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.     

Experimental Infection of Australian Freshwater Fish with Epizootic Haematopoietic Necrosis Virus (EHNV)

Abstract

The ranavirus, epizootic hematopoietic necrosis virus (EHNV), is endemic to southern Australia with natural outbreaks resulting in mass mortality events in wild Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and less severe disease in farmed Rainbow Trout Oncorhynchus mykiss. To further investigate the host range for EHNV, 12 ecologically or economically important freshwater fish species from southeastern Australia were exposed experimentally to the virus. A bath-challenge model at 18 ± 3°C was employed with limited use of intraperitoneal inoculation to determine if a species was likely to be susceptible to EHNV. Of the species tested, Murray–Darling Rainbowfish Melanotaenia fluviatilis and Dewfish Tandanus tandanus (also known as Freshwater Catfish) were considered to be potentially susceptible species. EHNV was isolated from approximately 7% of surviving Eastern Mosquitofish Gambusia holbrooki, indicating this widespread alien fish species is a potential carrier. The infection of Silver Perch Bidyanus bidyanus and Macquarie Perch Macquaria australasica and the lack of infection in Murray Cod Maccullochella peelii peelii and Golden Perch Macquaria ambigua ambigua after exposure to EHNV via water confirmed earlier data from Langdon (1989). Five other species of native fish were potentially not susceptible to the virus or the fish were able to recover during the standard 35-d postchallenge observation period. Overall, it appeared that EHNV was less virulent in the present experimental model than in previous studies, but the reasons for this were not identified.

Received May 21, 2012; accepted November 1, 2012     

Molecular Size,pH, Temperature Stability,and Ontogeny of Inhibitor(s) of Infectious Pancreatic Necrosis Virus (IPNV) in Normal Rainbow Trout Serum

Abstract

In this study, we investigated the characteristics of inhibitor(s) of infectious pancreatic necrosis virus (IPNV) found in the serum of normal rainbow trout Oncorhynchus mykiss (RTS). The molecular size, stability to pH and temperature, and ontogeny of the inhibitor in trout were studied, and the effect of cations (Ca²⁺ and Mg²⁺) on the activity of the inhibitor was tested. The strongest inhibition of virus was obtained at approximately 150 kDa as measured by ultracentrifugation, sieve gel chromatography, and ultrafiltration. The inhibition decreased significantly when RTS was dialyzed or filtered in the absence of divalent cations, but replacement of at least one cation restored activity. Activity was stable at temperatures ranging from 30°C to 50°C, but 55°C completely destroyed the inhibitory capacity of RTS. The inhibitory activity of RTS was not reduced between pH 4 and 10 but was diminished below pH 4 and above pH 10; such activity was not abrogated by proteases. Additionally, pretreatment of RTS with the polysaccharide mannan significantly reduced inhibition. Thus, the serum inhibitor(s) had many characteristics of a lectin. To determine the ontogeny of inhibition, serum samples were taken from normal rainbow trout, beginning at 2 weeks posthatch; consistent inhibition was not obtained until the rainbow trout had reached the age of 23 weeks posthatch.     

Regulatory Effects of Water Temperature on Loma salmonae (Microspora) Development in Rainbow Trout

Abstract

Water temperature, a pivotal factor influencing interactions between teleosts and pathogens, was examined to determine its effects on the kinetics of xenoma formation and dissolution subsequent to experimental exposure of rainbow trout Oncorhynchus mykiss to the microsporidian gill pathogen Loma salmonae. The permissive water temperature range in which xenomas developed was between 9° and 20°C. Parasite development was arrested at temperatures outside this range, as indicated by the absence of visible xenomas among exposed fish. In addition, when these trout were subsequently moved to temperatures within the permissive range, xenomas failed to develop. Water temperature, within the permissive range, had no significant effect on either the number of xenomas that formed or the proportion of fish that developed xenomas following gastric intubation with a standard dose of spores. The relationship between water temperature and xenoma onset-time was best described (R ² = 88.3%) by polynomial regression analysis: onset = 320 ? 33.4T + 0.9547T ², where T is temperature (°C). Xenoma onset rate was also described through a modified degree-days model, yielding a predictive equation appropriate for use under conditions of fluctuating temperature. The thermal units, expressed as days × (°C above 7°C) necessary for xenoma onset were 298.6 on average. Xenoma dissolution rates, from the time of onset, also appeared to have a trend; more rapid dissolution occurred as temperatures increased. However, this trend correlated minimally with regression models.     

