Equidae pythiosis in Brazil and the world: a systematic review of the last 63 years (1960-2023).

This systematic review compiles reports of clinical pythiosis in horses, mules and donkeys from 1960 to 2023 worldwide, focusing on Brazil. We searched databases and included 71 articles detailing clinical characteristics, geographic distribution, epidemiology, diagnostic methods, therapies, and outcomes. The results showed that publications on equine pythiosis have significantly increased since 2010. Brazil reported the highest incidence, comprising 55% of cases, predominantly in the southern, northeastern, and central-western regions during summer and autumn. Cutaneous pythiosis was the most prevalent form, generally presenting as single lesions in the appendicular region, and affected females more than males. Diagnosis typically involved histopathology, used alone or with other methods. Various treatments have been employed, with surgery, often combined with chemotherapy and immunotherapy, being the most common. Notably, 80.84% of treated animals recovered, highlighting the effectiveness of these therapies in enhancing survival rates. The limitations of the study included the lack of data in published case reports, which made it difficult to collect and calculate epidemiological data. Additionally, we recognize that pythiosis in Brazil is underreported, since this disease does not have mandatory notification and several cases are not registered and/or reported in the literature. Lastly, it is hypothesized that equid pythiosis may be more widespread than currently known, and its real occurrence in Brazil remains uncertain.

In vitro and ex vivo anti-Pythium insidiosum potential of ozonated sunflower oil.

This study sought to evaluate the in vitro and ex vivo susceptibility of Pythium insidiosum to ozonized sunflower oil (OSO) and verify the morphological alterations of OSO-exposed hyphae. Susceptibility assays were performed according to the broth microdilution protocol M38-A2/CLSI, and the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Non-ozonated sunflower oil (SO) was used as the oil control. Additionally, kunkers from equine pythiosis were exposed to OSO. Damages caused by OSO and SO on P. insidiosum hyphae ultrastructure were verified using scanning electron microscopy. The MIC range for OSO was 7000 to 437.5 mg/mL, and the values for SO were higher, ranging from 56000 to 14000 mg/mL. The MOC was equal to MIC for both oil formulations. The OSO fully inhibited the oomycete growth from kunkers, although there was P. insidiosum growth in the kunker control in 24 h of incubation. The SEM analyses showed that both OSO and SO caused morphological alterations in P. insidiosum hyphae, highlighting the presence of cavitation along the hyphae with loss of continuity of the cell wall, which was more evident in the OSO-treated hyphae. The OSO had the best oomicidal activity, leading us to believe that our findings may support future research containing this formulation to be applied in integrative medicine protocols to control pythiosis in animals and humans.

Melaleuca alternifolia formulations in the treatment of experimental pythiosis.

Essential oils (EO) are aromatic compounds from the plant secondary metabolism. Melaleuca alternifolia EO is well known for its medicinal properties and promising use as an antimicrobial agent. Pythiosis is a difficult-to-treat and emerging disease caused by the oomycete Pythium insidiosum. This study evaluated a nanoemulsion formulation of M. alternifolia (NEMA) in topical and intralesional application to treat experimental pythiosis. Dermal toxicity tests were performed on M. alternifolia EO in Wistar rats. Pythiosis was reproduced in rabbits (n = 9) that were divided into groups: group 1 (control), cutaneous lesions with daily topical application of a non-ionizable gel-based formulation and intralesional application of sterile distilled water every 48 h; group 2 (topical formulation), lesions treated daily with topical application of a non-ionizable gel-based formulation containing 5 mg/ml of NEMA; and group 3 (intralesional formulation), lesions treated with NEMA at 5 mg/ml in aqueous solution applied intralesionally/48 h. The animals were treated for 45 days, and the subcutaneous lesion areas were measured every 5 days. M. alternifolia EO showed no dermal toxicity. The lesion areas treated with intralesional NEMA reduced at the end of treatment, differing from groups 1 and 2 (P < 0.05). In the topically treated group, the lesion areas did not differ from the control group, although the number of hyphae significantly reduced (P < 0.05). Under the experimental conditions of this study, the NEMA formulations presented a favorable safety profile. However, further studies are required to evaluate if this safety applies to higher concentrations of NEMA and to validate its use in clinical pythiosis.

Biogenic silver nanoparticles in the treatment of experimental pythiosis Bio-AgNP in pythiosis therapy.

