Expression profiles of four Nile Tilapia innate immune genes during early stages of Aeromonas veronii infection.

OBJECTIVE: During Egypt's hot summer season, Aeromonas veronii infection causes catastrophic mortality on Nile Tilapia Oreochromis niloticus farms. Egypt is ranked first in aquaculture production in Africa, sixth in aquaculture production worldwide, and third in global tilapia production. This study aimed to investigate, at the molecular level, the early innate immune responses of Nile Tilapia to experimental A. veronii infection. METHODS: The relative gene expression, co-expression clustering, and correlation of four selected immune genes were studied by quantitative real-time polymerase chain reaction in four organs (spleen, liver, gills, and intestine) for up to 72 h after a waterborne A. veronii challenge. The four genes studied were nucleotide-binding oligomerization domain 1 (NOD1), lipopolysaccharide-binding protein (LBP), natural killer-lysin (NKL), and interleukin-1 beta (IL-1ß). RESULT: The four genes showed significant transcriptional upregulation in response to infection. At 72 h postchallenge, the highest NOD1 and IL-1ß expression levels were recorded in the spleen, whereas the highest LBP and NKL expression levels were found in the gills. Pairwise distances of the data points and the hierarchical relationship showed that NOD1 clustered with IL-1ß, whereas LBP clustered with NKL; both genes within each cluster showed a significant positive expression correlation. Tissue clustering indicated that the responses of only the gill and intestine exhibited a significant positive correlation. CONCLUSION: The results suggest that NOD1, LBP, NKL, and IL-1ß genes play pivotal roles in the early innate immune response of Nile Tilapia to A. veronii infection, and the postinfection expression profile trends of these genes imply tissue-/organ-specific responses and synchronized co-regulation.

Echocardiographic characterization of cardiac chambers and vasculatures in buffaloes (Bubalus bubalis) during diastolic and systolic phases.

Background: Ultrasonography had diagnostic importance in the evaluation of different diseases in buffaloes, including cardiovascular diseases. Aim: The current work describes the normal echocardiographic findings in healthy buffaloes, along with establishing reference values for echocardiographic dimensions for both sides of the heart, i.e., left and right ones. Methods: About 30 healthy adult buffaloes that belonged to private farms in Assiut, Egypt, were included in this study. Each animal underwent a complete clinical evaluation as well as hematological analyses, lipid profile indices, liver functions, cardio-thoracic radiography, and echocardiography to confirm no diseased conditions were detected. The study was conducted on healthy buffaloes (n = 30) in Assiut Governorate, Egypt. Results: The obtained results reported healthy buffaloes with normal clinical findings as well as indices of blood pictures and serum biochemicals that were within the reference intervals. Radiography revealed a free reticulum and a well-defined diaphragm. The heart was seen as a typical radio-opaque organ. Ultrasonographically, using grayscale B-mode and M-mode, the heart was commonly imaged from the left fourth intercostal space. Different echocardiographic views were described, including the four chamber view, i.e., right atrium (RA), right ventricle (RV), tricuspid valve (TCV), left atrium, left ventricle, mitral valve, and interventricular septum (IVS), and the right ventricular outflow tract, i.e., RA, TCV, RV, pulmonary artery (PA), and pulmonary valve. Cross sections in each of the apex and base of the heart were described. Echocardiographic dimensions during cardiac diastole and systole, including diameters and wall thickness of each of the atria and ventricles, were demonstrated. Interventricular septal thickness wall thickness as well as diameters of the aorta and PA, were stated. Conclusion: The work tried to put reference values on the normal echocardiographic dimensions using 2-D B-mode gray scale ultrasonography in healthy adult buffaloes. These echocardiographic reference dimensions with normal echocardiographic imaging will be very helpful in enhancing the diagnostic efficacy of ultrasounds for recognizing abnormal findings related to cardiac disorders.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Effect of different post-partum therapeutic protocols with intrauterine oxytetracycline, oxytocin and/or GnRH injection in post-kidding goats on oxytetracyclines residues in goat milk and postpartum ovarian resumption with referring to clinical and haematological pictures.

