Effects of Water Temperature on Experimental Transmission of Dermal Sarcoma in Fingerling Walleyes

Abstract

The effects of temperature on experimental transmission of dermal sarcoma, a spontaneous tumor of adult walleyes Stizostedion vitreum to fingerling walleyes was determined. Test temperatures were 10, 15, and 20°C. Three-month-old walleyes were inoculated intramuscularly with a cell-free filtrate from a dermal sarcoma collected from an adult walleye in the spring of the year. Tumors were first grossly visible in fish held at 15°C at 8 weeks postinoculation. At 12 weeks postinoculation, all fish were euthanized and examined for presence of tumors. Tumor transmission was most successful at 15°C, followed by that at 20°C; many fish maintained at these temperatures had grossly visible tumors. Although the majority offish held at 10°C also had tumors, tumors in these fish developed to a lesser degree than those observed in fish held at the higher temperatures.     

Optimization of a Plaque Neutralization Test (PNT) to Identify the Exposure History of Pacific Herring to Viral Hemorrhagic Septicemia Virus (VHSV)

Abstract

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations.

Received November 7, 2016; accepted January 14, 2017 Published online April 4, 2017     

Effects of temperature and hypoxic stress on the oxygen consumption rates of the mudsucker fish Labeo capensis (Smith)

The specific oxygen consumption rate (VO ₂ IMb) of Labeo capensis, the freshwater mudsucker, was determined for small and large fish at winter (8°C) and summer (23°C) temperatures. VO JMb was also determined during hypoxic conditions of the experimental water. It was found that VO JMb does not differ substantially compared with other active fish. The VO 4Mb for a fish with a body mass of 250 g is 0,053 ml 0 ₂ per gram wet body mass per hour for fish acclimated at 8°C and 0,127 ml 0 ₂ per gram per hour for fish acclimated at 23°C. The calculated mass exponent, b, is 0,71 at 23°C and 0,68 at 8°C. Hypoxic conditions are relatively well tolerated especially by 23°C- adapted fish.     

Serological Evidence of Infectious Hematopoietic Necrosis in Rainbow Trout from a French Outbreak of Disease

Abstract

Following the detection of infectious hematopoietic necrosis virus (IHNV) in France in April 1987, a serological survey was conducted of the rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) from an infected cultured stock previously known to be contaminated with viral hemorrhagic septicemia virus (VHSV) for 3 years. The work lasted from April to December 1987, at which time all the remaining fish were slaughtered. Serum samples were assayed by a plaque-reduction test and a simplified neutralization test that is more suitable for processing large numbers of serum samples. Such investigations revealed that IHNV neutralization by trout antibodies depended on trout complement, as did neutralization of VHSV. Incubation for 16 h at 4°C increased the sensitivity of the test compared to incubation for 1 h at 20°C. During the course of clinical IHN from April to June, young fish did not display any neutralizing activity, but in September, 29 of 50 of them exhibited significant anti-IHN neutralizing antibody titers ranging from 21 to over 160, and 18 of 46 of these same fingerlings did so in December. Similarly, fish that had undergone VHS infection in August began to develop anti-VHSV antibodies in December (5 of 50), demonstrating that one fish can harbor neutralizing antibodies to both IHNV and VHSV, and that these antibodies had required 14 weeks to appear under fish culture conditions at 10°C. As could be expected from seroneutralization tests, neutralizing antibodies to IHNV did not result in protection against VHS. Sera from 13 of 20 adult fish sampled in mid-June revealed neutralizing antibodies to IHNV, suggesting that they harbored the virus prior to the clinical infection that affected their progeny. Only two of the fish showed low anti-VHSV antibody titers. Similarly, neutralizing antibodies to IHNV were detected in 53 of 73 other adult fish sampled in late October, 10 months after they had spawned and 7 months after mortality had occurred among their progeny. Given the prevalence, level, and persistence of neutralizing antibody titers, the seroneutralization test would be worth investigating more thoroughly to define the conditions that could make it a reliable tool for checking the virus status of trout carriers.     