Pythiosis is a rapidly progressing disease that can be lethal to affected individuals due to resistance to available therapeutic protocols. The disease affects mammals, with the largest number of reports in horses and humans. The present study investigated the activity of biogenic silver nanoparticles (bioAgNP) in the treatment of experimental pythiosis. The disease was reproduced in nine female 90-day-old New Zealand rabbits. Animals were divided into three groups: group1 (control, n = 3) daily and topically treated with a nonionized gel-based formulation and 1 ml of sterile distilled water intralesion administered every 48 hours; group 2 (n = 3), daily and topically treated with gel-based formulation containing 1 µg/ml bio-AgNP; group 3 (n = 3), treated with 1 ml bio-AgNP in 1 µg/ml aqueous solution intralesion administered every 48 hours. Animals were treated for 45 days, and the area of subcutaneous lesions was measured every 5 days. Results showed that groups 2 and 3 differed from control group (P < .05) in the lesion area, as well as the amount of hyphae within the lesions. It was observed that lesions of treated animals (groups 2 and 3) did not differ from each other, showing that the application route did not influence the regression of lesions. However, it was observed that one animal from group 2 reached clinical cure at 35 days of treatment. This research is pioneer in the application of nanocomposites for the treatment of experimental pythiosis and showed that bio-AgNP can be powerful allies of integrative medicine and can be included in pythiosis therapeutic protocols.

Protein profile of Brazilian Pythium insidiosum isolates.

Pythium insidiosum is an important aquatic Oomycota that causes pythiosis in mammals, especially horses, dogs, and humans; these inhabit marshy environments in tropical and subtropical areas. The aim of this study was to determine the protein profile, as well as identify likely immunodominant proteins, of Brazilian P. insidiosum isolates from southern Brazil, an important equine pythiosis endemic area. P. insidiosum isolates (horses, n = 20 and dogs, n = 02) were analyzed by SDS-PAGE and Western blot techniques. Horse, cattle, dog, and rabbit sera of both diseased and healthy animals were used to identify P. insidiosum proteins. SDS-PAGE protein profile detected antigens of molecular weights ranging from 100 to 20 KDa. Dog isolates revealed a protein profile similar to that of horse isolates. Anti-P. insidiosum antibodies in the sera of the four species could recognize proteins of different molecular weights (∼74 KDa to ∼24 KDa), and proteins ∼50-55 KDa and ∼34 KDa were shown to be immunodominant. Furthermore, ∼74 KDa, ∼60 KDa, ∼30 KDa and ∼24 KDa proteins were poorly recognized by host species antibodies. The Brazilian P. insidiosum isolates analyzed showed a similar protein profile; however, further studies are essential for the identification and characterization of proteins expressed by P. insidiosum, and an evaluation of the immunological profile of hosts susceptible to this Oomycota is necessary.

Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum.

This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism.

In Vitro Susceptibility of Pythium insidiosum to Melaleuca alternifolia, Mentha piperita and Origanum vulgare Essential Oils Combinations.

Pythium insidiosum is the etiologic agent of pythiosis, a severe and emerging disease that affects mammals. Failure of conventional antifungal therapies is partially justified by the absence of ergosterol in the plasma membrane of this oomycete. Despite research advancement, the treatment of pythiosis has not been not fully established. The present study investigated the in vitro susceptibility profile of Brazilian isolates of P. insidiosum (n = 20) against Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils, and their combinations. Susceptibility tests were performed according to CLSI M38-A2 protocol, and combinations were evaluated by the microdilution cherkerboard method. All tested essential oils showed antimicrobial activity against P. insidiosum, and the greatest activity of O. vulgare was highlighted. Synergistic and/or indifferent effect was observed for all combinations evaluated, especially the M. piperita and O. vulgare combination, which showed 65 % synergism. This is the first study to report in vitro combinations of essential oils against P. insidiosum indicating the susceptibility of this oomycete to M. alternifolia, M. piperita and O. vulgare essential oils, as well as their combinations.

Treatment of experimental pythiosis with essential oils of Origanum vulgare and Mentha piperita singly, in association and in combination with immunotherapy.

This study investigated the in vivo antimicrobial activity of the essential oils of Origanum vulgare and Mentha piperita both singly, associated and in combination with immunotherapy to treat experimental pythiosis. The disease was reproduced in 18 rabbits divided into six groups (n=3): group 1, control; group 2, treated with essential oil of Mentha piperita; group 3, treated with essential oil of Origanum vulgare; group 4, treated with commercial immunotherapic; group 5, treated with a association of oils of M. piperita and O. vulgare and group 6, treated with a combination of both oils plus immunotherapy. Essential oils were added in a topical cream base formula, and lesions were treated daily for 45 days. The animals in groups 4 and 6 received a dose of immunotherapeutic agent every 14 days. The results revealed that the evolution of lesions in groups 5 and 6 did not differ from one another but differed from the other groups. The lesions of group 5 increased 3.16 times every measurement, while those of group 6 increased 1.83 times, indicating that the smallest growth of the lesions occurred when the combination of therapies were used. A rabbit from group 5 showed clinical cure at day 20 of treatment. This research is the pioneer in the treatment of experimental pythiosis using essential oils from medicinal plants and a combination of therapies. This study demonstrated that the use of essential oils can be a viable alternative treatment to cutaneous pythiosis, particularly when used in association or combination with immunotherapy.