Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao

Oxalic acid has been shown as a virulence factor for some phytopathogenic fungi, removing calcium from pectin and favoring plant cell wall degradation. Recently, it was published that calcium oxalate accumulates in infected cacao tissues during the progression of Witches’ Broom disease (WBD). In the present work we report that the hemibiotrophic basidiomycete Moniliophthora perniciosa, the causal agent of WBD, produces calcium oxalate crystals. These crystals were initially observed by polarized light microscopy of hyphae growing on a glass slide, apparently being secreted from the cells. The analysis was refined by Scanning electron microscopy and the compositon of the crystals was confirmed by energy-dispersive x-ray spectrometry. The production of oxalate by M. perniciosa was reinforced by the identification of a putative gene coding for oxaloacetate acetylhydrolase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate. This gene was shown to be expressed in the biotrophic-like mycelia, which in planta occupy the intercellular middle-lamella space, a region filled with pectin. Taken together, our results suggest that oxalate production by M. perniciosa may play a role in the WBD pathogenesis mechanism.     

Specific In Situ Visualization of the Pathogenic Endophytic Fungus Aciculosporium take,the Cause of Witches’ Broom in Bamboo

The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth in bamboo. The colonized shoot eventually results in witches’ broom formation but maintains normal leaf arrangement and branching pattern. To analyze the mechanism of well-regulated symptom development, the location of the fungal endophytic hyphae in host tissues was visualized. A colorimetric in situ hybridization technique using a species-specific oligonucleotide probe targeting the 18S rRNA of A. take was used. In situ hybridization was performed on tissue sections of diseased shoots with or without external signs of fungal colonization. Specific signals were detected in intercellular spaces of the bamboo tissues. Most signals were detected in the shoot apical meristem and the leaf primordia. In addition, fewer signals were detected in the lateral buds, juvenile leaves, and stems. These results indicate that A. take grows endophytically, particularly in the shoot apical meristem of the host. The location of A. take hyphae suggests that the mechanism of symptom development can be explained by the action of exogenous fungal auxin, which continuously induces primordium initiation within the host.The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth, but the host bamboo plant maintains normal leaf arrangement and branching pattern (28, 29). This fungus produces the plant hormone auxin, indole-3-acetic acid (IAA) in culture (28). Hypersynthesis of auxins by pathogenic microorganisms has often been associated with diseases in which hyperplasia is exhibited by the host plant (31). However, the well-regulated morphological changes of the A. take-colonized bamboo cannot be explained by a mere hormonal imbalance. The symptom of A. take-colonized bamboo is characterized by the successive growth of thin phytomers (developmental modules consisting of a node, a stem segment, a leaf with a sheath, and a lateral bud) from a normal shoot apex (Fig. 1a and b). Diseased shoots continue to grow without branching and have extraordinarily small leaves. Sometimes, a diseased shoot develops 30 to 40 leaves, whereas healthy shoots develop only 3 to 5 leaves (25). When the conidiostroma is formed at the apex, the shoots cease to grow, and lateral buds initiate outgrowth (Fig. ​(Fig.1c).1c). This process occurs repeatedly and eventually results in a typical witches’ broom morphology after a few years (Fig. 1d and e). Excluding stroma formation, the fungus presents no external signs of its presence on the diseased shoots. Accurate localization of the growing hyphae would aid in establishing the process of symptom development.Open in a separate windowFIG. 1.Bamboo shoot colonized by the endophytic fungus Aciculosporium take. (a to c) Schematic of witches’ broom symptom development on a bamboo species of Phyllostachys. (a) The normal shoot ceased to grow after three to five leaves developed. (b) An A. take-colonized shoot grew out with extraordinarily small leaves but maintained normal leaf arrangement. The shoot continued to grow with successive thin phytomers. (c) After stromata (arrows) were formed at the shoot apex, the lateral buds began to grow but maintained a normal branching pattern. This sequence repeats. (d to f) Bamboo (P. pubescens) shoot colonized by A. take. (d) Early symptom. Diseased shoots (DS) have leaves that are much smaller than normal leaves (NL). Bar, 1 cm. (e) Developed symptom shows typical witches’ broom appearance. Bar, 1 cm. (f) Ascostroma (AS) is formed on a conidiostroma (CS) at the apex of the diseased shoot. The conidiostroma is surrounded by a sheath. Bar, 1 mm.Many fungal endophyte species have been reported for bamboos (19), and therefore, species-specific detection techniques are needed to observe A. take in host tissues. A number of staining techniques utilizing lectins and other chemical compounds have been used to observe naturally growing hyphae in host tissues (13). However, these approaches cannot be used to distinguish particular species. Therefore, such staining techniques using lectins and other chemical compounds are effective to differentiate fungi possessing particular morphological characteristics, for example, arbuscules, appressoria, or haustoria (33). Unfortunately, fungal endophytes are difficult to identify in host tissues, as discussed previously by Schulz and Boyle (24), partly because the morphologies of endophytic hyphae are usually different from those of cultured hyphae. To differentiate specific fungal species from other fungi, green fluorescent protein or related reporter protein transformants can be used (18, 36). This approach requires the development of genetic modification techniques for individual fungal species. In addition, this method cannot be applied to natural samples because it is necessary to inoculate host plants with fungal transformants. Although immunostaining techniques are also an effective approach (7, 26), they require complicated preparations for generating individual species-specific antibodies.In situ hybridization (ISH) with a species-specific probe is a potentially powerful technique for directly detecting endophytic fungi in natural samples. Species-specific rRNA-targeted oligonucleotide probes have been developed to detect and identify microorganisms in the environment (1, 6). ISH has also been used to detect fungal pathogens in animal tissues (10, 12, 14, 17). Despite these uses, only a few studies have applied this technique for the detection of fungal endophytes or epiphytes (16, 20). This is partly because the detection of fluorescence-labeled probes was hampered by the strong autofluorescence from the plant cell walls (23). However, Pirttilä et al. (20) succeeded in detecting fungal endophytes in meristematic tissues of Scots pine buds by the colorimetric ISH technique. In this study, ISH methodology using a 18S rRNA-targeted oligonucleotide probe was applied to visualize the location of the endophytic fungus A. take in bamboo with witches’ broom disease.     

