Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Serum enolase: a non‐destructive biomarker of white skeletal myopathy during pancreas disease (PD) in Atlantic salmon Salmo salar L.

Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases in Atlantic salmon, Salmo salar L., aquaculture. Despite this, there are limited means of assessing fish health non‐destructively. Previous investigation of the serum proteome of Atlantic salmon, Salmo salar L., during pancreas disease (PD) has identified proteins in serum that have potential as biomarkers of the disease. Amongst these proteins, the enzyme enolase was selected as the most viable for use as a biomarker of muscle myopathy associated with PD. Western blot and immunoassay (ELISA) validated enolase as a biomarker for PD, whilst immunohistochemistry identified white muscle as the source of enolase. Enolase was shown to be a specific marker for white muscle myopathy in salmon, rising in serum concentration significantly correlating with pathological damage to the tissue.     

Studies with an experimental model for pancreas disease of Atlantic salmon Salmo Salar L.

This paper describes reproducing pancreas disease in Atlantic salmon, Salmo salar L., parr with intraperitoneal inoculation of homogenized kidney and spleen taken From affected fish. Pancreatic necrosis and cardiomyopathy changes were induced consistently in all experimental groups. A time-course study demonstrated that pancreatic changes occurred as early as 4 days post inoculation and a repair process was evident in some fish on day 7 post inoculation. Heart lesions were first observed on day 5 post inoculation and increased in prevalence and severity as the experiment continued. Both types of lesion were serially passaged. and passively transmitted to fish cohabited with inoculated parr. The incidence of these changes was unaffected by passage of the infective homogenate through a 0.2 μ filter. The homogenate was inactivated after treatment with chloroform. Passive immunization with serum from recovered farmed salmon protected parr against challenge. It is suggested that pancreas disease is caused by a virus with a lipid envelope.     

Sleeping disease and pancreas disease: comparative histopathology and acquired cross-protection

Sleeping disease (SD), a disease of freshwater-reared rainbow trout, Oncorhynchus mykiss (Walbaum), was histologically compared to pancreas disease (PD), a disease of sea water-reared Atlantic salmon, Salmo salar L. Some weeks after injection of kidney homogenates from SD- or PD-affected fish in rainbow trout, the fish successively developed pancreatic, heart and muscular lesions in both populations, although the SD-injected population was the most affected. An acquired protection was observed against PD and SD after an initial injection with PD- or SD-infective material. This acquired cross-protection and the similar histopathologies in PD and SD suggest the two diseases could be caused by a similar or identical agent.     

Surveillance of health status on eight marine rainbow trout, Oncorhynchus mykiss (Walbaum), farms in Denmark in 2006

The health status of eight marine rainbow trout farms was followed from mid-June to mid-September 2006 by sampling both dead and healthy fish approximately every 2 weeks for bacteriological and virological investigation. No fish pathogenic viruses were detected, but all farms experienced disease and mortality as a result of various bacterial infections. Yersinia ruckeri was found on four and Renibacterium salmoninarum on five of the farms, but only during the first part of the surveillance period. This indicates that the fish carried the infection from fresh water, and cleared the infection in salt water. Aeromonas salmonicida subsp. salmonicida caused mortality on five farms, but persisted throughout the sampling period. Although A. salmonicida was probably carried from fresh water, the fish were not able to clear the infection in the sea. Vibrio anguillarum caused mortality on six of the farms throughout the sampling period, O1 being the dominant serovar, and Photobacterium damselae subsp. damselae was found on seven farms as a cause of disease. During the period of highest water temperatures Vibrio parahaemolyticus and Vibrio vulnificus were detected in dead fish in five and two farms, respectively, although their significance as causative pathogens is questionable. Vibrio vulnificus has not previously been found in rainbow trout in Denmark. Both mortality and number of antimicrobial treatments during the period were considerably higher in unvaccinated compared with vaccinated fish. Resistance to commonly used antimicrobials was low or absent.     

Effect of pancreas disease caused by SAV 2 on protein and fat digestion in Atlantic salmon

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co‐varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies.     

