Development of a real-time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene.

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)-based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease-encoding (lldY) gene was selected as a target for the design of S. iniae-specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 101 amplicon copies per assay (equivalent to 2 × 10-9  ng/µl) using bacterial DNA and of 1.44 × 101 gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.

Rapid onsets of warming events trigger mass mortality of coral reef fish.

Our study reveals a hitherto overlooked ecological threat of climate change. Studies of warming events in the ocean have typically focused on the events' maximum temperature and duration as the cause of devastating disturbances in coral reefs, kelp forests, and rocky shores. In this study, however, we found that the rate of onset (Ronset), rather than the peak, was the likely trigger of mass mortality of coral reef fishes in the Red Sea. Following a steep rise in water temperature (4.2 °C in 2.5 d), thermally stressed fish belonging to dozens of species became fatally infected by Streptococcus iniae Piscivores and benthivores were disproportionately impacted whereas zooplanktivores were spared. Mortality rates peaked 2 wk later, coinciding with a second warming event with extreme Ronset The epizootic lasted ∼2 mo, extending beyond the warming events through the consumption of pathogen-laden carcasses by uninfected fish. The warming was widespread, with an evident decline in wind speed, barometric pressure, and latent heat flux. A reassessment of past reports suggests that steep Ronset was also the probable trigger of mass mortalities of wild fish elsewhere. If the ongoing increase in the frequency and intensity of marine heat waves is associated with a corresponding increase in the frequency of extreme Ronset, calamities inflicted on coral reefs by the warming oceans may extend far beyond coral bleaching.

Isolation and molecular characterization of streptococcal species recovered from clinical infections in farmed Nile tilapia (Oreochromis niloticus) in the Philippines.

Streptococcosis cause severe losses for global tilapia farming, especially in developing countries. The aim of this study was to identify and characterize streptococci recovered from Nile tilapia farmed in the Philippines. Moribund and apparently healthy fish were sampled from grow-out cages, ponds and hatcheries. Clinical signs observed included exophthalmia, eye opacity, ascites, lethargy, erratic swimming and haemorrhages. Results showed that both Streptococcus iniae and Streptococcus agalactiae were associated with disease in these sites. Consistent with global reports, including those from South-East Asia, S. agalactiae was more widespread than S. iniae. Molecular serotyping of the S. agalactiae isolates identified the serotype Ia and serotype Ib. Histopathological findings were meningitis, meningoencephalitis and septicaemia. Identical virulence profiles were found for all strains of S. iniae, while S. agalactiae strains were separated into virulence profile I and profile II. All strains were susceptible to the tested antibiotics and resistant to oxolinic acid. Only S. agalactiae serotype Ib showed resistance to sulphamethoxazole-trimethoprim. This is the first study from the Philippines to characterize the streptococci involved in disease outbreaks in tilapia aquaculture. Outputs from this study will promote the development of efficacious disease control strategies in tilapia farming for the Philippines and South-East Asia.

Phenotypic and genotypic characterization of Streptococcus spp. isolated from tilapia (Oreochromis spp.) cultured in river-based cage and earthen ponds in Northern Thailand.

Streptococcus spp. are major pathogenic bacteria associated with massive mortality in tilapia. This study investigated the phenotypic and genotypic characterization of Streptococcus agalactiae (GBS) and Streptococcus iniae (S. iniae) isolated from tilapia in river-based floating cage and earthen pond farms in northern Thailand. Isolates were identified by biochemical and molecular analyses. Capsular typing, enterobacterial repetitive intergenic consensus polymerase chain reaction and multilocus sequence typing were performed to investigate the genetic relatedness. Six and one isolates were confirmed as GBS and S. iniae, respectively. All Streptococcus spp. isolates were obtained from 4 river-based cage farms (4/33), while samples collected from earthen pond farms (N = 28) were negative for streptococcosis. All GBS with serotype Ⅲ and sequence type (ST) 283 was observed. The ß-haemolytic GBS isolates were resistant to five antimicrobials, while the S. iniae was susceptible to all antimicrobials. This study indicates both GBS and S. iniae are the major bacterial pathogens responsible for streptococcosis infection in farmed tilapia of northern Thailand with GBS as dominant species. This survey highlights that the river-based cage farms seriously impact on the healthy development of the tilapia industry.

