Wound healing and recovery from injury were investigated in eggs and larvae of herring (Clupea harengus L.), plaice (Pleuronectes platessa L.) and salmon (Salmo salar L.). The resistance of herring eggs to mecha.nical damage was first examined. The chorion of eggs before and just after fertilisation could be burst by loads of 4-30 g but eggs 5 h post-fertilisation could withstand over 1000 g without bursting. Resistance remained high until just before hatching when it decreased to 20-680 g. The vitelline membrane, however, showed a lower resistance at all stages. Early herring and plaice larvae were caught and eaten by medusae (Aurelia aurita, Tiaropsis multiserrata, Bougainvillea sp.), hydroids (Sarsia sp.), megalopa larvae of the prawn Nephrops norvegicus and adult mysids. Early herring larvae survived minor stings from an Aurelia ephyra, and also experimentally inflicted lesions such as superficial scratches, suction wounds and amputation of up to 2 mm of the tail in sea water. The caudal region of the primordial fin regenerated within a month when less than 1 mm was cut off. Yoll, sac and first feeding herring also survived an incision of 0.3 mm long through the body ventral to the notochord and dorsal to the gut; in starving larvae survival was poorer in the later stages of starvation. When skin was removed in larvae of all 3 species the mortality depended on the area of the lesion, thp maximum area tolerated increasing with larval size. In sea water the threshold area was 0.1-0.2 mm2 dO.r 6-8 mm long plaice, <: 0.3 mm for 10-13 mm long herring and 0.3-0.4 mm for 14-17 mm long herring. In river water the threshold was 1~2mm for 19':1l2m1m long saImon and 6.5-8 mm for 26-28 mm long salmon. The thresholds w ore about 1-3% of the total body surface area. Tolerance was increased in isosmotic salinities, the threshold area being as high as 10-14% of the body surface in 24-28 mm long salmon in 8%0. Healing of skin lesions was observed in vivo and by histology, the main response being a mass migration of epidermal cells from the periphery of the lesion. Wound areas of 0.1-7 mm closed in 4-12 h, the mean rates of cell migration being 40-110JUm/h at 10_11°C. The rate of migration was temperature dependent. The normal skin structure was restored with regeneration of a new basement membrane and dermis within 3 weeks. Older stage larvae showed an inflammatory response similar to adult fish.

Atlantic salmon, SaI1M salar. L., have been shown to be more susceptible to infections by the caligid copepod Lepeophtheirus salmonis (Kreyer, 1837) than other salmonid species. Atlantic salmon exhibit a reduced cellular immune response to the attached parasite which has led to the hypothesis of the presence of sea louse associated compounds that depressed the fish's normally efficient inflammatory mechanisms. The aim of the current study was to test this hypothesis. A biochemically defmed in vitro culture system was developed that would allow collection of the secreted/excreted products of the copepodid larvae of L. salmonis, and avoid their contamination by metabolites of the host from their collection, in vivo. Available tissue culture methods proved inadequate in supporting copepodid culture because of the louse requirement for a seawater maintenance medium that was osmotically unsuitable for cultured cells. Tissue engineering technologies developed in the construction of human living skin equivalents were successful in the development of an Atlantic Salmon Skin Equivalent (ASSE). ASSE is a novel organotypic tissue culture substrate that was constructed from Atlantic salmon fibroblasts (AS-6) and primary cultures of Atlantic salmon epithelial cells. Cells were supported in a matrix of collagen fibres, acid extracted from the tails of rats, and combined using a layering technique to create a substitute salmon skin. ASSE has a fibroblastic dermal equivalent overlaid by an epidermis-like layer and a layer of collagen, and was maintainable in a seawater media. During its development, the cells within ASSE showed signs of differentiation that included stratification, increased fibronectin production by cultured fibroblasts, and the formation of a basement membrane-like layer at the junction of the dermis and epidermis. In this environment, ASSE allowed the survival of copepodid larvae for an average of 12 days, a period approximately 5 days longer than that of their free-swimming counterparts. Furthermore, cultured copepodids were observed feeding, exhibiting the normal range of settlement behaviours, and also showed increases in their length. However, metamorphosis to the chalimus I stage did not occur and was not stimulated by the supplementation of ASSE with salmon mucus, salmon peptone or DL methionine. Nevertheless, copepodid maintenance on ASSE allowed the collection of the compounds they released into the culture media. These were assayed for their effects on the immunological functioning of salmon macrophages. These assays demonstrated that the chemotaxic ability of macrophages was significantly reduced following