Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

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.

Immunotherapy based on Pythium insidiosum mycelia drives a Th1/Th17 response in mice.

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.

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

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.

Screening of antibacterial drugs for antimicrobial activity against Pythium insidiosum.

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.

In vitro assessment of antifungal, antibacterial, and antiprotozoal drugs against clinical isolates of Conidiobolus lamprauges.

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.

In vitro combination of antifungal agents against Malassezia pachydermatis.

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.

In vitro combination between antifungals and diphenyl diselenide against Cryptococcus species.

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.

Effects of fed mycotoxin contaminated diets supplemented with spray-dried porcine plasma on cholinergic response and behavior in piglets.

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.

In Vitro Activities of Miltefosine and Antibacterial Agents from the Macrolide, Oxazolidinone, and Pleuromutilin Classes against Pythium insidiosum and Pythium aphanidermatum.

We tested 29 isolates of Pythium insidiosum and one isolate of Pythium aphanidermatum to investigate their susceptibility to miltefosine and antibacterial drugs from the macrolide, oxazolidinone, and pleuromutilin classes. We found that miltefosine, azithromycin, clarithromycin, josamycin, linezolid, sutezolid, retapamulin, tiamulin, and valnemulin had inhibitory and cidal activity against the pathogens at concentrations ranging from 0.25 to 64 µg/ml. Our results suggest that these antimicrobials are promising candidates for future studies on pythiosis in animals and humans.

Creatine kinase and ATPase activities in piglets fed a fungal mycotoxin co-contaminated diet: Consequences in the pathogenesis of subclinical intoxication.

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.

Changes of adenosinergic system in piglets fed a diet co-contaminated by mycotoxin and their effects on the regulation of adenosine.

The effects of diets co-contaminated with 300 µg/kg of aflatoxins and 8000 µg/kg of fumonisins on adenosinergic system of the pigs weaned at 15 days of age were studied. Piglets were inspected daily, and body weight measurement and blood collections were performed at every five days. Piglets intoxicated by mycotoxins presented lower weight gain (p < 0.001) in comparison to control. Intoxicated piglets also showed a reduction in the serum levels of zinc and adenosine and in adenosine deaminase (ADA) activity (p < 0.001). Positive correlations between zinc levels and ADA activity (p < 0.001) and between adenosine levels and ADA activity (p < 0.05) were observed. Ternary plot shows the influence of zinc levels on ADA activity and on adenosine levels, suggesting that low zinc levels, caused by subclinical mycotoxin intoxication, can cause immunomodulatory effects in piglets. We conclude that piglets intoxicated by fumonisins and aflatoxins have low ADA activity and adenosine levels in serum. This can be directly related to zinc reduction, which is a cofactor for ADA. The co-contamination by these mycotoxins in piglet feed impairs growth and immune defenses of the animals, adversely affecting animal health and production. Therefore, changes in the purinergic pathway may affect the pathogenesis of the disease.

An experimental murine model of otitis and dermatitis caused by Malassezia pachydermatis.

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.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection.

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

Spray-dried porcine plasma added to diets contaminated with aflatoxins and fumonisins shows beneficial effects to piglet health.

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.

Effects of supplementation with spray-dried porcine plasma on blood variables on piglets feed with diet contaminated by mycotoxins.

The aim of this study was to evaluate the supplementation spray-dried porcine plasma (SDPP) effects on body weight, glucose levels and hematological and immunology variables for piglets fed with diet contaminated with low or high aflatoxin and fumonisin levels. Thus, 24 piglets were allotted in four groups, and the group A received a control diet; the group B received a diet with 6% of SDPP; the group C 300 µg/kg of aflatoxins and 8000 µg/kg of fumonisin; the group D 300 µg/kg of aflatoxins, 8000 µg/kg of fumonisin and 6% of SDPP, respectively. A significant increase on hemoglobin concentration and hematocrit levels was observed between C and A groups, result similar also in group D to hemoglobin concentration. A significant reduction on leukocytes and monocytes levels was observed in the group C compared to group A. Most important finding was that SDPP prevented reduction on leukocytes, but not in the monocytes levels in animals of group D. Moreover, the animals of group C presented lower weight, in the same way presented lower on glucose levels. In the other hand, animals in the group C had higher levels of nitrate/nitrite (NOx) and C-reactive protein (CRP) compared other groups. Based on these evidences, the present study showed ingestion of diet contaminated with elevated aflatoxins and fumonisins levels may negatively affected the white cell count and the weight, increasing the seric biomarkers related to inflammatory response, and consequently impairs the immune system. Additionally, SDPP supplementation for piglets prevents the reduction on leukocytes levels and the negative effects associated with weight gain, as well as minimizing the inflammatory response, demonstrating that SDPP can modulate the inflammatory processes and consequently may improve the immune system due active proteins presents in SDPP.

Serum and brain purine levels in an experimental systemic infection of mice by Cryptococcus neoformans: Purinergic immunomodulatory effects.