Early Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) Infection in Rainbow Trout

Abstract

A series of experiments was carried out with infectious hematopoietic necrosis virus (IHNV; 193-110 isolate) in rainbow trout Oncorhynchus mykiss (weight, ～1.2 g) to determine the duration of the patent period and the timing of onset of the infectious periods. We first attempted to transmit IHNV to recipient fish from infected rainbow trout 2–3 d after they had been exposed. No infection transfer occurred despite high titers (10^4.79 to 10^4.91 plaque-forming units 5–8 d postexposure (dpe). To determine the number of secondary cases produced by one infectious individual, we exposed approximately 50 rainbow trout (weight, ～1.5 g) in each of seven replicate tanks to a donor fish that had been infected with virus by bath exposure 3 d earlier. The prevalence of infection in recipient fish rose from 0.84% at 2 dpe to 7.9% at 6 dpe. Maximum incidence (22 cases) occurred between 2 and 4 dpe. No disease-specific mortalities occurred in recipient fish during the experiment. The titer of virus in both recipient and donor fish increased from 2 to 4 dpe. There was a positive correlation between the level of infection among donors and prevalence values among recipient fish (r ² = 0.60). The level of challenge by one infectious fish under the conditions provided was enough for infection transfer from sick cohabitant to susceptible fish but was not enough for initiation of a full-scale epizootic among recipients.     

Pharmacokinetics of sulphadimidine in carp (Cyprinus carpio L.) and rainbow trout (Salmo gairdneri Richardson) acclimated at two different temperature levels

Summary

The influence of temperature (10° C and 20° C) on pharmacokinetics and metabolism of sulphadimidine (SDM) in carp and trout was studied.

At 20° C a significantly lower level of distribution (Vd_area ) and a significantly shorter elimination half‐life (T _(½>) β) was achieved in both species compared to the 10° C level. In carp the body clearance parameter (Cl_B (SDM) was significantly higher at 20° C compared to the value at 10° C, whereas for trout this parameter was in the same order of magnitude for both temperatures.

N₄‐acetylsulphadimidine (N₄‐SDM) was the main metabolite of SDM in both species at the two temperature levels. The relative N₄‐SDM plasma percentage in carp was significantly higher at 20° C than at 10° C, whereas there was in trout no significant difference.

In neither species was the peak plasma concentration of N_4‐SDM (C_maxN4‐SDM)) significantly different at two temperatures.

The corresponding peak time of this metabolite (T_{max (N4‐SDM)}) was significantly shorter at 20° C compared to 10° C in both carp and trout.

In carp at both temperatures, acetylation occurs to a greater extent than hydroxylation. Only the 6‐hydroxymethyl‐metabolite (SCH₂OH) was detected in carp, at a significant different level at the two temperatures. Concentrations of hydroxy metabolites in trout were at the detection level of the HPLC‐method (0.02‐μg/ml). The glucuronide metabolite (SOH‐gluc.) was not detected in either species at the two temperatures.     

Treatment of Ichthyophthiriasis in Rainbow Trout and Common Carp with Common and Alternative Therapeutics

Abstract

The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 μL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 μL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 ± 1°C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 ± 1°C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 ± 1°C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.     

Experimentally Induced Whirling Disease II. Determination of Longevity of the Infective Triactinomyxon Stage of Myxobolus cerebralis by Vital Staining

Abstract

Effects of temperature and aging on viability and infectivity of laboratory-produced actinosporean triactinomyxon spores (infective stage of the organism causing whirling disease) were studied. In vitro staining of triactinomyxon spores with vital fluorescein diacetate correlated with the ability of the spores to infect fry of rainbow trout Oncorhynchus mykiss. The experimentally produced actinosporean stage of Myxobolus cerebralis was short-lived, persisting for only 3–4 d at 12.5°C and for less time at warmer temperatures. The vital staining method has potential for screening therapeutants intended to control myxosporean infection of fish.     

