Transcriptome analysis of Pacific white shrimp (Liptopenaeus vannamei) after exposure to recombinant Vibrio parahaemolyticus PirA and PirB proteins.

Vibrio parahaemolyticus is a Gram-negative bacterium commonly found in marine and estuarine environments and is endemic among the global shrimp aquaculture industry. V. parahaemolyticus proteins PirA and PirB have been determined to be major virulence factors that contribute significantly to the development of acute hepatopancreatic necrosis disease. Our previous work had demonstrated the lethality of recombinant PirA and PirB proteins to Pacific white shrimp (Liptopenaeus vannamei). To understand the host response to these proteins, recombinant PirA and PirB proteins were administered using a reverse gavage method and individual shrimp were then sampled over time. Shrimp hepatopancreas libraries were generated and RNA sequencing was performed on the control and recombinant PirA/B-treated samples. Differentially expressed genes were identified among the assayed time points. Differentially expressed genes that were co-expressed at the later time points (2-, 4- and 6-h) were also identified and gene associations were established to predict functional physiological networks. Our analysis reveals that the recombinant PirA and PirB proteins have likely initiated an early host response involving several cell survival signaling and innate immune processes.

Probiotics enhance resistance to Streptococcus iniae in Nile tilapia (Oreochromis niloticus) reared in biofloc systems.

Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1ß, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.

Toxicity of recombinant PirA and PirB derived from Vibrio parahaemolyticus in shrimp.

Acute hepatopancreatic necrosis disease (AHPND), caused by emerging strains of Vibrio Parahaemolyticus, is of concern in shrimp aquaculture. Secreted proteins PirA and PirB, encoded by a plasmid harbored in V. parahaemolyticus, were determined to be the major virulence factors that induce AHPND. To better understand pathogenesis associated with PirA and PirB, recombinant proteins rPirA and rPirB were produced to evaluate their relative toxicities in shrimp. By challenging shrimp at concentration of 3 µM with reverse gavage method, rPirA and rPirB (approximately 0.4 and 1.5 µg per g of body weight, respectively) caused 27.8 ± 7.8% and 33.3 ± 13.6% mortality, respectively; combination of 3 µM rPirA and rPirB resulted in 88.9 ± 7.9% mortality. Analysis of protein mobility in native gel revealed that rPirB was apparently in the form of monomer while rPirA was oligomerized as an octamer-like macromolecule, suggesting that inter- and intra-molecular interactions between rPirA and rPirB enhanced the toxic effect. An attempt to block or reduce rPirA activity with a putative receptor, N-acetyl-galactosamine, was unsuccessful, implying that remodeling analysis of PirA molecule, such as the octamer observed in this study, is necessary. Results of this study provided new insight into toxic mechanism of PirA and PirB and shall help design strategic antitoxin methods against AHPND in shrimp.

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.

l-rhamnose-binding lectins (RBLs) in Nile tilapia, Oreochromis niloticus: Characterization and expression profiling in mucosal tissues.

Rhamnose-binding lectins (RBLs) are crucial elements associated with innate immune responses to infections and have been characterized from a variety of teleost fishes. Given the importance of RBL in teleost fishes, we sought to study the diversity and expression profiles of RBLs in an important cultured fish, Nile tilapia (Oreochromis niloticus) following experimental infection with Streptococcus agalactiae, a major cause of streptococcosis in farmed tilapia. In this study, four predicted RBL genes were identified from Nile tilapia and were designated as OnRBL3a, OnRBL3b, OnRBL3c, and OnRBL3d. These OnRBLs were composed of two tandem-repeated type five carbohydrate recognition domains (CRDs), classified as type IIIc, and all clustered together phylogenetically. OnRBL-CRDs shared conserved topology of eight cysteine residues, characteristic peptide motifs of -YGR- and -DPC- (or -FGR- and -DTC-), and similar exon/intron organization. OnRBLs had the highest expression in immune-related tissues, gills, intestine or liver. However, the changes of OnRBL expression in the gills and intestine at 2 h, 4 h and 24 h post S. agalactiae challenge were modest, suggesting that tilapia may not mediate the entry or confront the infection of S. agalactiae through induction of RBL genes. The observed expression pattern may be related to the RBL type and CRD composition, S. agalactiae pathogenesis, the accessibility of ligands on the bacterial surface, and/or the species of fish. OnRBLs characterized in this study were the first RBL members identified in Nile tilapia and their characterization will expand our knowledge of RBLs in immunity.