A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches’ broom disease in cacao

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches’ broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.     

The Activity of TcCYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa,Caused by the Fungus Moniliophthora perniciosa

The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.     

Inhibitory Role of β-Casein on the α-Synuclein Aggregation Associated with Parkinson’s Disease In Vitro

Large soluble oligomeric species are observed as probable intermediates during fibril formation in aggregations of Parkinson’s disease (PD). Fibrillar deposits occur in PD. Amyloid forms α-Synuclein is one of the main compounds aggregations. β-Casein, a member of the Casein family, has been demonstrated to inhibit α-Synuclein aggregation by chaperone-like activity. In this study, we investigated the effect of chaperone activity of β-Casein in preventing the aggregation of α-Synuclein protein. We have examined the effect of β-Casein in preventing α-Synuclein aggregation by using from Thioflavin T-binding assay, transmission electron microscopy, ANS-binding assay, circular dichroism spectroscopy and FTIR spectroscopy. Results from the ThT binding assay demonstrated an increase in the ThT fluorescence intensity of α-Synuclein incubated in absence of β-Casein but in its presence fluorescence intensity is decreased. Electron microscopy data also indicated that β-Casein decreased the aggregation content of α-Synuclein. ANS results also showed that β-Casein significantly decreased the the hydrophobic area in α-Synuclein incubated. Circular dichroism spectroscopy (CD) results also showed that β-sheet structures of α-Synuclein incubated change to structural α-helical and β-turn in presence of β-Casein. FTIR spectroscopy indicates the presence of β-sheet structures in α-Synuclein incubated in absence of β-Casein and β-sheet structures decreased in its presence. Thus, our results suggest that in vitro, β-Casein interacts with α-Synuclein fibrils, changes the α-Synuclein structure and prevents amyloid fibril formation. This means that β-Casein could be essential for therapies inhibiting aggregation and to be an important therapeutic drug against PD.     

