Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety‐three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.     

Development of attenuated erythromycin‐resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re‐attenuated S. agalactiae (named TFJ‐ery) was developed from a natural low‐virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ‐ery were determined. The results indicated that TFJ‐ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ‐ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ‐ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ‐ery at a dose of 5.0 × 10⁷ CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post‐vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post‐vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ‐ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.     

Genomic comparison of virulent and non‐virulent Streptococcus agalactiae in fish

Streptococcus agalactiae infections in fish are predominantly caused by beta‐haemolytic strains of clonal complex (CC) 7, notably its namesake sequence type (ST) 7, or by non‐haemolytic strains of CC552, including the globally distributed ST260. In contrast, CC23, including its namesake ST23, has been associated with a wide homeothermic and poikilothermic host range, but never with fish. The aim of this study was to determine whether ST23 is virulent in fish and to identify genomic markers of fish adaptation of S. agalactiae. Intraperitoneal challenge of Nile tilapia, Oreochromis niloticus (Linnaeus), showed that ST260 is lethal at doses down to 10² cfu per fish, whereas ST23 does not cause disease at 10⁷ cfu per fish. Comparison of the genome sequence of ST260 and ST23 with those of strains derived from fish, cattle and humans revealed the presence of genomic elements that are unique to subpopulations of S. agalactiae that have the ability to infect fish (CC7 and CC552). These loci occurred in clusters exhibiting typical signatures of mobile genetic elements. PCR‐based screening of a collection of isolates from multiple host species confirmed the association of selected genes with fish‐derived strains. Several fish‐associated genes encode proteins that potentially provide fitness in the aquatic environment.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

Experimental early pathogenesis of Streptococcus agalactiae infection in red tilapia Oreochromis spp.

Streptococcus agalactiae causes a severe systemic disease in fish, and the routes of entry are still ill‐defined. To address this issue, two groups of 33 red tilapia Oreochromis spp. each of 10 g were orally infected with S. agalactiae (n = 30), and by immersion (n = 30), six individuals were control‐uninfected fish. Three tilapias were killed at each time point from 30 min to 96 h post‐inoculation (pi); controls were killed at 96 h. Samples from most tissues were examined by haematoxylin–eosin (H&E), indirect immunoperoxidase (IPI) and periodic acid‐Schiff; only intestine from fish infected by gavage was evaluated by transmission electron microscopy. The results of both experiments suggest that the main entry site of S. agalactiae in tilapia is the gastrointestinal epithelium; mucus seems to play an important defensive role, and environmental conditions may be an important predisposing factor for the infection.     

Virulence regulation of cel‐EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel‐EIIB protein‐mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel‐EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel‐EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel‐EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel‐EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel‐EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel‐EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel‐EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel‐EIIB strain (p < 0.05). In conclusion, cel‐EIIB protein‐mediated cel‐PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.     

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real‐time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS‐s/IGS‐a, which targets the 16S‐23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post‐injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post‐injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish.     

CD40 induces an antimicrobial response against the intracellular pathogen Streptococcus agalactiae in Nile tilapia,Oreochromis niloticus

Streptococcus agalactiae is a Gram‐positive facultative intracellular bacterium that leads to severe economic loss of tilapia worldwide. Previous studies demonstrated that CD40 contributes to host protection against intracellular injection. In this study, CD40 was characterized from Nile tilapia (Oreochromis niloticus), named OnCD40. Sequence analysis showed that open reading frame of OnCD40 was 933 bp, containing a single peptide, a transmembrane domain and four cysteine‐rich domains. The qRT‐PCR revealed that OnCD40 was expressed in all examined tissues with the most abundant ones in spleen and thymus. After S. agalactiae stimulation, the expression of OnCD40 was significantly induced in most of the detected organs. Moreover, OnCD40‐overexpressing fish elicited significant protection against subsequent S. agalactiae challenge; approximately 10000‐fold fewer bacteria were detected in spleen of OnCD40‐overexpressing fish in comparison with control fish. Thus, CD40 had protecting function in Nile tilapia against intracellular pathogens.     