treatment with louse culture supernatants . (LCS), as was their phagocytic ability. In both assays, the activity of each function was approximately 50% lower than that recorded in untreated cells. Intracellular respiratory burst and the phagocytic index of active phagocytes, however, was not affected. The biological activity of LCS was inhibited following heating, dilution, and treatment with proteinase K, indicating that the active immunosuppressive compounds were either themselves proteins, or required protein to be effective. The active substance was designated Louse Immunomodulatory Factor (LIF). The enzyme profiles of culture supernatants were investigated using API ZYM test strips. The profiles of LCS supernatants were significantly different to those of control supernatants from the early stages of louse incubation with ASSE. LCS supernatants showed elevated levels of leucine aminopeptidase, C4 and C8 esterases, alkaline phosphatase, P-glucuronidase, and Nacetyl- j-glucosaminidase. The involvement of these enzymes in the digestive processes of arthropods is well documented. However, the presence of the chitin hydrolysing enzyme Nacetyl- Beglucosaminidase, may also be suggestive of the preparation of copepodids for moulting. This, and the involvement of these enzymes in digestion in copepodids, and as possible immunomodulatory compounds is discussed. Gel filtration chromatography identified 14 proteins in the LCS that were not present in the control supernatants. These proteins were in the molecular weight range <1 kOa to 2665 kOa. No biological activity was attributed to these proteins when isolated by chromatography and assayed for their effects on macrophage chemotaxis. This lack of activity may be associated with their dilution during the chromatography process in which the supernatants were diluted approximately 5000 times. Experimental evidence showed that the activity of LIF was absent when supernatants were diluted to 1:1000, and so methods of concentration may be required in order to establish the immunological activity of these proteins. This study has developed an Atlantic salmon skin substitute that could have a great number of applications in the study of salmonid metabolism, cellular communication, immunology, and drug and chemical testing. Here, it supported the extended maintenance of sea lice larvae in vitro and allowed the collection of the products of their culture. The findings have shown that the copepodid stage of L. salmonis produces substances, LIF, that depress the chemotaxic and phagocytic activity of salmon macrophages, in vitro. These active substances may also be responsible for the depression of the inflammatory responses of sea lice infected Atlantic salmon, in vivo.

This study investigated the use of non-specific immunostimulants to alleviate stress-mediated suppression of defence mechanisms and subsequent susceptibility to bacterial pathogens in rainbow trout (Oncorhynchus mykiss). One yeast (1-3),)1-6)-β-glucan and a bacterial peptidoglycan were selected as immunostimulants from a panel of test substances on the basis of enhanced intracellular superoxide generation by kidney macrophages stimulated in vitro. Kidney macrophage effector activity was not affected after 1, 2, 3 or 4 weeks of in-feed treatment with 0.05% or 5% of glucan or peptidoglycan. However, production of bactericidal superoxide by inflammatory peritoneal macrophages did increase significantly after four weeks of oral treatment with 0.05% peptidoglycan. Although a single confinement of fish (93% reduction of water volume for five minutes) caused a physiological stress response, as indicated by hyperglycaemia in plasma, kidney and inflammatory macrophage activities were only affected after six daily confinements. Phagocytosis, intracellular superoxide production and killing of Aeromonas salmonicida in vitro by kidney macrophages were significantly reduced. Conversely, production of extracellular superoxide, which may be associated with damage to self, was enhanced. Peritoneal macrophages displayed a similar but less marked respiratory burst response after repeated confinement. Some of the alterations in macrophage function caused by daily confinement were prevented by feeding 0.05% peptidoglycan four weeks before the first confinement. The increase in kidney macrophage extracellular superoxide production caused by repeated confinement was significantly alleviated by in-feed peptidoglycan. Similarly, the decrease in intracellular production by peritoneal macrophages caused by repeated confinement was prevented by in-feed treatment with peptidoglycan. Neither peptidoglycan nor repetitive confinement had any effect on complement lytic activity. These results indicate that dietary peptidoglycan was able to reduce, by regulating macrophage function, the impact of stress on certain bactericidal defences and potential damage to self. However, there was no significant difference in the persistence of viable A. salmonicida in the spleen or blood of infected fish in any of the experimental treatments.

