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Development of vaccines against sea lice
Abstract
A review of efforts to develop a vaccine against sea lice is presented together with analysis of the rationale behind the approaches and potential future directions. Vaccines against the caligid copepod, Lepeophtheirus salmonis , have the potential to be a cost‐effective means of controlling the infection and avoid many of the disadvantages of medicine treatments. However, research towards such vaccines is in its infancy and approaches so far used have met with little or no success. Most strategies for sea louse vaccines have adopted methods used for vaccines against other ectoparasites. A vaccine against the cattle tick ( Boophilus microplus ) is in field use while other vaccines such as the sheep blowfly ( Lucilia cuprina ) vaccine are at an earlier stage of development. These haematophageous parasites ingest host antibody as part of a large blood meal which can target antigenic sites in the gut. However, the assumption that arachnid and insect physiology are directly comparable with that of sea lice is not proven, and this may partly explain the slow progress this approach has had with sea lice. Success in developing a louse vaccine will depend upon a better understanding of louse digestive biology, particularly an evaluation of whether the cattle tick model is applicable to the development of a louse vaccine. If the louse gut is to be targeted immunologically, critical antigens will need to be identified and evaluated, bearing in mind that an economic vaccine must include recombinant proteins or be a DNA vaccine. Alternatives to the louse gut as a target are also worth consideration. Antibodies could target critical host–parasite interactions that are amenable to disruption, although no such targets have been identified.
© 2002 Society of Chemical Industry
... Several strategies have been used or are being developed for the control of sea lice in salmon farms (reviewed by Rae, 2002; Table 1) and include biological predators (Bjordal, 1990;Deady et al., 1995), vaccines (Raynard et al., , 2002Grayson et al., 1995;Ross et al., 2006;Frost et al., 2007;Carpio et al., 2011), immunostimulants (Covello et al., 2011;Purcell et al., 2013;Poley et al., 2013), drugs and chemicals (Burka et al., 1997, as well as good management practices such as disinfectants (Pietrak and Opitz, 2004), fallowing, single-year class, and removal of sick or dead salmon (Bron et al., 1993). Despite the importance of salinity to this marine parasite, freshwater bath treatment has not been successful in controlling the parasite (Stone et al., 2002). ...
... Not very effective; poor antibody response in Atlantic salmon probably due to lack of isolation of potent antigens within the sea louse Raynard et al., 1994Raynard et al., , 2002Grayson et al., 1995;Ross et al., 2006;Frost et al., 2007;Carpio et al., 2011 Drugs and chemicals Selectively kill sea lice usually through neurotoxicity or disruption of molting cycle ...
... This is because if a particular drug is continually employed for sea lice control, surviving parasites are favored to continue propagating and will eventually become the dominant strain of parasite in the population. Widespread parasiticide resistance in L. salmonis underscores the need for an integrated sea lice management strategy achievable through combined use of drugs, chemicals, and nonchemical alternatives including manipulation of host immunity using vaccines and/or immunostimulants (Raynard et al., 2002). Innate immunity provides protection against pathogen invasion in fish (Magnadóttir, 2006;Whyte, 2007), and its manipulation is currently being explored as a sea lice control strategy. ...
... Thus, immunoprophylaxis (vaccination), is potentially the most efficacious strategy against L. salmonis (Jenkins et al., 1993). There are a number of problems associated with chemotherapeutants that can be overcome with vaccines (Ellis, 2001; Raynard et al., 2002): Furthermore, any vaccine would specifically target salmon parasites whereas chemotherapeutants do not discriminate between its targets. From a financial perspective, the cost of vaccination would be lower than the cost of buying and storing chemotherapeutants, assuming antigens could be expressed as recombinant proteins or DNA vaccines were used (Raynard et al., 2002). ...
... There are a number of problems associated with chemotherapeutants that can be overcome with vaccines (Ellis, 2001; Raynard et al., 2002): Furthermore, any vaccine would specifically target salmon parasites whereas chemotherapeutants do not discriminate between its targets. From a financial perspective, the cost of vaccination would be lower than the cost of buying and storing chemotherapeutants, assuming antigens could be expressed as recombinant proteins or DNA vaccines were used (Raynard et al., 2002). For a review of DNA vaccines in aquaculture see Heppell and Davis (2000). ...
... They form envelopes that surround the luminal contents of the midgut or hepatopancreas and appear necessary to the digestive process. It is hypothesised that they: (i) protect the epithelium against mechanical abrasion, (ii) support enzymes, (iii) perform a filter function for osmotic regulation and (iv) act as a selectively permeable barrier against macromolecules (Brunet et al., 1994; Raynard et al., 2002). This may prevent an antibody binding to an antigen (Raynard et al., 2002). ...
