Aeromonas shuberti as a cause of multi‐organ necrosis in internal organs of Nile tilapia,Oreochromis niloticus

A disease with white spots in internal organs of Nile tilapia occurred in Zhanjiang, southern China. Multiple, white nodules, 0.8–2.2 mm in diameter, were scattered throughout the liver, spleen and kidney of diseased fish. Signs of nodules reproduced after artificial infection with the isolated strain. Isolated bacteria were Gram‐negative, facultative anaerobic, motile, short rod‐shaped, with a length of 1.2–2.2 μm. Morphological and biochemical tests, as well as phylogenetic analysis, all strongly indicated that the isolate from tilapia is identical to Aeromonas schubertii (A. schubertii) which temporary named LF1708 strain. Antibiotic sensitivity assays showed the LF1708 is sensitive to 24 of 27 tested antibiotics. Pathogenicity test revealed that the isolate at the dose of 3.75 × 10⁶ CFU/g killed 100% of experimental tilapia within 2 days and the dose of 1 × 10⁷ CFU/g killed 100% of experimental zebrafish within 1 day. Histopathology of diseased tilapia infected with A. schubertii showed numerous necrotic lesions widely distributed in spleen, liver and kidney, and infiltration with a large number of bacteria. To our knowledge, this was the first report that associated A. schubertii with mortality in tilapia.     

Growth response and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection

This study determined the growth performance and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection. Tilapia were challenged with three doses of S. iniae (8.8 × 10³, 8.8 × 10⁴ and 8.8 × 10⁵ CFU fish^?1 for low, medium and high challenges respectively). Groups of non‐injected and tryptic soy broth‐injected fish were maintained as controls. Significantly (P<0.05) higher mortality (45.0%) occurred in the high challenge treatment than in the low challenge treatment group (29.6%). The medium challenge group had mortality (36.3%) that did not differ significantly from the high or low treatment. Few fish died in the non‐injected and broth‐injected treatments (3.4% and 0.8% respectively). The tilapia that survived S. iniae infection used to assess growth performance were selected from survivors without gross clinical signs of disease. These fish were randomly stocked at a rate of 30 fish into each 57 L aquarium in triplicate and fed to apparent satiation for 8 weeks. No significant differences were detected in weight gain, feed intake, feed efficiency ratio or survival between S. iniae‐survived tilapia and the control treatments following the 8‐week growth performance trial. Following the 8‐week feeding study, tilapia were challenged with 1 × 10⁶ CFU fish^?1 of S. iniae to assess acquired immunity. Mean cumulative mortality was significantly higher (P<0.05) in the control treatments (41.7% for the non‐injected and 43.3% for the broth‐injected fish) than in the low, medium and high challenge treatments (7.4%, 3.3% and 8.3% respectively). Serum protein was significantly (P<0.05) elevated in the S. iniae‐survived tilapia that were subsequently challenged when compared with controls challenged for the first time. Agglutinating antibody titre was significantly higher in the fish in the medium and high challenge treatments, compared with the control fish challenged for the first time. The results suggest tilapia that survive S. iniae challenge without showing overt disease signs performed as well as non‐infected tilapia. Further, the S. iniae‐survived tilapia challenged following the 8‐week growth performance trial gained acquired resistance to homologous S. iniae challenge.     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

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.     

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.     

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.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

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.     

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia,Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 μL of the vaccine or sham vaccinated with 100 μL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2–3 × 10⁷ CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS–PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22–35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia,Oreochromis niloticus (L.)

Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non‐A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport‐induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 10⁶ CFU fish^?1) and A. veronii (8.9 × 10⁶ CFU fish^?1) killed 100% of experimental fish within 24 h, while a 10‐fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100‐fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.     

Virulence assay of rhizoid and non‐rhizoid morphotypes of Flavobacterium columnare in red tilapia,Oreochromis sp., fry

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris‐like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non‐rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non‐rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post‐challenge as determined by whole fish bacterial loads. At 24 and 48 h post‐challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non‐rhizoid morphotype. The results suggested that an inability of the non‐rhizoid morphotype to persist in tilapia fry may explain lack of virulence.     