BACKGROUND: The post-parturient period in goat had marked changes in an animal's endocrine and metabolic status as well as by reduction in feed intake when the nutrient demand for impending lactogenesis was increasing. The current study aimed to monitor the residues of oxytetracycline in Baladi goat milk and their hazards on public health as well as the time required until complete disappearance of this medicament from milk through following up periods included 0, 12, 24, 36, 48, 60, 72, 84, 96 and 120 h in post-kidding goat following intrauterine application of oxytetracycline. The study also compared between the efficacy of oxytetracycline only, oxytetracycline with oxytocin, or oxytetracycline with GnRH, through monitoring the clinical findings and haematological pictures at days 0, 5 and 7 post-partum as well as studying the changes in numbers and size of follicles at days 15, 30 and 45 postpartum after different treatments strategies in different groups i.e. Control healthy goat (Contgr), Oxytetracycline treated goat (Oxytetgr), Oxytetracycline-oxytocin treated goat (Oxytet-Oxytogr) and Oxytetracycline-GnRH treated goat (Oxytet-GnRHgr). The study was carried out on clinically healthy Baladi goats (n = 40) that gave birth recently. They were divided into 4 equal groups (n = 10 goats for each); Contgr which received no medication after birth, Oxytetgr which administrated oxytetracycline tablets intrauterine at day of birth, Oxytet-Oxytogr which treated by oxytetracycline tablets intrauterine at day of birth followed by oxytocin injection at 3rd day after birth, and Oxytet-GnRHgr which treated by oxytetracycline tablets intrauterine at day of birth followed by GNRH injection at 3rd day after birth. RESULTS AND CONCLUSIONS: The study concluded the highest oxytetracyclines residues in goats' milk were reported after 36 h following intrauterine oxytetracycline application where complete disappearance of oxytetracyclines residues in goats' milk required 120 h elapsed after intrauterine oxytetracycline application in which the goats milk became safe for human consumption. The study also reported powerful influence of the applied variable therapeutic regimens on post-partum ovarian resumption through clear significant variations in numbers and sizes of follicles either between different goats' groups within the same day, or between days 15, 30 and 45 post-partum within each independent goat group.

Comparative study between efficacy of dexamethasone-prostaglandin-receptal combination and mechanical correction in uterine torsion cases in Egyptian buffalo-cows (Bubalus bubalis).

BACKGROUND: According to reports, the majority of domesticated species exhibited uterine torsion. It was occasionally noted as a cause of dystocia in buffaloes. The uterus might twist more frequently late in pregnancy because of certain animal traits. The current research monitored the clinical findings and laboratory assays associated with uterine torsion cases in pregnant buffalo-cows through comparing between normal labored buffalo-cows (Norm-Labgr; n = 20), mechanically corrected uterine torsed animals without medicament interference (UtrTorsgr; n = 160), and mechanically corrected uterine torsed animals with medicament interference (UtrTors-Medgr; n = 40) through focusing on placental characterization, calves body weight, milk constituents and milk somatic cell count (SCC) in normal labored buffaloes and uterine torsed ones. Through clinical and laboratory investigations of these buffaloes (N = 220) had been conducted 3 times; 7 h pre-calving and post calving (Post uterine correction) i.e. 48 and 96 h. Uterine torsion prevalence parameters, placental characterization, calves body weight, milk constituents and milk somatic cell counts were evaluated in normal labored buffaloes and uterine torsed ones. RESULTS AND CONCLUSIONS: The study concluded pre-calving remarkable variations in clinical findings, leukogram picture, calf birth weight and some placental characterization parameters between Norm-Labgr and each of UtrTorsgr and UtrTors-Medgr whereas these variations disappeared post-partum as a result to either only mechanical correction or mechanical correction plus medicaments interference. No pre-or post-calving significant changes between UtrTorsgr and UtrTors-Medgr except for the abnormal clinical findings were more representative in UtrTors-Medgr than those in UtrTorsgr particularly pre-calving. The applied pre-calving therapeutic regimen including dexamethasone-prostaglandin-receptal combination had a powerful potential efficacy that induced vaginal delivery of calves in UtrTors-Medgr as well as prepartum mechanical correction of torsed uterus approved higher efficacy in UtrTorsgr. The applied prepartum mechanical correction of torsed uterus and/or pre-calving therapeutic regimen as well as subsequent post-calving, post uterine correction applied medicament treatment accelerated rapid recovery of affected buffalo-cows through achieving rapid restoring of their physiological parameters. Buffalo-cow's milk composition, milk pH and milk SCC were not affected whereas no significant variations were reported between Norm-Labgr, UtrTorsgr and UtrTors-Medgr.