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity.

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)-containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha-D-mannose/alpha-D-glucose >N-acetyl-beta-D-glucosamine >N-acetyl-beta-D-glucosamine/N-acetylneuraminic acid >N-acetyl-D-galactosamine >alpha-D-galactose/N-acetyl-alpha-D-galactosamine >beta-D-galactose = alpha-L-fucose. Virulence studies demonstrated isolate AL-02-36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%-100% versus 60% for isolate ALG-00-530 at equivalent doses (~3 × 106  CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2-3 logs) and survived (28 days) in formulated water-containing catfish mucus. Highly virulent isolate AL-02-36 possessed at least 2.5- to fivefold higher protease activity following growth in mucus than the less virulent ALG-00-530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.

Expression of immune genes in systemic and mucosal immune tissues of channel catfish vaccinated with live theronts of Ichthyophthirius multifiliis.

Ichthyophthiriasis caused by Ichthyophthirius multifiliis (Ich) has a worldwide distribution and affects most freshwater fishes. Fish surviving natural infection and/or immunized with Ich develop strong innate and adaptive immune responses. However, there is a lack of the knowledge regarding immune gene expression patterns in systemic and mucosal immune tissues, and how immune genes interact and lead to innate and adaptive immune protection against Ich infection in fish. The objective of this study was to investigate the expression of innate and adaptive immune-related genes in systemic (liver, spleen) and mucosal (gill, intestine) tissues of channel catfish over time following vaccination with live Ich theronts. The vaccinated fish showed significantly higher antibody titers and survival (95%) than those of mock immunized fish. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 h (h4) to 2 days (d2) post-vaccination in systemic immune tissues. Immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) were more highly upregulated and remained upregulated for longer duration in systemic tissues than in mucosal tissues of the vaccinated fish. The cytokine genes IL-1ßa and IFN-γ were rapidly upregulated in both systemic and mucosal tissues of vaccinated fish, with peak expression from h4 to d1 post-vaccination. Toll-like receptor genes TLR-1 and TLR-9 showed relatively stable upregulation in the gill of immunized fish following vaccination. Results of this study revealed the molecular immune responses in mucosal and systemic tissues of vaccinated fish and demonstrated that Ich vaccination resulted in innate and adaptive immune responses against Ich infection.

Molecular immune response of channel catfish immunized with live theronts of Ichthyophthirius multifiliis.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. The fish surviving natural infections or immunized with live theronts develop strong specific and non-specific immune responses. Little is known about how these immune genes are induced or how they interact and lead to specific immunity against Ichthyophthirius multifiliis in channel catfish Ictalurus punctatus. This study evaluated the differential expression of immune-related genes, including immunoglobulin, immune cell receptor, cytokine, complement factor and toll-like receptors in head kidney from channel catfish at different time points after immunization with live theronts of I. multifiliis. The immunized fish showed significantly higher anti-Ich antibody expressed as immobilization titer and ELISA titer than those of control fish. The vast majority of immunized fish (95%) survived theront challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hour (h4) to 2 days (d2) post immunization. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II ß, TcR-α, and TcR-ß) showed up-regulation from h4 to d6 post immunization, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1ßa, IL-1ßb, IFN-γ and TNF-α) increased rapidly at h4 post immunization and were at an up-regulated level until d2 compared to the bovine serum albumin control. Expression of complement factor and toll-like receptor genes exhibited a rapid increase from h4 to d2 post immunization. Results of this study demonstrated differential expression of genes involved in the specific or non-specific immune response post immunization and that the vaccination against Ich resulted in protection against infection by I. multifiliis.

Whole-Genome Sequencing and Annotation of Aeromonas veronii Isolates from Channel Catfish.