An effective established biosensor of bifunctional probes-labeled AuNPs combined with LAMP for detection of fish pathogen Streptococcus iniae.

In purpose of valid Streptococcus iniae detection, we established a colorimetric biosensor using gold nanoparticles (AuNPs) labeled with dual functional probes and along with loop-mediated isothermal amplification (LAMP) assay (LAMP-AuNPs). Based on the characteristics of self-aggregation and bio-conjugation with ligands, AuNPs were chosen for observable color change in tandem with LAMP amplification method to reach high sensitivity and easy operation. Meanwhile, the improvement of dual probes that could fully utilize the LAMP product gave the biosensor a stable result exhibition. LAMP-AuNPs targeting gene ftsB, one of the ATP transporter-related genes, turned out favorable specificity in cross reaction among other fish pathogens. The detect limit of 102 CFU revealed a better sensitivity compared with polymerase chain reaction (PCR) method and AuNPs lateral flow test strip (LFTS). It was also proved to be effective by zebrafish infection model trials with less than 2-h time consumption and nearly no devices which make it a convenient biosensor for point-to-care S. iniae detection.

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico.

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S-1 and S-2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP-PCR and ERIC-PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 104 and 106  CFU per fish with the S-1 isolate, while 100% mortality rates were recorded in zebrafish at 102 and 104  CFU per fish with the S-2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.

Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia.

This study describes the isolation and pathogenicity of Streptococcus iniae in cultured red hybrid tilapia (Nile Tilapia Oreochromis niloticus × Mozambique Tilapia O. mossambicus) in Malaysia. The isolated gram-positive S. iniae appeared punctiform, transparently white, catalase and oxidase negative and produced complete ß-hemolysis on blood agar, while a PCR assay resulted in the amplification of the 16 S rRNA gene and lactate oxidase encoded genes. The isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin. Pathogenicity trials conducted in local red hybrid tilapia (mean ± SE = 20.00 ± 0.45 g) showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 104, 106, and 108 CFU/mL of the pathogen, respectively. The clinical signs included erratic swimming, lethargy, and inappetance at 6 h postinfection, while mortality was recorded at less than 24 h postinfection in all infected groups. The LD50-336 h of S. iniae against the red hybrid tilapia was 102 CFU/mL. The post mortem examinations revealed congested livers, kidneys, and spleens of the infected fish. This is the first report of S. iniae experimental infection in cultured red hybrid tilapia in Malaysia. Received January 20, 2017; accepted July 16, 2017.

Fatal bacterial septicemia in a bottlenose dolphin Tursiops truncatus caused by Streptococcus iniae.

A captive 8 yr old male bottlenose dolphin Tursiops truncatus succumbed to septicemia with multisystemic inflammation including suppurative enteritis, encephalitis, and pneumonia with chronic pancreatitis. A pure culture of beta-hemolytic, catalase- and oxidase-negative, Gram-positive cocci was isolated from the hilar lymph nodes and pancreas. The isolate was identified by 16S rDNA sequencing as Streptococcus iniae. Histological examination of the digestive system revealed a mixed infection of both bacteria and fungus. Recognized as a pathogen in fish, dolphins, and humans, this is the first report of S. iniae in a dolphin in mainland China. As the number of managed animals in oceanariums is increasing, so is the frequency of contact with fish used as food for marine mammals and humans, highlighting the importance of education and appropriate personal protective protocols to minimize the risk of transmission. An understanding of marine mammal infectious disease organisms is essential to ensuring the health of marine mammals and humans coming into contact with such animals and their food. This study illustrates a systematic clinical, microbiological, and pathological investigation into a septicemic bottlenose dolphin infected with S. iniae. Our findings provide useful information for those involved in the diagnosis and control of infectious diseases in marine mammals and offer insight into an important zoonotic pathogen.