Monitoring antimicrobial residues in table eggs in Aswan governorate markets and their impact on egg quality and public health.

Background: Organic egg is among the most common organic foods offered for sale in Egyptian markets in recent years, and consumers buy them at a higher price because they believe organic eggs are safer and have superior nutritional value than conventional eggs. Aim: The present work aimed to monitor antimicrobial residues in brown table eggs, whether conventional or organic type, in Aswan governorate markets and assessed their physical and chemical quality and their public health hazards. Methods: Brown table egg samples (n = 400 total) were randomly selected in the present study, in which they represented two equal groups (n = 200 each) including conventional eggs and organic eggs. Eggs were collected from different retail stores in the Aswan governorate, Egypt. Egg samples were subjected to thorough physical and chemical quality evaluation as well as an assessment of antimicrobial residues. Results: The results reported that organic eggs were cleaner and had a better odor, less blood, and meat spots, but smaller with more shell cracks than conventional eggs. Chemical analysis of some nutrient contents in the egg yolk revealed significantly higher nutritive values of organic eggs than that of conventional ones as the organic eggs contain significantly higher levels of vitamin A and vitamin D/D3 and significantly lower values of cholesterol, calcium, magnesium, and zinc than those in conventional eggs. Disc diffusion assay has been used for monitoring antimicrobial residues in egg samples. The results have shown that all examined organic eggs were free from antimicrobial residues, while 12% and 8% of conventional egg yolk and white were positive for antimicrobial residues, respectively. Conclusion: The study concludes the higher nutritive value of organic eggs compared with the conventional type because of their significantly higher contents of vitamins A and D and their significantly lower contents of cholesterol. Moreover, organic eggs were free from antimicrobial residues which maximizes their public health benefits..

Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare.

Flavobacterium columnare causes columnaris disease in freshwater fish in both natural and aquaculture settings. This disease is often lethal, especially when fish population density is high, and control options such as vaccines are limited. The type IX secretion system (T9SS) is required for F. columnare virulence, but secreted virulence factors have not been fully identified. Many T9SS-secreted proteins are predicted peptidases, and peptidases are common virulence factors of other pathogens. T9SS-deficient mutants, such as ΔgldN and ΔporV, exhibit strong defects in secreted proteolytic activity. The F. columnare genome has many peptidase-encoding genes that may be involved in nutrient acquisition and/or virulence. Mutants lacking individual peptidase-encoding genes, or lacking up to ten peptidase-encoding genes, were constructed and examined for extracellular proteolytic activity, for growth defects, and for virulence in zebrafish and rainbow trout. Most of the mutants retained virulence, but a mutant lacking 10 peptidases, and a mutant lacking the single peptidase TspA exhibited decreased virulence in rainbow trout fry, suggesting that peptidases contribute to F. columnare virulence.

Corynebacterium conjunctivae: A New Corynebacterium Species Isolated from the Ocular Surface of Healthy Horses.

Twenty-two unidentified Gram-positive, rod-shaped organisms were recovered from the conjunctival surface of apparently healthy horses and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolates were tentatively assigned to the genus Corynebacterium, although they did not match any recognized species. Comparative 16S rRNA gene sequencing studies demonstrated that all of the isolates were phylogenetically members of the genus Corynebacterium. The isolates shared 99.4 to 100% 16S rRNA gene sequence similarity among the strains and 96.5% similarity with Corynebacterium tapiri 2385/12T, which was the closest phylogenetically related species. The DNA G+C content was 58.4 mol%. The major fatty acids were C15:0, C16:0, C17:1 ω8c and C18:1 ω9c, while the predominant mycolic acids consisted of C30:0, C32:0 and C34:0. The isolates were distinguished from related Corynebacterium species by a number of phenotypic properties. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from horses be classified in the genus Corynebacterium as Corynebacterium conjunctivae sp. nov. The type strain of C. conjunctivae is ICM19-01138T (DSM 109759T = CCUG 73728T).

