Human iPSC-derived renal organoids engineered to report oxidative stress can predict drug-induced toxicity.

Advances in regenerative medicine have led to the construction of many types of organoids, which reproduce important aspects of endogenous organs but may be limited or disorganized in nature. While their usefulness for restoring function remains unclear, they have undoubted usefulness in research, diagnostics, and toxicology. In toxicology, there is an urgent need for better models for human kidneys. We used human iPS-cell (hiPSC)-derived renal organoids to identify HMOX1 as a useful marker of toxic stress via the oxidative stress pathway, and then constructed an HMOX1 reporter in hiPSCs. We used two forms of hiPSC-derived HMOX1-reporter renal organoids to probe their ability to detect nephrotoxicants in a panel of blind-coded compounds. Our results highlight the potential usefulness, and some limitations, of HMOX1-reporter renal organoids as screening tools. The results may guide development of similar stress-reporting organoid assays for other stem-cell-derived organs and tissues.

The role of denitrification genes in anaerobic growth and virulence of Flavobacterium columnare.

AIMS: Comparative genomics analyses indicated that the Flavobacterium columnare genome has unique denitrification genes relative to Flavobacterium psychrophilum and Flavobacterium johnsoniae, including nasA (nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase) and nosZ (nitrous oxide reductase). The current study determines the roles of nasA, nirS, norB and nosZ in anaerobic growth, nitrate reduction, biofilm formation and virulence. METHODS AND RESULTS: Four in-frame deletion mutants in virulent F. columnare strain 94-081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94-081, FcΔnasA,FcΔnirS and FcΔnosZ mutants did not grow as well anaerobically, whereas the growth of FcΔnorB strain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions in FcΔnasA, FcΔnirS and FcΔnosZ compared to parent strain 94-081. Under anaerobic conditions, Fc∆nasA, Fc∆norB and Fc∆nosZ formed significantly less biofilm than the wild type strain at 24 and 96 h, but FcΔnirS was not significantly affected. The nitrite reductase mutant FcΔnirS was highly attenuated in catfish, whereas FcΔnasA, FcΔnorB and FcΔnosZ had similar virulence to FcWT. CONCLUSIONS: These results show, for the first time, that denitrification genes enable F. columnare to grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes to F. columnare virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate potential for F. columnare to grow in nitrate-rich anaerobic zones in catfish production ponds, and they suggest that a Fc∆nirS strain could be useful as a safe live vaccine if it protects catfish against columnaris disease.

Construction and evaluation of type III secretion system mutants of the catfish pathogen Edwardsiella piscicida.

Catfish is the largest aquaculture industry in the United States. Edwardsiellosis is considered one of the most significant problems affecting this industry. Edwardsiella piscicida is a newly described species within the genus Edwardsiella, and it was previously classified as Edwardsiella tarda. It causes gastrointestinal septicaemia, primarily in summer months, in farmed channel catfish in the south-eastern United States. In the current study, we adapted gene deletion methods used for Edwardsiella to E. piscicida strain C07-087, which was isolated from a disease outbreak in a catfish production pond. Four genes encoding structural proteins in the type III secretion system (T3SS) apparatus of E. piscicida were deleted by homologous recombination and allelic exchange to produce in-frame deletion mutants (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT). The mutants were phenotypically characterized, and virulence and vaccine efficacy were evaluated. Three of the mutants, EpΔssaV, EpΔyscR and EpΔesaM, were significantly attenuated compared to the parent strain (p < .05), but EpΔescT strain was not. Vaccination of catfish with the four mutant strains (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT) provided significant protection when subsequently challenged with wild-type strain. In conclusion, we report methods for gene deletion in E. piscicida and development of vaccine candidates derived from a virulent catfish isolate.

Identification of high-risk Listeria monocytogenes serotypes in lineage I (serotype 1/2a, 1/2c, 3a and 3c) using multiplex PCR.

