Pond-level risk factors associated with columnaris disease on Mississippi commercial catfish farms

A large commercial catfish enterprise encompassing over 500 food fish ponds from five farms covering multiple counties in the Mississippi Delta was included in this analysis of columnaris risk factors. A gram-negative bacterium, Flavobacterium columnare, is the cause of columnaris disease and is considered the second-most prevalent bacterial disease in farm-raised catfish. The objective of this study was to determine if pond-level risk factors reported by farm personnel were associated with columnaris disease mortalities. To identify risk factors affecting susceptibility of farm-raised channel catfish Ictalurus punctatus to columnaris disease, a Catfish Management database was developed. Logistic regression was used to model the relationships between probability of columnaris in ponds and risk factors examined. Generalized linear mixed models incorporating hierarchically structured random effects of ponds and one or more fixed-effects risk factors were fitted. In the screening process, each risk factor was evaluated in the basic model as a single fixed-effects factor, and if associated with the outcome (P ≤ 0.20), was retained for development of multivariable models. Two multivariable logistic regression models were constructed from data collected at the pond level by producers. The first was constructed from data in which water quality was not considered. Pond depth and reduced feed consumption for a 14-d period prior to disease outbreaks measured on a per hectare basis were significantly (P ≤ 0.05) associated with columnaris disease. The second, in which water quality variables were also considered, pond depth, reduced feed consumption, shorter intervals from stocking to disease outbreaks, and total ammonia nitrogen were significantly (P ≤ 0.05) associated with columnaris occurrence. This study showed some commonly recorded production variables were associated with columnaris disease outbreaks and, if monitored, could help identify "at risk" ponds before disease outbreaks occur.     

Modulary effects of temperature on antibody response and specific resistance to challenge of channel catfish, Ictalurus punctatus, immunized against Edwardsiella ictaluri

The effects of various temperature treatments on the level of the humoral antibody response in channel catfish immunized with formalin killed Edwardsiella ictaluri was determined in laboratory controlled experiments. Immunized fish that were held at 25 degrees C for 30 days and 12 degrees C for an additional 30 days had higher antibody titers, and were more protected upon challenge, than immunized fish held at 25 degrees C for 60 days. Also immunized catfish held at 25 degrees C for 5 or 10 days followed by 12 degrees C water had higher antibody titers than immunized fish held at 12 degrees C or 25 degrees C for 60 days. In a field experiment carried out during winter and spring (February-May) fingerling channel catfish were vaccinated with E. ictaluri using intraperitoneal injection or immersion with either sonicated or whole cell preparations. Following challenge, the fish vaccinated by immersion in the sonicated preparation had 11.8% mortality whereas the groups immersed in whole cell bacterin, injected with the whole cell bacterin in adjuvant, or injected with sonicate showed 24.6, 57.9 and 41.7% mortality, respectively. Although the fish vaccinated by immersion with the sonicated bacteria had lower antibody titers than those vaccinated by the other methods the immersion vaccinates were more protected against challenge with the pathogen.     

Effects of variable periods of food deprivation on the development of enteric septicemia in channel catfish

Enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri, is the most significant bacterial disease affecting channel catfish Ictalurus punctatus. Withholding feed during outbreaks of ESC is a widely accepted industry practice used to control losses from the disease. Scientific evidence concerning the validity of the practice is contradictory. Two studies were conducted to further evaluate the survival of channel catfish fingerlings following variable periods of feed deprivation before and after exposure to E. ictaluri in controlled aquarium experiments. In the first study, feed was withheld for varying time periods before bacterial challenge. After bacterial challenge, feed was either withheld or fish were fed daily. The second study utilized fish fed daily or fish deprived of feed 7 d before bacterial challenge. Daily feeding was resumed 4, 48, and 96 h after fish were exposed to E. ictaluri. In both experiments, the prechallenge feed treatments did not affect mortality. In contrast, withholding feed after bacterial challenge reduced mortalities by 52% in experiment 1 and by 45% in experiment 2. The highest mortality was observed when fish were fed immediately after immersion exposure and the lowest when fish were completely denied feed or fed daily starting 96 h after challenge. This reduction in mortality occurred when the concentration of E. ictaluri in aquarium water was negligible. These data suggest that when E. ictaluri is present in the water, feeding fish increases mortality by enhancing oral exposure to the pathogen.     

Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine–feed mixture, equating to estimated doses of 5 × 10⁷ and 5 × 10⁶ CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 10⁶ CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ～5 × 10⁶ CFU/g of feed) offered exceptional protection (relative percent survival = 82.6–100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ～5 × 10⁷ CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Received July 31, 2014; accepted March 2, 2015     

Attempts to Control Flexibacter columnaris Epizootics in Pond-Reared Channel Catfish by Vaccination

Abstract

Evaluation of bacterins for the immunization of channel catfish Ictalurus punctatus against Flexibacter columnaris was carried out over 5 years. Groups of 60,000–100,000 pondreared channel catfish, weighing an average of 1.5–4.1 g, were vaccinated each year with formalininactivated Flexibacter columnaris bacterins by immersion. Bacterins were prepared from isolates of the preceding year's epizootic. Equal numbers of nonvaccinated channel catfish reared under similar conditions served as controls. Fish were vaccinated during May each year when water temperatures ranged from 16 to 18°C. Data were recorded daily from early June through midSeptember as water temperatures rose to 24–26°C. Deaths were recorded daily, and the number of hours required for antibiotic treatments were monitored. Analysis of the data indicated a general trend toward a beneficial effect of vaccination, particularly for larger fish. Mortality and hours of antibiotic treatment were significantly higher for most control groups than for vaccinated groups.     

Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus)

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.     

Pond-Level Risk Factors Associated with Columnaris Disease on Mississippi Commercial Catfish Farms

Abstract

A large commercial catfish enterprise encompassing over 500 food fish ponds from five farms covering multiple counties in the Mississippi Delta was included in this analysis of columnaris risk factors. A gram-negative bacterium, Flavobacterium columnare, is the cause of columnaris disease and is considered the second-most prevalent bacterial disease in farm-raised catfish. The objective of this study was to determine if pond-level risk factors reported by farm personnel were associated with columnaris disease mortalities. To identify risk factors affecting susceptibility of farm-raised channel catfish Ictalurus punctatus to columnaris disease, a Catfish Management database was developed. Logistic regression was used to model the relationships between probability of columnaris in ponds and risk factors examined. Generalized linear mixed models incorporating hierarchically structured random effects of ponds and one or more fixed-effects risk factors were fitted. In the screening process, each risk factor was evaluated in the basic model as a single fixed-effects factor, and if associated with the outcome (P ≤ 0.20), was retained for development of multivariable models. Two multivariable logistic regression models were constructed from data collected at the pond level by producers. The first was constructed from data in which water quality was not considered. Pond depth and reduced feed consumption for a 14-d period prior to disease outbreaks measured on a per hectare basis were significantly (P ≤ 0.05) associated with columnaris disease. The second, in which water quality variables were also considered, pond depth, reduced feed consumption, shorter intervals from stocking to disease outbreaks, and total ammonia nitrogen were significantly (P ≤ 0.05) associated with columnaris occurrence. This study showed some commonly recorded production variables were associated with columnaris disease outbreaks and, if monitored, could help identify “at risk” ponds before disease outbreaks occur.

Received September 16, 2011; accepted February 9, 2012     

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

Studies on the live-virus vaccine against infectious laryngotracheitis of chickens. II. Evaluation of the tissue-culture-modified strain C7 in laboratory and field trials

Laboratory and field tests were conducted to evaluate the immunogenicity of a live-virus vaccine against infectious laryngotracheitis (ILT) of chickens that was prepared using tissue-culture-modified strain C7. Eighty-three percent or more of chickens vaccinated by the ocular (OC) or intranasal (IN) route with 10(4.75) TCID50 of the attenuated strain C7 at 50 days of age were protected against challenge with a virulent strain of ILT virus without showing any clinical signs for 4 weeks post-vaccination (PV). When vaccine was administered by aerosol, however, only 65% of vaccinated chickens were protected against challenge. Fifty-seven percent of chickens vaccinated at 70 days of age maintained immunity for 6 months PV. Immune response in younger chickens was inferior to that in older ones. In the field trials, clinical observation revealed no adverse reactions caused by the vaccination, and 60% or more of broilers and 80% or more of layers vaccinated by the OC route were protected against challenge at 4 weeks PV. These results confirmed the safety and efficacy of the vaccine.     

