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.     

Susceptibility of freshwater rearing Asian seabass (Lates calcarifer) to pathogenic Streptococcus iniae

Asian seabass (Lates calcarifer) has been recognized as an economically important aquaculture species which can be adapted to and cultivated in wide range of salinities. The number of freshwater intensive seabass farms in Thailand is increasing annually. Here, we first describe the susceptibility of Asian seabass, which were cultured in freshwater, to Streptococcus inae (SI) and their pathological changes. Three isolates of putative SI were identified using a combination of standard biochemical assays and species‐specific PCR prior subjected to in vivo challenge. Accumulated mortalities of the fish which received 10⁷ CFU fish^?1 of either SI1J, SI SGSA or SI2J were 90%, 90% and 100% at 7 days‐post infection (dpi), respectively, and mortalities increased sharply between 3 and 5 dpi. Clinical signs such as erratic swimming and opaque eyes were identified from a few infected fish, while most died rapidly without any abnormal signs. Histopathological manifestations were observed in the multiple organs (kidney, liver and brain). Haemorrhage, hyperhemia, cellular degeneration and inflammatory cells infiltration were commonly found within the internal organs. Notably, the formation of numerous encyst‐like lesion aggregated by eosinophilic cells, resembling macrophages, were typically found in the brain of the infected fish. Summarily, this study first revealed that freshwater reared Asian seabass is highly susceptible to SI infection and haemorrhagic septicaemia was a major pathological change that could be found in the infected fish.     

Outbreaks of Streptococcosis associated with Streptococcus iniae in Siberian sturgeon (Acipenser baerii) in China

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses in aquaculture farms all over the world. At least 27 species of fish have been documented to be infected by S. iniae, including cultured and wild populations. In August and October 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhages and nodules showing on epicardium occurred on the Acipenser baerii farms in Ya'an country, China. Histological examination revealed a multisystemic, necrotising inflammatory response that was particularly marked in liver, kidney, heart and brain. Mass mortality (>40%) was observed in infected fish and two Gram‐positive cocci (Ab130920 and Ab131025) were obtained from kidneys and livers of diseased fish. Experimental infections with these two isolates resulted in marked symptoms in the sturgeons similar to those observed in natural outbreaks, and the LD₅₀ values of the two isolates were 5.1 × 10⁵ and 6.4 × 10⁵ cfu per fish respectively. The two microorganisms were identified as S. iniae through physiological and biochemical tests, 16S rRNA and lctO gene sequence analysis. Both two isolates showed a similar antibiotic susceptibility, which were sensitive to ampicillin, amoxicillin, cefazolin, amikacin, deoxycycline, florfenicol, azithromycin, ciprofloxacin, vancomycin and resistant to streptomycin, gentamicin, kanamycin, norfloxacin and sinomin (SMZ/TMP). Multiplex PCR assay for virulence genes showed both isolates possessed six main virulence genes: simA, scpI, pdi, pgm, cpsD and sagA genes. These results indicated that S. iniae could act as a pathogen of farmed A. baerii. This is the first report of S. iniae infection associated with mass mortality in A. baerii.     

Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae

The antimicrobial activity and mode of action of chitosan were evaluated against Streptococcus iniae, a pathogenic Gram‐positive bacterium of fish worldwide. Cell proliferation kinetics were examined following exposure to varying concentrations of chitosan. The action of chitosan on S. iniae was also investigated by measuring agglutination activity, conductivity, and extracellular and intracellular bacterial adenosine triphosphate (ATP) levels. Chitosan exhibited antibacterial activity against S. iniae at concentrations of 0.1% and above and was lethal at a concentration of 0.4% and higher. The mechanism of antibacterial activity of chitosan at the inhibitory level of bacterial growth appears to hinge upon the interaction between chitosan and the oppositely charged bacterial surface. This interplay causes agglutination, which was readily observed grossly and microscopically. After interacting with the cell surface via adsorption, an efflux of intracellular ATP was documented, which suggests that chitosan disrupts the bacterial cell causing leakage of cytosolic contents and ultimately cell death. Results suggest chitosan may be worth evaluating as a natural alternative to antibiotic against S. iniae infection of fish.     

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.     

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty‐two Chilean isolates and the DSM20767^T type strain grew in the presence of 2,2′‐Dipyridyl at varying concentrations (250–330 μm ), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 μm of 2,2′‐Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron‐limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 μm ), it did facilitate growth at lower iron concentrations (10 μm and 1 μm ). An exception was the H‐2 isolate, which only grew with 100 μm of iron sulphide. In turn, 100 μm of haemin was toxic when isolates were grown in normal KDM‐2. In silico R. salmoninarumATCC 33209^T genome analysis detected various genes coding iron uptake‐related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.     