The genomes of seven Aeromonas veronii strains isolated from tissues of healthy or diseased channel catfish obtained from Alabama, USA, fish farms were sequenced and annotated. These genome sequences will enable comparative analyses to determine the roles these bacteria play in catfish aquaculture and the development of new preventative or management strategies.

Genome-wide association study for Streptococcus iniae in Nile tilapia (Oreochromis niloticus) identifies a significant QTL for disease resistance.

Streptococcus iniae is a problematic gram-positive bacterium negatively affecting Nile tilapia (Oreochromis niloticus), one of the main aquacultural species produced worldwide. The aim of this study was to identify the genetic architecture of survival to S. iniae and identify single nucleotide polymorphism (SNPs) linked to quantitative trait loci (QTL) related to survival to S. iniae challenge. With this purpose, Nile tilapia from the Spring Genetics breeding program were sent to a controlled S. iniae challenge test where phenotypes were scored as dead for fish that died during challenge test and survivors for the fish alive at the termination of the test. Additionally, fin-clip samples from all fish in the test were collected for DNA extraction. Out of 1904 fish in the challenge test, tissue samples of 321 fish were sent for genotyping using double digest restriction site associated DNA sequencing (ddRADseq). After quality control and filtering, 9,085 SNPs were used to perform a genome-wide association study (GWAS). A significant signal in LG8 was observed indicating association with survival to S. iniae challenge, with SNPs explaining from 12% to 26% of the genetic variance. To demonstrate the usefulness of marker assisted selection (MAS) to selectively breed fish for survival to S. iniae, offspring of breeding candidates classified as "resistant" and "susceptible" based on haplotypes of the four most significant markers were sent to a controlled S. iniae challenge test. At the end of the test, the differences in mortality between the two groups were strikingly different with a final cumulative percent mortality of less than 1% and 73% for offspring from "resistant" and "susceptible" parents, respectively. These results demonstrate that MAS for improved resistance to S. iniae is feasible.

Global gene expression in channel catfish after vaccination with an attenuated Edwardsiella ictaluri.

To understand the global gene expression in channel catfish after immersion vaccination with an attenuated Edwardsiella ictaluri (AquaVac-ESC™), microarray analysis of 65,182 UniGene transcripts was performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 2, a total of 52 unique transcripts were found to be upregulated in vaccinated fish at 48 h post vaccination, whereas a total of 129 were downregulated. The 52 upregulated transcripts represent genes with putative functions in the following seven major categories: (1) hypothetical (25%); (2) novel (23%); (3) immune response (17%); (4) signal transduction (15%); (5) cell structure (8%); (6) metabolism (4%); and (7) others (8%). The 129 downregulated transcripts represent genes with putative functions in the following ten major categories: (1) novel (25%); (2) immune response (23%); (3) hypothetical (12%); (4) metabolism (10%); (5) signal transduction (7%); (6) protein synthesis (6.2%); (7) cell structure (5%); (8) apoptosis (3%); (9) transcription/translation (2%); and (10) others (6%). Microarray analysis revealed that apolipoprotein A-I was upregulated the most (8.5 fold, P = 0.011) at 48 h post vaccination whereas a novel protein (accession no. CV995854) was downregulated the most (342 fold, P = 0.001). Differential regulation of several randomly selected transcripts in vaccinated fish was also validated by quantitative PCR. Our results suggest that these differentially regulated genes elicited by the vaccination might play important roles in the protection of channel catfish against E. ictaluri.

Effect of Ichthyophthirius multifiliis parasitism on the survival, hematology and bacterial load in channel catfish previously exposed to Edwardsiella ictaluri.