Spatial and Temporal Variation of Whirling Disease Risk in Montana Spring Creeks and Rivers

Abstract

Spring creeks are important spawning and rearing areas for wild trout, but the stable flows, cool temperatures, and high nutrient levels that characterize these unique habitats may also make them highly susceptible to establishment and proliferation of the whirling disease pathogen Myxobolus cerebralis. We evaluated the spatial and temporal dynamics in whirling disease risk by using sentinel rainbow trout Oncorhynchus mykiss fry in nine different spring creeks and their conjoining rivers or reservoirs in Montana over a 20-month period. Whirling disease risk was high in five of the seven pathogen-positive spring creek study sites; at these sites, prevalence levels exceeded 90% and over 50% of sentinel fry had moderate to high infection severity scores. Spring creeks generally had higher disease prevalence and severity than paired river or reservoir sites. Fine sediment levels varied widely among springs creeks with high and low whirling disease risk, and we found no significant association between fine sediment level and infection severity. The low risk measured for some spring creeks was likely attributable to the pathogen invasion being in its early stages rather than to environmental characteristics limiting the severity of infection. High whirling disease risk occurred over a wide range of temperatures at spring creek sites (4.5–13°C) and river sites (1.7–12.5°C). There was an unusual seasonal cycle of infection in spring creeks, with peak infection levels occurring from late fall to early spring and declining to near zero in late spring to early fall. The low infection risk during spring suggests that spring-spawning trout would be at a low risk of infection, even in spring creeks with otherwise high disease severity. In contrast, fry of fall-spawning trout may be much more susceptible to infection in spring creek environments.

Received November 22, 2011; accepted May 7, 2012     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

A Comparison of Three Techniques for Detecting Bovine Herpesvirus Type 1 (BHV-1) in Naturally and Experimentally Contaminated Bovine Semen

Contents: Immune electron microscopy, hybridization (Sandwich, Southern-blot and dot-blot hybridization) as well as two cell culture inoculation techniques to detect bovine herpesvirus 1 (BHV-1) in naturally and artificially contamined bovine semen were compared. Immune electron microscopy was not a suitable method because of its low limit of detection. Dot-blot hybridization, as the most sensitive hybridization technique, could detect 150 pg BHV-1 DNA/semen straw (～ 10⁶TCID₅₀/ 500 μl semen). Best results were obtained by using cell culture techniques. With the first technique embryonic bovine lung cells (EBLC) on microtiterplates were inoculated with an ultracentrifuged pellet of seminal plasma (pelleting method). The second procedure comprised a dilution of 250 μl (1/2 content of a straw) in 6,25 ml cell culture medium which were overlayed on a confluent layer of EBLC in a 25 cm² flask. After an incubation of 4 hours at 37°C the medium was decanted and replaced by 6,5 ml fresh medium (dilution method). With both tissue culture methods as little as 5 TCID₅₀ of BHV-1 in 500 μl of artificially contaminated semen were detectable. However the dilution method was better suited than the pelleting method because toxic effects to cells were less pronounced. It was even possible to detect BHV-1 in naturally contaminated semen samples where all other methods failed. The DNA of virus isolates from semen showed three different restriction patterns. Inhalt: Vergleich dreier Methoden zum Nachweis von bovinem Herpesvirus Typ 1 (BHV-1) in natürlich und experimentell kontaminiertem Rindersamen Die Immunelektronenmikroskopie, die Hybridisation (Sandwich-, Southern-Blot- und die Dot-Blot Hybridisation) sowie zwei Zellkultur-Inokulationstechniken wurden zum Nachweis von bovinem Herpesvirus Typ-1 (BHV-1) in natürlich und künstlich kontaminiertem Stierensamen verglichen. Die Immunelektronenmikroskopie erwies sich dabei als zu wenig empfindliche Methode. Die Dot-Blot Hybridisation war die beste der Hybridbationsmethoden. Es lieβen sich damit bis zu 150 pg BHV-1 DNA/Paillette (～ 10⁶ TCID ₅₀ /500 μl Samen) nachweisen. Am besten eigneten sich Zellkulturmethoden. Eine erste Methode bestand darin, embyonale bovine Lungenzellen in Microtiterplatten mit einem Ultrazentrifugensediment des Samenplasmas zu inokulieren (pelleting method). In einem zweiten Verfahren wurde 1/2 Inhalt einer Pailette (250 μl) in 6,25 ml Zellkulturmedium verdünnt und damit ein Zellrasen von embyonalen bovinen Lungenzellen überschichtet. Nach 4 h bei 37°C wurde das Medium abgegossen und durch 6,5 ml neues Medium ersetzt (Verdünnungsmethode). Mit beiden Methoden lieβen sich 5 TCID₅₀ BHV-1 in 500 μl Rünstlich kontaminiertem Samen nachweisen. Die Verdünnungsmethode war aber besser geeignet, weil der toxische Effekt auf die Zellen geringgradiger war. Mit der Verdünnungsmethode lieβ sich aus Ejakulaten BHV-1 isolieren, auch in Fällen, in denen alle andern Methoden versagten. Die DNA's von Virusisolaten aus Rindersamen wiesen drei verschiedene Restriktionsmuster auf.     

