New insights on evolutionary aspects of Pythium insidiosum and other peronosporaleans.

BACKGROUND: The evolution of pathogenic mechanisms is a major challenge, which requires a thorough comprehension of the phylogenetic relationships of pathogens. Peronosporaleans encompasses a heterogeneous group of oomycetes that includes some animal/human pathogens, like Pythium insidiosum. OBJECTIVE: We analysed here the phylogenetic positioning and other evolutionary aspects related to this species and other peronosporaleans, using a multi-locus approach with one mitochondrial and three nuclear genes. METHODOLOGY: Phylogenetic patterns of 55 oomycetes were inferred by maximum likelihood and Bayesian analysis, and a relaxed molecular clock method was applied to infer the divergence time of some peronosporaleans branches. RESULTS: Pythium insidiosum was monophyletic with a major and polytomous clade of American isolates; however, Pythium spp. was found to be paraphyletic with Phytopythium sp. and Phytophthora spp. In general, peronosporaleans subdivided into four lineages, one of which evidenced a close relationship of P insidiosum, P aphanidermatum and P arrhenomanes. This lineage diverged about 63 million years ago (Mya), whereas P insidiosum diversified at approximately 24 Mya. The divergence of American and Thai isolates seems to have occurred at approximately 17 Mya, with further American diversification at 2.4 Mya. CONCLUSION: Overall, this study clarifies the phylogenetic relationships of P insidiosum regarding other peronosporaleans in a multi-locus perspective, despite previous claims that phylogenomic analyses are needed to accurately infer the patterns and processes related to the evolution of different lineages in this group. Additionally, this is the first time that a molecular clock was applied to study the evolution of P insidiosum.

Intradermal injection of Pythium insidiosum protein antigens for improved diagnosis and treatment of pythiosis in an experimental model.

The oomycetous pathogen Pythium insidiosum is the causative agent of pythiosis, a life-threatening disease that affects animals and humans. This infectious disease is difficult to treat, and early and accurate diagnosis is critical for effective treatment. In this sense, this study aimed to evaluate the intradermal (ID) injection of P. insidiosum protein antigens (PiPA) for the diagnosis and treatment of pythiosis using an experimental model. For diagnostic purposes, PiPA were injected by the ID route in the following groups of rabbits: (a) control; (b) previously immunized with PiPA injected by the subcutaneous (SC) route; and (c) infected with P. insidiosum zoospores. For treatment purposes, rabbits with pythiosis were also treated with PiPA by the ID or SC routes. Mean induration sizes were different at 24 h and 72 h readings when compared to the control group. Sensitivity of the protocol was 100% at 24 h and 80% at 72 h, with 100% specificity in both readings. PiPA treatment using ID or SC routes did not result in significant differences in lesion sizes and cure rates; however, serum levels of interferon-gamma were higher in SC route. This study demonstrates the applicability of PiPA ID for diagnosis and treatment of pythiosis in an experimental model.

In vitro susceptibility of the oomycete Pythium insidiosum to metallic compounds containing cadmium, lead, copper, manganese or zinc.

Pythium insidiosum is an aquatic oomycete that causes pythiosis, an important and severe disease of difficult treatment that affects humans, domestic and wild animals. This infection is often described in horses in Brazil and humans in Thailand. In clinical practice, we have observed many cases that do not respond to available therapies, indicating the need to explore alternative therapeutic approaches. In this sense, studies using metal compounds in conjunction with available antimicrobial agents have been demonstrated greater antimicrobial activity. Thus, in this research, we tested in vitro activities of metallic compounds containing cadmium, lead, copper, manganese, or zinc against 23 isolates of P. insidiosum. The assays were performed by broth microdilution based on CLSI M38-A2 document. The minimum inhibitory and fungicidal concentrations were established for all isolates. Copper acetate and cadmium acetate showed the highest inhibitory effects, with minimal inhibitory concentration ranging from 4-64 µg/ml and 16-256 µg/ml, respectively. The mean geometric for minimal fungicidal concentrations were, respectively, 26 µg/ml and 111.43 µg/ml for copper acetate and cadmium acetate. These results suggest that copper and cadmium can inhibit P. insidiosum growth, highlighting the greater inhibitory activity of copper acetate. In addition, our results propose that copper and/or cadmium compounds can be used in upcoming researches to formulate effective new complexed drugs against P. insidiosum in in vitro and in vivo experimental models.