Atlantic salmon (Salmo salar) were experimentally infected with Lepeophtheirus salmonis copepodids and aspects of the host’s immune response investigated. Copepodid secretory/excretory product (SEP) produced during early settlement was analysed using fast-protein liquid chromatography (FPLC), sodium dodecyl sulphate (SDS)- electrophoresis and zymography. Following establishment and the appearance of the chalimus stages, the expression of the chemokine interleukin-8 (IL-8) in the heart, spleen, head kidney, fins, liver and pyloric cæca was investigated using real-time (quantitative) PCR (qPCR). Furthermore, the secretions of L. salmonis chalimus were analysed for the presence of the prostanoid PGE2 using commercially available enzyme-linked immunoassay (EIA) kits. Analysis of copepodid secretory/excretory product suggested that any immunosuppressive component is not proteinaceous in nature. Whilst there was a definite increase in protein concentration of SEP relative to control SEP, further analysis using subtractive chromatographic analysis did not reveal any unique fraction present in either SEP or CSEP that was absent in the other. Interleukin-8 expression levels in tissues changed following L. salmonis infection, with heart and spleen showing significant increases in IL-8 gene expression, whilst the head kidney, fins, liver and pyloric cæca showed no significant increase. The increase in splenic IL-8 expression may be linked to its role as one of the major secondary lymphoid organs. However, this is the first record of increase in IL-8 expression in cardiac tissue. The secretions of L. salmonis chalimus were found to contain quantifiable levels of PGE2, albeit in highly variable quantities. This concurs with already published findings for adult L. salmonis (see Fast, et al. 2004). It is proposed that the chalimus states us the PGE2 to modulate the hosts’ immune response at the site of attachment and feeding.
... These studies report parasite burden reduction in S. salar, with efficacy between 30 to 57%. Furthermore, in response to ectoparasite vaccination, fish-host humoral and cellular components have been observed [36][37][38]. Notably, has been observed the expression modulation of IgM and IgT, antioxidant response, and related inflammatory genes in fish immunized and exposed to the ectoparasite infestation [37,39,40]. Moreover, the use of antigens associated with the host-iron metabolism modulation was recently reported [41]. ...
... Furthermore, in response to ectoparasite vaccination, fish-host humoral and cellular components have been observed [36][37][38]. Notably, has been observed the expression modulation of IgM and IgT, antioxidant response, and related inflammatory genes in fish immunized and exposed to the ectoparasite infestation [37,39,40]. Moreover, the use of antigens associated with the host-iron metabolism modulation was recently reported [41]. ...
The sea louse Caligus rogercresseyi genome has opened the opportunity to apply the reverse vaccinology strategy for identifying antigens with potential effects on lice development and its application in sea lice control. This study aimed to explore the efficacy of three sea lice vaccines against the early stage of infestation, assessing the transcriptome modulation of immunized Atlantic salmon. Therein, three experimental groups of Salmo salar (Atlantic salmon) were vaccinated with the recombinant proteins: Peritrophin (prototype A), Cathepsin (prototype B), and the mix of them (prototype C), respectively. Sea lice infestation was evaluated during chalimus I-II, the early-infective stages attached at 7-days post infestation. In parallel, head kidney and skin tissue samples were taken for mRNA Illumina sequencing. Relative expression analyses of genes were conducted to identify immune responses, iron transport, and stress responses associated with the tested vaccines during the early stages of sea lice infection. The vaccine prototypes A, B, and C reduced the parasite burden by 24, 44, and 52% compared with the control group. In addition, the RNA-Seq analysis exhibited a prototype-dependent transcriptome modulation. The high expression differences were observed in genes associated with metal ion binding, molecular processes, and energy production. The findings suggest a balance between the host’s inflammatory response and metabolic process in vaccinated fish, increasing their transcriptional activity, which can alter the early host–parasite interactions. This study uncovers molecular responses produced by three vaccine prototypes at the early stages of infestation, providing new knowledge for sea lice control in the salmon aquaculture.
... However, L. salmonis has developed resistance to many of these products and only some of them are still used in the salmon industry . The resistance and the toxicity of these products has led researchers to begin developing alternative methods to fight against the parasite (Raynard et al. 2002;Treasurer 2002;Erikson and Misimi 2008a;). Among these alternative methods, the use of semiochemicals to repel or to attract the parasite into a trap appears to be a promising approach . ...
... Le saumon étant un poisson à viande riche en lipides, des résidus de ces produits phytosanitaires peuvent se retrouver dans la viande destinée à la consommation (Berntssen et al. 2011;Nostbakken et al. 2015). Des stratégies alternatives sont donc développées pour lutter contre ce parasite (Deady et al. 1995;Raynard et al. 2002;Bricknell et al. 2003;Molloy et al. 2011;Gharbi et al. 2015). La sémiochimie apparait alors comme un moyen de lutte intéressant abordé par . ...
