zootechnical performance, health, or meat quality. In addition, our goal was to report the anti-aflatoxin effect of this phytogenic blend. Four treatments were used: TC, or control; T250, T500, and T1000, representing test doses of 250, 500, 1000 mg PB/kg of feed, respectively, or a 34-day experiment (initial and growth phases). On day 22 of the study and age of the birds, 500 ppb of aflatoxin was included in the diet to represent an intestinal challenge and to evaluate the growth-promoting effects of PB. In the initial phase (up to 21 days), there were no differences between groups in weight gain, feed intake, or feed conversion. After adding an aflatoxin-contaminated feed, doses of 250 and 500 mg/kg minimized the adverse effects on feed consumption and feed conversion caused by aflatoxin; but 1000 mg/kg did not differ between groups. In birds that consumed PB (T250, T500, and T1000) compared to the control, there were the following changes: 1) lower counts of heterophiles, lymphocytes, and monocytes; 2) lower lipid peroxidation and high non-protein thiols levels in breast meat; 3) lower bacteria counts in broiler litter; and 4) lower ALT levels. Greater intestinal villus/crypt ratios were observed at T250 and T500. The dose of 250 mg/kg reduced saturated fatty acids and increased unsaturated fatty acids. The chemical-physical composition of the meat did not differ between treatments. The findings suggest that the addition of a PB has a high potential to improve performance for chickens in the growing stage and minimize the adverse effects of aflatoxicosis.

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Mycotoxins are toxic secondary metabolites produced by fungus which cause worldwide concern regarding food and feed safety. Ochratoxin A (OTA), fumonisin B1 (FB1) and deoxynivalenol (DON) are some of the main mycotoxins and oxidative stress is the main mechanism of toxicity. Thereby, this study investigates the in vitro cytoprotective effects of curcumin (CUR) and silymarin (SIL) - known for their strong antioxidant activity - in PK-15 cells exposed to OTA, FB1 and DON. Pretreatment with CUR and SIL enhanced the viability of cells exposed to the mycotoxins (P < 0.001) and attenuated reactive oxygen species (ROS) formation by DON (P < 0.01), partially reduced ROS formation by FB1 (P < 0.001), but not OTA. CUR significantly decreased apoptosis in cells exposed to DON (P < 0.01) but was not able to prevent apoptosis in cells exposed to OTA and FB1. Whereas SIL was able to prevent apoptosis in PK-15 cells exposed to FB1 and DON (P < 0.01) but was not able to decrease apoptosis in cells exposed to OTA. In summary, these data indicate that curcumin and silymarin are able to provide cytoprotection against toxicity induced by OTA, FB1 and DON in PK-15 cells.

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Pythium insidiosum is an oomycete that affects mammals, especially humans and horses, causing a difficult-to-treat disease. Typically, surgical interventions associated with antimicrobial therapy, immunotherapy, or both are the preferred treatment choices. PitiumVac® is a therapeutic vaccine prepared from the mycelial mass of P. insidiosum and is used to treat Brazilian equine pythiosis. To better understand how PitiumVac® works, we analyzed the composition of PitiumVac® and the immune response triggered by this immunotherapy in mice. We performed an enzymatic quantification that showed a total glucan content of 21.05% ± 0.94 (α-glucan, 6.37% ± 0.77 and (1,3)(1,6)-ß-glucan, 14.68% ± 0.60) and mannose content of 1.39% ± 0.26; the protein content was 0.52 mg ml-1 ± 0.07 mg ml-1. Healthy Swiss mice (n = 3) were subcutaneously preimmunized with one, two, or three shots of PitiumVac®, and immunization promoted a relevant Th1 and Th17 responses compared to nonimmunization of mice. The highest cytokine levels were observed after the third immunization, principally for IFN-γ, IL-17A, IL-6, and IL-10 levels. Results of infected untreated (Pythiosis) and infected treated (Pythiosis + PVAC) mice (n = 3) showed that PitiumVac® reinforces the Th1/Th17 response displayed by untreated mice. The (1,3)(1,6)-ß-glucan content can be, at least in part, related to this Th1/Th17 response.