Longitudinal serological surveys of Atlantic salmon, Salmo salar L., using a rapid immunoperoxidase-based neutralization assay for salmonid alphavirus

Longitudinal serological surveys for salmon pancreas disease virus (SPDV), the causal agent of pancreas disease (PD), were conducted on multiple caged populations of Atlantic salmon, Salmo salar L., on two farms over a 77-week period (farm 1, freshwater and marine stages) and a 36-week period (farm 2, marine stage only), using a microtitre-based virus neutralization (VN) assay. Collected sera were also screened for viraemia with SPDV, and pancreas, heart and muscle tissues were examined for lesions consistent with PD. Outbreaks of PD occurred during the marine phase on both farms, as demonstrated by seroconversion, the isolation of virus and progressive histopathological changes consistent with a PD outbreak. All populations monitored showed a progressive increase in seroprevalence of 90-100%, typically accompanied by rises in geometric mean antibody titres. With the exception of one caged population, which showed a marked biphasic seroprevalence pattern, the seroprevalence figures in the remaining four monitored populations remained high (> or =70%) until the end of the study period. Peak VN titres of > or =1/1280 were detected on both farms. The results provide essential baseline information for the interpretation of SPDV VN serology results, and indicate that this methodology is suited to both the diagnosis and seroepidemiology of SPDV infections.     

Prevalence of piscine orthoreovirus and salmonid alphavirus in sea‐caught returning adult Atlantic salmon (Salmo salar L.) in northern Norway

Heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) and pancreas disease (PD) caused by salmonid alphavirus (SAV) are among the most prevalent viral diseases of Atlantic salmon farmed in Norway. There are limited data about the impact of disease in farmed salmon on wild salmon populations. Therefore, the prevalence of PRV and SAV in returning salmon caught in six sea sites was determined using real‐time RT‐PCR analyses. Of 419 salmon tested, 15.8% tested positive for PRV, while none were positive for SAV. However, scale reading revealed that 10% of the salmon had escaped from farms. The prevalence of PRV in wild salmon (8%) was significantly lower than in farm escapees (86%), and increased with fish length (proxy for age). Sequencing of the S1 gene of PRV from 39 infected fish revealed a mix of genotypes. The observed increase in PRV prevalence with fish age and the lack of phylogeographic structure of the virus could be explained by virus transmission in the feeding areas. Our results highlight the need for studies about the prevalence of PRV and other pathogens in Atlantic salmon in its oceanic phase.     

Pathology of red spot disease in sea mullet, Mugil cephalus L., from eastern Australia

Abstract. Lesions present on sea mullet, Mugil cephalus L., during the first 3 weeks of red spot disease outbreaks are described. Necrotizing dermatitis is a severe, locally extensive, granulomatous lesion associated with invasion of dermis and underlying skeletal muscle by numerous, non-septate, fungal hyphae 12–18 μm in diameter. Erythematous dermatitis is a mild to severe, focal, chronic active dermatitis without fungal involvement. Lesions intermediate between these two forms, with small to moderate numbers of fungal hyphae in dermis and skeletal muscle also occur. Findings indicate that erythematous dermatitis lesions and intermediate-type lesions subsequently resolve, while necrotizing dermatitis lesions consistently develop into dermal ulcers, with associated severe necrotizing granulomatous myositis. Generally, dermal ulcers occur significantly more often on posterior and dorsal areas of the body surface than on anterior and ventral areas. Lesions heal by combinations of epidermal and dermal repair, fibroplasia, destruction of fungi, removal of necrotic skeletal muscle and regeneration of myofibres. Atrophy of exocrine pancreas occurs in both diseased and clinically normal fish, but is generally more severe in diseased fish. The possible roles of suspected disease determinants, including falls in dissolved oxygen concentrations to sub-lethal levels prior to red spot disease outbreaks, are discussed.     

Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater^® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120–2,343 bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.     