Discovery of Dihydrochalcone as Potential Lead for Alzheimer’s Disease: In Silico and In Vitro Study

By the virtual screening method we have screened out Dihydrochalcone as a top-lead for the Alzheimer’s disease using the database of about 32364 natural compounds. The binding affinity of this ligand to amyloid beta (A) fibril has been thoroughly studied by computer simulation and experiment. Using the Thioflavin T (ThT) assay we have obtained the inhibition constant IC50 M. This result is in good agreement with the estimation of the binding free energy obtained by the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulation with the force field CHARMM 27 and water model TIP3P. Cell viability assays indicated that Dihydrochalcone could effectively reduce the cytotoxicity induced by A. Thus, both in silico and in vitro studies show that Dihydrochalcone is a potential drug for the Alzheimers disease.     

Ligustrazine Phosphate Ethosomes for Treatment of Alzheimer’s Disease, In Vitro and in Animal Model Studies

In the present study, we have investigated transdermal administration of ligustrazine phosphate (LP), as an antioxidant, for the treatment of Alzheimer's disease (AD). The LP transdermal ethosomal system was designed and characterized. Franz-type diffusion cells and confocal laser scanning microscopy were used for the in vitro permeation studies. Furthermore, the effect of LP transdermal ethosomal system on AD was evaluated in the scopolamine-induced amnesia rats by evaluating the behavioral performance in the Morris water maze test. The activities of the antioxidant enzymes and the levels of the lipid peroxidation product malondialdehyde (MDA) in the brain of rats were also determined. The results showed that both the penetration ability and the drug deposition in skin of the LP ethosomal system were significantly higher than the aqueous one. The LP transdermal ethosomal system could recover the activities of the antioxidant enzymes and the levels of MDA in the brain of the amnesic rats to the similar status of the normal rats, which was also indirectly reflected by the improvement in the behavioral performance. In conclusion, LP might offer a potential alternative therapeutic drug in the fight against AD, and ethosomes could be vesicles of choice for transdermal delivery of LP.     

Moniliophthora perniciosa,the mushroom causing witches’ broom disease of cacao: Insights into its taxonomy,ecology and host range in Brazil

Witches' broom caused by Moniliophthora perniciosa is the main disease of cacao (Theobroma cacao) in Brazil. The fungus is known to occur on other host families and these populations have been addressed in the literature as biotypes: C (Malvaceae); H (Malpighiaceae); L (Bignoniaceae) and S (Solanaceae). No complete elucidation of the phylogenetic relationships of isolates obtained from this disparate host range appears in the literature. One member of H (ex Heteropterys acutifolia) has been described as a distinct species. But should other biotypes be also recognized as distinct taxa? In the present study, a survey yielding 24 isolates of M. perniciosa from ten hosts and covering a wide range of geographic regions in Brazil was undertaken. These isolates were compared with those from T. cacao using three DNA regions for the phylogenetic analyses: ITS, LSU and RPB1. Morphology was also examined. All isolates in this study were found to belong to M. perniciosa, including the population from H. acutifolia, formerly treated as Moniliophthora brasiliensis but reduced here to a synonym of M. perniciosa. This species ranged from pathogenic to a previously unreported occurrence as a non-pathogenic endophyte in the Atlantic rainforest tree Allophylus edulis (Sapindaceae). M. perniciosa was recorded on a range of solanaceous hosts (16 species) over a wide variety of ecosystems. The ecological and evolutionary significance of these novel findings are discussed.     

The Expression and Release of Hsp60 in 6-OHDA Induced In Vivo and In Vitro Models of Parkinson’s Disease

In Parkinson’s disease, dopaminergic neuron damage/death causes the release of soluble substances that are selectively toxic to neighboring/additional dopaminergic neurons through the activation of microglia. Hsp60 can be released from injured cells of central nervous system to activate microglia. However, its expression and role in Parkinson’s disease has not been well understood. Here, we performed a 6-OHDA treated Parkinson’s disease model in adult rats. Western blot analysis showed a time-course expression of Hsp60, which decreased gradually and then rose back. Immunofluorescence staining showed that Hsp60 was decreased in dopaminergic neuron, and most Hsp60 located on the surface of activated microglia. Furthermore, in cellular Parkinson’s disease model, Hsp60 was obviously detected in the culture supernatants after 6-OHDA treatment, and a concomitant decrease in cell extracts. Taken together, our results suggested that Hsp60 could be released extracellularly to activate microglia in Parkinson’s disease model.     

