Studies on the pathogenesis of streptococcal infection in cultured yellowtails Seriola spp.: effect of the cell free culture on experimental streptococcal infection

Abstract. A previous paper has revealed that experimental streptococcal infection was associated with a rapid growth of the intestinal bacteria, suggesting association of the condition with an exotoxin. The present study was undertaken to see the effect of the exotoxin on the streptococcal infection by administering cell free culture before the bacterial challenge. Cell free culture of Streptococcus sp. strain YT-3 was inoculated intramuscularly at a dosage of 0–5 ml per 100 g body weight 1 h prior to bacterial challenge. Three fish were killed at intervals after challenge for viable counting of bacteria in the viscera and blood. Intramuscular inoculation with either low virulent or virulent bacteria alone at dosages of 10⁶ to 10⁷ cells did not produce the disease in the fish, with almost complete clearance of the bacteria from the viscera and blood within 120 h. When the exotoxin was inoculated intramuscularly prior to the bacterial challenge, however, either low virulent or virulent bacteria at 10⁶ to 10⁷ cells could produce fatal infection with prominent streptococcal clinical signs.     

Spring viraemia of carp virus (SVCV): comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen

Abstract. The sensitivity of the immunoperoxidase (IP) and fluorescent antibody (FA) techniques applied to frozen sections of organs of carp infected with spring viraemia virus (SVCV) was similar, both in respect of the intensity of the reaction and in the detection rate of the antigen. Seven days after a waterborne infection both methods detected the antigen in the kidneys and to a lesser extent in the liver and spleen. Quantification of virus gave a titre of 10^2.8 to 10^5.5 TCID₅₀/g of kidney, whereas the agent could not be isolated from liver and spleen tissue. In contrast, at 24¹/₂ days post–infection the viral antigen could be readily detected in liver and spleen but only occasionally in the kidneys using the IP and FA techniques. At this time, liver and spleen showed infectivity titres of 10^3.8 to 10^7.2 TCID₅₀/g tissue, whereas the kidneys were found to be free of infective virus. It is concluded that using the IP and FA techniques SVCV can be detected most frequently in the kidneys from 7 to 14 days post–infection and in the liver and spleen from 14 to 24 days post-infection.     

Protective properties of egg yolk IgY containing anti-Edwardsiella tarda antibody against paracolo disease in the Japanese eel, Anguilla japonica Temminck & Schlegel

Abstract. Immunized hens are known to contain a high level of immunoglobulin Y (IgY) in their egg yolk. In this study, the present authors obtained anti- Edwardsiella tarda IgY (containing 20% specific IgY, agglutination titre, 1:128) from hens vaccinated by injection with formalin-killed bacterin. The IgY was stable against eel digestive factors, and therefore, was orally administered with viable E. tarda to the Japanese eels and the efficacy of protection against E. tarda infection was evaluated. Orally administered IgY at a dose of 400mg fish^-1 cleared E. tarda inoculated simultaneously at 10^5–6 CFU fish^-1 from the intestine within 24h. Moreover, orally administered IgY at doses of 200 and 400mg fish^-1 inhibited the penetration of E. tarda inoculated simultaneously at 10^4–6 CFU fish^-1 into the liver and kidney via the damaged intestine. The fish orally administered with IgY showed reduced mortality. These results suggest that egg yolk containing anti- E. tarda IgY is effective in preventing edwardsiellosis.     

The infectivity by different routes of exposure and shedding rates of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon, Salmo salar L., held in sea water

Abstract. The infectivity of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida to Atlantic salmon, Salmo salar L., in sea water was investigated and found to be similar to that reported for fresh water. The minimal infective dose in short duration bath exposures (1–3 days) was 10⁴ colony-forming units (cfu) per ml, while prolonged exposure for three weeks, but not for 1 week, produced infection with 10² cfu/ml. Intragastric intubation of A. salmonicida established infection with doses of >10⁵ cfu. Release of bacteria from dead or morbid infected fish was monitored and found to be in the order of 10⁵–10⁸ cfu/fish/h. These results emphasize the importance of removing dead fish from farm sites.     