Comparison between the intestinal microbiome of healthy fish and fish experimentally infected with Streptococcus agalactiae

Nile tilapia (Oreochromis niloticus) farming is an economic activity that is soaring in the whole world. Septicemia due to Streptococcus agalactiae is the main disease impacting fish farming. The aim of this study was to compare the gut microbiome of healthy animals and animals experimentally infected with S. agalactiae strain 21171A. The microbiome was established with 16S ribosomal DNA next‐generation sequencing (NGS). One hundred Nile tilapias, with an average weight of 35 g, were distributed into two groups. Fifty fish from the challenged group were orally inoculated with 100 μl of a bacterial solution containing 1.98 × 10³ CFU/ml of S. agalactiae strain 21171A, while 50 controls were orally inoculated with sterile saline. After the experiment, 24 fish from the challenged group and 27 fish from the control group were analysed. For both groups, bacteria attached to the mucosa (M) and present in faeces (F) were analysed. The mean of the number of taxa identified in the infected group (M + F) (45.87 ± 30.13) was lower than in the control (M + F) (67.70 ± 21.10) (p < .01). Nineteen bacterial taxa were more abundant in faecal samples from the infected group when compared with the control group (p < .01). Thirty‐nine taxa were associated with mucosa samples from the challenged group when compared to the control samples (p < .01). No OTU was associated with healthy samples. The results demonstrate that the infection with S. agalactiae reduces the variability of the gut microbiota. Moreover, some bacteria proliferate during the infection.     

Maternal immunity of tilapia broodstock vaccinated with polyvalent vaccine and resistance of their offspring against Streptococcus agalactiae

This study aimed to examine the use of Streptococcus agalactiae polyvalent vaccine in tilapia broodstock and the effect of maternal immunity and resistance on their offspring against S. agalactiae strain. The broodstock was injected with polyvalent vaccine of S. agalactiae at a dose of 10⁸ CFU per fish at 2nd gonadal maturity until spawning. Challenge test was carried out on the offspring at the 5, 10, 15 and 20 days after hatching using NK₁, N₁₇O, N₁₄G, N₃M, N₄M strain respectively and combination of them. We observed immunological parameters in broodstock, eggs and larvae and relative per cent survival (RPS) of larvae after challenged with pathogenic S. agalactiae. The results showed that the leukocytes, phagocytic activity, respiratory burst, lysozyme activity and antibody levels of vaccinated broodstock had higher level compared with unvaccinated broodstock. The high level of the lysozyme activity, antibody levels and recombination activating gene 1 (RAG1) were also observed in eggs and larvae from vaccinated broodstock. Larvae produced from vaccinated broodstock when challenged with variety strain of pathogenic S. agalactiae had RPS value more than 50% until 20 days after hatching. In conclusion, polyvalent vaccine of S. agalactiae administrated in the broodstock could enhance immunity in the broodstock and protect their offspring from pathogenic S. agalactiae.     

Identification and typing of Vagococcus salmoninarum using genomic and proteomic techniques

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross‐reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC‐PCR and REP‐PCR) and MALDI‐TOF‐MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI‐TOF‐MS is the most promising method for typing and characterization of V. salmoninarum.     

Water quality influences the presence of Streptococcus agalactiae in cage cultured red hybrid tilapia,Oreochromis niloticus × Oreochromis mossambicus

