Characterization of Lactococcus garvieae and Streptococcus agalactiae in cultured red tilapia Oreochromis sp. in Thailand.

OBJECTIVE: Acute mortality with clinical symptoms of streptococcal-like infections was observed in red tilapia Oreochromis sp. cultured in floating cages in Prachin Buri Province, Thailand, during May 2023. Herein, we identified an emerging pathogen, Lactococcus garvieae, as the etiological agent. METHODS: After bacterial isolation from the brain and kidney of diseased fish, identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK 2 system. Sequencing of the 16S ribosomal RNA (rRNA) gene and phylogenetic analysis were applied to confirm bacterial species. Antimicrobial susceptibility testing was conducted. Histopathological findings in the brain, kidney, spleen, liver, and heart were evaluated. RESULT: From 20 fish samples, L. garvieae (n = 18 isolates) and Streptococcus agalactiae (n = 2 isolates) were identified. A phylogenetic tree of the 16S rRNA gene revealed that Thai isolates of either L. garvieae or S. agalactiae clustered with reference piscine isolates from intercontinental locations. Our isolates showed resistance against quinolones while being susceptible to other antimicrobials. Histopathological changes demonstrated severe septicemic conditions, with more invasive lesions-especially in the heart and liver-being apparent in L. garvieae-infected fish compared to S. agalactiae-infected fish. CONCLUSION: This study represents the first reported outbreak of L. garvieae with a concurrent S. agalactiae infection in farmed red tilapia in Thailand.

Immune Activation Following Vaccination of Streptococcus iniae Bacterin in Asian Seabass (Lates calcarifer, Bloch 1790).

Juvenile Asian seabass (Lates calcarifer) (body weight 10 ± 0.7 g) were intraperitoneally injected with 1012 CFU fish-1 of formalin-killed Streptococcus iniae. The protective efficacy of the vaccine on survival and infection rate was assessed upon challenge at 4, 8, 12, 20, and 28 weeks post-vaccination. The results revealed that the challenged vaccinated fish showed no mortality at all time points, and the control fish presented 10-43.33% mortality. The infection rate at 2 weeks post-challenge was 0-13.33% in the vaccinated fish and 30-82.35% in the control group. At 8 weeks post-vaccination, the vaccinated fish showed comparable ELISA antibody levels with the control; however, the antibody levels of the vaccinated fish increased significantly after the challenge (p < 0.05), suggesting the presence of an adaptive response. Innate immune genes, including MHC I, MHC II, IL-1ß, IL-4/13B, and IL-10, were significantly upregulated at 12 h post-challenge in the vaccinated fish but not in the control. In summary, vaccination with S. iniae bacterin provided substantial protection by stimulating the innate and specific immune responses of Asian seabass against S. iniae infection.

Streptococcus agalactiae infection caused spinal deformity in juvenile red tilapia (Oreochromis sp.).

A case of juvenile red tilapia (Oreochromis sp.) showing body deformity due to spinal curvature was investigated. Approximately 20% of the crop (4,000 fish/crop) was affected. Bacterial isolation from the kidney and tissue surrounding the spinal lesion of the affected fish was negative. Histopathology revealed granulomatous inflammation and Gram-positive cocci in connective tissues around the bone and notochord. PCR assay confirmed the presence of S. agalactiae in the spinal tissue lesion. Spinal deformity in red tilapia observed in our study may be associated with the inflammatory process and granuloma that compress the skeleton structure. The present study highlights chronic streptococcosis in tilapia culture that may be unnoticed.

Identification of bacterial pathogens in cultured fish with a custom peptide database constructed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: The majority of infectious diseases of cultured fish is caused by bacteria. Rapid identification of bacterial pathogens is necessary for immediate management. The present study developed a custom Main Spectra Profile (MSP) database and validate the method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of fish bacterial pathogens. Streptococcus agalactiae, Streptococcus iniae, Aeromonas hydrophila, Aeromonas veronii, and Edwardsiella tarda obtained from diseased fish were used as representative bacterial pathogens in this study. Bacterial peptides were extracted to create a Main Spectra Profile (MSP), and the MSPs of each bacterial species was added into the MALDI Biotyper database. Fifteen additional isolates of each bacterial species were tested to validate the utilized technique. RESULTS: The MSPs of all field isolates were clearly distinguishable, and the MSPs of the same species were clustered together. The identification methodology was validated with 75 bacterial isolates. The reliability and specificity of the method were determined with MALDI Biotyper log score values and matching results with 16 s rDNA sequencing. The species identification using the public MALDI Biotyper library (Bruker MALDI Biotyper) showed unreliable results (log score < 2.000) with 42.67% matching result with the reference method. In contrast, accurate identification was obtained when using the custom-made database, giving log score > 2.115, and a 100% matching result. CONCLUSION: This study demonstrates an effective identification of fish bacterial pathogens when a complete custom-made MSP database is applied. Further applications require a broad, well-established database to accommodate prudent identification of many fish bacterial pathogens by MALDI-TOF MS.