The aim of this study was to evaluate the purine levels in serum and brains of mice experimentally infected by Cryptococcus neoformans. Twenty-four mice were divided into the following groups: a control group (n = 12; Group A) and an infection group with animals that were infected (n = 12; Group B) with a 0.3-mL intraperitoneal injection containing 1.7 × 107C. neoformans cells. Blood and brains were collected on days 20 (n = 6 per group) and 50 (n = 6 per group) post-infection (PI). Histopathology and lung and brain cultures were performed to confirm fungal infection and tissue injuries. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HYPO), xanthine (XAN) and uric acid (UA) in brains and serum were measured by high-performance liquid chromatography (HPLC) analysis. At both time points, histopathology analysis revealed inflammatory infiltrates in the brains and lungs of infected mice; clinical signs, such as piloerection and clinical respiratory distress, were also observed. ATP levels were significantly increased on days 20 and 50 PI (P < 0.01) in brains and serum, while brain ADO levels were increased on day 20 PI; brain and serum ADO levels were decreased on day 50 PI. Levels of ADP and AMP did not differ between groups (P > 0.05). Serum levels of INO of infected mice increased only on day 50 PI (P < 0.05). HYPO levels were reduced in the brains of infected animals at both experimental time points and were decreased in serum at day 50 PI (P < 0.05). XAN levels increased in infected mice only in serum on day 50 PI (P < 0.05). The endogenous anti-oxidant uric acid was significantly increased in brain (days 20 and 50 PI) and decreased in serum. It is possible that C. neoformans infection in mice leads to a high ATP/ADO ratio that may improve the brain pro-inflammatory response during both periods, while high ATP levels in serum act as a systemic signal to improve the immune response. Moreover, the anti-oxidant uric acid may increase in the brain to protect inflamed tissue from oxidative stress.

Aflatoxins produced by Aspergillus parasiticus present in the diet of quails increase the activities of cholinesterase and adenosine deaminase.

The aim of this study was to evaluate the effects of aflatoxins on cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and adenosine deaminase (ADA) activities in quails. For this, twenty male quails were randomly distributed into two groups (n = 10 each): the group A was composed by quails that received feed without aflatoxin (the control group); while the group B was composed by quails that received feed contaminated with 200 ppm/kg of feed of aflatoxin. On day 20, the animals were euthanized to measure the activities of AChE (total blood and brain), BChE (serum) and ADA (serum, liver, and brain), as well as for histopathological analyses (liver and intestine). AChE, BChE, and ADA levels increased in animals intoxicated by aflatoxin compared to the control group. The presence of aflatoxin lead to severe hydropic degeneration of hepatocytes and small focus of hepatocyte necrosis. In conclusion, aflatoxins poisoning increased AChE, BChE, and ADA activities, suggesting the involvement of these enzymes during this type of intoxication, in addition to the fact that they are well known molecules that participate in physiological and pathological events as inflammatory mediators. In summary, increased AChE, BChE and ADA activities contribute directly to the inflammatory process and tissue damage, and they might be involved in disease development.

In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum.

We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 µg∕ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.

In Vitro and In Vivo Antimicrobial Activities of Minocycline in Combination with Azithromycin, Clarithromycin, or Tigecycline against Pythium insidiosum.

The present study investigated the in vitro and the in vivo interactions among azithromycin, clarithromycin, minocycline, and tigecycline against Pythium insidiosum. In vitro antimicrobial activities were determined by the broth microdilution method in accordance with CLSI document M38-A2, and the antibiotic interactions were assayed using the checkerboard MIC format. In vivo efficacy was determined using a rabbit infection model. The geometric mean MICs of azithromycin, clarithromycin, minocycline, and tigecycline against P. insidiosum were, respectively, 1.91, 1.38, 0.91, and 0.79 µg/ml. By checkerboard testing, all combinations resulted in in vitro synergistic interactions (>60%). Antagonism was not observed. The in vivo studies showed that azithromycin (20 mg/kg/day twice daily) alone or in combination with minocycline (10 mg/kg/day twice daily) significantly decreased the fungal burden. This study demonstrates that azithromycin possesses potent curative efficacy against subcutaneous pythiosis in the rabbit model.

Participation of purines in the modulation of inflammatory response in rats experimentally infected by Cryptococcus neoformans.

The present study was carried out to assess the participation of purines in the activation and modulation of inflammatory response of rats experimentally infected by Cryptococcus neoformans. Twenty four Wistar rats were divided into two groups of 12 animals each: Group A - uninfected control group and Group B - infected by C. neoformans. Blood was collected 20 and 50 days post-infection (PI) from six animals of each group in order to verify purine levels (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HYPO), xanthine (XAN) and uric acid (URIC)). ATP levels were significantly increased (P < 0.05) in serum from infected animals on days 20 and 50 PI, as well as adenosine levels after 20 days PI on rats. On day 50 PI, levels of adenosine and uric acid were also reduced, but the levels of inosine and xanthine increased in animals infected by the fungus (P < 0.05). Therefore, it was possible to conclude that the purine levels in serum were altered and that these changes may be able to influence the pathogenesis of the disease caused by C. neoformans due the participation of purines (ATP and adenosine main) in the activation and modulation of inflammatory response.