ABSTRACT
Objective: The aerolysin (aerA) is a virulence indicator used to identify the pathogenicity of the Aeromonas strain. Targeting a pathogen's crucial virulence gene for detection is essential, as it determines the potential threat to the host. This study aimed to develop a gold nanoparticle (AuNP) probe for detecting the gene aerA in Aeromonas hydrophila among field samples. Materials and Methods: Kidney samples among both healthy and sick Nile tilapias in five provinces of Luzon Island were collected for bacterial analysis. Screening using specific primers targeting aerA was conducted in parallel with testing the AuNPs probe on the same sample set. The positive control provided by BFAR-NFLD, confirmed by polymerase chain reaction (PCR) assay, was used as a positive sample containing the target gene. Results: The AuNP probe demonstrated a computed accuracy of 81.32%, sensitivity of 100%, and specificity of 81.26%. Among the 257 reactions, 59 were false positives, while no false negative results were observed. The AuNP probe could detect aerA at levels as low as 30 ng/µl. The low prevalence of the target gene may be attributed to the use of general media instead of specific media like Rimler-Shotts agar. Conclusion: The established colorimetric detection method for A. hydrophila with the aerA gene offers a swift alternative to PCR, negating the requirement for advanced equipment like a thermal cycler.
ABSTRACT
OBJECTIVE: Acute hepatopancreatic necrosis disease is an emerging infectious disease of Penaeus species. The causative agent is Vibrio species, which dispels binary toxin similar to pirA and pirB, which causes mortality in infected shrimp. The aim of this research was to investigate the evolutionary relationship of pirA and pirB homologous genes present in this Asia and Costa Rica in silico. MATERIALS AND METHODS: The sequences for in silico analysis were all retrieved from the Basic Local Alignment Search Tool Nucleotide (BLASTN) tool of the National Center for Biotechnology Center. For pirA, a total of 25 sequences submitted from different Asian countries and Costa Rica were retrieved for analysis. Meanwhile, for pirB, a total of 11 sequences submitted from five Asian countries were retrieved. Sequences were aligned using the CLUSTAL W alignment tool under Molecular Evolutionary Genetics Analysis (MEGA) 7 software. The evolutionary history was then estimated using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) method, whereas the evolutionary distances were determined using the maximum composite likelihood model with 1,000 bootstrap replications. RESULTS AND DISCUSSION: The results show that, among 27 DNA sequences analyzed for pirA gene, three groups were generated, while for pirB, 13 DNA sequences yielded only one group. The analysis revealed low genetic variation among isolates for both pirA and pirB genes. CONCLUSION: This result suggests that the low frequency of polymorphism and geographic location cannot be attributed to the differences in V. parahaemolyticus isolates in Asian countries relative to Costa Rican isolates in pirA and pirB genes.
ABSTRACT
OBJECTIVE: Major histocompatibility complex (MHC) is a set of molecular proteins on the surface of antigen presenting cells encoded by a large gene family which are important parts of the immune system. This study was conducted to convey information on the genetic characteristics of the MHC II DRB3 gene in riverine and swamp buffaloes. MATERIALS AND METHODS: Characterization of MHC II DRB3 gene was carried out using polymerase chain reaction (PCR)-based assay. Thirty-milliliter milk samples were collected from 10 swamp-type and 10 riverine-type buffaloes. RNA from milk samples were extracted using Trizol and then followed by reverse transcription-PCR (RT-PCR). RESULTS: The phylogenetic analysis with 1,000 bootstrap replications clearly showed complex parsimony in MHC II DRB3 gene between 10 riverine- and 10 swamp-type but also confirmed that the samples are similar to Bubalus bubalis. Aligned sequences of the 20 water buffaloes were compared with three other ruminants (Bos taurus, Ovis aries, and Capra hircus) and non-ruminant (Sus scrofa) that serve as an outgroup. MHC sequences from GenBank show that there was an average of 705 identical pairs, with 22 transitional pairs and 30 transversional pairs with a ratio of 0.7. CONCLUSION: Based on the molecular data, the current study conforms to other works of literature that this gene is highly polymorphic which can be due to its function in the immune responsiveness and disease resistance. Further study on the immunological response of MHC II DRB3 to infection may elucidate its underlying function and role in the protection against specific disease of animals.
