Haitham Mohammed

Characterization of the gut microbiota of three commercially valuable warmwater fish species

Due to the strong influence of the gut microbiota on fish health, dominant bacterial species in the gut are strong candidates for probiotics. This study aimed to characterize the gut microbiota of channel catfish Ictalurus punctatus, largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus to provide a baseline for future probiotic studies.

New attenuated vaccine against columnaris disease in fish: Choosing the right parental strain is critical for vaccine efficacy

Flavobacterium columnare, the causative agent of columnaris disease, is a highly diverse species comprised by three genomovars. Genomovar II strains are more virulent toward catfishes than genomovar I isolates. The objective of this study was to compare the vaccine efficacy of avirulent mutants derived from genomovars I and II using a rifampicin-resistance strategy. First, we compared the efficacy of 13 genomovar II mutants in channel catfish (Ictalurus punctatus) fingerlings and identified mutant 17-23 as the best vaccine candidate based on their relative percent survival (RPS) against a highly virulent genomovar II strain (BGFS-27). In the second experiment, we vaccinated zebrafish (Danio rerio) with two genomovar II mutants (17-23 and 16-534) and FCRR (genomovar I mutant) followed by exposure to BGFS-27 strain. RPS values were 28.4, 20.3 and 8.1% for 17-23, 16-534, and FCRR, respectively. For experiments 3 and 4, we tested both 17-23 and FCRR in channel catfish fry and Nile tilapia (Oreochromis niloticus). In both experiments, vaccinated fish were divided in two groups and each challenged with either a genomovar I (ARS-1) or a II (BGFS-27) strain. Channel catfish fry vaccinated with 17-23 and FCRR followed by challenge with BGFS-27 resulted in RPS values of 37.0% and 4.4%. When fish were challenged with ARS-1, RPS values were 90.9% and 72.7% for fish vaccinated with 17-23 and FCRR, respectively. Nile tilapia vaccinated with 17-23 and FCRR followed by challenged with BGFS-27 had RPS values of 82.1% and 16.1%, respectively. When fish were challenged with strain ARS-1, RPS values were 86.9% and 75.5%. Overall, our results demonstrated that vaccination with genomovar II mutant 17-23 confers better protection in channel catfish and Nile tilapia than FCRR against columnaris disease caused by genomovar II. Both mutants were equally protective against columnaris caused by genomovar I showing that 17-23 mutant cross-protected against both genomovars.

Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes

This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20 °C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis.

Epidemiology of columnaris disease affecting fishes within the same watershed

In the southeastern USA, columnaris disease (caused by Flavobacterium columnare) typically affects catfish raised in earthen ponds from early spring until late summer. Recently, unusually severe outbreaks of columnaris disease occurred at the E. W. Shell Fisheries Center located in Auburn, AL, USA. During these outbreaks, catfish and other aquaculture and sport fish species that were in ponds located within the same watershed were affected. Our objective was to investigate the genetic diversity among F. columnare isolates recovered from different sites, sources, and dates to clarify the origin of these outbreaks and, ultimately, to better understand the epidemiology of columnaris disease. A total of 102 F. columnare isolates were recovered from catfishes (channel catfish Ictalurus puntactus, blue catfish I. furcatus, and their hybrid), bluegill Lepomis microchirus, Nile tilapia Oreochromis niloticus, largemouth bass Micropterus salmoides, egg masses, and water during columnaris outbreaks (from spring 2010 to summer 2012). Putative F. columnare colonies were identified following standard protocols. All isolates were ascribed to Genomovar II following restriction fragment length polymorphism analysis of the 16S rRNA gene. Genetic variability among the isolates was revealed by amplified fragment length polymorphism. Date of isolation explained most of the variability among our isolates, while host was the least influential parameter, denoting a lack of host specificity within Genomovar II isolates. The susceptibility of each of the isolates against commonly used antibiotics was tested by antibiogram. Our data showed that 19.6 and 12.7% of the isolates were resistant to oxytetracycline and kanamycin, respectively.

Potassium permanganate elicits a shift of the external fish microbiome and increases host susceptibility to columnaris disease

The external microbiome of fish is thought to benefit the host by hindering the invasion of opportunistic pathogens and/or stimulating the immune system. Disruption of those microbial communities could increase susceptibility to diseases. Traditional aquaculture practices include the use of potent surface-acting disinfectants such as potassium permanganate (PP, KMnO4) to treat external infections. This study evaluated the effect of PP on the external microbiome of channel catfish and investigated if dysbiosis leads to an increase in disease susceptibility. Columnaris disease, caused by Flavobacterium columnare, was used as disease model. Four treatments were compared in the study: (I) negative control (not treated with PP nor challenged with F. columnare), (II) treated but not challenged, (III) not treated but challenged, and (IV) treated and challenged. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze changes in the external microbiome during the experiment. Exposure to PP significantly disturbed the external microbiomes and increased catfish mortality following the experimental challenge. Analysis of similarities of RISA profiles showed statistically significant changes in the skin and gill microbiomes based on treatment and sampling time. Characterization of the microbiomes using 16S rRNA gene pyrosequencing confirmed the disruption of the skin microbiome by PP at different phylogenetic levels. Loss of diversity occurred during the study, even in the control group, but was more noticeable in fish subjected to PP than in those challenged with F. columnare. Fish treated with PP and challenged with the pathogen exhibited the least diverse microbiome at the end of the study.