Piscine francisellosis, caused by Francisella noatuenensis subsp orientalis (Fno), is an emerging infectious disease in the tilapia industry, but no effective commercial treatments or vaccines are available. The use of immunostimulants is a promising method to control diseases in aquaculture, and various algae and algal-derived compounds are potent immunostimulants for improving immune status. Algae produce a great variety of secondary metabolites that exert a broad spectrum of biological activities. The aim of this thesis was to evaluate the effectiveness of algal compounds against Fno in vitro and in vivo and determine their potential to control francisellosis infection in Nile tilapia Oreochromis niloticus L. under experimental conditions, and in an alternative host, namely the greater wax moth Galeria mellonella. Some of the algae and their compounds (Chlorella sp., alginic acid, and ß-glucan) exerted antimicrobial activity in vitro against Fno, Aeromonas hydrophila and Streptococcus agalactiae and stimulated responses of Nile tilapia macrophages (Chapter 2). An immersion challenge model for Fno STIR-GUS-F2f7 was developed in two genetic groups of Nile tilapia, and the homo gold strain was more susceptible to infection than wild type (Chapter 3). In vivo trials were conducted in Nile tilapia homo gold where fish were fed diets supplemented with 10% Scenedesmus quaricauda, 10% Haematococcus pluvialis, and 0.1% or 0.2% alginic acid or ß-glucan, and then challenged with Fno and co-infected with S. agalactiae (Chapter 4). The Fno challenge failed to produce mortality; however, co-infection resulted in high mortalities in all groups. As the in vivo trial in tilapia could not be to repeated, a G. mellonella model for Fno was validated. Fno doses between 0.7–1.7 x 108 CFU mL-1 killed G. mellonella, while tetracycline, alginic acid and ß-glucan rescued the wax moth from lethal doses of bacteria (Chapter 5).

Li Jun. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 189-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of infectious pancreatic necrosis (IPN), a disease associated with serious economic loss in Atlantic salmon (Salmo salar). The interaction between IPNV and the host is poorly characterised. IPNV has been detected within macrophages in natural and experimental infections. The macrophage is an important component of the host immune system, participating in innate and adaptive immune responses. The overarching objective of this project was to study aspects of the interaction between IPNV and innate immune responses in the Atlantic salmon macrophage. Methods were developed for the isolation and in vitro culture of Atlantic salmon macrophages. These cells were isolated from head kidney using percoll gradients and subsequently cultured in 24 well plates using Leibovitz L-15 medium containing penicillin, streptomycin and foetal calf serum. This procedure enabled the in vitro culture of macrophages for 9 days post isolation. Real time RT-PCR assays were developed to quantitate the expression of IPNV, Interferon (IFN), Mx, and Elongation factor 1 (ELF-1) in IPNV-infected macrophages and uninfected controls. ELF-1 is utilised as a control gene for relative quantitation in RT-PCR studies. The RT-PCR assays utilised targetspecific primers, and MGB probes. Assay efficiencies varied from 0.85 to 0.99, these were suitable for quantitative RT-PCR analyses. IPNV was demonstrated to replicate in macrophages cultured in vitro as assessed by quantitative RT-PCR. IPNV levels in macrophages were greatest at the early stages of infection. Virus was detected in infected macrophages throughout the nine day period of investigation. Quantitative RT-PCR analyses of the expression of the immune response genes IFN and Mx suggested that IPNV blocks IFN production, as opposed to blocking IFN signalling. The ability of three immunostimulants, Lipopolysaccharide (LPS), macrophage activating factor (MAF), and glucan to up regulate immune responses in IPNV-infected macrophages was also investigated. None of these immunostimulants were able to enhance expression of IFN and Mx, suggesting that these substances may not represent useful therapeutic means of mitigating IPN in Atlantic salmon.

