Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.     

Epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection in farmed rainbow trout, Oncorhynchus mykiss (Walbaum): findings based on virus isolation, antigen capture ELISA and serology

Abstract. The epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection was studied in farmed rainbow trout, Oncorhynchus mykiss (Walbaum). Estimates of mortality during five outbreaks on a commercial farm from 1986 to 1992 ranged from 0033 to 0.2% per day and total mortality did not exceed 3–4% in any outbreak. Affected fish were 0+ and less than 125 mm forklength. Clinical signs were non-specific, and laboratory examination was required to confirm the diagnosis. At the height of the outbreak in 1992, EHNV was demonstrated by virus isolation and antigen capture ELISA in 89% of clinically affected fish and 51% of dead fish, while the prevalence of infection in apparently healthy in-contact fish was 4%. Two and 4 months later the virus was not detected in a group of apparently healthy fish that had been affected earlier. Antibodies specific for EHNV were not found in rainbow trout from the infected farm; however, strong humoral responses were detected by ELISA in two immunized fish, indicating that the virus was immunogenic. These data suggested that EHNV was poorly infective but highly virulent in rainbow trout. Clinical EHNV infection was positively correlated with high rearing density and a low rate of water exchange, and therefore, with presumed poor water quality. Water temperature, which ranged from 11 to 17°C during outbreaks, did not appear to determine the incidence of clinical infection. EHNV infection in farmed rainbow trout was preceded by infection in free-living redfin perch, Perca fluviatilis L., in the water catchment, but it was uncertain whether this represented the source of infection for rainbow trout.     

Co‐infection of rainbow trout (Oncorhynchus mykiss) with infectious hematopoietic necrosis virus and Flavobacterium psychrophilum

Co‐infection of rainbow trout with infections haematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum is known to occur, and it has been speculated that a combined infection can result in dramatic losses. Both pathogens can persist in fish in an asymptomatic carrier state, but the impact of co‐infection has not been well characterized or documented. In this study, it was hypothesized that fish co‐infected with F. psychrophilum and IHNV would exhibit greater mortality than fish infected with either pathogen alone. To test this, juvenile rainbow trout were co‐infected with low doses of either IHNV or F. psychrophilum, and at 2 days post‐initial challenge, they were given a low dose of the reciprocal pathogen. This combined infection caused high mortality (76.2%–100%), while mortality from a single pathogen infection with the same respective dose was low (5%–20%). The onset of mortality was earlier in the co‐infected group (3–4 days) when compared with fish infected with F. psychrophilum alone (6 days) or IHNV (5 days), confirming the synergistic interaction between both pathogens. Co‐infection led to a significant increase in the number of F. psychrophilum colony‐forming units and IHNV plaque‐forming units within tissues. This finding confirms that when present together in co‐infected fish, both pathogens are more efficiently recovered from tissues. Furthermore, pathogen genes were significantly increased in co‐infected groups, which parallel the findings of increased systemic pathogen load. Extensive tissue necrosis and abundant pathogen present intracellularly and extracellularly in haematopoietic tissue. This was pronounced in co‐infected fish and likely contributed to the exacerbated clinical signs and higher mortality. This study provides novel insight into host–pathogen interactions related to co‐infection by aquatic bacterial and viral pathogens and supports our hypothesis. Such findings confirm that mortality in fish exposed to both pathogens is greatly elevated compared to a single pathogen infection.     

Immunohistochemical detection of neuromodulators in the intestine of Salmo trutta L. naturally infected with Cyathocephalus truncatus Pallas (Cestoda)

Pathological and immunohistochemical investigations were carried out on the middle intestine of uninfected and parasitized brown trout, Salmo trutta L., from the Ceresone Channel in North Italy. Eighty-six brown trout were sampled by electrofishing, and 32 (37.2%) were infected with Cyathocephalus truncatus Pallas, 1781 (Cestoda). The intensity of infection ranged from 1 to 82 parasites per host and the most infected segments were the anterior (near the pyloric caeca region) and the central part of the middle intestine. Immunohistochemical tests were applied on sections of intestinal tissue of healthy and infected fish, and the presence of substance P (SP), calcitonin gene related peptide (CGRP), met-enkephalin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and serotonin (5-HT) was documented. Endocrine epithelial cells of the tunica mucosa were positive to SP-, CGRP-, met-enkephalin-, and NPY-like peptides and 5-HT antisera; moreover, a higher number of these cells were recorded in the intestine of infected trout in comparison to uninfected fish. In addition, in parasitized S. trutta , SP-like and 5-HT immunoreactivities were found in likely immuno-related cells of the tunica propria-submucosa. Nerve cell bodies and terminals in the myenteric plexus were immunoreactive to almost all the tested peptides and 5-HT antisera. These data provide evidence for the role of the neuroendocrine system of S. trutta in the modulation of inflammatory responses to C. truncatus . Results are discussed with respect to a peptidergic involvement and host immune response to an intestinal tapeworm.     