Attempts were made to identify the association between water quality parameters and the presence of Streptococcus agalactiae in cage cultured red hybrid tilapia, Oreochromis niloticus × O. mossambicus. Fish from commercial floating net cage‐culture systems in a river and lake were randomly sampled over a 24‐month period. Swabs from the brains, eyes and kidneys were streaked directly onto blood agar to isolate S. agalactiae. Water temperature, dissolved oxygen, pH, clarity, ammonia, nitrite, sulfide, rate of water flow and depth of water at sampling sites were measured at the same time of fish sampling. The prevalence of fish that were cultured positive to S. agalactiae was significantly higher in lake compared with river. The length and weight of the infected fish were between 9 and 33 cm, and between 20 and 760 g respectively. There was a significant and positive strong correlation between the presence of S. agalactiae and fish mortalities in lake. All water quality parameters showed significant differences between river and lake. However, only water temperature, clarity and pH of lake and the ammonia, temperature and dissolved oxygen in river showed significant correlation with the presence of S. agalactiae in the cultured fish. It was concluded that several unfavourable water quality in the fish farm influencing the presence of S. agalactiae in cultured red hybrid tilapia.     

Roles of water quality and disinfectant application on inactivation of fish pathogenic Streptococcus agalactiae with povidone iodine,quaternary ammonium compounds and glutaraldehyde

Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low‐ [LS] and high‐level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100?; AD), benzalkonium chloride (Better BKC 80%?; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde?; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher‐level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5‐log inactivation at 1‐min exposure at 20°C under HS conditions, and also with ten‐fold‐diluted concentrations at 60‐min exposure time at 30°C. However, AD required 10‐min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity.     

Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.)

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non‐encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild‐type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host–pathogen interactions.     

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.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

Dietary supplementation with Bacillus subtilis LT3‐1 enhance the growth,immunity and disease resistance against Streptococcus agalactiae infection in genetically improved farmed tilapia,Oreochromis niloticus

A feeding trial was conducted to investigate the effect of different levels of Bacillus subtilis LT3‐1 in diets on growth, immune parameters, intestinal morphology and disease resistance in genetically improved farmed tilapia, Oreochromis niloticus. Fish (46.91 ± 0.17 g) were fed with a basal diet supplemented with B. subtilis LT3‐1 at 0 (B0), 3.8 × 10¹⁰ (B1), 7.6 × 10¹⁰ (B2), 1.14 × 10¹¹ (B3) and 1.52 × 10¹¹ (B4) CFU kg^?1 for 6 weeks. The results showed that the weight gain of fish in B1 group was significantly enhanced compared to that in B0 group (p < 0.05). The addition of B. subtilis significantly affected serum biochemical indices (total protein, albumin, aspartate aminotransferase, alkaline phosphatase). Besides, the haematocrit, total counts of red and white blood cells, as well as the serum catalase and lysozyme activities, were increased, whereas the serum malondialdehyde, the serum immunoglobulin M and complement three contents were reduced. Parameters for intestinal morphology suggested a healthier intestine for the fish fed B. subtilis‐supplemented diets than fish fed the control diet. The survival rate after Streptococcus agalactiae challenge increased in tilapia fed with B. subtilis. The present study demonstrated B. subtilis can effectively improve growth, immunological status and resistance against S. agalactiae infection in tilapia farming.     

Recruitment sources and spatial patterns of population demographics of spotted bass in a large river–tributary network

Understanding spatial patterns in population characteristics and the principal natal environments supporting riverine fish populations are important for fisheries management. Fin ray microchemistry was used to identify natal environment, and age estimates from sectioned fin rays were used to estimate growth and mortality rates for spotted bass, Micropterus punctulatus (Rafinesque), in a segment of the Ohio River (Smithland Pool) and three tributaries. Differences in water Sr:Ca and Ba:Ca among the Ohio River and tributaries were reflected in fin ray edge Sr:Ca and Ba:Ca. Fourteen percent of spotted bass ≥ age 2 captured in the Ohio River originated in tributaries, whereas 10% captured in tributaries originated in the Ohio River. Spotted bass in the Ohio River reached larger maximum size (L∞ = 448.7) than conspecifics in tributaries (L∞ = 324.4), although mortality rates were not different. Although 86% of spotted bass were collected in their inferred natal environment, small tributaries may be a supplemental source of recruitment for the spotted bass stock in Smithland Pool.