Isolation and Characterization of Potentially Human Pathogenic,Cytotoxin‐producing Aeromonas Strains from Retailed Seafood in Berlin,Germany

The presence of potentially human pathogenic strains of Aeromonas was investigated in 84 samples of seafood which were purchased from retail traders in Berlin, Germany in spring 2000. A total of 134 Aeromonas strains were isolated on selective [GSP agar and Aeromonas (Ryan) agar] and unselective (standard count agar and enterohaemolysin agar) media from 27 (32.1%) of the samples and were classified as Aeromonas hydrophila (67.9%), A. caviae (26.1%) and A. sobria (6.0%) by biotyping. Thirteen (48.1%) of the 27 positive samples contained more than one species of Aeromonas. Production of haemolysins on enterohaemolysin agar was found with 132 (98.5%) of the strains at 28°C and with 130 strains (97.0%) at 37°C growth temperature. Vero cytotoxins were produced by 99 (73.9%) of the strains when grown at 28°C but only by 24 of the strains (17.9%) at 37°C. The latter strains were identified as A. hydrophila (n = 22) and A. sobria (n = 2) which came from 17 (20.2%) samples of raw seafood and from ready‐to‐eat salted herring ‘Matjes’ products. Cytotoxin‐encoding genes for aerolysin (aer) and haemolysin A (hlyA) were investigated by PCR. Aer and hlyA genes were detected in both, strains which produced toxins only at 28°C and strains which produced toxins at 37°C. Our data indicate that raw seafood and ready‐to‐eat fish products can harbour potential human pathogenic, cytotoxin producing Aeromonas strains.     

Passive Immunization of Pacific Herring against Viral Hemorrhagic Septicemia

Abstract

The plasma of Pacific herring Clupea pallasii that survived laboratory-induced viral hemorrhagic septicemia (VHS) epizootics contained humoral substances that, when injected into naïve animals, conferred passive immunity against the disease. Among groups exposed to viral hemorrhagic septicemia virus (VHSV), injection of donor plasma from VHS survivors resulted in significantly greater survival (50%) and significantly lower tissue titers (1.5 × 10⁵ plaque-forming units [PFU]/g) than the injection of plasma from VHSV-naïve donors (6% survival; 3.7 × 10⁶ PFU/g). Additionally, the magnitude of the protective immune response increased during the postexposure period; plasma that was collected from survivors at 123 d postexposure (931 degree-days) provided greater protection than plasma collected from survivors at 60 d postexposure (409 degree-days). These results provide proof of concept that the VHSV exposure history of Pacific herring populations can be determined post hoc; furthermore, the results can be used as the foundation for developing additional high-throughput diagnostic techniques that may be effective at quantifying herd immunity and forecasting the potential for future VHS epizootics in populations of wild Pacific herring.

Received October 20, 2010; accepted April 7, 2011     

Longevity of Bolbophorus damnificus Infections in Channel Catfish

Abstract

The digenean Bolbophorus damnificus infects commercial channel catfish Ictalurus punctatus, causing mortality, lower feed consumption, and reduced growth in surviving fish. The purpose of this study was to determine the length of time for which B. damnificus prodiplostomulum metacercariae (juvenile trematode stage that infects fish) would remain viable (parasite appearing to be intact or exhibiting movement) in channel catfish. Fish (n = 210) were infected with molecularly confirmed B. damnificus cercariae harvested from naturally infected marsh rams-horn snails Planorbella trivolvis. During the first sampling (at 20 d postinfection), 8.3 ± 3.6 metacercariae/fish (mean ± SD) were found in the host muscle and visceral organs. The channel catfish were then acclimated to a water temperature of either 18°C or 28°C. After 11 months, 6.8 ± 3.5 and 5.9 ± 3.0 metacercariae/fish were found in groups held at 18°C and 28°C, respectively. The mean number of parasites per fish did not significantly differ between fish held at the two temperatures and did not significantly decline over time at either temperature. Fish examined from 13 to 30 months postinfection all contained viable metacercariae that were morphologically and molecularly identified as B. damnificus. At 18 months, 12 metacercariae (of which 11 were intact and 10 displayed movement) were found in the one fish sampled; at 30 months, the last fish sampled contained three intact metacercariae (one displayed slight movement). Our results indicate that B. damnificus metacercariae can remain viable in channel catfish for at least an 18–30-month production cycle during which they have the potential to affect fish growth; in addition, infected fish may serve as intermediate hosts for these metacercariae for at least 2.5 years postinfection.