Recovery of Serratia marcescens in Hemolymph of Macrobrachium rosenbergii from Experimentally Seeded Water

Seratia marcescens was used as an indicator to determine if different concentrations of bacteria in water relate to concentrations in the hemolymph of Macrobrachium rosenbergii . Prawns were exposed to high (10⁵ to 10⁶ cells/ml), low (10³ to 10⁴ cells/ml) and control (no S. marcescens ) treatments with S. marcescens inoculated in aquaria water. S. marcescens ranged from 10 to 9.9 ± 10³ cells/ml from the high concentration group; a single isolate and zero S. marcescens were recovered from the low and control groups, respectively.     

Histopathology, electron microscopy and isolation of channel catfish virus in experimentally infected European catfish, Silurus glanis L.

Abstract Two groups of European catfish, Silurus glanis L., fingerlings were infected with channel catfish virus (CCV) by either intraperitoneal injection with 10⁵ TCID₅₀ of CCV, or bathing in water containing 10⁵ TCID₅₀ of CCV per 1·0 ml. The virus was isolated from spleen, intestine and brain of CCV-injected fish at day 1 and the titres ranged from 10^2·1 to 10^3·3 TCID₅₀/g. However, the tissue distribution of CCV was irregular and no virus was isolated after day 3 post-exposure. In CCV-bathed fish, the virus was isolated only from the liver of one specimen at day 3 post-exposure. No clinical signs of CCV disease developed in any of the fish. Specimens in each regime from all sampling periods showed some minor histopathological changes, but there were no differences between treatments. Lesions included oedema and focal haemorrhage in the liver and the spleen was congested. Electron micrographs of tissue samples showed the presence of a few virus particles around the nuclei of kidney, spleen and intestinal cells, and in or around a myelinated nerve within the optic lobes of infected fish during the first 4 days of infection.     

Immune Responses and Resistance against Vibrio parahaemolyticus Induced by Probiotic Bacterium Arthrobacter XE-7 in Pacific White Shrimp, Litopenaeus vannamei

A 63-d feeding experiment was conducted to determine the effects of dietary supplementation of probiotic bacterium Arthrobacter XE-7 on immune responses and resistance against Vibrio parahaemolyticus in the Pacific white shrimp, Litopenaeus vannamei . The probiotic bacteria were administered orally at four different doses of 0, 10⁶, 10⁸, and 10¹⁰ colony-forming unit (CFU)/g feed for shrimp. On Day 50, the shrimp were challenged with Vibrio parahaemolyticus by bath. On Days 7, 21, 49, and 63, six shrimp per tank were sampled to take intestine and hemolymph. With increasing dietary supplementation of probiotic bacteria, shrimp mortality decreased from 63.16% (the control) to 55.9% (10⁶ CFU/g feed), 51.75% (10⁸ CFU/g feed), and 51.78% (10¹⁰ CFU/g feed), respectively. Vibrio counts in intestine of shrimp fed probiotic bacterium Arthrobacter XE-7 was generally lower than that in the control shrimp ( P  < 0.05). The probiotic bacteria generally increased the immune parameters in shrimp, that is, total hemocyte counts, percentage phagocytosis, respiratory burst activity, and serum phenoloxidase activity. The results showed that probiotic bacterium Arthrobacter XE-7 can be used as probiotic in shrimp feed.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Experimental pathogenicity in rainbow trout, Oncorhynchus mykiss (Walbaum), of two distinct morphotypes of long-spined Saprolegnia isolates obtained from wild brown trout, Salmo trutta L., and river water

Juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), were experimentally infected to investigate the pathogenicity of 20 isolates of two morphotypes of long-haired Saprolegnia obtained from wild brown trout, Salmo trutta L., and river water in Spain. The trout were exposed to 2 × 10⁵ and 3 × 10⁵ L^–1 zoospores. Saprolegnia infection could not occur without 'ami-momi' treatment. Pathogenicity varied greatly among isolates as mortality ranged from 0 to 100% of the fish. There was a statistically significant difference ( P  < 0.001) between the mortality caused by morphotype I isolates and that produced by those of morphotype II. The most pathogenic isolates usually belonged to morphotype II, consisting of isolates which had secondary cysts with bundles of hooked hairs which were shorter and less numerous than those of morphotype I; the morphotype I isolates usually had low pathogenicity. Lesions were most frequently found on the fins. Cultures detected the presence of Saprolegnia in internal organs. Histopathology of the intestine suggests that Saprolegnia may reach this and other organs via the blood stream from surface lesions.     