The effect of Ichthyophthirius multifiliis (Ich) parasitism on survival, hematology and bacterial load in channel catfish, Ictalurus punctatus, previously exposed to Edwardsiella ictaluri was studied. Fish were exposed to E. ictaluri 1 day prior to Ich in the following treatments: (1) infected by E. ictaluri and Ich at 2,500 theronts/fish; (2) infected by E. ictaluri only; (3) infected by Ich at 2,500 theronts/fish only; and (4) non infected control. Mortality was significantly higher in fish previously exposed to E. ictaluri and then infected by Ich (71.1 %). Mortalities were 26.7 %, 28.9 % and 0 % for fish infected by E. ictaluri only, by Ich only and non-infected control, respectively. Quantitative polymerase chain reaction demonstrated the presence of E. ictaluri in the brain, gill, kidney and liver of fish infected with E. ictaluri regardless of Ich parasitism. At day 8, E. ictaluri parasitized fish had significantly more bacteria present in the brain, gill and liver, with no bacteria detected in these organs in the E. ictaluri-only treatment, suggesting that the bacteria persisted longer in parasitized fish. Decreased red blood cells count and hematocrit in fish at days 8 and 19 after co-infection suggests chronic anemia. Lymphocyte numbers significantly decreased in all infected treatments versus the non-infected controls at days 2, 8 and 19. Lymphopenia suggests that lymphocytes were actively involved in the immune response. Bacterial clearance was probably influenced by the stress of parasitism and/or the mucosal response induced by ectoparasitic Ich that resulted in the higher mortality seen in the co-infected treatment.

Efficacy of a modified live Flavobacterium columnare vaccine in fish.

Flavobacterium columnare is an aquatic bacterium that is responsible for columnaris disease. This aquatic pathogen has a worldwide distribution and is highly infectious to both warm and cold water fish. A modified live F. columnare vaccine was developed by repeated passage of a virulent strain on increasing concentrations of rifampicin that resulted in attenuation. Here we report vaccination/challenge trials to evaluate efficacy and safety. In separate laboratory trials, immersion vaccination of channel catfish (Ictalurus punctatus) fry between 10 to 48 days post hatch (DPH) with experimental vaccine or licensed product resulted in relative percent survival (RPS) between 57-94% following challenge. Similarly, a vaccination/challenge trial using largemouth bass (Micropterus salmoides) fry at 10 DPH was performed using various doses of licensed product under laboratory conditions. Results demonstrated safety of the vaccine and significant protection following challenge with RPS values between 74-94%, depending on vaccine dose. Together, these trials demonstrate the vaccine administered to early life-stage channel catfish and largemouth bass is safe and reduces mortality following challenge with F. columnare.

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine.

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.

Proteome analysis of virulent Aeromonas hydrophila reveals the upregulation of iron acquisition systems in the presence of a xenosiderophore.

The Gram-negative bacterium, Aeromonas hydrophila, has been responsible for extensive losses in the catfish industry for over a decade. Due to this impact, there are ongoing efforts to understand the basic mechanisms that contribute to virulent A. hydrophila (vAh) outbreaks. Recent challenge models demonstrated that vAh cultured in the presence of the iron chelating agent deferoxamine mesylate (DFO) were more virulent to channel catfish (Ictalurus punctatus). Interestingly, differential gene expression of select iron acquisition genes was unremarkable between DFO and non-DFO cultures, posing the question: why the increased virulence? The current work sought to evaluate growth characteristics and protein expression of vAh after the addition of DFO. A comparative proteome analysis revealed differentially expressed proteins among tryptic soy broth (TSB) and TSB + DFO treatments. Upregulated proteins identified among the TSB + DFO treatment were enriched for gene ontology groups including iron ion transport, siderophore transport and siderophore uptake transport, all iron acquisition pathways. Protein-protein interactions were also evaluated among the differentially expressed proteins and predicted that many of the upregulated iron acquisition proteins likely form functional physiological networks. The proteome analysis of the vAh reveals valuable information about the basic biological processes likely leading to increased virulence during iron restriction in this organism.

Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis.