Epizootic haematopoietic necrosis virus—An assessment of the likelihood of introduction and establishment in England and Wales

Epizootic haematopoietic necrosis virus (EHNV) is an iridovirus that affects perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss). It emerged in Australia in the 1980s and has not been discovered elsewhere. It causes a high level of mortality in perch resulting in steep population declines. The main possible routes of introduction of the virus to England and Wales are the importation of infected live fish or carcasses. However, no trade in live susceptible species is permitted under current legislation, and no importation of carcasses currently takes place. The virus is hardy and low levels of challenge can infect perch. Therefore, mechanical transmission through the importation of non-susceptible fish species should be considered as a potential route of introduction and establishment. Carp (Cyprinus carpio) have been imported to the UK from Australia for release into still-water fisheries. A qualitative risk assessment concluded that the likelihood of EHNV introduction and establishment in England and Wales with the importation of a consignment of carp was very low. The level of uncertainty at a number of steps in the risk assessment scenario tree was high, notably the likelihood that carp become contaminated with the virus and whether effective contact (resulting in pathogen transmission) is made between the introduced carp and susceptible species in England and Wales. The virus would only establish when the water temperature is greater than 12 °C. Analysis of 10 years of data from two rivers in south-west England indicated that establishment could occur over a period of at least 14 weeks a year in southern England (when average water temperature exceed 12 °C). Imports of live fish from Australia need to be evaluated on a case-by-case basis to determine which, if any, sanitary measures are required to reduce the assessed risk to an acceptable level.     

Effect of temperature on the pharmacokinetics of benzocaine in rainbow trout (Oncorhynchus mykiss) after bath exposures

The pharmacokinetics of benzocaine during bath exposures at 1 mg/L were determined in rainbow trout acclimated at 6 °C, 12 °C or 18 °C for at least 1 month. Individual fish were exposed to benzocaine in a recirculating system for 4 h and pharmacokinetic parameters were estimated in a unique manner from the concentration of benzocaine in the bath water vs. time curve. Elimination from plasma was also determined after the 4 h exposure. The uptake clearance and metabolic clearance increased with increased acclimatization temperatures (uptake clearance 581 ± 179 mL/min/kg at 6 °C and 1154 ± 447 mL/min/kg at 18 °C; metabolic clearance 15.2 ± 4.1 mL/min/kg at 6 °C and 22.3 ± 4.2 mL/min/kg at 18 °C). The apparent volume of distribution had a trend for increasing with temperature that was not significant at the 5% level (2369 ± 678 mL/kg at 6 °C to 3260 ± 1182 mL/kg at 18 °C). The elimination half-life of benzocaine in plasma was variable and did not differ significantly with temperature (60.8 ± 30.3 min at 6 °C to 35.9 ± 13.0 min at 12 °C). Elimination of benzocaine from rainbow trout is relatively rapid and even more rapid at higher acclimatization temperatures based on calculated metabolic clearances and measured plasma concentrations, but was not evident by measurement of terminal plasma half-lifes.     

Susceptibility of Selected Inland Salmonids to Experimentally Induced Infections with Myxobolus cerebralis,the Causative Agent of Whirling Disease

Abstract

Laboratory exposures to the infectious stages (triactinomyxons) of Myxobolus cerebralis demonstrated a range of susceptibility to whirling disease among four species of inland salmonids. Replicate groups of each species were exposed to two concentrations of triactinomyxons, a low dose (100–200 per fish) and a high dose (1,000–2,000 per fish). Exposed fish were evaluated for clinical signs, for severity of microscopic lesions at 35 d, 2 and 5 months, and for spore concentrations in the head cartilage at 5 months. A standard strain of rainbow trout Oncorhynchus mykiss matched for age served as a susceptible species control. Rainbow trout, westslope cutthroat trout O. clarki lewisi, Yellowstone cutthroat trout O. clarki bouvieri, and bull trout Salvelinus confluentus were susceptible to M. cerebralis infections. Clinical signs, including radical swimming (“whirling”) and black tails, were observed at 7 weeks postexposure among rainbow and cutthroat trout challenged at 3 weeks of age. Clinical signs were rare among bull trout exposed at an age of 4 weeks and absent among rainbow and cutthroat trout exposed at 3 months posthatch. Most rainbow, cutthroat, and bull trout were found to be infected when examined at 5 months postexposure. The most severe microscopic lesions among infected fish at 5 months postexposure were found among rainbow trout. Cutthroat trout had less severe lesions, bull trout had mild infections, and no evidence of infection was found among Arctic grayling Thymallus arcticus. Mean spore concentrations among infected fish correlated with the severity of microscopic lesion scores. Rainbow trout had mean concentrations of spores in head cartilage reaching 10⁶, whereas more resistant species such as bull trout had 10⁴ spores; no spores were found among Arctic grayling at 5 months postexposure.     