Microevolutionary analyses of Pythium insidiosum isolates of Brazil and Thailand based on exo-1,3-ß-glucanase gene.

Pythium insidiosum is an important oomycete due to its ability to infect humans and animals. It causes pythiosis, a disease of difficult treatment that occurs more frequently in humans in Thailand and in horses in Brazil. Since cell-wall components are frequently related to host shifts, we decided here to use sequences from the exo-1,3-ß-glucanase gene (exo1), which encodes an immunodominant protein putatively involved in cell wall remodeling, to investigate the microevolutionary relationships of Brazilian and Thai isolates of P. insidiosum. After neutrality ratification, the phylogenetic analyses performed through Maximum parsimony (MP), Neighbor-joining (NJ), Maximum likelihood (ML), and Bayesian analysis (BA) strongly supported Thai isolates being paraphyletic in relation to those from Brazil. The structure recovered by these analyses, as well as by Spatial Analysis of Molecular Variance (SAMOVA), suggests the subdivision of P. insidiosum into three clades or population groups, which are able to explain almost 81% of the variation encountered for exo1. Moreover, the two identified Thai clades were almost as strongly differentiated between each other, as they were from the Brazilian clade, suggesting an ancient Asian subdivision. The derived positioning in the phylogenetic tree, linked to the lower diversity values and the recent expansion signs detected for the Brazilian clade, further support this clade as derived in relation to the Asian populations. Thus, although some patterns presented here are compatible with those recovered with different molecular markers, exo1 was revealed to be a good marker for studying evolution in Pythium, providing robust and strongly supported results with regard to the patterns of origin and diversification of P. insidiosum.

Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil.

This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

Echinococcus granulosus sensu lato and Taenia hydatigena in pig in southern Brazil / Echinococcus granulosus sensu lato e Taenia hydatigena em suínos no sul do Brasil

This study aimed to identify the parasitical etiologic agents of visceral cysts in pigs from the central/northern region of Rio Grande do Sul State, Brazil. Fifty-eight cysts were found in livers during veterinary inspection of swine slaughtered from January 2008 to 2012. Collected samples were submitted to macroscopic and molecular analyzes. Polymerase chain reaction (PCR), DNA sequencing and BLAST alignment of sequences was used to molecular characterization of the samples. By PCR 10.3% (6/58) of tested samples were positive for Echinococcus granulosus sensu lato and 56.9% (33/58) for Cysticercus tenuicollis. In this study, it was verified the occurrence of larval forms of E. granulosus sensu lato and Taenia hydatigena in pig herds from the central/northern region of Rio Grande do Sul State. The presence of both parasites is relevant due to the economic losses for the meat industry. Additionally, E. granulosus sensu lato has zoonotic importance and may be infecting pig herds in southern Brazil.

Neste estudo, buscou-se identificar os agentes etiológicos de origem parasitária em cistos nas vísceras de suínos oriundos sa região centro/norte do sEstado do Rio Grande do Sul, Brasil. Cinquenta e oito cistos foram encontrados durante a inspeção veterinária em fígados de suínos abatidos entre janeiro de 2008 a 2012. As amostras foram submetidas às análises macroscópicas e moleculares. Reação em cadeia da polimerase (PCR), sequenciamento de DNA e alinhamento das sequências no BLAST (bases de dados do GenBank) foram utilizados na caracterização molecular das amostras. Das amostras analisadas, 10,3% (6/58) foram positivas para Echinococcus granulosus sensu lato e 56,9% (33/58) para Cysticercus tenuicollis. Evidenciou-se a ocorrência de larvas de E. granulosus sensu lato e Taenia hydatigena em rebanhos suínos na região centro/norte do RS. A presença de ambos os agentes é relevante devido às perdas econômicas para a indústria de carnes. Além disso, destaca-se que E. granulosus sensu lato possui importância zoonótica e pode contaminar os rebanhos suínos no sul do Brasil.