Two trials were conducted to determine the effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis Fouquet (Ich). In trial I, catfish were immunized intraperitoneally (IP) with: 1) 1% formalin-inactivated trophonts, 2) 3% formalin-inactivated trophonts and 3) freeze-thawed trophonts. Positive and negative control catfish were immunized with live theronts and 5% bovine serum albumin (BSA), respectively. At day 14, 28 and 50 post-immunizations, no statistical difference was noted in serum or cutaneous anti-Ich antibody titers to formalin-inactivated trophonts or freeze-thawed trophonts. The survival of catfish challenged with live theronts ranged from 33.3% to 43.3% for the formalin-inactivated or freeze-thawed trophonts at 50 d post-immunization. The survival of catfish immunized with live theront and BSA was 93.3 and 0%, respectively. In trial II, catfish were IP immunized with sonicated trophonts at doses of 1) 5 trophonts or 10.2 microg protein g(-1) fish, 2) 10 trophonts or 20.4 microg protein g(-1) fish, 3) 20 trophonts or 40.8 microg protein g(-1) fish, and 4) 5% BSA as the control. Fish immunized with 10 or 20 trophonts g(-1) fish showed highest serum (1/210 to 1/480) and cutaneous antibody titers (1/48 to 1/52), respectively, at 22 d post-immunization and survival (63.3-60.0%). The fish immunized with 5 trophonts g(-1) fish had titers of 1/52 and 1/12 for serum and cutaneous antibody and survival of 23.3%. BSA immunized catfish had background titers and a survival of 6.7%. There was a significant correlation between doses of sonicated trophonts used to immunize and catfish survival (correlation coefficient = 0.859, p < 0.01). These results indicate that doses of sonicated trophonts, but not formalin-inactivated or freeze-thawed trophonts provided both serum and cutaneous antibody responses and survival to live trophont challenge.

In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri.

Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from -0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly (p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly (p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.

Enhanced mortality in Nile tilapia Oreochromis niloticus following coinfections with ichthyophthiriasis and streptococcosis.

Ichthyophthirius multifiliis Fouquet (Ich) and Streptococcus iniae are 2 major pathogens of cultured Nile tilapia Oreochromis niloticus (L). Currently there is no information available for the effect of coinfection by Ich and S. iniae on fish. The objective of this study was to determine the effects of parasite load and Ich development size on fish mortality following S. iniae infection. Low mortality (< or =20%) was observed in tilapia exposed to Ich or S. iniae alone. Mortalities increased from 38% in tilapia exposed to Ich at 10,000 theronts fish(-1) to 88% in fish at 20,000 theronts fish(-1) following S. iniae exposure. The median days to death were significantly fewer (7 d) in fish exposed to Ich at 20,000 theronts fish(-1) than fish exposed to 10,000 theronts fish(-1) (10 d). A positive correlation (correlation coefficient = 0.83) was noted between tilapia mortality and size of Ich trophonts at the time of S. iniae challenge. Fish parasitized with well-developed trophonts (Day 4, 2 x 10(7) microm3 in volume) suffered higher mortality (47.5%) than fish (10.0%) infested by young trophonts (Hour 4, 1.3 x 10(4) microm3 in volume) after S. iniae challenge. The results of this study demonstrated that both parasite load and trophont size increased susceptibility and mortality of tilapia to S. iniae infection.

Flavobacterium columnare chemotaxis to channel catfish mucus.

Flavobacterium columnare is a Gram-negative pathogen of many species of wild and cultured fish. Isolates from diseased channel catfish belong to either genomovar I or II. Genomovar II isolates were found to be more virulent than genomovar I isolates. The objective of the present study was to determine whether differences exist in the chemotactic response of these genomovars to mucus obtained from the skin, gills and intestines of healthy channel catfish using the capillary chemotaxis assay. Mucus from the skin and gill induced a greater chemotactic response by F. columnare than mucus from the intestine. Sixty percent of mucus from the skin of individual catfish yielded a positive chemotactic response from F. columnare. Finally, skin mucus induced a greater chemotactic response in genomovar II F. columnare than in genomovar I F. columnare isolates. The data indicate that mucus from channel catfish results in a chemotactic response by F. columnare. This positive chemotactic response may be an important first step for F. columnare colonization of channel catfish skin or gills. Although the role that chemotaxis plays in the virulence of F. columnare is not fully defined, the chemotactic response of genomovar ll isolates suggests that chemotaxis is associated with virulence.