Puffy skin disease (PSD) in rainbow trout,Oncorhynchus mykiss (Walbaum): a case definition

Puffy skin disease (PSD) is a disease that causes skin pathology in rainbow trout, Oncorhynchus mykiss (Walbaum). Incidence of PSD in UK fish farms and fisheries has increased sharply in the last decade, with growing concern from both industry sectors. This paper provides the first comprehensive case definition of PSD, combining clinical and pathological observations of diseased rainbow trout from both fish farms and fisheries. The defining features of PSD, as summarized in the case definition, were focal lateral flank skin lesions that appeared as cutaneous swelling with pigment loss and petechiae. These were associated with lethargy, poor body condition, inappetance and low level mortality. Epidermal hyperplasia and spongiosis, oedema of the dermis stratum spongiosum and a mild diffuse inflammatory cellularity were typical in histopathology of skin. A specific pathogen or aetiology was not identified. Prevalence and severity of skin lesions was greatest during late summer and autumn, with the highest prevalence being 95%. Atypical lesions seen in winter and spring were suggestive of clinical resolution. PSD holds important implications for both trout aquaculture and still water trout fisheries. This case definition will aid future diagnosis, help avoid confusion with other skin conditions and promote prompt and consistent reporting.     

Epidemiological observations of pancreas disease of farmed Atlantic salmon, Salmo salar L., in Ireland

Epidemiological investigations into the pancreas disease (PD) of farmed salmon were conducted on populations of Atlantic salmon reared in Ireland during 2003 and 2004. The investigations surveyed all marine salmon farms operating in Ireland through a detailed questionnaire with follow-up farm visits. Information was gathered on 21 populations of fish in 2003 and 14 populations in 2004. Thirteen of the 21 populations suffered PD in 2003 and 12 of the 14 in 2004. The mean mortality due to PD on affected farms was 18.8% in 2003 and 14.8% in 2004 and the loss of growth due to PD was estimated at 11.4% over the 2-year period. The highest risk periods for outbreaks of PD were early summer and early autumn and the farms most seriously affected by PD mortality were in the western counties of Ireland. Factors which showed an indication of association with a PD outbreak or high mortality during a PD outbreak were: livestock movement to another sea site, high feeding rate prior to any PD outbreak, the presence of another PD positive farm in the same water body, greater than 250000 fish on a site, a previous history of PD on a site, a high sea lice burden, and sites located in the western regions of Ireland which reared a specific strain of salmon.     

Observations on the pathogenicity of Haemogregarina sachai Kirmse, 1978, in farmed turbot Scophthalmus maximus (L.)

Abstract. Cultured turbot Scophthalmus maximus (L.) from three farms at Hunterston, Scotland were found to be infected with Haemogregarina sachai . Up to 6% of some populations were affected with gross tumorous lesions of the musculature and viscera. The lesions consisted of necrotic tissue with a caseous centre. Histologically, there was an accumulation of parasitized reticulo-endothelial cells, cell debris and pyknotic nuclei. Parasitaemias of up to 36% of all blood cell types were observed. The majority of infected cells were neutrophils and monocytes. Infected leucocytes were hypertrophied. There was an erythrocytopenia of infected fish with increases in erythroblasts, monocytes and neutrophils but a decrease in thrombocytes and lymphocytes. Transmission experiments between fish were unsuccessful.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

Health status of two salmonid aquaculture facilities in North Portugal: characterization of the bacterial and viral pathogens causing notifiable diseases

A comparative bacteriological and virological survey was conducted in two fish farms in the North of Portugal. The fish species examined included cultured rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., as well as wild fish captured near both facilities. The microbial load in the internal organs of apparently healthy fish was nonitored over a year, an all the disease problems occurring during this period were investigated. Although both farms presented intermediate levels of infection(30–40% infected fish), farm B showed the poorest microbiological quality since constant but low mortalities were observed throughout the year. Flavobacterium and Psedomonas-Xanthomonas were the predominant bacterial groups, comprising around 40–50% of the isolates from each farm. In farm B, members of the Enterobacteriaceae and mortile Aeromonas also showed significant prevalence (about 20%). The only outbreak of a notifiable disease was an occurrence of furunculosis, caused by Aeromonas salmonicida subsp. salmoncida, in farm A. However, Yersinia ruckeri was isolated not only from diseased fish, but also from asymptomatic fish, usually in mixed infections with motile Aeromonas or infections with motile Aeromonas or infectious pancreatic necrosis virus (IPNV). While Y. ruckeri isolates associated with mortalities belonged to the serotype O1 (subgroup a), those isolated from asymtomatic fish corresponded to serotype O3. Two strains of IPNV (serotype Ab) were isolated in farm B, which represents the first viral agent detected in Portuguese aquaculture. Qualitative and quantitative differences in microbial load were observed between cultured and wild fish. No notifiable bacterial or viral pathogens were detected in any of the feral species studied.     

