Antimicrobial susceptibility and enrofloxacin resistance of streptococcal bacteria from farmed Nile tilapia,Oreochromis niloticus (Linnaeus 1758) in Thailand

This study examined the antimicrobial susceptibility and mutation(s) in quinolone resistance‐determining regions (QRDRs) in streptococcal pathogens isolated from farmed Nile tilapia Oreochromis niloticus in Thailand. Surveillance of antimicrobial susceptibility in tilapia streptococcal pathogens reveals that Streptococcus agalactiae (n = 97) and Streptococcus iniae (n = 3) from diseased tilapia were susceptible to amoxicillin, florfenicol, sulfamethoxazole/trimethoprim and sulfadimethoxine/ormetoprim, however, only 78 isolates were susceptible to enrofloxacin. Twenty‐two enrofloxacin‐resistant S. agalactiae isolates were further examined for mutations in the QRDRs of gyrA, gyrB, parC and parE genes. Twenty isolates had single base pair changed in the gyrA sequence, C‐242‐T. Point mutations in gyrB, GC‐1135, 1136‐AA and T‐1466‐G, were identified in one isolate. All resistant isolates harboured a mutation in the parC gene, C‐236‐A, while no mutations were observed in the parE gene. The study represented mutations of gyrA and parC genes as marked modification of the enrofloxacin‐resistant S. agalactiae from farmed tilapia. This study is a primary report of the QRDRs mutations associated with fluoroquinolone resistance from streptococcal pathogen in the cultured fish. The phenotypic and genotypic characterization of enrofloxacin resistance S. agalactiae evident in this study has led to an improved regulation of antimicrobial use in Thai aquaculture.     

The molecular detection of Taura syndrome virus emerging with White spot syndrome virus in penaeid shrimps of Thailand

This study is to investigate the outbreak of the Taura syndrome (TS) in penaeid shrimps of Thailand. Following the outbreak in 2003, the Taura syndrome virus (TSV) was detected in two closely related penaeids; the Pacific white shrimp, Penaeus vannamei, and the black tiger prawn, Penaeus monodon. Multiple viral infections were detected, we found that the TSV-infected P. vannamei also presented evidence of the White spot syndrome virus (WSSV) as evidenced by histological examination and molecular diagnostic methods. Infections of TSV and WSSV were investigated over a period of ten months between October 2003 and July 2004. TSV infection in shrimps was diagnosed by RT-PCR using the OIE recommended 9195/9992 primers. Positive shrimp gave the expected amplicon of 231 bp. We also amplified and sequenced a fragment of the vp1 gene (354 bp). The pairwise comparisons of vp1 revealed that Thai TSV was phylogenetically related to the Taiwanese strains (98.3% identity).     

Class 1 integrons in Aeromonas hydrophila isolates from farmed Nile tilapia (Oreochromis nilotica)

The aim of this study was to determine antimicrobial resistance of Aeromonas hydrophila isolated from farmed Nile Tilapia. A total of 50 A. hydrophila isolates from clinical cases were screened for the presence of class 1, 2 and 3 integrons and all the strains resistant to enrofloxacin and/or ciprofloxacin (n=19) examined for mutation in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The intI1 gene was detected in 23 A. hydrophila strains (46%) but no intl2 and intl3 were detected. Among these, 14 isolates (60.8%) carried gene cassettes inserted in variable regions i.e., partial aadA2, aadA2, dfrA1-orfC and dfrA12-aadA2, of which the most common gene cassette array was dfrA12-aadA2 (26.09%). Conjugal transfer of class 1 integrons with resistance gene array was detected. All the A. hydrophila strains resistant to enrofloxacin and/or ciprofloxacin possessed mutations in the QRDRs of gyrA and parC. Only a Ser-83-Ile substitution was identified in GyrA and only a Ser-80-Ile amino change was found in ParC. The data confirms that A. hydrophila from farm-raised Nile Telapia serve as a reservoir for antimicrobial resistance determinants.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

Comprehensive Investigation of Streptococcosis Outbreaks in Cultured Nile Tilapia,Oreochromis niloticus,and Red Tilapia,Oreochromis sp., of Thailand

Streptococcosis causes economic losses due to mass mortality at all culturing stages in Nile tilapia, Oreochromis niloticus, and red tilapia, Oreochromis sp., farming throughout Thailand. Diseased tilapia collected from outbreak areas during 2003–2012 were examined using histopathological, biochemical, and molecular tools. Infected fish showed clinical signs of septicemia, and bacteria were found in visceral organs. All gram‐positive cocci isolates were negative to catalase and oxidase, and exhibited β‐hemolysis; however, they possessed various biochemical profiles. PCR amplification of the 16S rRNA gene was used for 165 samples, and resulted in identification of 143 (86.67%) with Streptococcus agalactiae and 14 (8.48%) with Streptococcus iniae, and 8 (4.85%) with mixed infection. High similarity (≥98%) of 16S rRNA gene sequences to the reference strain S. agalactiae (accession no. EF092913) and S. iniae ATCC29178 type strain was observed in the typing of S. agalactiae and S. iniae from Thai farmed tilapia. This investigation documented that at least two species of streptococcal bacteria, S. agalactiae and S. iniae, were involved in tilapia streptococcal infection in Thailand. The molecular recognition of the etiologic agents showed that S. agalactiae was the dominant species that cause disease in all culture areas, whereas S. iniae were discovered only in cases from the northeastern and central regions.