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Susceptibility testing is essential to inform the correct management of Aspergillus infections. In this study we present antifungal susceptibility profile of A. fumigatus isolates recovered from lungs of birds with and without aspergillosis. Fifty three isolates were tested for their antifungal susceptibility to voriconazole (VRC), itraconazole (ITZ), amphotericin (AMB) and caspofungin (CSP) using the M38-A2 broth microdilution reference method. Five isolates were resistant to more than one antifungal drug (CSP + AMB, VRC + ITZ and AMB + ITZ). Fifteen (28%) isolates with susceptible increased exposure (I) to ITZ were sensible to VRC. Resistance to AMB (>2µg/mL) was observed in only four isolates. Eleven (21%) A. fumigatus present resistance to ITZ (13%) and VRC (8%). Fungal isolation from respiratory samples has been regarded as being of limited usefulness in the ante mortem diagnosis of aspergillosis in birds. However, the results suggest that the detection and antifungal susceptibility profile may be helpful for monitoring of therapy for avian species and where antifungal resistance might be emerging and what conditions are associated to the event.(AU)

Os testes de suscetibilidade são essenciais para informar o correto manejo das infecções por Aspergillus. Neste estudo apresentamos o perfil antifúngico de isolados de A. fumigatus provenientes de pulmões de aves com e sem aspergilose. Cinqüenta e três isolados foram testados quanto à susceptibilidade antifúngica ao voriconazol (VRC), itraconazol (ITZ), anfotericina B (AMB) e caspofungina (CSP) pelo método de referência de microdiluição do caldo M38-A2. Cinco isolados foram resistentes a mais de um antifúngico (CSP + AMB, VRC + ITZ e AMB + ITZ). Quinze (28%) isolados suscetíveis - com exposição aumentada (I) ao ITZ foram sensíveis ao VRC. A resistência ao AMB (>2µg/mL) foi observada em apenas quatro isolados. Onze (21%) A. fumigatus apresentaram resistência a ITZ (13%) e VRC (8%). O isolamento de fungos de amostras respiratórias tem sido considerado de utilidade limitada no diagnóstico ante mortem de aspergilose em aves. No entanto, os resultados sugerem que a detecção e o perfil de suscetibilidade a antifúngicos podem ser úteis para o monitoramento da terapia de espécies aviárias, assim como a emergência da resistência antifúngica e quais condições podem estar associadas ao evento.(AU)

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Susceptibility testing is essential to inform the correct management of Aspergillus infections. In this study we present antifungal susceptibility profile of A. fumigatus isolates recovered from lungs of birds with and without aspergillosis. Fifty three isolates were tested for their antifungal susceptibility to voriconazole (VRC), itraconazole (ITZ), amphotericin (AMB) and caspofungin (CSP) using the M38-A2 broth microdilution reference method. Five isolates were resistant to more than one antifungal drug (CSP + AMB, VRC + ITZ and AMB + ITZ). Fifteen (28%) isolates with susceptible increased exposure (I) to ITZ were sensible to VRC. Resistance to AMB (>2µg/mL) was observed in only four isolates. Eleven (21%) A. fumigatus present resistance to ITZ (13%) and VRC (8%). Fungal isolation from respiratory samples has been regarded as being of limited usefulness in the ante mortem diagnosis of aspergillosis in birds. However, the results suggest that the detection and antifungal susceptibility profile may be helpful for monitoring of therapy for avian species and where antifungal resistance might be emerging and what conditions are associated to the event.(AU)

Os testes de suscetibilidade são essenciais para informar o correto manejo das infecções por Aspergillus. Neste estudo apresentamos o perfil antifúngico de isolados de A. fumigatus provenientes de pulmões de aves com e sem aspergilose. Cinqüenta e três isolados foram testados quanto à susceptibilidade antifúngica ao voriconazol (VRC), itraconazol (ITZ), anfotericina B (AMB) e caspofungina (CSP) pelo método de referência de microdiluição do caldo M38-A2. Cinco isolados foram resistentes a mais de um antifúngico (CSP + AMB, VRC + ITZ e AMB + ITZ). Quinze (28%) isolados suscetíveis - com exposição aumentada (I) ao ITZ foram sensíveis ao VRC. A resistência ao AMB (>2µg/mL) foi observada em apenas quatro isolados. Onze (21%) A. fumigatus apresentaram resistência a ITZ (13%) e VRC (8%). O isolamento de fungos de amostras respiratórias tem sido considerado de utilidade limitada no diagnóstico ante mortem de aspergilose em aves. No entanto, os resultados sugerem que a detecção e o perfil de suscetibilidade a antifúngicos podem ser úteis para o monitoramento da terapia de espécies aviárias, assim como a emergência da resistência antifúngica e quais condições podem estar associadas ao evento.(AU)