Bacterial meningitis in farmed rainbow trout Salmo gairdneri Richardson affected with chronic pancreatic necrosis

Abstract. Chronic mortalities associated with meningeal and pancreatic lesions are described, from two trout farms in Scotland. Histopathological and microbiological studies suggested that bacterial infection with strains of Pseudomonas fluorescens of low pathogenicity was developing in fish already affected by the lesions of chronic infectious pancreatic necrosis virus infection (IPN). Antibiotic therapy reduced losses temporarily; eradication of the virus resulted in complete elimination of the condition.     

Investigation of outbreaks of a novel disease, 'Sleepy Grouper Disease', affecting the brown-spotted grouper, Epinephelus tauvina Forskal

Abstract. A novel disease affecting the brown-spotted grouper, Epinephelus tauvina Forskal, described here as Sleepy Grouper Disease¹ (SGD), resulted in significant economic losses in some Singaporean marine net-cage farms from April to August 1992. Investigations suggested that the aetiological agent was a virus, probably introduced with imported groupers. The virus was provisionally identified as an iridovirus on morphological evidence. The disease caused extreme lethargy in affected fish with few visible external signs. Mortalities either occurred gradually over the week from onset of clinical signs, or over a shorter period and in large numbers if fish were stressed. Consistent tissue changes were seen by light microscopy in the spleen, heart and kidney of affected fish. Electron microscopy showed viral particles associated with damage to cell organelles. In an experimental infection, apparently healthy fish cohabiting with an infected fish developed similar lesions and died. The significance of SGD in grouper culture is discussed.     

Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salar L.

Abstract. Two different commercial vaccines against furunculosis, caused by Aeromonas salmonicida subsp. salmonicida , were tested in Atlantic salmon on seven fish farms. Both vaccines were based on formalin-inaclivated bacterins containing aluminium salts as adjuvants. The fish were vaccinated by intraperitoneal injection in the spring approximately one month prior to transfer to sea water, and they were challenged by natural outbreaks of furunculosis. During the first year, six of the farms experienced disease outbreaks. The overall mortality was 7·14% in vaccinated fish and 21·7% in unvaccinated controls, giving a relative percentage survival (RPS) of 67%. In the seventh farm, outbreaks of furunculosis more than one year after vaccination revealed that there was still a trend towards lower mortality in vaccinated fish, though the mean RPS fell to 22%. The use of adjuvants in the vaccines resulted in local lesions in the abdominal cavity of vaccinated fish. However, the severity of the lesions declined gradually, and they did not influence fish quality at the time of slaughtering. Vaccination also had a moderately adverse impact on fish weight gain in most cases.     

Hole‐in‐the‐head disease in discus fish,Symphysodon (Heckel, 1840): Is it a consequence of a dietary Ca/P imbalance?

Hole‐in‐the‐head (HITH) disease‐affected fish develop characteristic lesions in the skin above sensory pores of the head and the trunk. This study investigated whether an unfavourable Ca/P ratio in the diet could provoke lesions consistent with HITH disease in discus fish Symphysodon (Heckel, 1840) as a comparable condition to secondary hyperparathyroidism of tetrapod species. Two groups of five fish were fed a plain beef heart diet (Ca/P of 0.03), whereas two other groups were kept on commercial discus feed (Ca/P of 2.73). Each feeding group was submitted to two different water hardness regimes (35.66–71.39 mg/L CaCO₃ and 124.94–196.33 mg/L CaCO₃, respectively). All fish were observed for the development of the characteristic lesions for 16 weeks. At the end of the study, histological, bacteriological and parasitological examinations were conducted and plasma Ca, P and Mg values were determined. Diplomonad flagellates were detected in two fish. Isolated bacteria of all groups mostly belonged to Aeromonadales and Pseudomonadales. No significant difference of plasma mineral values between the groups was observed. Compared to the results of other authors, Ca stayed mainly in the range and P exceeded the reference values. Histological examinations did not indicate HITH disease, and no fish developed signs of the disease during the study. Clinical trial registration number GZ 68.205/0135‐WF/V/36/2014.