Effects of dietary inclusion of various concentrations of Scutellaria baicalensis Georgi extract on growth,body composition,serum chemistry and challenge test of far eastern catfish (Silurus asotus)

Effects of various concentrations of Scutellaria baicalensis (SB) extract in diets on growth, body composition, serum chemistry and disease challenge test of far eastern catfish (Silurus asotus) were determined and compared with a commercially available immune enhancer. Eight experimental diets were prepared in triplicate: control (Con) diet without supplementation of SB and SB‐0.25, SB‐0.5, SB‐1, SB‐2, SB‐3 and SB‐5 diets containing SB at concentrations of 0.25, 0.5, 1, 2, 3 and 5%, respectively. In addition, 0.1% of a commercial immune enhancer product (CP) was also tested. No significant difference in weight gain of fish was found. Feed consumption, feed efficiency ratio and protein retention of fish were not affected by the experimental diets. At the end of the 8‐week feeding trial, 10 externally normal fish from each tank were infected by Vibrio anguillarum or Strepotococcus iniae. Cumulative mortality of fish fed the Con diet was higher than that of fish fed the all other diets in 10 and 25 days after V. anguillarum or S. iniae infection. Results of this study indicated that dietary inclusion of SB extract was effective in improving survival of eastern catfish after V. anguillarum and S. iniae infection, but the various concentrations of SB did not affect fish performance.     

Resistance to Streptococcus iniae and its genetic associations with traits of economic importance in Asian seabass (Lates calcarifer)

Streptococcus iniae is one of the most serious aquatic pathogens, causing significant economic losses in marine and freshwater species, including Asian seabass (Lates calcarifer). Controlling this gram‐positive bacterial pathogen has been an issue in aquaculture systems, due to the combined effects of aquaculture intensification and climatic impacts. To date, there have not been any genetic parameter estimates for S. iniae resistance in Asian seabass. The main aim of this study was to examine genetic variation in S. iniae resistance and its genetic correlations with growth and cannibalism in Asian seabass families produced from a breeding programme for high growth in 2016 and 2017. The study included a total of 5,835 individual fish that were offspring of 41 sires and 60 dams (31 half‐sib and 34 full‐sib families). The experimental fish were challenged by intraperitoneal injection with a volume containing 10⁵ CFU (colony‐forming unit)/fish. Resistance to S. iniae was measured as survival rate at 6 hr, 3, 5, 7, 10 and 15 days post‐challenge test. There were significant variations in S. iniae resistance among families at different observation periods (ranging from 24.4% to 80%). Restricted maximum‐likelihood method and mixed model analysis were applied to estimate heritability for S. iniae resistance. The heritability for S. iniae resistance ranged from 7% to 18% across different statistical models used. The common full‐sib effects accounted for 0.1%–2% of the total variation in resistance to S. iniae. Genetic correlations of the S. iniae resistance at 6 hr and 3 days with later post‐challenge test periods were low to moderate. However, these estimates for S. iniae resistance between successive measurement times (5, 7, 10 and 15 days) were high and close to 1. The genetic correlations of resistance with body weights at 180, 270 and 360 days post‐hatch were not significant as well with cannibalism. It is concluded that there is substantial additive genetic variation in resistance to S. iniae, suggesting there is potential for genetic improvement of Asian seabass for resistance to S. iniae through selective breeding.     

Effects of Bacillus subtilis Strains on Growth,Immune Parameters,and Streptococcus iniae Susceptibility in Nile Tilapia,Oreochromis niloticus

This study was conducted to evaluate the effects of probiotic‐amended diets fed to juvenile Nile tilapia, Oreochromis niloticus, on growth and susceptibility to Streptococcus iniae infection. Fish (average weight 16.5 ± 0.2 g) were fed five diets formulated with Bacillus subtilis strains SB3086, SB3295, SB3615, or AP193 either individually or in combination of strains SB3086 and SB3615 at a targeted concentration of approximately 4 × 10⁷ colony‐forming units (CFU)/g of feed or with a basal control diet with no additives for 21 d. After the 21‐d growth trial, no significant difference in growth performance was observed with any probiotic‐amended diet. Results from serum bactericidal activity showed a significant difference between treatments and the control (P = 0.0002), except for the SB3295‐amended diet (P = 0.9020). Lysozyme activity was also significantly different in fish fed probiotic diets from those fed control diet (P = 0.0001). After 21 d of feeding, fish were challenged with S. iniae by intraperitoneal injection at a dosage of 8 × 10⁶ CFU per fish. Results from the challenge also showed a significant difference between treatments and control (P = 0.0001). Overall, fish fed with strain SB3615 showed the lowest percent mortality (44.0 ± 7.2%) and those fed the control diet showed the highest mortality (77.3 ± 7.0%). The combined feeding with strains SB3086 and SB3615 did not result in any significant difference in reducing mortality because of S. iniae infection in juvenile Nile tilapia when compared with the individual probiotic treatments.     

