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Current microbiology2010; 62(4); 1225-1229; doi: 10.1007/s00284-010-9781-4

Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

Abstract: Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.
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Publication Date: 2010-12-25 PubMed ID: 21188592DOI: 10.1007/s00284-010-9781-4Google Scholar: Lookup
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Summary

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This study examines the use of nested polymerase chain reaction as a means to identify Pythium insidiosum in cutaneous lesions of horses and rabbits. Pythium insidiosum, typically found in subtropical and tropical areas, is a fungus-like organism and the cause of pythiosis, a disease characterized by cutaneous and subcutaneous lesions primarily in horses.

Introduction

The research focuses on Pythium insidiosum, a pathogen that causes pythiosis in animals and humans. Although horses are the majorly affected species, the disease can also infect humans, making its detection and identification significant for both veterinary and medical science.

Challenge in Conventional Detection

  • Traditional diagnostic methods for pythiosis, including microbiological and serological tests, often yield inconclusive results, particularly for horses and humans. This poses a significant challenge for the effective treatment and control of the disease.

Methodology

  • The study employs a previously established nested polymerase chain reaction (PCR) assay, a molecular biology technique used to amplify a desired piece of DNA. The method was applied directly to clinical and experimental samples to detect P. insidiosum DNA.
  • Researchers used universal fungal primers during the first round of PCR and P. insidiosum-specific primers during the second round to select and amplify the target DNA.
  • A total of 49 samples, involving 21 clinical samples from horses and 28 from experimentally infected rabbits, were analysed using this method.

Results

  • The first round of PCR resulted in 800-base pair products, while the second round created smaller, 105-base pair amplicons specific to P. insidiosum.
  • No amplicons were found in negative control samples, validating the specificity of the nested PCR strategy.
  • The successful amplification of P. insidiosum DNA in both clinical equine samples and experimental rabbit samples shows that the nested PCR method is effective at identifying this pathogen.

Conclusion

  • Based on these findings, the research concludes that nested PCR is a powerful and efficient tool in diagnosing pythiosis, both in endemic cases (as seen in the horse samples) and experimental cases (as encountered in rabbit samples).

Implication

  • The research implies that the use of nested PCR for diagnosing pythiosis could lead to more effective treatment and control of the disease. Furthermore, the method could potentially be expanded to detect other pathogens in future studies.

Cite This Article

APA
Botton SA, Pereira DI, Costa MM, Azevedo MI, Argenta JS, Jesus FP, Alves SH, Santurio JM. (2010). Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits. Curr Microbiol, 62(4), 1225-1229. https://doi.org/10.1007/s00284-010-9781-4

Publication

Current microbiology
ISSN: 1432-0991
NlmUniqueID: 7808448
Country: United States
Language: English
Volume: 62
Issue: 4
Pages: 1225-1229

Researcher Affiliations

Botton, Sonia A
  • Laboratório de Pesquisas Micológicas, Departamento de Microbiologia e Parasitologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Campus Universitário, Prédio 20, Sala 4139, Santa Maria, RS 97105-900, Brazil.
Pereira, Daniela I B
    Costa, Mateus M
      Azevedo, Maria Isabel
        Argenta, Juliana S
          Jesus, Francielli P K
            Alves, Sydney Hartz
              Santurio, Janio Morais

                MeSH Terms

                • Animals
                • Brazil
                • Horse Diseases / diagnosis
                • Horse Diseases / microbiology
                • Horses
                • Polymerase Chain Reaction / methods
                • Pythiosis / microbiology
                • Pythiosis / veterinary
                • Pythium / genetics
                • Pythium / isolation & purification
                • Rabbits

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