From hatching to slaughter, Atlantic salmon and Rainbow trout are subjected to several stressful events. Stress impairs physiological, behavioral and zootechnical performances of animals. The public interest in animal welfare brings us to study the assessment and control methods of stress in farmed salmonids. To do that, we studied the consequences of two different stressful events and proposed a method for assessing welfare of freshwater Rainbow trout. We also studied the interaction between stress and pathology with the salmon louse Lepeophtheirus salmonis. After developing an attractiveness test of salmon for the parasite, we investigated the influence of short- and long-term stress on salmon attractiveness for the parasite. Handling stress increased attractiveness of salmon for the parasite, while there was no difference between chronically stressed and control salmon. We proposed then a method to control this parasite by isolating a semiochemical which inhibit the hooking behavior of the parasite on the host. These results open interesting prospects for improving the assessment and control methods of stress in farmed salmonids. The investigation of stress consequences on animal welfare and on production performances is crucial. The semiochemical inhibiting the hooking behavior of the parasite on the salmon needs more studies to identify its mechanisms of action for use in farms.
... host mucus, skin, and blood have only limited contact with the host immune system. Pike and Wadsworth (1999) summarized studies of immune modulation and noted that the younger stages, which have a more intimate association with host tissues, might be a target for vaccines. They also report on immunohistochemical screening of monoclonal antibodies. Raynard et al. (2002) noted that research to develop vaccines against sea lice is still in its infancy. A vaccine has been developed against the blood-feeding cattle tick, Boophilus microplus, but the assumption that arachnid and insect physiology are directly comparable with that of sea lice is not proven, and success in developing a sea louse vaccine will ...
... Raynard et al. (2002) noted that research to develop vaccines against sea lice is still in its infancy. A vaccine has been developed against the blood-feeding cattle tick, Boophilus microplus, but the assumption that arachnid and insect physiology are directly comparable with that of sea lice is not proven, and success in developing a sea louse vaccine will depend on a better understanding of sea louse digestive biology (Raynard et al., 2002). Trypsins in the gut L. salmonis consistently consume significant blood meals, as suggested by the red gut seen in adult females. ...
Studies of the biology of sea lice have been conducted from various perspectives for two decades. For Lepeophtheirus spp., most of the published literature has centred on the economically important Lepeophtheirus salmonis, while for Caligus spp., research has focused on a wider range of species. The most numerous species of Caligus in North Atlantic waters, however, is Caligus elongatus, which is also economically important to salmon fanning. Since the last review by Pike, A. W., and Wadsworth, S. L. (1999. Sea lice on salmonids: their biology and control. Advances in Parasitology, 44: 234-337.), research on sea lice has developed considerably, including the application of genetic methods. This new research has focused on life history biology, studying developmental stages under different environmental conditions (e.g. temperature and salinity), behaviour, distribution and the dispersal of free-living stages, monitoring practices, population structure, and modelling. The results of this research have informed risk analyses and allowed the refinement of management strategies to reduce sea lice infestations in wild and fanned populations of anadromous salmonids. Molecular techniques have been used to describe population structure and identify differences in genetic characterization of geographically separate populations and population markers. Research has been initiated to understand the parasite-host relationship at a molecular level and to develop a vaccine against sea lice. (c) 2006 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
... However, research toward such vaccines is at an early stage and approaches thus far have met with little or no success. Most strategies for sea lice vaccines have adopted methods used for vaccines against other ectoparasites but the assumption that arachnid and insect physiology are directly comparable with that of sea lice is not proven [35]. ...
... These advantages include sustained action and no withdrawal period or residual drugs within the flesh. A generic approach to the development of effective vaccines against ectoparasites arthropods has been described previously [12,31,35]. The range of target antigens mentioned included a variety of components associated with the gut, parasite attachment, feeding, hormonal regulation and reproduction. ...
... Moreover, the percent of loss in total biomass growth per production cycle due to average infestations varied from 3.62 to 16.55% [3]. Similarly, Lepeophtheirus salmonis (sea lice), is an economically important fish ectoparasite in other European countries and America, and it's infestations in salmonid culture are worrisome in nature that demand effective management strategies [4]. ...
•Argulosis, a disease caused by Argulus spp. of ectoparasites in scaly fish, is a global concern for aquaculture industry.•The resistance of the parasite to anti-parasitic drugs and the quantum of loss has been felt world-wide.•The current scenario of management and the development in vaccination are discussed herewith.
... Vaccines against ectoparasites are still in their infancy, with a few successful commercial products providing reliable protection, such as against cattle tick (Willadsen et al., 1995). The complexity of interactions between vertebrate host and arthropod parasites is yet to be explored for potential vaccine targets (Raynard et al., 2002;Fuente et al., 2016). Translated research from advances in the tick vaccine have already provided promising results in peptide vaccines against salmon lice. ...