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The aim of this study was to evaluate the effect of spray-dried porcine plasma (SDPP) supplementation on cholinesterase enzymes and its relationship with animal behavior of weaning piglets exposed to mycotoxin contaminated diets. To achieve these objectives, two experimental design approaches were used. Male piglets (7.15±0.61kg) were allocated in four groups: CTL group received a regular diet; SDPP group received a regular diet and 6% SDPP; MYC group received a diet containing desired contamination of 210 µg/kg aflatoxins and 6.690 µg/kg fumonisins; group MYC+SDPP received 253 µg/kg aflatoxins, 6930 µg/kg fumonisins and 6% SDPP. The animals treated with mycotoxin co-contaminated diets showed an increase in AChE and BChE activities in peripheral system (MYC) when compared to control (CTL). Furthermore, supplementation with SDPP (MYC+SDPP group) prevented the mycotoxin-related reduction of AChE in blood and brain. Behavioral tests showed that sleeping and resting behaviors were more often observed in the MYC group; this group also fed fewer times when compared to the other groups, characterizing the deleterious effect of mycotoxins. Taken together, the data suggest changes in AChE and BChE activities may indicate alterations in cholinergic neurotransmission and consequently in the behavior of piglets.

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We tested 25 isolates of Pythium insidiosum to investigate their susceptibility to antibacterial drugs that act through inhibition of protein synthesis or other mechanisms of action. We observed that tetracycline, erythromycin, linezolid, nitrofurantoin, Synercid (quinupristin and dalfopristin), chloramphenicol, clindamycin, cetrimide, and crystal violet had inhibitory activity against P. insidiosum. Those in vitro results suggest that antibacterials that inhibit protein synthesis should be the primary antimicrobials investigated for the treatment of pythiosis in animals and humans.

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Cryptococcus species are an encapsulated fungal pathogen that cause cryptococcal meningitis. There are limited therapeutic options for this infection. The management includes the use of different antifungals such as amphotericin B, flucytosine, or fluconazole, either alone or in combination. However, numerous therapeutic failures, as well as the limited effectiveness of such therapeutics, have been described. Diphenyl diselenide is a chemically synthesised molecule with was found to have antimicrobial activity. In this study, we evaluated the antifungal activities of fluconazole, amphotericin B and flucytosine, in combination with diphenyl diselenide against 30 clinical isolates of Cryptococcus spp. using CLSI M27-A3 method and the checkerboard microdilution technique. Our results show that the combination of flucytosine and diphenyl diselenide displayed 100% of synergism. However, when we analysed (PhSe)2 plus AMB or FLZ we observed around 70% of indifference. Our results suggest that the combination of diphenyl diselenide with other antifungal agents deserves attention as a new option for the development of alternative therapies for cryptococcosis.

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We have determined the in vitro activity of antifungal, antibacterial, and antiprotozoal drugs alone and in combination against seven Conidiobolus lamprauges clinical isolates. The assays were based on the M38-A2 protocol and the checkerboard microdilution method. The lowest inhibitory concentrations were observed for amphotericin B, miconazole (MCZ), terbinafine, and miltefosine (MTF) (MIC range 0.25-1; 2-8; 0.25-2; 2-16 µg/ml, respectively). The main synergism observed was through the combination of azithromycin (AZI)+MTF and dapsone (DAP)+MTF (100%), AZI+DAP (85.7%), AZI+MCZ (57.1%) as well as MCZ plus CTX and DAP (42.9%). The in vitro activities suggest that the combination of MTF and AZI or DAP are promising candidate therapies for conidiobolomycosis.

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The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions.

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We evaluated the efficacy of azithromycin (50 mg/kg, every 12 h [q12h] orally) and miltefosine (25 mg/kg, q24h orally) treatments in an experimental model of vascular/disseminated pythiosis in immunosuppressed mice. Azithromycin was the only treatment able to reduce mortality. The histopathological findings showed acute vascular inflammation, pathogen dissemination, necrotizing myositis, neuritis, and arteritis. The results suggest that azithromycin, but not miltefosine, may have clinical relevance in the treatment of vascular/disseminated pythiosis.