Diets containing corn naturally contaminated with deoxynivalenol reduces the susceptibility of rainbow trout (Oncorhynchus mykiss) to experimental Flavobacterium psychrophilum infection

The objective of this study was to determine if deoxynivalenol (DON) exposure alters the susceptibility of rainbow trout to bacterial coldwater disease caused by Flavobacterium psychrophilum. Rainbow trout were fed a nutritionally complete diet containing corn that was naturally contaminated with DON at a desired concentration of <0.5 (control and pair‐fed treatments), 4 or 6 ppm over 7 weeks to apparent satiation. After 4 weeks, fish were infected by intraperitoneal injection with F. psychrophilum (3.03x10⁶ CFU mL^?1) via intraperitoneal injection and monitored for morbidity and mortality. A significant linear reduction in feed intake was associated with increasing dietary levels of DON contamination over the initial 4 weeks. There was a significant reduction (P < 0.05) in cumulative per cent mortality in DON‐fed groups (4.1 ppm, 11%; 5.9 ppm, 7%) in comparison to control (46%) and pair‐fed (25%) groups at 21 days post infection. Mortality of trout pair‐fed the control diet was also significantly lower (P < 0.05) than the control group fed to apparent satiation. A replicate trial using genetically similar fish and the same experimental design produced similar results. These results suggest that DON exposure and restricted feed intake provided a protective effect for rainbow trout infected with F. psychrophilum.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

Investigation of wild caught whitefish, Coregonus lavaretus (L.), for infection with viral haemorrhagic septicaemia virus (VHSV) and experimental challenge of whitefish with VHSV

Abstract One hundred and forty-eight wild whitefish, Coregonus lavaretus (L.), were caught by electrofishing and sampled for virological examination in December 1999 and 2000, during migration from the brackish water feeding grounds to the freshwater spawning grounds, where the whitefish may come into contact with farmed rainbow trout. All samples were examined on cell cultures. No viruses were isolated. Three viral haemorrhagic septicaemia virus (VHSV) isolates of different origin were tested in infection trials by immersion and intraperitoneal (IP) injection, using 1.5 g farmed whitefish: an isolate from wild caught marine fish, a farmed rainbow trout isolate with a suspected marine origin and a classical freshwater isolate. The isolates were highly pathogenic by IP injection where 99-100% of the whitefish died. Using an immersion challenge the rainbow trout isolates were moderately pathogenic with approximately 20% mortality, whereas the marine isolate was virtually non-pathogenic. At the end of the experiment it was possible to isolate VHSV from survivors infected with the marine and suspected marine isolates. Because of the low infection rate in wild whitefish in Denmark, the role of whitefish in the spread of VHSV in Denmark is probably not significant. The experimental studies, however, showed that whitefish are potential carriers of VHSV as they suffer only low mortality after infection but continue to carry virus.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

Efficacy studies of passive immunization against Aeromonas salmonicida infection in brook trout, Salvelinus fontinalis (Mitchill)