Geraniol Protects Against the Protein and Oxidative Stress Induced by Rotenone in an In Vitro Model of Parkinson’s Disease

Dysfunction of autophagy, mitochondrial dynamics and endoplasmic reticulum (ER) stress are currently considered as major contributing factors in the pathogenesis of Parkinson’s disease (PD). Accumulation of oxidatively damaged cytoplasmic organelles and unfolded proteins in the lumen of the ER causes ER stress and it is associated with dopaminergic cell death in PD. Rotenone is a pesticide that selectively kills dopaminergic neurons by a variety of mechanism, has been implicated in PD. Geraniol (GE; 3,7-dimethylocta-trans-2,6-dien-1-ol) is an acyclic monoterpene alcohol occurring in the essential oils of several aromatic plants. In this study, we investigated the protective effect of GE on rotenone-induced mitochondrial dysfunction dependent oxidative stress leads to cell death in SK-N-SH cells. In addition, we assessed the involvement of GE on rotenone-induced dysfunction in autophagy machinery via α-synuclein accumulation induced ER stress. We found that pre-treatment of GE enhanced cell viability, ameliorated intracellular redox, preserved mitochondrial membrane potential and improves the level of mitochondrial complex-1 in rotenone treated SK-N-SH cells. Furthermore, GE diminishes autophagy flux by reduced autophagy markers, and decreases ER stress by reducing α-synuclein expression in SK-N-SH cells. Our results demonstrate that GE possess its neuroprotective effect via reduced rotenone-induced oxidative stress by enhanced antioxidant status and maintain mitochondrial function. Furthermore, GE reduced ER stress and improved autophagy flux in the neuroblastomal SK-N-SH cells. The present study could suggest that GE a novel therapeutic avenue for clinical intervention in neurodegenerative diseases especially for PD.     

The Chemical Molecule B355252 is Neuroprotective in an In Vitro Model of Parkinson’s Disease

6-Hydroxydopamine (6-OHDA) is a neurotoxin frequently used to create in vitro and in vivo experimental models of Parkinson’s disease (PD), a chronic neurodegenerative disorder largely resulting from damage to the nigrostriatal dopaminergic pathway. No effective drugs or therapies have been developed for this devastating disorder, and current regimens of symptomatic therapeutics only alleviate symptoms temporarily. Therefore, effective treatments that reverse or cure this disorder are urgently needed. The aim of the study described in this report was to investigate the therapeutic impact of B355252, an aryl thiophene sulfonamide chemical entity, in the widely recognized in vitro model of PD, and to characterize the molecular signaling pathways. We show here that 6-OHDA-induced cell death in HT22, a murine neuronal cell model, through a pathway that involves the mitochondria by increasing the levels of reactive oxygen species (ROS), raising intracellular calcium ([Ca²⁺]i), enhancing the release of cytochrome c to the cytosol, and promoting activation of stress-activated protein kinase/c-Jun NH₂-terminal kinase (SAPK/JNK) signaling pathway. More importantly, we found that B355252 protected HT22 neurons against 6-OHDA toxin-induced neuronal cell death by significant attenuation of ROS production, blocking of mitochondrial depolarization, inhibition of cytochrome c release, sequestration of [Ca²⁺]i, modulation of JNK cascade, and strong inhibition of caspase 3/7 cleavage. Overall, this study demonstrates that death of neurons under toxic conditions characteristic of PD can be efficiently halted by B355252 and suggests that further development of the molecule could be potentially beneficial as a therapeutic prevention or treatment option for PD.     

Iron Chelation Inhibits Osteoclastic Differentiation In Vitro and in Tg2576 Mouse Model of Alzheimer’s Disease

Patients of Alzheimer’s disease (AD) frequently have lower bone mineral density and higher rate of hip fracture. Tg2576, a well characterized AD animal model that ubiquitously express Swedish mutant amyloid precursor protein (APPswe), displays not only AD-relevant neuropathology, but also age-dependent bone deficits. However, the underlying mechanisms remain poorly understood. As APP is implicated as a regulator of iron export, and the metal chelation is considered as a potential therapeutic strategy for AD, we examined iron chelation’s effect on the osteoporotic deficit in Tg2576 mice. Remarkably, in vivo treatment with iron chelator, clinoquinol (CQ), increased both trabecular and cortical bone-mass, selectively in Tg2576, but not wild type (WT) mice. Further in vitro studies showed that low concentrations of CQ as well as deferoxamine (DFO), another iron chelator, selectively inhibited osteoclast (OC) differentiation, without an obvious effect on osteoblast (OB) differentiation. Intriguingly, both CQ and DFO’s inhibitory effect on OC was more potent in bone marrow macrophages (BMMs) from Tg2576 mice than that of wild type controls. The reduction of intracellular iron levels in BMMs by CQ was also more dramatic in APPswe-expressing BMMs. Taken together, these results demonstrate a potent inhibition on OC formation and activation in APPswe-expressing BMMs by iron chelation, and reveal a potential therapeutic value of CQ in treating AD-associated osteoporotic deficits.     