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This study was aimed to analyze the effects of spray-dried porcine plasma (SDPP) on the health of post weaning piglets challenged with diets contaminated with aflatoxins and fumonisins. Fifty-six male piglets (7.15 ± 0.61 kg) were allocated in four groups: CTL group received a regular diet; SDPP group received a regular diet and 6% SDPP; MYC group received a diet containing 300 µg/kg aflatoxins and 8,000 µg/kg fumonisins; group MYC+SDPP received 300 µg/kg aflatoxins, 8,000 µg/kg fumonisins and 6% SDPP. The animals that received the experimental diet containing mycotoxins (MYC group) had lower weight gain at the end of the experiment compared to the other treatments. Animals receiving SDPP showed decreased urea levels throughout the experiment (P<0.05). Animals from MYC group presented an increased on reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) levels and decreased catalase activity (P<0.05). In contrast, SDPP prevented the increase of ROS and TBARS and stimulated superoxide dismutase activity (P<0.05). In conclusion, diet contaminated with mycotoxins (group MYC) caused subclinical intoxication in the piglets, as observed by the increase on free radical's production and lipid peroxidation. Conversely, SDPP presented a protective effect, minimizing the effects of oxidative stress caused by aflatoxins and fumonisins ingestion.

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We report a malasseziosis model in immunocompromised Swiss mice. For this model, the mice were immunosuppressed with a combination of cyclophosphamide at 150 mg/kg and hydrocortisone acetate at 250 mg/kg. Two groups were formed according to the site of inoculation. Dermatitis group received an intradermal injection of 5 × 106 cell/mouse at a shaved dorsal region, while the otitis group received the same inoculum in the middle ear. Five animals/group were euthanised at different times, and the skin and ear were histopathologically analysed. During the first euthanasia, which occurred after inoculation, microscopic examination showed that all mice presented budding yeast-like in a tissue sample. The presence of yeasts decreased over time being undetected on the 17th day (dermatitis group) and the 21st day (otitis group) after inoculation. This is the first murine model for malasseziosis that can be useful for evaluating new treatment approaches.

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Creatine kinase (CK) activity, through the creatine-kinase-phosphocreatine (CK/PCr) system, provides a temporal and spatial energy buffer to maintain cellular energetic homeostasis, being responsible to provide adenosine triphosphate (ATP) to the proper function of ATPases enzymes, such as the sodium-potassium (Na+, K+-ATPase) and hydrogen (H+-ATPase) pumps. Thus, the aim of this study was to evaluate the involvement of CK/PCr system in the impairment of energetic homeostasis in piglets fed with a diet co-contaminated with mycotoxins, as well as the effects on ATPases enzymes. Animals were randomly divided in two groups (eight repetitions with two animals each): CON (basal diet) and MYC (mycotoxin diet; 9300 µg/kg of aflatoxins and 8000 µg/kg of fumonisins) which were feed during 15 days. Piglets that received a diet containing 300 µg/kg of aflatoxins and 8000 µg/kg of fumonisins (MYC group) presented lower body weight on days 10 and 15 of experiment when compared to control (CON group). Serum CK activity was lower on days 5, 10 and 15 of experiment in the MYC group. The same occurred for serum Na+, K+-ATPase and H+-ATPase activities on days 10 and 15 when compared to CON group. Moreover, serum calcium levels were superior on day 15 of experiment in the MYC group, while serum potassium and sodium levels were lower in comparison to CON group. Based on these evidences, a diet co-contaminated by aflatoxins and fumonisins inhibits serum CK activity, impairing the energetic homeostasis. This inhibition alters the activities of ATPases (Na+, K+-ATPase and H+-ATPase), contributing to the imbalance of Na+, K+ and Ca+ ionic levels. In summary, the cascade of alterations contributes directly to disease pathogenesis of piglets intoxicated by mycotoxins.

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Pythiosis is a life-threatening disease caused by the fungus-like microorganism Pythium insidiosum that can lead to death if not treated. Since P. insidiosum has particular cell wall characteristics, pythiosis is difficult to treat, as it does not respond well to traditional antifungal drugs. In our study, we investigated a new immunotherapeutic approach with potential use in treatment and in the acquisition of immunity against pythiosis. Dendritic cells from both human and mouse, pulsed with P. insidiosum heat-inactivated zoospore, (1,3)(1,6)-ß-glucan and the immunotherapeutic PitiumVac® efficiently induced naïve T cell differentiation in a Th1 phenotype by the activation of specific Th1 cytokine production in vitro. Heat-inactivated zoospores showed the greatest Th1 response among the tested groups, with a significant increase in IL-6 and IFN-γ production in human cells. In mice cells, we also observed a Th17 pathway induction, with an increase on the IL-17A levels in lymphocytes cultured with ß-glucan pulsed DCs. These results suggest a potential use of DCs pulsed with P. insidiosum antigens as a new therapeutic strategy in the treatment and acquisition of immunity against pythiosis.

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