Abstract. Aeromonas salmonicida , the aetiologic agent of furunculosis, causes high mortality in cultured salmonids. Experiments were conducted to determine the therapeutic and prophylactic efficacy of passive immunization against furunculosis in brook trout, Salvelinus fontinalis (Mitchill), infected by immersion. Rabbit hyperimmune serum was produced against a virulent strain of A. salmonicida and an aliquot of this serum was absorbed with cells of a non-virulent strain of A. salmonicida. Immunoglobulins from aliquots of the absorbed and non-absorbed serum were purified using affinity chromatography. Each serum or immunoglobulin preparation was tested in passive immunization experiments. Brook trout were infected by immersion in a suspension of virulent A. salmonicida , and passively immunized by intraperitoneal injection at the time of experimental infection, or at various periods after experimental infection. Passive immunization of brook trout against furunculosis was therapeutically efficacious when effected either at zero, 24 or 48h post-infection, but not at 72 or 96h. Purified rabbit immunoglobulins specific to virulent A. salmonicida were as protective as the initial rabbit hyperimmune serum in protecting brook trout against furunculosis. To determine the prophylactic efficacy of this treatment, the groups of fish passively immunized at the time of the experimental infection were challenged a second time at either 14, 35, 41 or 56 days after passive immunization. Brook trout were protected against a second experimental bath challenge with virulent A. salmonicida for a period of 35–41 days.     

氟苯尼考在感染鲁氏耶尔森氏菌的西伯利亚鲟体内的药效学

通过构建鲁氏耶尔森氏菌(Yersinia ruckeri)感染西伯利亚鲟(Acipenser baeri)病理模型,设立阴性对照组(不攻菌不给药)及阳性对照组(攻菌不给药),比较研究了高(50 mg.kg-1)、中(20 mg.kg-1)、低(5 mg.kg-1)氟苯尼考剂量治疗组和氯霉素(10 mg.kg-1)对照组西伯利亚鲟体内的药效学。连续给药3天后,阴性对照组和阳性对照组累计死亡率分别为0%和50%,表明病理模型构建成功;而氟苯尼考低、中、高剂量组和治疗对照组累计死亡率分别为30%、0%、0%、10%。愈后各实验组鱼血液中均无细菌感染;除阴性对照组外,鱼肝、肾组织中均可分离到鲁氏菌,但不致鱼死亡。实验结果表明,氟苯尼考对感染鲁氏耶尔森氏菌的西伯利亚鲟有较好的治疗作用,药效优于氯霉素,可用于治疗鱼类细菌性疾病。     

Hexamitiasis leads to lower metabolic rates in rainbow trout Oncorhynchus mykiss (Walbaum) juveniles

This study assessed the effects of Hexamita salmonis (Moore) on metabolism of rainbow trout Oncorhynchus mykiss (Walbaum) and its effect on the host's susceptibility to infectious pancreatic necrosis virus (IPNV) after antiparasitic treatment. Rainbow trout naturally infected with H. salmonis were treated with 10 mg metronidazole kg fish^?1 per day, and their physiological recovery was assessed through measuring resting metabolism on the 7th, 14th, 21st and 28th day after treatment. In addition, we exposed the naïve fish to H. salmonis and measured the resting metabolism (oxygen consumption as mg O₂ kg^?1 per hour) on the 10th, 20th and 30th day after the exposure to assess the variation in metabolic rates after infection. Significantly lower rates of metabolic activity (P < 0.05) were anticipated 20 days after infection with H. salmonis compared with the fish infected with H. salmonis for 10 days or with the parasite‐free fish. Similarly, the treated fish needed about 20 days to fully recover from hexamitiasis. The susceptibility of rainbow trout to IPNV remained unchanged in the presence of H. salmonis. Weight loss was significantly higher (P < 0.05) in infected than that in the parasite‐free fish. Fish should be examined regularly for H. salmonis and treated immediately whether found to prevent economic losses and excessive size variation.     

Scanning and transmission electron microscopical observations on Hexamita salmonis (Moore, 1922) related to mortalities in rainbow trout fry Salmo gairdneri Richardson

Abstract. Examination of the digestive tract of rainbow trout fry infected with Hexamita salmonis revealed no pathological changes in the epithelial cells of the pyloric caeca or upper intestine even where the parasites were closely apposed to the brush border of these cells. No invasion of the epithelium by the parasites was observed, although examples of shrinkage necrosis (apoptotic bodies) of epithelial cells were often found. The ultrastructure of the parasite resembled that of the closely related Giardia lamblia . Despite the lack of pathological changes to the intestinal epithelium, treatment of the fish to remove the parasites greatly reduced mortalities.     