The anaesthetic potency of benzocaine-hydrochloride in three freshwater fish species

Anaesthesia was induced in the common carp, Cyprinus carpio, tiiapia, Oreochromis mossambicus and rainbow trout, Salmo gairdneri, at concentrations of 25; 50; 75 and 100 mg/1 of benzocaine-hydrochloride as well as neutralized benzocaine-hydrochloride at water temperatures of 15; 20 and 25 °C. The results obtained indicated intra-and interspecific differences in the susceptibility of fish to anaesthesia due to metabolic, chemoreceptive and temperature tolerance differences in and amongst the three species.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

Vaccination of Rainbow Trout against Infectious Hematopoietic Necrosis (IHN) by Using Attenuated Mutants Selected by Neutralizing Monoclonal Antibodies

Abstract

A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P < 0.001) attenuated compared with parental strains. Vaccination of rainbow trout by waterborne exposure to the mutants conferred solid protection against challenge with wild-type virus. In some trials, fish vaccinated with the RB-1 mutant at 50% tissue culture infectious doses (TCID50) of 1 × 10⁴–1 × 10⁵ TCID50/mL or with the 193-110-4 mutant at 1 × 10²–1 × 10³ TCID50/mL, held for 14 d, then challenged with the homologous wild-type strain at 1 × 10⁵ TCID50/mL showed relative percent survival of 95–100% (P < 0.005). There was no significant difference (P > 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 × 10⁵ TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 × 10³–1 × 10⁵ TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease.     

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.     

Comparison of the susceptibility of brown trout (Salmo trutta) and four rainbow trout (Oncorhynchus mykiss) strains to the myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD)

Differences in susceptibility to the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), between four strains of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were evaluated. Fish were exposed to water enzootic for the parasite in the field for 5 days and were subsequently transferred to the laboratory. Relative parasite load was determined after 2, 3 and 4 weeks post-exposure (wpe) by quantitative real-time PCR (qPCR) of kidney samples and number of parasite stages was determined in immunohistochemical stained sections of kidney, liver and spleen tissues. According to qPCR results, the highest amount of parasite DNA per equal amount of host tissue at all time points was measured in brown trout. Two of the rainbow trout strains showed lower relative parasite load than all other groups at the beginning of the experiment, but the parasite multiplied faster in these strains resulting in an equal level of relative parasite load for all rainbow trout strains at 4 wpe. A weak negative correlation of fish size and parasite load was detected. Only in samples of a few fish, single stages of T. bryosalmonae were found in sections stained by immunohistochemistry impeding quantitative evaluation of parasite numbers by this method. The results indicate a differential resistance to T. bryosalmonae between the rainbow trout strains investigated and between rainbow trout and brown trout.     

Metabolism and thermoregulation of individual and clustered long-fingered bats,Miniopterus schreibersii,and the implications for roosting

Oxygen consumption of individual long-fingered bats, Miniopterus schreibersii, was measured at air temperatures (T_r) between 2 and 42°C and that of clusters of four and six bats between 5 and 30°C. BMR of individuals was estimated to be 2.29 ml O₂ g ¹ h^-1 between 34 and about 38°C. M. schreibersii showed two different responses to T_a, either maintaining body temperature (T_b) by increasing metabolic rate, or allowing T_b to fall close to ambient temperature and conserving energy (torpor). Euthermic clusters of four and six bats had lower rates of mass-specific oxygen consumption and lower thermal conductances than individuals at equivalent temperatures and torpid clusters maintained a greater temperature differential between T_b and T_a. Lowest rates of metabolism were measured for euthermic bats at air temperatures higher than those available to them in their natural roost in summer, so typical roost temperatures result in metabolic rates that are about 2 4–3.1 times estimated BMR. Roost temperatures of M. schreibersii in South Africa are substantially higher in winter than are generally accepted as being suitable for hibernation but these do not substantially affect torpid metabolic rates, which are low and independent of ambient temperature below 22°C. Clearly at least some species of insectivorous bats are capable of hibernating at temperatures generally considered to be too warm for this activity.