Development of a quantitative polymerase chain reaction assay for detection of the aetiological agents of piscine lactococcosis.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

Type IX Secretion System Effectors and Virulence of the Model Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here, we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted 10 genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding 10 secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum causes bacterial cold-water disease in wild and aquaculture-reared fish and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, Flavobacterium columnare, requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The F. psychrophilum genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the Bacteroidetes to delete a core F. psychrophilum T9SS gene, gldN The ΔgldN mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type gldN on a plasmid restored secretion. Compared to wild-type and complemented strains, the ΔgldN mutant was deficient in adhesion, gliding motility, and extracellular proteolytic and hemolytic activities. The ΔgldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease.IMPORTANCE Bacterial cold-water disease, caused by F. psychrophilum, is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to F. psychrophilum and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors and targets for the development of control measures.

Insights into myxozoan composition and physiology revealed by histochemical properties of myxospores.

Myxozoa (phylum Cnidaria) are a diverse group of metazoan parasites that predominately infect fish. Little is known regarding the composition and physiology of their myxospore life stage. The objective of this work was to investigate the composition of myxospores and extrasporogonic stages of nine myxozoan species infecting various teleost fish using histochemical staining techniques. Thirty histochemical stains were applied to formalin-fixed, paraffin-embedded tissues processed routinely for light microscopic evaluation. The polar capsules were the most consistent stain target across the taxa examined. Polar capsule staining with Alizarin red, von Kossa and methyl green-pyronin suggests the presence of intracapsular calcium and phosphate, which may contribute to polar filament discharge or pathogenesis of host invasion. The shell valves and suture lines of most myxozoans were stained with Luna and phosphotungstic acid haematoxylin stains, consistent with the presence of chitin and microfibrils, respectively. Vacuoles were consistently highlighted by diastase-susceptible periodic acid-Schiff and Grocott's methenamine silver staining, indicating glycogen. Other histochemical stains exhibited inconsistent staining across the taxa, suggesting differences in myxospore composition potentially reflective of physiologic variations and tissue tropisms. This work provides some information on conserved features and taxa-associated composition of myxospores and lends insight into myxozoan physiology and host-parasite interactions.

Characterization of the normal equine conjunctival bacterial community using culture-independent methods.

BACKGROUND: The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. OBJECTIVES: This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. STUDY DESIGN: Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. METHODS: Conjunctival biopsies (culture-independent) were collected, and DNA was extracted from the tissues. Bacterial communities in conjunctival biopsies were characterized by next-generation sequencing of the 16S rRNA genes. Individual reads were ascribed to operational taxonomic units (OTUs) using BLASTn and Greengenes databases. Species richness, evenness, and Good's coverage were determined for each conjunctiva-associated microbial community. RESULTS: Culture-independent samples produced a total of 329 bacterial OTUs. The main OTUs identified in the study belonged to the Gram-negative species Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). CONCLUSIONS: Contrary to previously published data based on culture-dependent methods, the horse eye microbial community was dominated by Gram-negative bacteria of the phylum Proteobacteria.

Molecular confirmation of Henneguya adiposa (Cnidaria: Myxozoa) and associated histologic changes in adipose fins of channel catfish, Ictalurus punctatus (Teleost).

Henneguya adiposa is one of ten known, closely related myxozoan species that parasitize a variety of tissue sites in the channel catfish, Ictalurus punctatus. Reported to specifically target the adipose fin, H. adiposa is not associated with morbidity or mortality, although detailed descriptions of its associated histologic pathology are lacking. The objective of this work was to confirm the presence of H. adiposa within fin lesions of affected channel catfish using DNA sequenced from histologic sections obtained by laser capture microdissection, as well as to describe pathologic changes induced by infection. The parasite formed large, white, elongate, nodular plasmodia that caused localized tissue damage and incited a granulomatous inflammatory response within a deep connective tissue layer at the base of the adipose fin. Myxospores released from ruptured plasmodia into adjacent tissue were observed to migrate superficially in tracts through the skin, indicating a portal of exit for environmental dispersal. Defects in the connective tissue layer created by ruptured plasmodia were infiltrated by granulomatous inflammation and fibroplasia, suggesting lesion resolution by scar formation over time. Sequencing of the 18S rRNA gene amplified from excised myxospores confirmed the myxozoan's identity as H. adiposa, with 100% similarity to the reference sequence from previous published work.

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

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

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.

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.