The effect of high total ammonia concentration on the survival of channel catfish experimentally infected with Flavobacterium columnare

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with E columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean +/- SE) was noted between the channel catfish in treatment 1 (95.2 +/- 1.2%) and those in treatment 2 (95.6 +/- 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 + 4.5%) and 4 (41.8 +/- 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F columnare.     

Using Diagnostic Laboratory Records to Monitor Occurrence of Enteric Septicemia of Channel Catfish in Mississippi

Abstract

Diagnostic laboratory information was used to monitor the temporal and spatial occurrence of enteric septicemia of catfish (ESC) in Mississippi. From 1980 through 1988, the Mississippi Cooperative Extension Service (MCES) entered information on 15,649 fish-kill investigations into a computerized data base. Analysis of the records revealed the following trends and patterns. From 1980 through 1985, there was an increase in laboratory submissions and in the ESC proportional morbidity rate (PMR; proportion of diagnosed epizootics that were specific for ESC. However, from 1986 to 1988, although the total yearly number of laboratory submissions had stabilized, there was a decrease in the PMR of ESC cases. During the 9-year interval, 427 producers from 56 counties had ESC diagnosed in their ponds. Twenty-eight producers from four counties were responsible for about 40% of all ESC cases.     

Preliminary Studies of a Newly Developed Subunit Vaccine for Channel Catfish Virus Disease

Abstract

The soluble channel catfish virus (CCV) envelope was harvested and used as a vaccine for channel catfish virus disease. Three- to four-day-old eggs of channel catfish Ictalurus punctatus and 1-week-old fry were vaccinated by immersion. A booster was given to subgroups of fry 2 weeks after vaccination. Vaccinated and nonvaccinated control groups were challenged with viable CCV 8 weeks after vaccination. All challenged nonvaccinated control fry died during the first experiment, and 56% died in the second experiment. Survival offish vaccinated as eggs or fry was 31 and 82%, respectively; survival of groups given a booster dose was 81 and 89%, respectively.     

Distribution of Edwardsiella ictaluri in California

Abstract

Wild and domestic populations of channel catfish Ictalurus punctatus were examined to determine the distribution of the disease called enteric septicemia of catfish (ESC) in California. The causative agent of ESC, Edwardsiella ictaluri, was isolated from five separate sites in California. Two of these isolations were from rectal swabs of asymptomatic fish, confirming that a carrier state may exist. Normal-appearing fish with serum antibody titer to E. ictaluri were commonly found in domestic channel catfish populations, suggesting that many fish become infected but recover. Wild channel catfish with antibody to E. ictaluri were also found in major reservoirs and water distribution canals. Edwardsiella ictaluri appears to be widely distributed within California.     

The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection.

The level of antigen-specific interferon-gamma (IFN-gamma) production can be used as an indicator of cellular immunity. In this study, we investigated the role of cellular immune response in protection against classical swine fever virus (CSFV). Pigs were vaccinated once with CSFV vaccine and challenged 6 days post-vaccination (dpv). Vaccinated animals had significantly higher CSFV-specific IFN-gamma secreting cells than the unvaccinated pigs (p<0.05) at the time of challenge and were protected against CSFV infection, whereas the control pigs died within 14 days post-infection (dpi). In the second experiment, pigs were vaccinated once with either CSFV vaccine or CSFV vaccine combined with Aujeszky's disease (AD) vaccine and challenged at 140 dpv. All vaccinated pigs developed both CSFV-specific, cellular and antibody responses and were protected against CSFV infection. However, differences in cellular, but not antibody, responses were observed in the two vaccinated groups. The group vaccinated with CSFV vaccine developed a significantly higher number of CSFV-specific, IFN-gamma secreting cells (p<0.05), exhibited a shorter fever period and less pathological changes, when compared with the group vaccinated with the combined vaccine. The kinetics of IFN-gamma production, following challenge in the two vaccinated groups, were also different. Taken together, our results indicated that CSFV-specific, IFN-gamma production could be detected early after antigen exposure and correlated with protection against CSFV challenge. Our findings highlight the role of cellular immune responses in porcine anti-viral immunity.     