Selection of Aptamers for Amyloid β-Protein,the Causative Agent of Alzheimer's Disease

Alzheimer''s disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult¹. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies^2,3.Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD^4,5. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood⁶. Specific molecular recognition tools may unravel the relationships amongst Aβ assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies^7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)^9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).Despite emergence of aptamers as tools in modern biotechnology and medicine¹¹, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)^12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-β¹⁷, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils¹⁸. Aptamers generated using monomeric and several forms of fibrillar β₂-microglobulin (β₂m) were found to bind fibrils of certain other amyloidogenic proteins besides β₂m fibrils¹⁹. Ylera et al. described RNA aptamers selected against immobilized monomeric Aβ40²⁰. Unexpectedly, these aptamers bound fibrillar Aβ40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Aβ40²¹ generated using photo-induced cross-linking of unmodified proteins (PICUP)^22,23. Similar to previous findings^17,19,20, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope²¹. Here, we present the SELEX methodology used in production of these aptamers²¹.Download video file.^{(175M, mp4)}     

Oxidative Stress in Skin Fibroblasts Cultures of Patients with Huntington’s Disease

Oxidative stress and mitochondrial dysfunction should play a role in the neurodegeneration in Huntington’s disease (HD). The most consistent finding is decreased activity of the mitochondrial complexes II/III and IV of the respiratory chain in the striatum. We assessed enzymatic activities of respiratory chain enzymes and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with HD. We studied respiratory chain enzyme activities, activities of total, Cu/Zn- and Mn-superoxide-dismutase, glutathione-peroxidase (GPx) and catalase, and coenzyme Q₁₀ (CoQ₁₀) levels in skin fibroblasts cultures from 13 HD patients and 13 age- and sex-matched healthy controls. When compared with controls, HD patients showed significantly lower specific activities for catalase corrected by protein concentrations (P < 0.01). Oxidized, reduced and total CoQ₁₀ levels (both corrected by citrate synthase (CS) and protein concentrations), and activities of total, Cu/Zn- and Mn-superoxide-dismutase, and gluthatione-peroxidase, did not differ significantly between HD-patients and control groups. Values for enzyme activities in the HD group did not correlate with age at onset and of the disease and with the CAG triplet repeats. The primary finding of this study was the decreased activity of catalase in HD patients, suggesting a possible contribution of catalase, but not of other enzymes related with oxidative stress, to the pathogenesis of this disease.     

In Vitro Reproduction of the Life Cycle of Pythium insidiosum from Kunkers’ Equine and Their Role in the Epidemiology of Pythiosis

Pythium insidiosum is an important pathogen of mammals’ species, including humans. Equine is the main species affected by this oomycete. P. insidiosum requires an aquatic environment to develop its life cycle, and the susceptible hosts are contaminated when they contact the microorganism in swampy areas. The equine pythiosis is characterized by the formation of irregular masses within the cutaneous lesions, called kunkers, which easily detach from the lesion. From these structures, it is possible to isolate P. insidiosum in pure cultures. The present study aimed to reproduce in vitro the life cycle of P. insidiosum from kunkers of equine clinical lesions. Fifteen kunkers from different horses were tested. It was observed that the discharge of zoospores occurred after 24–48 h of incubation at 37 °C in, respectively, 40 and 47 % of the kunkers evaluated. Only two samples showed no development of the asexual cycle of P. insidiosum under the conditions tested. It was possible to demonstrate that kunkers are able to restart the asexual cycle of P. insidiosum. Based on our in vitro results, we highlight the importance of these structures in the epidemiology of the pythiosis, since kunkers can be a potential source of contamination of this oomycete for aquatic environments.