Francisella Infection in Cultured Tilapia in Thailand and the Inflammatory Cytokine Response.

Francisella infections developed in freshwater Nile Tilapia Oreochromis niloticus and red tilapia Oreochromis spp. farms in Thailand during 2012-2014. The diseased fish were lethargic and pale in color and showed numerous white nodules in their enlarged spleens. Histopathological examination and electron microscopy suggested that the white nodules were multifocal granulomas consisting of coccobacilli within vacuolated cells. Isolation of Francisella-like bacteria was achieved from 42 of 100 samples, while polymerase chain reaction confirmed Francisella infections in all samples. Analysis of the 16S rRNA gene from samples obtained from three different geographical culture areas revealed more than 99% similarity with F. noatunensis subsp. orientalis. The influence of Francisella infection on inflammatory cytokines was determined on splenic cells of fish intraperitoneally injected with the bacteria (0.8 × 10(5) colony-forming units per fish). Infected tilapia showed significantly greater expression of the pro-inflammatory genes interleukin-1ß (IL-1ß) and tumor necrotic factor-α (TNF-α) within 24 h postinjection (hpi) and for up to 96 hpi. However, down-regulation of an anti-inflammatory gene, transforming growth factor-ß (TGF-ß) was observed as early as 24 hpi. This investigation demonstrates that an imbalance between pro- and anti-inflammatory cytokines in response to the infection may account for the substantial number of granulomas in fish hematopoietic tissues that was found in the later stage of the disease. Received September 9, 2015; accepted December 13, 2015.

White spot disease risk factors associated with shrimp farming practices and geographical location in Chanthaburi province, Thailand.

Over the past 2 decades, shrimp aquaculture in Thailand has been impacted by white spot disease (WSD) caused by white spot syndrome virus (WSSV). Described here are results of a survey of 157 intensive shrimp farms in Chanthaburi province, Thailand, to identify potential farm management and location risk factors associated with the occurrence of WSD outbreaks. Logistic regression analysis of the survey responses identified WSD risks to be associated with farms sharing inlet water and culturing shrimp year round and with a single owner operating more than 1 farm. The analysis also showed WSD risks to be reduced at farms that used probiotics and applied lime to pond bottoms when fallow to neutralize acidity and kill microorganisms. Regression modeling identified no association of geographical location with WSD. The data should assist shrimp farms in mitigating the effects of WSD in Thailand.

Comparative sequence analysis of a multidrug-resistant plasmid from Aeromonas hydrophila.

Aeromonas hydrophila is a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated from A. hydrophila obtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1_Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn21 type transposon. This transposon contains the drug resistance genes qacH, bla(OXA-10), aadA1, and sul1 in a class 1 integron; tetA and tetR in transposon Tn1721; and catA2 and a duplicate sul1 in a locus showing 100% similarity to IncU plasmids isolated from fish. The bla(OXA-10) and aadA1 genes showed 100% similarity to those from the Acinetobacter baumannii AYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred.

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.

Characterization of antibiotic resistance in Vibrio spp. isolated from farmed marine shrimps (Penaeus monodon).

A total of 83 Vibrio isolates from farmed marine shrimps (Penaeus monodon) were tested for the presence of class 1, 2 and 3 integrons, SXT constin and tetracycline resistance-encoding genes. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were determined in fluoroquinolone-resistant Vibrio strains (n=17). Five isolates were found to carry class 1 integrons, of which only one contained the partial rumA gene in the variable region. All the Vibrio strains were devoid of class 2 and 3 integrons. Seven isolates harbored SXT constin. None of the Vibrio isolates were positive to the tet(K), tet(L), tet(M), tet(O) and tet(S) genes. Ten fluoroquinolone-resistant Vibrio strains carried a point mutation G-248-T in the gyrA QRDR, leading to a Ser-83-Ile substitution in GyrA, but none of these strains had mutations in the QRDR of the parC gene.