Received July 14, 2010; accepted March 6, 2011     

Relation of Water Temperature to Bacterial Cold-Water Disease in Coho Salmon,Chinook Salmon,and Rainbow Trout

Abstract

The effect of water temperature on the progress of experimentally induced Cytophaga psychrophila infection was investigated in juveniles of coho salmon Oncorhynchus kisutch, chinook salmon O. tshawytscha, and rainbow trout O. mykiss (formerly Salmo gairdneri). A virulent strain of C. psychrophila was administered to fish by subcutaneous injection. Infected fish were held in tanks containing pathogen-free well water at temperatures ranging from 3 to 23°C. Mean times from infection to death of the fish were shortest at 12–15°C, which were the temperatures associated with the shortest time for doubling the population of this bacterium in vitro. Juvenile steelhead (anadromous rainbow trout) injected with viable C. psychrophila cells and held in 22°C water did not become diseased.     

Isolation of Bdellovibrio as Biological Therapeutic Agents Used For the Treatment of Aeromonas hydrophila Infection in Fish

Fourteen strains of Bdellovibrio‐like organisms were isolated from cultured fish ponds using Aeromonas hydrophila J‐1 as host, one of them formed large plaques after 48 h of incubation at 28°C on a double layer plate, designated as Bdellovibrio C‐1. The Bdellovibrio was confirmed by electron microscopy and PCR amplification of Bdellovibrio‐specific 16S rDNA. The optimum temperature for the growth of BdC‐1 was between 15–37°C and with optimal activity at temperatures of 25–30°C. The ability of BdC‐1 to lyse A. hydrophila was similar in the pH range of 6.5 to 8.5. It can lyse 23 Gram‐negative bacterial strains comprising three genera of fish pathogens and one strain of Escherichia coli but cannot lyse Gram‐positive bacteria such as Bacillus subtillis and Staphylococcus aureus. Immersion of fish in water containing different concentrations of BdC‐1 was used in protection against an experimental infection of A. hydrophila J‐1. Results show that the mortality of groups immersed with BdC‐1 was lower than the group without BdC‐1. These results suggest that it may be possible to use Bdellovibrio to control the disease caused by A. hydrophila.     

A Viral Disease in Larvae and Juveniles of the Japanese Flounder Paralichthys olivaceus

Abstract

From 1985 to 1987, outbreaks of a disease resulting in mass mortality occurred in larvae and juveniles of the Japanese flounder Paralichthys olivaceus cultured at prefectural and private hatcheries in Hiroshima Prefecture, Japan. The disease occurred in 10-30-d-old fish that were reared at about 18–20°C, and mortality usually reached 80–90% in a few weeks. The affected fish had opaque fins and a hyperplastic epidermis on the fins and skin. Electron microscopy revealed hexagonal virus particles in the nucleus (100–140 nm in diameter without an envelope) and cytoplasm (190–230 nm with an envelope) of the affected epidermal cells. Although isolation of the causative agent by the use of five fish-cell cultures was not successful, the disease was transmitted to healthy larval flounder by exposing them to a 0.45-μm filtrate of diseased fish homogenate. The agent's morphological features and its sensitivity to ether, to a pH of 3, and to a 30min exposure to 50°C indicate it is a herpesvirus.     

Virulence of five live vaccines against Avian Infectious Laryngotracheitis and their immunogenicity and spread after eyedrop or spray application

Summary

Five live virus vaccines against avian infectious laryngotracheitis were studied with regard to safety, immunogenicity and route of administration. Significant differences in virulence between the vaccine strains were found. Reduced virulence was accompanied by a reduction of immunogenicity and capacity to spread.

After eyedrop application, a low virulent vaccine induced 90‐ 100% flock immunity for the first 10 weeks after vaccination (PV), followed by a slow decline to 50% at 31 weeks PV, whereas flock immunity induced with the more virulent types remained at about 90% till the end of the experiments (24 and 48 weeks PV).

Aerosol vaccination induced 70 ‐ 100% flock immunity but vaccine reactions were severe. Application of vaccine in a coarse spray did not result in adverse vaccine reactions but induced a maximal protection rate of only 50%.