Herpesvirus cyprini: lethality and oncogenicity

Abstract. Cyprinid herpesvirus 1 (CHV) or Herpesvirus cyprini was virulent for carp, Cyprinus carpio L., fry following 1 h immersion in water at 20 °C. Cumulative mortality for carp fry was 86–97% in 2-week-old common carp, 20% in 4-week-old fancy carp, and 0% in both 8-week-old common and fancy carp. The virus did not produce mortality in fry of crucian carp, grass carp or other cyprinids. It was also oncogenic in carp, inducing papillomas to the extent of 55% among both common and fancy carp fry. The neoplasms appeared 5–6 months after carp had been exposed to the virus by immersion and recurred at an incidence of 83% in carp 7·5 months post-desquamation of the tumour. The CHV was reisolated from all moribund fish and from all survivors. It also induced papillomas at an incidence of 13% in adult mirror carp and at 10% in adult fancy carp 5 months after intraperitoneal inoculation of 10⁵ TCID₅₀ ml^-1 fish. The virus was rcisolated only from the ncoplastic tissue and not from internal organs. The neoplasms were normally located on fin, skin or mandible, at the intraperitoneal inoculation site. Specific fluorescence for CHV antigen was frequently detected in the gills, liver, kidneys and intestine of 2-week-old fry from 3 to 21 days following challenge with CHV. It was found in greater concentrations in experimentally induced papillomata on 2-week-old carp fry survivors examined 24 weeks after challenge than in naturally occurring neoplasms.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

The specific detection of infectious pancreatic necrosis virus in infected cells and fish by the immuno dot blot method

Abstract. The immuno dot blot technique is a rapid, sensitive and quantitative method for the diagnosis of infectious pancreatic necrosis virus ( ipnv ) in both infected tissue culture lysates and tissue homogenates of fish. Uninfected tissue culture lysates and fish homogenates do not react in the peroxidase dot blot system after appropriate treatment. The immuno dot blot method takes 4h to complete, and the dot blot membrane can be stored in the dark for a long period. The viral proteins in the infected tissue culture cells could be detected at 3h post-infection if an initial high MOI was used. The minimum dose of ipnv-ab strain which could be detected using homologous serum was 2·5 ng purified ipnv protein/dot. The smallest infectious dose of VR-299, SP, AB, EVE and CY-1 ipnv which could be detected by the immuno dot blot system was 10⁴ to 10⁵ TCID₅₀. When one μg of purified ipnv was used, a 1·6 × 10⁴ dilution of rabbit anti-AB antisera could be detected.     

Uptake and Clearance of Edwardsiella ictaluri in the Peripheral Blood of Channel Catfish Ictalurus punctatus Fingerlings during Immersion Challenge

Uptake and clearance of Edwardsiella ictaluri in the peripheral blood of channel catfish Ictalurus punctatus fingerlings were monitored for 216 h after exposure to E. ictaluri for 4 h and 8 h under static conditions. Most fish exposed to E. ictaluri developed bacteriemia 24 h post-exposure, and by 72 h post-exposure E. ictaluri was recovered from all the blood of all exposed fish. The number of E. ictaluri colony forming units (CFU) in the blood of moribund fish ranged between 1.7 × 10³ to 1.6 × 10⁵ CFU/50 μL whole blood. Clearance of bacteria from the blood was observed by 216 h post-exposure and all fish surviving bacterial exposure developed agglutinating antibody against E. ictaluri . The pathogenesis of the infection was accompanied by the shedding of viable E. ictaluri into the water which may serve as a mechanism by which fish to fish transmission occurs.     

Optimum Feeding Rate of the Rotifer Brachionus plicatilis on the Marine Alga Nannochloropsis sp.

The optimum feeding rate of the rotifer Bruchionus plicutilis was investigated to determine the best conditions for growth of the rotifer, and also in order to maintain good water quality of the culture. Fifty rotifers of a large size strain were cultured individually at five food density levels of Nannoehloropsis sp. (0.5 ± 10⁶, 1.5 ± 10⁶, 3 ± 10⁶, 5 ± 10⁶, and 8 ± 10⁶ cells/ml). At each level, daily survival and offspring production were recorded until the death of the final individual. The data obtained were analyzed by the life table method. The maximum value of three growth indices (the net reproduction rate, mean life expectancy at age 0, and intrinsic rate of increase) was obtdned at the food density of 1.5 ± 10⁶ cells/ml. The ration size was calculated to be 325% (dry weight)/ day, which is equivalent to about 70% of the saturated feeding rate. It was suggested that the feeding rate should be controlled to lower than the saturated value for an efficient mass culture.     