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.     

Effect of Lactobacillus acidophilussupplementation on growth performances,digestive enzyme activities and gut histomorphology of striped catfish (Pangasianodon hypophthalmusSauvage, 1878) juveniles

The influence of dietary supplementation of Lactobacillus acidophilus, on growth performance, digestive enzyme activities, gut histomorphology and gut microflora were evaluated in juveniles striped catfish (Pangasianodon hypophthalmus). Five experimental diets were formulated by supplementing 0, 10³, 10⁵, 10⁷ and 10⁹ CFU/g L. acidophilus in fishmeal and casein‐based semi‐purified diet. Triplicate groups of striped catfish (21.69 ± 0.18 g) were stocked in 15 fiberglass tanks with stocking density of 25 individuals per tank and fed twice daily at 2.5% of the fish body weight for 12 weeks. Weight gain, specific growth rate, feed conversion ratio and the protein efficiency ratio were significantly higher in fish fed with 10⁵ and 10⁷CFU/g L. acidophilussupplemented diets compared with the other treatment groups. Compared with the control and fish fed low (10³ CFU/g) L. acidophilus supplementation, those fed with 10⁵ and 10⁷ CFU/g had significantly higher (p < 0.05) apparent protein digestibility. Inclusion of L. acidophilus at 10⁷ CFU/g diet significantly increased amylase, protease and lipase activities. Microscopic analysis showed that the villi length in both the anterior and posterior gut and microvilli length in the posterior gut increased significantly in fish fed L. acidophilus supplementation at 10⁵ and 10⁷ CFU/g of diet. The fish fed L. acidophilus supplemented diets significantly increased the total lactic acid bacteria counts in the gut of striped catfish compared with the control‐fed group. Based on gut histomorphology and growth performance, inclusion of L. acidophilus at 10⁵ CFU/g appears to have the most positive effect on striped catfish farming.     

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.     

Effects of potential probiotic Bacillus subtilis KADR1 and its subcellular components on immune responses and disease resistance in Labeo rohita

The present study was conducted to determine the effects of Bacillus subtilisKADR1 and its subcellular components on immunity and disease resistance in Labeo rohita against Aeromonas hydrophila infection. Fish were fed diet containing different concentrations of live bacterial cells (DI‐10⁶, DII‐10⁸ and DIII‐10¹⁰ CFU/g) and another group of fish were immunized intraperitoneally with cellular components (WCPs, CWPs and ECPs) of Bacillus subtilisKADR1. After 4 weeks of trial, fish were challenged intraperitoneally with Aeromonas hydrophila cell suspension and survival percentage was recorded. Significantly higher post‐challenge survivability was recorded in fish groups fed 10⁸ CFU/g of KADR1 (80.24%; RPS = 75.76%) or immunized with WCPs (77.77%; RPS = 72.73%), compared with the control (18.51%). Analysis of immunological parameters viz. serum lysozyme, phagocytosis, serum total protein, respiratory burst, serum IgM levels, superoxide dismutase and alternative complement pathway activity reflected significant enhancement (p < .05) of immune response in fish fed 10⁶, 10⁸ and 10¹⁰ CFU/g of live cells, or immunized with cellular components of Bacillus subtilisKADR1, with the highest values were observed DII fed group, followed by the group immunized with WCPs. Our results suggest that dietary administration of Bacillus subtilisKADR1 at 10⁸ CFU/g can effectively enhance the immune responses and disease resistance in Labeo rohita against Aeromonas hydrophila.