Microneutralisation titres provided a useful indicator of immunity from the onset of immunity until immunity started to decline. A vaccine virus carrier state was demonstrated by means of sentinel birds.     

The effect of processing and marketing procedures on the bacteriological condition and shelf life of eviscerated turkeys

Experiments carried out in a turkey processing plant showed that there was a fivefold increase in the number of psychrophilic bacteria present after holding eviscerated carcasses in static slush ice tanks for 24 hr. The predominant bacteria in the 24 hr chill tanks were strains of Flavobacterium, Cytophaga and Pseudomonas.

The carcasses were wrapped in heat‐shrunk‐oxygen‐impermeable film and after freezing packed in individual boxes and held at ‐20° C. The storage life at 1°, 10° and 20° C. was determined together with the time taken for the frozen carcasses to equilibrate to these temperatures. It was found that at 1° C. the carcasses kept for about 3 weeks, but at 10° C. they spoiled within 7 days and at 20° G. within 3 days.

An analysis of the spoilage flora showed that although Pseudomonas strains predominated on the turkeys stored at 1° C. fewer were isolated from turkeys stored at 10° C. and 20° C. At these two temperatures an organism identified as Enterobacter liquefaciens predominated together with atypical lactobacilli resembling unidentified strains previously described by Thornley and Sharpe (1959).     

Optimal Dosage of Erythromycin Thiocyanate in a New Feed Additive to Control Bacterial Kidney Disease

Abstract

An experimental feed additive containing erythromycin thiocyanate was formulated into fish feeds and tested to determine the optimal dosage and length of administration to treat acute infections of Renibacterium salmoninarum in chinook salmon Oncorhynchus tshawytscha. Trials were conducted on groups of yearling salmon acclimated and held in the laboratory at 10°C or 14°C in both the winter and spring. Parametric and nonparametric statistical analyses were used to assess survival rates during the tests and to evaluate the condition of fish alive at the completion of the trials. Survival was highest in trials of fish held at 10°C and among those fish that consumed higher quantities of erythromycin for 28 d of continuous therapy. Because portions of the daily rations were more likely to be refused when target dosages exceeded 100 mg/kg body weight, particularly in winter conditions, we recommend a standard therapeutic dosage of 100 mg/kg for 28 d.     

North American Strain of Viral Hemorrhagic Septicemia Virus is Highly Pathogenic for Laboratory-Reared Pacific Herring

Abstract

Specific-pathogen-free Pacific herring Clupea pallasi were reared in the laboratory from eggs and then challenged at 5, 9, and 13 months of age by waterborne exposure to low (10^1.5–2.5 plaque-forming units [PFU] per milliliter), medium (10^3.5–4.5 PFU/mL), or high (10^5.5–6.5 PFU/mL) levels of a North American isolate of viral hemorrhagic septicemia virus (VHSV). The fish were extremely susceptible to the virus, showing clinical disease, mortality approaching 100%, and only a limited increase in resistance with age. Mortality began 4–6 d after exposure and peaked at approximately day 7 in fish exposed to high levels of virus. Whereas the mean time to death showed a significant dose response (P < 0.001), the percent mortality and virus titers in dead fish were generally high in all groups regardless of initial challenge dose. External signs of disease were usually limited to 1–2-mm hemorrhagic areas on the lower jaw and isthmus and around the eye, but 2 of 130 infected fish exhibited extensive cutaneous hemorrhaging. Histopathologic examination of tissues from moribund fish sampled at 2–8 d after exposure revealed multifocal coagulative necrosis of hepatocytes, diffuse necrosis of interstitial hematopoietic tissues in the kidney, diffuse necrosis of the spleen, epidermis, and subcutis, and occasional necrosis of pancreatic acinar cells. Virus titers in tissues of experimentally infected herring were first detected 48 h after exposure and peaked 6-8 d after exposure at 10^7.7 PFU/g. Fish began shedding virus at 48 h after exposure with titers in the flow-through aquaria reaching 10^2.5 PFU/mL at 4–5 d after exposure, just before peak mortality. When the water flow was turned off for 3 h, titers in the water rose to 10^3.5 PFU/mL, and the amount of virus shed by infected fish (on average, greater than 10^6.5 PFU/h per fish) appeared sufficient to sustain a natural epizootic among schooling herring. Taken together, these data suggest that VHSV could be a significant limiting factor for populations of Pacific herring.     