Glochidia (larvae) of freshwater mussels are obligate parasites which attach to and become encysted in the gills or fins of host fish species. The immune responses of the host fish to the parasite affects the susceptibility of the fish to glochidia of different mussels. The immune response provides an opportunity to identify which fish species are hosts. The number and variety of mussels in rivers and lakes has sharply declined since the last century due to various anthropogenic factors, and some mussels species are facing extinction. It is an urgent task to preserve these vanishing mussels, or extinction will be inevitable. An attempt was made to develop an assay, using the immunological response to glochidia, to screen fish species for appropriate hosts. This would facilitate the production and rearing of juveniles. In order to design these assays, reagents such as anti-immunoglobulins which can react with antibodies from many different fish species have to be developed. This work was carried out to develop such reagents. Host and non-host fish were immunized with killed bacteria (Brucella abortus) to study their humoral immune response to an antigen. All fish were able to respond well, as measured by agglutination and Western Blot assays. Antibodies produced by the Brucella injections were used to stimulate anti-fish immunoglobulins in goats, and the antisera were tested for their ability to recognize immunoglobulins from different host fish species. The specificities of these reactions were compared to the reactivity of Protein A. Goat antisera were able to cross-react with different fish antibodies, but it was found that Protein A was a more suitable reagent. Protein A is seemingly suitable to identify the host-fish species and could be used as a reagent for the serological diagnosis of various fish diseases. / Master of Science

The expanding aquaculture industry continues to encounter major challenges in the form of highly contagious aquatic viruses. Control and eradication measures targeting the most lethal and economically damaging virus-induced diseases, some of which are notifiable, currently involve ‘stamping out’ policies and surveillance strategies. These approaches to disease control are performed through mass-culling followed by restriction in the movement of fish and fish products, resulting in considerable impacts on trade. Although effective, these expensive, ethically complex measures threaten the sustainability and reputation of the aquatic food sector, and could possibly be reduced by emulating innovative vaccination strategies that have proved pivotal in maintaining the success of the terrestrial livestock industry. DIVA ‘differentiating infected from vaccinated animal’ strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced specifically to infection can be distinguished from those induced in vaccinated animals. Various approaches were carried out in this study to assess the feasibility of marker/DIVA vaccination for two of the most important disease threats to the global Atlantic salmon and common carp/koi industries, i.e. infectious salmon anaemia (ISA) and koi herpesvirus disease (KHVD), respectively. Antibody responses of Atlantic salmon (Salmo salar L.), following immunisation with an ISA vaccine, administered with foreign immunogenic marker antigens (tetanus toxoid (TT), fluorescein isothiocyanate (FITC) and keyhole limpet hemocyanin (KLH)) were assessed by antigen-specific enzyme linked immunosorbent assay (ELISA). Although antibodies were induced to some markers, these were unreliable and may have been affected by temperature and smoltification. Detectable antibodies to ISAV antigen were also largely inconsistent despite low serum dilutions of 1/20 being employed for serological analysis. The poor antibody responses of salmon to the inactivated ISA vaccine suggested that DIVA vaccination is not feasible for ISA. A similar approach for KHV, utilising green fluorescent protein (GFP) as the marker, similarly failed to induce sufficiently detectable antibody responses in vaccinated carp (Cyprinus carpio L.). However, as high anti-KHV antibody titres were obtained with an inactivated KHV vaccine (≥1/3200), alternative approaches were carried out to assess the feasibility of DIVA vaccination for carp. Investigations of early KHV pathogenesis in vivo and antigen expression kinetics