Parasitism by protozoan Ichthyophthirius multifiliis enhanced invasion of Aeromonas hydrophila in tissues of channel catfish

Protozoan Ichthyophthirius multifiliis Fouquet (Ich) and bacterium Aeromonas hydrophila are two common pathogens of cultured fish, which cause high fish mortality. Currently there is no information available for the effect of parasitism by Ich on survival of channel catfish and invasion of A. hydrophila in fish tissues following exposure to A. hydrophila. A trial was conducted in this study to: (1) determine whether A. hydrophila increased fish mortality in Ich-parasitized channel catfish; and (2) compare the bacterial quantity in different tissues between non-parasitized and Ich-parasitized catfish by real-time polymerase chain reaction (qPCR). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (80%) when exposed to A. hydrophila by immersion than non-parasitized fish (22%). Low mortality was observed in catfish exposed to Ich alone (35%) or A. hydrophila alone (22%). A. hydrophila in fish tissues were quantified by qPCR using a pair of gene-specific primers and reported as genome equivalents per mg of tissue (GEs/mg). Skin, gill, kidney, liver and spleen in Ich-parasitized fish showed significantly higher load of A. hydrophila (9400-188,300 GEs/mg) than non-parasitized fish (4700-42,100 GEs/mg) after exposure to A. hydrophila. This study provides evidence that parasite infections enhance bacterial invasion and cause high fish mortality.     

Transmission of Edwardsiella ictaluri from Infected,Dead to Noninfected Channel Catfish

Abstract

Enteric septicemia of catfish (ESC) was transmitted horizontally from channel catfish Icialurus punctatus that had died from Edwardsiella ictaluri infection to contact channel catfish during 2 d of habitation in a tank. The contact channel catfish became positive for E. ictaluri antibody, became infected with this bacterium, and had signs of ESC and died within 12 d postexposure. Edwardsiella ictaluri was recovered from 24 of the 30 contact channel catfish that died from ESC, as well as from 9 of the 25 tested contact survivors. The cannibalizing of E. ictaluri-infected fish, or the shedding of E. ictaluri from dead fish, or both, were shown to be mechanisms of horizontal transmission of ESC among channel catfish.     

Effect of short-term feeding duration of diets containing commercial whole-cell yeast or yeast subcomponents on immune function and disease resistance in channel catfish, Ictalurus punctatus

Juvenile channel catfish, Ictalurus punctatus, were fed diets supplemented with yeast or yeast subcomponents (YYS) as commercial preparations of β‐glucan (MacroGard^® and Betagard A^®), mannan oligosaccharide (Bio‐Mos^® Aqua Grade), or whole‐cell Saccharomyces cerevisiae (Levucell SB20^®) at the manufacturers’ recommended levels. Fish were fed experimental diets for 1 or 2 weeks prior to disease challenge (pre‐challenge feeding periods) and sampled at the end of each feeding period to measure haematological and immune parameters and to determine the effects of dietary YYS on resistance to Edwardsiella ictaluri, the causative agent of enteric septicaemia disease (ESC). Feeding of experimental diets continued for 3 weeks post‐challenge. In channel catfish fed diets supplemented with MacroGard^®, Betagard A^®, or Levucell SB20^®, survival in the 1 week pre‐challenge feeding group and antibody titres in the 2 week feeding group were significantly higher post‐E. ictaluri challenge in relation to catfish fed with the control diet. In fish fed these same three diets, survival to ESC was significantly higher after 1 week vs. 2 weeks feeding, while the antibody response was significantly higher after 2 weeks vs. 1 week. Lysozyme activity was also higher in the 1 week feeding group, but the increased activity was unrelated to diet. Feeding YYS‐supplemented diets for a shorter duration of 1 week prior to challenge may prove beneficial in increasing resistance to ESC in channel catfish. However, we cannot discount that feeding YYS diets during the recovery period may have contributed to ‘glucan overload’ and reduced survival in the 2 week feeding group.     

Evaluation of a bovine virus diarrhea vaccine.

Singer Strain bovine virus diarrhea (BVD) modified live-virus vaccine, produced in a continuous bovine cell line using equine serum in the growth medium, evoked a high level of serum antibodies and protected against virulent challenge in vaccinated calves. Transmission of vaccinal virus from vaccinated cattle to susceptible controls did not occur when vaccinated and nonvaccinated cattle were kept in constant contact for 23 days. Postvaccinal reactions to the viral vaccine were not observed in vaccinated cattle from 10 feedlots or in cattle vaccinated with multiple doses of the experimental vaccine.