Upper and Lower Temperature Tolerance Limits for Juvenile Red Drums from Texas and South Carolina

Abstract

Upper and lower temperature tolerance limits, reported as temperatures lethal to 50% of the test fish (LT50s), were determined for juveniles of red drum Sciaenops ocellatus obtained from Texas and South Carolina brood stock. This information on red drum thermal tolerance limits was used to determine whether a more northerly distributed population exhibits greater cold tolerance and could therefore be used to improve success of stocking programs. A 96-h temperature tolerance test was performed on juveniles (71–155 mm total length) acclimated to water temperatures ranging from 12.0 to 28.0°C at a salinity of 20‰. Test fish were transferred directly into a series of high-temperature (27.0–37.0°C) or low-temperature (2.0–16.0°C) water baths. The upper LT50 for Texas red drums acclimated to 28.0°C was within 1.1 °C of that calculated for South Carolina red drums acclimated to the same temperature (upper LT50s were 35.7 and 34.6°C). Upper LT50s for fish acclimated to 20.0 and 12.0°C were also similar between the two stocks. Likewise, lower LT50s were similar between the two stocks of red drums: as determined from fish acclimated to 14.0 and 28.0°C, ranges of lower LT50s were 2.8–9.0°C for the Texas stock and 2.69.3°C for the South Carolina stock. The lower LT50 could not be determined for red drums acclimated to 12.0°C, because only three fish died. No consistent differences in temperature tolerance limits were found between the two hatchery stocks, precluding any recommendation to stock one group instead of the other.     

Experimental Induction of Proliferative Gill Disease in Specific-Pathogen-Free Channel Catfish

Abstract

Specific-pathogen-free channel catfish Ictalurus punctatus were exposed to sediment and mud from a pond containing channel catfish with proliferative gill disease. In one experiment, fish were to exposed to mud and sediment for 2 months in water maintained at 19°C. Fish were necropsied weekly, and certain tissues were examined histologically and ultrastructurally. Four trials were conducted with sediment samples from different epizootics of proliferative gill disease. In a second experiment, fish were exposed to sediment for 7 d in water maintained at 16, 19, or 26°C; the fish were then moved to clean water held at 16, 19, or 26°C. Fish were necropsied before transfer to clean water and weekly thereafter for 2 months. Channel catfish held at 19°C developed proliferative gill disease within 2 d of exposure to sediment. Primary cells of a uninucleate myxosporean parasite were present in the gills at the base of lamellae. These developed into plasmodia with numerous secondary cells, and some primary cells disintegrated, releasing their internal secondary cells. Similar development was observed in internal organs 1 week after appearance of the parasite in gills. Complete sporogony did not occur over the 2 months of this study. Plasmodia became necrotic and were not detected after 60 d. In fish exposed to sediment for 7 d at 16, 19, and 21°C, similar organisms were detected, but clinical disease occurred only at 19 and 26°C. Proliferative gill disease may be attributed to extrasporogonic stages of a myxosporean resembling Sphaerospora spp.     

The effect of temperature on the rate of passage of food in the smallmouth yellowfish,Barbus aeneus Burchell (Teleostei,Cyprinidae)

The effect of temperature on the rate of gut evacuation in Barbus aeneus was investigated in two ways. In the first, juvenile fish, kept under conditions of constant temperature, were fed marked food items and the time elapsing until these appeared in the faeces was noted. In the second, fish of a range of sizes were caught in the P.K. le Roux dam and immediately placed in cages in the dam to prevent their access to food. Groups of fish were sacrificed at intervals and the contents of standardized gut sections weighed. The field experiment was carried out in summer and in winter. The laboratory experiment yielded the relationship: P = 6270 × t^-2,11 where P is the passage time in hours and T is the temperature in °C. This gives passage times of the order of 8 h at 25°C and 39 h at 11,5°C. The field experiment indicated that there was an exponential relationship between degree of fullness and rate of evacuation, and that complete evacuation required about 24 h in summer, but more than 5 days in winter.     

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