in vitro (0-10 days post infection (dpi)) provided valuable data for the diagnostics necessary for DIVA surveillance strategies. Following viral infection, molecular methods were shown to be the most effective approach for early detection of KHV infected fish prior to sero-conversion, during which time antibodies are not detectable. An experimental immersion challenge with KHV, however, revealed complications in molecular detection during early infection. The KHV DNA was detected in external biopsies of skin and gills, but also internally in gut and peripheral blood leukocytes ≤ 6 hours post infection (hpi), suggesting rapid virus uptake by the host. The gills and gut appeared to be possible portals of entry, supported by detection of DNA in cells by in situ hybridisation (ISH). However, many false negative results using organ biopsies occurred during the first 4 dpi. The gills were the most reliable lethal biopsy for KHV detection by various polymerase chain reaction (PCR) assays, with a PCR targeting a glycoprotein-gene (ORF56) and a real-time PCR assay being the most sensitive of the 7 methods investigated. Importantly, non-lethal mucus samples reduced the number of false negative results obtained by all KHV PCR assays during the earliest infection stages with large levels of viral DNA being detected in mucus (up to 80,000 KHV DNA genomic equivalents 200 μL-1). KHV DNA was consistently detected in the mucus as a consequence of virus being shed from the skin. Determining the expression kinetics of different viral structural proteins can be useful for DIVA serological tests. Analysis of KHV antigen expression in tissues by immunohistochemistry and indirect fluorescent antibody test was inconclusive, therefore 2 novel semi-quantitative immunofluorescence techniques were developed for determining KHV antigen expression kinetics in susceptible cell lines. During the course of KHV infection in vitro, a greater abundance of capsid antigen was produced in infected cells compared to a glycoprotein antigen (ORF56), as determined by detection with antigen-specific monoclonal antibodies (MAbs). The capsid antigen was characterised as a ~100 kDa protein by SDS-PAGE and identified as a product of KHV ORF84 by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). This antigen was subsequently detected in the serum of >25% of KHV infected/exposed carp (6/17), as well as in carp vaccinated with a live attenuated vaccine (3/4), but not with an inactivated vaccine (0/7), by Western blot making it a potential DIVA target for an inactivated vaccine. Attempts were made to improve the sensitivity of KHV serological testing by taking advantage of recombinant proteins specific for KHV (CyHV-3), rORF62 and rORF68 and eliminating any interference by cross-reacting antibodies to carp pox (CyHV-1). These proteins successfully reacted with anti-KHV antibodies. The feasibility of DIVA strategies for KHVD was determined using these recombinant antigens to coat ELISA plates. Differential antibody responses were detected from carp sera to an internal virus tegument protein (rORF62) and external region of a transmembrane protein (rORF68). Fish vaccinated with an inactivated vaccine produced significantly lower antibody responses to rORF62 than to rORF68, whereas infected, exposed and live attenuated vaccinated fish recognised both proteins allowing differentiation between vaccinated and infected carp. However, the sensitivity of the assay was limited, possibly by high levels of natural antibodies detected at the relatively low serum dilutions (1/200) used. As the capsid antigen (ORF84) and tegument protein (ORF62) are derived from internal KHV structural proteins, they induce non-neutralising antibodies, which may be useful for DIVA strategies. Such antibodies are longer lasting than neutralising antibodies and often comprise the majority of fish anti-viral antibodies. This was noted in a fish surviving experimental challenge, which had an antibody titre of 1/10,000, but neutralising titre of 1/45. Such antigens may therefore hold potential for developing effective serological diagnostic tests for KHV and provide the potential for DIVA strategies against KHVD. Natural antibodies will, however, continue to present a challenge to the development of sensitive and reliable KHV serological tests, and hence the application of DIVA strategies.