AIMS: Using molecular subtyping techniques, Listeria monocytogenes is divided into three major phylogenetic lineages, and a multiplex PCR method can differentiate five L. monocytogenes subgroups: 1/2a-3a, 1/2c-3c, 1/2b-3b-7, 4b-4d-4e and 4a-4c. In this study, we conducted genome comparisons and evaluated serotype-associated genes for their utility as a multiplex PCR-based method for distinguishing high-risk serotypes 1/2a and 1/2c in lineage I from low-risk serotypes 3a and 3c. METHODS AND RESULTS: Primer sets were developed that are specific for serotype 1/2c (LMOSLCC2372_0308) and serotype 3a (LMLG_0742). These primers were then tested in a multiplex format with primers specific for serotype 1/2a (flaA) to separate serotypes 1/2a, 1/2c, 3a and 3c using 25 strains of lineage I L. monocytogenes. CONCLUSIONS: Here, for the first time, we report primers specific for L. monocytogenes serotype 1/2c and serotype 3a, and we demonstrate a multiplex PCR method for separating the four serotypes of lineage I L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The described multiplex PCR assay consistently showed successful separation of 1/2a and 1/2c strains from 3a and 3c strains. PCR is routinely performed in many diagnostic and epidemiologic investigations for L. monocytogenes, and these primers should increase the feasibility and accessibility of L. monocytogenes serotyping.

Effect of multiple mutations in tricarboxylic acid cycle and one-carbon metabolism pathways on Edwardsiella ictaluri pathogenesis.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC). We have shown recently that tricarboxylic acid cycle (TCA) and one-carbon (C1) metabolism are involved in E. ictaluri pathogenesis. However, the effect of multiple mutations in these pathways is unknown. Here, we report four novel E. ictaluri mutants carrying double gene mutations in TCA cycle (EiΔmdhΔsdhC, EiΔfrdAΔsdhC), C1 metabolism (EiΔglyAΔgcvP), and both TCA and C1 metabolism pathways (EiΔgcvPΔsdhC). In-frame gene deletions were constructed by allelic exchange and mutants' virulence and vaccine efficacy were evaluated using in vivo bioluminescence imaging (BLI) as well as end point mortality counts in catfish fingerlings. Results indicated that all the double gene mutants were attenuated compared to wild-type (wt) E. ictaluri. There was a 1.39-fold average reduction in bioluminescence, and hence bacterial numbers, from all the mutants except for EiΔfrdAΔsdhC at 144 h post-infection. Vaccination with mutants was very effective in protecting channel catfish against subsequent infection with virulent E. ictaluri 93-146 strain. In particular, immersion vaccination resulted in complete protection. Our results provide further evidence on the importance of TCA and C1 metabolism pathways in bacterial pathogenesis.

Genome sequence of Edwardsiella ictaluri 93-146, a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish.

Edwardsiella ictaluri is the cause of extensive mortalities and economic losses to the channel catfish industry of the southeast United States. Here we report the complete genome of Edwardsiella ictaluri 93-146. Whole-genome sequence analysis of E. ictaluri provides a tool for understanding the genomic regions specific to the species and the Edwardsiella genus.

Verification of an Edwardsiella ictaluri-specific diagnostic PCR.

AIMS: To verify the specificity of a PCR assay for the identification and diagnosis of Edwardsiella ictaluri. METHODS AND RESULTS: An Edwardsiella ictaluri-specific PCR assay was developed utilizing two features of the ribosomal DNA gene clusters. The first feature is the presence of two ribosomal gene clusters located in tandem to one another (the inter-ribosomal spacer, IRS). This characteristic is present in the Edwardsiella genus but absent in the other sequenced members of the Enterobacteriaceae. The second feature is the presence of an intervening sequence (IVS) in the 23S rRNA gene of Edw. ictaluri. To verify the specificity of this assay, we tested genomic DNA from a variety of bacterial species. The IVS/IRS PCR assay results in an c. 2000-bp product from all Edw. ictaluri isolates tested, but not from any other species including Edwardsiella tarda. CONCLUSIONS: The IVS/IRS PCR assay is highly specific for Edw. ictaluri and useful as a tool for identifying this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This research verifies the specificity of PCR-based assay for Edw. Ictaluri, and we describe this assay as a highly versatile diagnostic tool for its identification.

Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri.

AIMS: To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons. METHODS AND RESULTS: The Edw. ictaluri rrn operons were identified from a 5-7 kbp insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences and analysis of all eight Edw. ictaluri rrn operons and unique regions located upstream and downstream. Two rrn operons were located in tandem with 169 bp separating them, which is apparently a conserved feature between Edw. ictaluri and Edwardsiella tarda. I-CeuI enzyme digestion of Edw. ictaluri genomic DNA and analysis by pulsed field gel electrophoresis indicated that rrn operon number and chromosomal locations are conserved within the species Edw. ictaluri. CONCLUSIONS: The rrn operons of Edw. ictaluri have similar structure and flanking regions compared with other members of the family Enterobacteriaceae; however, the presence of eight copies of the rrn operon makes Edw. ictaluri unique within the family. SIGNIFICANCE AND IMPACT OF THE STUDY: This research clarifies previous phylogenetic analyses of Edw. ictaluri and provides support for the Edw. ictaluri genome sequencing project. In addition, we identified a unique feature of two rrn operons that shows potential for the development of a diagnostic PCR method.

Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus).

AIM: To develop a method for conducting pulsed-field gel electrophoresis (PFGE) on Flavobacterium columnare, to use PFGE to characterize F. columnare channel catfish isolates, and to determine whether variation in pathogenic potential exists in F. columnare isolates from channel catfish. METHODS AND RESULTS: On the basis of PFGE-derived profiles, similarity dendrograms constructed for more than 30 F. columnare isolates showed two major genetic groups with more than 60% similarity. Channel catfish fingerlings challenged with PFGE group A isolates by bath immersion had significantly higher average mortalities (>60%) than fish challenged with PFGE group B isolates (<9%). However, abrasion and skin mucus removal made channel catfish fingerlings susceptible to disease caused by group B isolates following immersion exposure. CONCLUSION: Our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, and that isolates in PFGE group A isolates tend to be more pathogenic to immunocompetent channel catfish fingerlings than PFGE group B isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE is a potentially useful tool for determining whether F. columnare isolates are more likely to be primary or secondary pathogens. Pathogenesis research for columnaris disease in catfish should focus on pathogenic isolates from PFGE group A.

Use of a putative transcriptional regulator gene as target for specific identification of Staphylococcus epidermidis.

AIMS: To investigate the use of a Staphylococcus epidermidis transcriptional regulator gene as target for species-specific determination. METHODS AND RESULTS: Staph. epidermidis genes encoding putative transcriptional regulators were retrieved from GenBank and those showing no homology with other bacterial sequences were selected. Of the four PCR primer sets analysed, the primers Serp0107F/R from serp0107 amplified a specific product of 581 bp from Staph. epidermidis DNA only, and they did not cross-react in PCR with nonepidermidis staphylococci and other common bacteria. CONCLUSION: Being uniquely present in Staph. epidermidis, putative transcriptional regulator gene serp0107 offers a valuable target for specific identification of Staph. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: As a member of a specialized gene group, putative transcriptional regulator gene serp0107 may be important to Staph. epidermidis adaptation to its niche environment. Further analysis of serp0107 and its related protein may help reveal new insights on the molecular regulation of Staph. epidermidis survival and virulence.

Identification and characterization of a two-component hemolysin from Edwardsiella ictaluri.

The channel catfish pathogen Edwardsiella ictaluri possesses hemolysin activity, and strains that are adapted for growth in fish tend to have greater hemolysin activity than strains that are adapted for in vitro growth conditions. To investigate its potential role in virulence, an isogenic hemolysin mutant strain of E. ictaluri R4383 was constructed by transposon mutagenesis. Sequencing of the chromosomal insertion site identified two genes, designated eihA and eihB, that encode proteins with homology to the Serratia family of two-component hemolysins. EihB is similar to the secretion/activation proteins from this family, and EihA is similar to the cytolysin proteins from this family. Bacterial challenge in channel catfish fingerlings did not show a significant difference in virulence between the wild type E. ictaluri strain and the hemolysin deficient E. ictaluri mutant strain.