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum. The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA-DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.

First record of the fish pathogen Flavobacterium columnare genomovar II from bluegill, Lepomis macrochirus (Rafinesque), with observations on associated lesions

Flavobacterium columnare is the causative agent of ‘columnaris disease’ (Davis 1922), a disease that kills or debilitates a wide ecological and phylogenetic spectrum of temperate and tropical freshwater fish (Bernardet 1989; Bernardet & Grimont 1989; Bernardet et al. 1996; Olivares-Fuster et al. 2007; Bullard, McElwain & Arias 2011). It is regarded as a predominant pathogen of fish maintained in both food production aquaculture systems and the ornamental fish trade (Decostere et al. 1999a,b; Schneck & Caslake 2006), and it is of special interest in warm-water aquaculture ponds in the southeastern US, where it is regarded as one of the primary bacterial pathogens that constrains culture of channel catfish, Ictalurus punctatus (Rafinesque) (see Thune, Stanley & Cooper 1993; Plumb 1999). Much of the biological information about this bacterium derives from aquaculture epizootics (Olivares-Fuster et al. 2007, 2011), wherein columnaris disease is marked by epidermal sloughing and scale loss concomitant with a symmetrical body discolouration that has been called ‘saddleback’ lesion (Cone, Miller & Austin 1980; Bullard et al. 2011). The 3 genomovars (Ursing et al. 1995) of F. columnare (see Triyanto & Wakabayashi 1999) exhibit differential virulence (Arias et al. 2004; ThomasJinu & Goodwin 2004; Darwish & Ismaiel 2005; Shoemaker et al. 2008), but information on their prevalence, host distribution and pathobiology is largely indeterminate. Bluegill, Lepomis macrochirus Rafinesque, is a widely introduced centrarchid that ranges throughout the Mississippi River and adjacent basins in North America. This fish was co-introduced as a forage fish for the recreationally prized largemouth bass, Micropterus salmoides (Lacepede), and its populations thrive along with those of largemouth bass in lakes, ponds and creek pools as well as densely vegetated shorelines of impoundments (Boschung & Mayden 2004). Bluegill is a generalist omnivore that tolerates a wide range of salinities and thermal regimes, which has contributed to its high level of invasiveness: now a documented invasive species in >20 countries (Kawamura et al. 2006; Fuller, Nico & Williams 1999). During 19–23 April 2012, a significant bluegill kill occurred throughout a series of hydrologically linked earthen ponds located in east-central Alabama (32°40′51.89′′N; 85°30′39.73′′W). Moribund bluegill were sampled for bacterial analysis with a sterile loop and cultured on Shieh agar supplemented with tobramycin at a concentration of 1 lg mL 1 (Decostere, Haesebrouck & Devriese 1997) at 28 °C for 48 h. Rhizoid, yellowpigmented colonies were considered putative F. columnare cultures and were re-isolated on Shieh agar lacking antibiotic. Pure cultures were incubated in Shieh broth at 28 °C with shaking Correspondence S A Bullard Department of Fisheries and Allied Aquacultures, 203 Swingle Hall, Auburn University, Auburn, Alabama 36849, USA (e-mail: ash.bullard@auburn. edu)

More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare.

Abstract A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post‐vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF‐27) and gill tissues collected pre‐challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host‐pathogen interactions. Following RNA‐sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast‐like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100‐fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro‐inflammatory cytokines (IL‐1b, IL‐8, IL‐17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge. HighlightsVaccinated catfish had a higher basal expression of many preformed mediators.Proteases and neuropeptide level in vaccinated fish fell rapidly following challenge.Collagen deposition and tissue remodeling processes were induced in vaccinated fish.Pro‐inflammatory genes were upregulated in unvaccinated fish post challenge.

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

ABSTRACT 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. HighlightsFour predicted RBL genes were identified from Nile tilapia.OnRBLs are type IIIc with two type five carbohydrate recognition domains.Changes of OnRBL expression post Streptococcus agalactiae infection were modest.

The effects of dietary inclusion of a Saccharomyces cerevisiae fermentation product in a commercial catfish ration on growth, immune readiness, and columnaris disease susceptibility

ABSTRACT We evaluated a Saccharomyces cerevisiae fermentation product (Diamond V Original XPC) in hybrid catfish (Ictalurus furcatus x I. punctatus) for its potential effects on growth, blood parameters, and disease resistance. The trial featured four levels of inclusion that were added to a commercial 32% protein floating catfish ration. Following six weeks of feeding, we observed marginally heightened resistance to columnaris disease and saw significant changes in the levels of immune effectors in the serum, including lysozyme, complement, and immunoglobulin.