Piscine francisellosis in an infectious emerging bacterial disease that affects several marine and fresh water fish species worldwide, including farmed salmon, wild and farmed cod, farmed tilapia and several ornamental species, for which no commercial treatment or vaccine exists. During 2011 and the first semester of 2012, chronic episodes of moderate to high levels of mortality with nonspecific clinical signs, and widespread multifocal white nodules as the most consistent gross pathological lesion were experienced by farmed tilapia fingerlings at two different locations in Northern Europe. In this study such outbreaks of granulomatous disease were diagnosed as francisellosis with a genus-specific PCR, and 10 new isolates of the bacterium including the one named STIR-GUS-F2f7, were recovered on a new selective “cysteine blood-tilapia” agar and cysteine heart agar with bovine haemoglobin. Ultrastructural observations of the pathogen in Nile tilapia (O. niloticus) tissues suggested the secretion of outer membrane vesicles (OMVs) by the bacterial cells during infection in these fish. This represented the first documented report of isolation of pathogenic Francisella strains from tilapia in Europe. The phenotypic characterisation indicated that isolates recovered were able to metabolise dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate and glucose-6-phosphate. The predominant structural fatty acids of the isolates were 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%) and 18:0 (5.5%). Anti-microbial resistance analyses indicated that STIR-GUS-F2f7 was susceptible to neomycin, novobiocin, amikacin, ciprofloxacin, imipenem, gatifloxacin, meropenem, tobramycin, nitrofurantoin, and levofloxacin using the quantitative broth micro-dilution method, while the qualitative disc diffusion method indicated susceptibility to enrofloxacin, kanamycin, gentamicin, tetracycline, oxytetracycline, florfenicol, oxolinic acid and streptomycin. The use of the following housekeeping genes: mdh, dnaA, mutS, 16SrRNA-ITS-23SrRNA, prfB putA rpoA, rpoB and tpiA indicated 100% similarity with other isolates belonging to the subspecies F. noatunensis orientalis (Fno). Koch’s postulates were successfully fulfilled by establishing an intraperitoneal injection (IP) challenge model with STIR-GUS-F2f7 in Nile tilapia. Moreover, the challenge model was used to investigate the susceptibility of 3 genetic groups of tilapia to STIR-GUS-F2f7. The lowest amount of bacteria required to cause mortality was 12 CFU/ml and this was seen as early as only 24 hours post infection in the red Nile tilapia and in the wild type after 26 days, no mortalities were seen in the species O. mossambicus with this dose. The mortality in red O. niloticus was significantly higher than that of the other two tilapia groups when 12 and 120 CFU/fish were injected. It was also observed that when a dose of 1200 CFU/ml was used, the mortality in O. niloticus wild type was significantly lower than that of the other two tilapia groups and no differences were seen among the 3 groups when the highest dose (1.2 x105 CFU/fish) was used. The median lethal dose (LD50) of O. niloticus wild type was the most stable during the experiment (values around 104 CFU/ml) and the highest of the three groups after day 25 post infection. At the end of the experiment (day 45) the LD50 was 30 CFU/ml in the red Nile tilapia, 2.3x104 CFU/ml for the wild type and 3.3x102 CFU/ml for O. mossambicus. This pattern, where the LD50 of the red tilapia was lower than that of the other two groups, was observed during the whole experiment. The outcomes of these experiments suggested that the red Nile tilapia family appeared to be the most susceptible while the wild type Nile tilapia family the most resistant. The complete genome of STIR-GUS-F2f7 was sequenced using next generation sequencing (NGS) Illumina Hi-Seq platform™, and the annotation of the assembled genome predicted 1970 protein coding sequences and 63 non-coding rRNA sequences distributed in 328 sub-systems. The taxonomy of the species Francisella noatunensis was revised using genomic-derived parameters form STIR-GUS-F2f7 and other strains in combination with a polyphasic approach that included ecologic, chemotaxonomic and phenotypic analyses. The results indicated that STIR-GUS-F2f7 and all the other strains from warm water fish represent a new bacterial species for which the name Francisella orientalis was assigned. Moreover the description of F. noatunensis was emended and the creation of a new subspecies within this taxon i.e. Francisella noatunensis subsp. chilense was proposed. The results of this study led to the development of a highly efficacious vaccine to protect tilapia against francisellosis.