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus , caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10⁷ copies μg⁻¹ DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10⁶–10⁸ copies of CyHV-2 μg⁻¹ DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22–10 °C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (±SE) of 7.3 ± 11 to 394 ± 55 μg⁻¹ DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Abstract. A natural bath challenge method has been developed for furunculosis using Atlantic salmon, Salmo salar L., and rainbow trout, Salmo gairdneri Richardson. Fish were placed in an enclosed, continuously circulating tank system, supplemented with additional oxygen, the temperature raised gradually (overnight) to 15–16°C, a low dose of Aeromonas salmonicida (strain 184/186) introduced into the tank and the challenge maintained for 14 days. The challenge strain was characterized with respect to possible virulence factors and possessed an A-layer, ability to auto-agglutinate, haemagglutinate, adhere to Atlantic salmon cells and resist destruction by serum. No caseinase activity was detected. LD₅₀ for salmon using this method was 1.8×10³ cells per millilitre while trout had an LD₅₀ of 9.5×10⁴ cells per millilitre. Onset of the disease appeared to depend on fish size with larger trout (50 g) taking up to 10 days to show signs of the disease while mortalities in smaller trout (8.5g) commenced on day 1 post-challenge.     

Infectious pancreatic necrosis virus: experimental infection of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae)

The use of wrasse as cleaner fish in farming of Atlantic salmon, Salmo salar L., is now widespread in Scotland, Ireland and Norway, but little is known about the susceptibility of these fish to the common pathogens of cage-cultured salmon. The susceptibility of goldsinny wrasse, Ctenolabrus rupestris L., to infectious pancreatic necrosis virus (IPNV) was investigated. Captive-bred C. rupestris were infected with IPNV-Sp (Shetland strain) using a bath challenge method. Two infection doses were used, low (4.1 × 10⁵ pfu mL⁻⁻¹) and high (2.4 × 10⁶ pfu mL⁻⁻¹), with two replicates for each experiment. Bathing times were 1 h for the low dose and 5 h for the high dose. A maximum prevalence of infection (30%) was seen 2 weeks post-infection in replicate 1 of the low dose experiment and was accompanied by low tissue titres. The tissue titres dropped to an undetectable level by 4 weeks post-infection. In the high dose experiment, a high prevalence of infection was seen along with moderate titres in tissues as well as a marked pathological response. Both prevalence and intensity declined rapidly and the fish recovered. In the high dose experiment, faecal titres followed the same pattern as those in the tissues. The implications for the use of wrasse in the farming of Atlantic salmon are discussed.     

Enrichment of Rotifers Brachionus plicatilis with Eicosapentaenoic Acid and Docosahexaenoic Acid Produced by Bacteria

Abstract— Two bacterial strains, rich in either eicosapentaenoic acid [EPA, 20:5(n-3)] ( Shewanella gel-idimarina ACAM 456) or docosahexaenoic acid [DHA, 22:6(n-3)] ( Colwellia psychroeryrhrus ACAM 605) were tested for their ability to enrich rotifers Erachionus plicatilis in these polyunsaturated fatty acids. Rotifers were exposed for 24 h to each bacterial strain and to a mixture of the two strains. They were then harvested and their fatty acid compositions were analysed and compared to those of rotifers that had been either starved or fed yeast Saccharomyces cerevisiae or microalgae Tetraselmis suecica in 2-L glass flasks. Exposure to 1.4 × 10⁹ cells/ml of the EPA-producing bacterium only resulted in rotifer EPA levels increasing from 0.1% to 1.2% of total dry weight (%dw). Similarly, following exposure to 1.0 × 10⁹ cells/mL of the DHA-producing bacterium only, rotifer DHA levels increased from below detection to 0.1% dw. When exposed to a mixture of the two bacterial strains, containing 7.0 × 10⁸ celldml of the EPA producer and 5.0 × 10⁸ cells/mL of the DHA producer, the rotifers'final EPA and DHA levels were 0.5% dw and 0.3% dw respectively. Although feeding strategies need refining, these results show, for the first time, that rotifers can be enriched with DHA from bacteria, and that rotifers can be enriched simultaneously with both DHA and EPA from different bacterial strains.