Evaluation of PCR primers from putative transcriptional regulator genes for identification of Staphylococcus aureus.

AIMS: To examine if PCR primers derived from putative transcriptional regulator genes can be useful for Staphylococcus aureus identification. METHODS AND RESULTS: Staphylococcus aureus gene sequences that encode transcriptional regulators were retrieved from GenBank and compared with other DNA sequences via BLAST searches. Two uniquely present, putative transcriptional regulator genes (i.e. Sa0836 and Sa0856) were selected as a consequence and PCR primers (Sa0836F/R and Sa0856F/R) were then designed from these genes for evaluation. A total of 84 bacterial strains/isolates including 23 Staph. aureus, 18 nonaureus Staphylococcus and 43 other common bacterial isolates were examined. The results indicated that PCR primers from Sa0836 and Sa0856 recognized genomic DNA from Staph. aureus only, but not from other non-aureus Staphylococcus or common bacteria. CONCLUSIONS: PCR detection of the putative transcriptional regulator genes Sa0836 and Sa0856 represents a useful means of identifying Staph. aureus from other bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of species-species transcriptional regulator genes may be a common phenomenon in bacteria. Besides their value as novel diagnostic markers, further investigation on the putative transcriptional regulator genes Sa0836 and Sa0856 and their related products may shed light on the molecular mechanisms of Staph. aureus adaptation and virulence.

Identification of a gene encoding a putative phosphotransferase system enzyme IIBC in Listeria welshimeri and its application for diagnostic PCR.

AIMS: To identify a Listeria welshimeri-specific gene that can be used for identification of this species by PCR. METHODS AND RESULTS: Through comparative analysis of genomic DNA from Listeria species using dot blot hybridization, an L. welshimeri-specific clone was isolated that contained a gene segment whose translated protein sequence is similar to enzyme IIBC from phosphotransferase systems in other bacteria. Using oligonucleotide primers derived from this L. welshimeri-specific clone, a 608-bp fragment was amplified from L. welshimeri genomic DNA and not from other Listeria species or other Gram-negative and Gram-positive species. CONCLUSION AND SIGNIFICANCE: The PCR employing L. welshimeri-specific primers shows promise as a useful method for differentiating L. welshimeri from other Listeria species and related bacteria.

Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes sustained constriction of the pulmonary artery and modulates normal vascular tone. Endothelial cells were thought to be the major source of ET-1, but recent studies show that vascular smooth muscle cells (SMCs) are also capable of its synthesis. We examined the ET-1 and endothelin-converting enzyme-1 (ECE-1) system in cells cultured from two adjacent layers, subendothelial (L1) and inner medial (L2), of normal sheep main pulmonary artery and the response of this system to exogenous ET-1 and transforming growth factor-beta1 (TGF-beta1). End points include assessment of preproET-1 (ppET-1) and ECE-1 gene coexpression, measurement of intracellular and released ET-1, and ECE-1 activity. RT-PCR analysis revealed that ppET-1 and ECE-1 transcripts were greater in L1 than in L2 cells. The L1 cells also synthesized (L1, 3.2 +/- 0.1; L2, 1.2 +/- 0.1 fmol/10(6) cells) and released (L1, 9.2 +/- 0.5; L2, 2.3 +/-0.1 fmol/ml) greater amounts of ET-1 than L2 cells. The L2 cells internalized exogenous ET-1 in a dose-dependent manner (EC(50) 8 nmol/l) and were more responsive to exogenous ET-1 than L1 cells, showing upregulation of both the ppET-1 and ECE genes. TGF-beta1 downregulated ET-1-stimulated ppET-1 and ECE-1 transcripts but only in L2 cells. In addition, L1 cells showed greater ECE-1 activity than L2 cells, and in both, the activity was sensitive to the metalloprotease inhibitor phosphoramidon. We conclude that the ET-1 system in L1 and L2 cells is distinct. The data suggest that the two cell types have diverse functions in the arterial wall; the L1 cells, like endothelial cells, provide a local source of ET-1; and since the L2 cells are more responsive to exogenous ET-1, they are likely to affect normal pulmonary vascular tone.

Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a: concomitant expression of serotype 5a and 1 capsular polysaccharides in recombinant A. pleuropneumoniae serotype 1.

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.

Regional variability in preproEndothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension. Participation Of arterial smooth muscle cells.

We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.

Attenuation, persistence, and vaccine potential of an Edwardsiella ictaluri purA mutant.

In this study, an adenine-auxotrophic strain of Edwardsiella ictaluri was constructed and its virulence, tissue persistence, and vaccine efficacy were evaluated. A clone containing the purA gene was isolated from an E. ictaluri genomic library, sequenced, and shown to have an overall sequence identity of 79.3% at the nucleotide level and 85.7% at the amino acid level with the Escherichia coli purA gene. The cloned E. ictaluri purA gene was mutated by deleting a 598-bp segment of the gene and inserting the kanamycin resistance gene from Tn903 into the gap. The delta purA::Km(r) gene was subcloned into the suicide plasmid pGP704, and the resulting plasmid was used to deliver the modified gene into a virulent strain of E. ictaluri by conjugation. Homologous recombination replaced the chromosomal purA gene with the mutated gene to create an adenine-auxotrophic strain (LSU-E2). Compared to wild-type E. ictaluri, LSU-E2 was highly attenuated by the injection, immersion, and oral routes of exposure. By the injection route, LSU-E2 had a 50% lethal dose (LD50) that was greater than 5 logs10 higher than the LD50 for wild-type E. ictaluri. In a tissue persistence study, LSU-E2 was able to invade channel catfish by the immersion route and persist in internal organs for at least 48 h. Channel catfish that were vaccinated with a single immersion dose of LSU-E2 had mortality significantly lower (P < 0.01) following a wild-type E. ictaluri challenge than that of nonvaccinated fish.

Immunization with bacterial antigens: edwardsiellosis.

Enteric septicaemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most serious disease affecting commercial catfish culture in the United States. ESC is generally an acute septicaemia that develops very quickly, especially in the temperature range of 22-28 degrees C, with a more chronic disease presentation outside this range. The ability of E. ictaluri to avoid the host's immune system and proliferate into a systemic infection is impressive. Catfish kidney tissue cultured positive for E. ictaluri as soon as 15 minutes following gastric lavage and signs of disease are observed microscopically within two days of immersion challenge, with reported mortalities as early as five days following immersion challenge. Analysis of E. ictaluri antigens by several investigators using SDS-PAGE and colorimetric western blotting with immune catfish has identified as many as 15 immunogenic bands. Analysis using two-dimensional SDS-PAGE and chemiluminescent western blotting identified 14 bands and 25 spots as consistently immunogenic. The strongest immunodominant antigens were reported as 34-37 KD and 60 KD, depending on the study. Lipopolysaccharide is the only purified component of E. ictaluri tested for fish vaccination, and results indicated that very poor protection was induced unless Freund's Complete Adjuvant was used. Because E. ictaluri strains are serologically homogeneous, most studies on vaccination have emphasized killed whole cell preparations and have delivered equivocal results. Although antibodies are produced to a variety of preparations, a positive antibody response does not correlate with protection unless very high titres are achieved. Efficacy of killed products has been demonstrated in field trials, and an orally delivered product has been licensed. However, protection probably relies on booster exposure of the host to E. ictaluri during non-permissive temperatures. As a facultative intracellular pathogen, further studies on vaccination of catfish against E. ictaluri should target products and delivery methods that favour induction of cell mediated immunity.