Efficacy of feed‐based adjuvant vaccine against Streptococcus agalactiae in Oreochromis spp. in Malaysia

This study was conducted to determine the systemic, mucosal immunity and protective capacity of the feed‐based adjuvant vaccine (FAV) of Streptococcus agalactiae following oral vaccination against streptococcosis in tilapias. Two hundred and sixteen red tilapia fish were divided into three major groups. Each major group consisted eight tilapia kept in nine 2000 L glass aquaria. At day 0, all fish from the FAV group were fed with feed that had been incorporated with an adjuvant, while fish in the feed‐based vaccine (FNV) group were fed with vaccine incorporated into the pellet without adjuvant. Fish in the control‐unvaccinated group, FC, were fed with normal commercial pellet. Booster dose was performed on day 14 post immunization. Fish from each group were sacrificed on a weekly basis for the entire 7 weeks. Serum, body mucus and gut lavage fluid were evaluated for antibody responses by indirect ELISA, while histological examination was carried out on the gut following intraperitoneal challenge. The FAV group had a significantly higher protection (P < 0.05) following challenge with 3.4 × 10⁹ CFU mL^?1 of live S. agalactiae than FNV group. This level of protection may be due to high antibody responses, increase in size of gut‐associated lymphoid tissue and high number of lymphocytes in the FAV group.     

Winter kill in intensively stocked channel catfish (Ictalurus punctatus): Coinfection with Aeromonas veronii,Streptococcus parauberis and Shewanella putrefaciens

Unusual persistent natural mortality occurred in a floating in‐pond raceway system intensively stocked with channel and hybrid catfish beginning in early November 2016 up until March 2017. The temperature during the period of outbreak ranged from 7.2 to 23.7°C. Gross examination of freshly dead and moribund fish revealed pale gills, slight abdominal distension and swollen inflamed vents. Comprehensive necropsy of 20 fish demonstrated vast amounts of bloody ascitic fluid in the coelomic cavity, visceral congestion, splenomegaly and pale friable livers but macroscopically normal kidneys, suggesting systemic bacterial infection. Bacterial cultures were initiated from skin, gills and major internal organs. Following incubation, a mixture of three bacterial colony phenotypes was observed on agar plates. Presumptive biochemical characterization of the isolates followed by 16S‐rRNA sequence analysis resulted in the identification of Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens. Channel catfish juveniles were experimentally infected with the recovered isolates to fulfil Koch's postulates. Moreover, an antibiogram was used to evaluate the susceptibility of the isolates to antimicrobial drugs approved for use in aquaculture. Aquaflor was used successfully for treatment. Here, we report bacterial coinfection lead by A. veronii and the first identification of S. parauberis and S. putrefaciens from cultured catfish in North America.     

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.     

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.     

Differential pathogenicity of five Streptococcus agalactiae isolates of diverse geographic origin in Nile tilapia (Oreochromis niloticus L.)

Streptococcus agalactiae is an emerging pathogen of fish and has caused significant morbidity and mortality worldwide. The main objective of this study is to assess whether pathogenic differences exist among isolates from different geographic locations. Nile tilapia (Oreochromis niloticus L.) were administered an intraperitoneal injection of suspension containing USA, Brazil, Honduras, Israel, or Kuwait S. agalactiae isolates at concentrations ranging from 10² to 10⁷ cfu mL^?1. The LD₅₀ values 7 days after challenge were as follows: USA (1.0 × 10² cfu mL^?1), Brazil (1.5 × 10³ cfu mL^?1), Honduras (6.8 × 10³ cfu mL^?1), Israel (1.0 × 10⁴ cfu mL^?1) and Kuwait (7.2 × 10⁵ cfu mL^?1). Fish from all groups exhibited lethargy, anorexia, exophthalmia, corneal opacity, erratic swimming, petechiae and mortality. Opercular clearing and ascites were only found after infection with certain geographic isolates. The findings in this study indicate that S. agalactiae isolates of different geographic origin can cause significant mortalities after experimental challenge and can have different pathogenic capacities. Isolates from the Americas (USA, Brazil and Honduras) were more pathogenic to Nile tilapia than isolates from the Middle East/Asia (Israel and Kuwait).     

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.     

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.