Replication of the infectious salmon anaemia virus (ISAV) in rainbow trout, Oncorhynchus mykiss (Walbaum)

Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon, Salmo salar L., in Norway. However, in laboratory experiments, the virus has also been found to propagate in trout, Salmo trutta L., where it is a persistent infection. It is crucial for the management of ISA in Norway, and for the prevention of spread to fish in other countries, that possible carriers of the virus are found. Another possible salmonid reservoir species is the rainbow trout, Oncorhynchus mykiss (Walbaum). After experimental infection, the ISA virus was found to be present in O. mykiss as late 28 days after infection, with a peak around day 20. The infection resulted in a significant drop in the haematocrit and haemorrhages on the liver in some specimens. The ISA virus was seen budding from endothelial cells lining blood vessels in the heart ventricle of challenged rainbow trout. It is concluded that the ISA virus is able to propagate in O. mykiss and that this species may function as a reservoir when cultured in areas where the virus is found. The virus does not seem to cause any significant mortalities of infected rainbow trout.     

Digestive capacity and compensatory growth in Atlantic cod (Gadus morhua)

The objective of this study was to examine whether digestive capacity correlates with growth rate in Atlantic cod (Gadus morhua). Ninety fish (1035±240 g; 478±36 mm) were assigned to one of three treatments: deprived of food for 5 weeks and then re-fed for 24 days, deprived for 10 weeks and then re-fed for 24 days, or fed for 24 days without deprivation (controls). Three times a week during feeding, the fish were provided with meals of capelin (Mallotus villosus) in excess. Within each treatment, ten fish were sacrificed before and twenty after the feeding period. The relative masses of the pyloric caeca, intestine, and white muscle were determined. Cytochrome c oxidase (CCO), citrate synthase (CS), and nucleoside diphosphate kinase (NDPK) activities were assayed in the pyloric caeca, intestine, and muscle as was trypsin activity in the pyloric caeca. During the re-feeding period, fish that had been deprived of food for 10 weeks showed compensatory growth, growing 1.9 times faster than the controls (0.94±0.26 versus 0.50±0.31% body mass ⋅ day⁻¹). Fish that displayed compensatory growth had a lower relative white muscle mass than controls (66.5±4.1% vs. 69.6±7.4%) while the relative masses of the pyloric caeca (2.21±0.49% vs. 1.78±0.31%) and intestine (0.95±0.20% vs. 0.77±0.15%) were greater than the controls, suggesting that the sizes of these digestive tissues might correlate with compensatory growth capacity. Citrate synthase in the pyloric caeca was the only enzyme that showed higher activity during compensatory growth (8.57±0.94 U ⋅ g tissue⁻¹ compared to 7.13±1.03 U ⋅ g tissue⁻¹ in the control group). This suggests that aerobic catabolic capacity of pyloric caeca could be related to growth capacity during recovery, possibly via the energetic cost of digestive enzyme synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induction of infectious pancreatic necrosis (IPN) in covertly infected Atlantic salmon, Salmo salar L., post-smolts by stress exposure, by injection of IPN virus (IPNV) and by cohabitation

Atlantic salmon post-smolts were given an intraperitoneal (ip) injection of tissue homogenate of Atlantic salmon fry from an outbreak of infectious pancreatic necrosis (IPN), and cohabitants were given an ip injection of Earle's balanced salt solution (EBSS). Parallel treatment groups were exposed to recurrent episodes of environmental stress by water drainage twice a week. Fish injected with EBSS and non-injected fish were exposed to water drainage. The control fish were left untreated. Mortality due to IPN started 3 weeks after challenge in non-injected and EBSS-injected fish that had been exposed to water drainage. This showed that the fish used in the experiment were covertly infected with IPN virus (IPNV) prior to challenge, although no virus was detected in the fish sampled before the experiment. In fish that received an injection of IPNV, mortality started 5-6 days after challenge, regardless of the presence or absence of stress exposure. The EBSS-injected cohabitants started to die after an additional 5-6 days, also regardless of the presence or absence of stress exposure. The final cumulative mortality in the IPNV-injected fish was significantly lower than in the EBSS-injected cohabitants, thus suggesting that the secondary immune response after injection of IPNV provided more protection than the response after a water-borne infection. No disease outbreak was observed in the control fish.     

Experimental Infection of Rainbow Trout,Oncorhynchus mykiss,and Hybrid Striped Bass,Morone chrysops ♂ × Morone saxatilis ♀, with Viral Hemorrhagic Septicemia Virus Genotype IVb

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD₅₀ for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 10⁴ pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.