The arthropod salmon louse (Lepeophtheirus salmonis) is a major threat to Atlantic salmon aquaculture and wild salmonids. Essentially like in monoculture, very high concentrations of susceptible hosts may result in high reproduction and severe production of waves of pests. Pest management is crucial both for fish health and protection of wild fish populations from aquaculture influence. Various methods have been utilized to control salmon lice infestations, such as pesticide use, physical treatments, construction modifications, fallowing, breeding, vaccination, and biological control. Most of the methods are partially successful, but none completely fulfills the necessary pest control strategy. Like in agriculture, lice/pest management is an arms race, but the marine environment makes it even more difficult to precisely hit the target pest and avoid unintended negative effects on general wildlife. In this study, we provide an overview of the methods and principles of salmon lice management and address current possibilities and limitations. We also highlight the potential of emerging strategies and enabling technologies, like genome editing, RNA interference, and machine learning, in arthropod management in aquaculture.
... The success and sustainability of the Atlantic salmon farming industry, and the welfare of wild salmon populations, are dependent on effective disease prevention, control, and health management regimes. Many such strategies are currently in use or being developed, including chemotherapeutants (e.g., 'in-feed' immunostimulants [14] and probiotics [15] or in situ 'baths' with parasiticides [16]), vaccination [17], and cleaner fish [2,5,18,19]. Since each of these sea lice control strategies has advantages and disadvantages, an Integrated Pest Management (IPM) approach has been employed [20]. ...
Parasitic sea lice (e.g., Lepeophtheirus salmonis) cause costly outbreaks in salmon farming. Molecular insights into parasite-induced host responses will provide the basis for improved management strategies. We investigated the early transcriptomic responses in pelvic fins of Atlantic salmon parasitized with chalimus I stage sea lice. Fin samples collected from non-infected (i.e. pre-infected) control (PRE) and at chalimus-attachment sites (ATT) and adjacent to chalimus-attachment sites (ADJ) from infected fish were used in profiling global gene expression using 44 K microarrays. We identified 6568 differentially expressed probes (DEPs, FDR < 5%) that included 1928 shared DEPs between ATT and ADJ compared to PRE. The ATT versus ADJ comparison revealed 90 DEPs, all of which were upregulated in ATT samples. Gene ontology/pathway term network analyses revealed profound changes in physiological processes, including extracellular matrix (ECM) degradation, tissue repair/remodeling and wound healing, immunity and defense, chemotaxis and signaling, antiviral response, and redox homeostasis in infected fins. The QPCR analysis of 37 microarray-identified transcripts representing these functional themes served to confirm the microarray results with a significant positive correlation (p < 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon’s fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon’s attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for L. salmonis.
... The number of reported non-chemical delousing methods has increased since 2016 in Norway [7]; technical innovations, such as surrounding the upper layer of a cage with an impermeable skirt and the use of snorkel cages, have been introduced as preventive measures to hinder the spread of salmon lice both within and between salmon farms. Research has also been carried out on selective breeding for salmon, and on the development of functional feeds and vaccines to combat the salmon louse [8][9][10][11]. ...
The salmon louse (Lepeophtheirus salmonis) causes problems in Atlantic salmon (Salmo salar) aquaculture in the Northern Hemisphere, because infestations can result in both a loss of production and in fish mortality. Several types of treatment have been used to control louse infestations, but these have seen varying success. The aim of this review is to examine the efficacy and safety of commonly used treatments (chemical, biological, mechanical, and preventive measures) as documented in peer-reviewed publications. Efficacy is assessed in relation to a reduction in numbers of lice, and safety is assessed as a lack of negative treatment-associated effects on fish health and welfare (Atlantic salmon and/or cleaner fish). Most chemical treatments showed decreasing efficacy over time, together with the use of increasing concentrations as a result of the development of resistance to the treatments by lice. The need for a restrictive use of pesticides to preserve treatment efficacy has been emphasized. The use of cleaner fish was suggested to be effective, with few or no negative effects towards Atlantic salmon. The use of cleaner fish would be preferable to chemical treatment if the farmed fish health and welfare criteria are met. At present, the number of peer-reviewed publications relating to other forms of treatment and prevention are sparse.
... However, no commercially available anti-sea lice vaccines exist. Previously published results suggest that the discovery of protective antigens, vaccine formulation, safety, administration, and efficacy need to be addressed to obtain regulatory approval and advance in the implementation of vaccination strategies for the control of salmon lice and other fish ectoparasites [15][16][17][18][19][20]. ...
Infestation with the salmon louse Lepeophtheirus salmonis (Copepoda, Caligidae) affects Atlantic salmon (Salmo salar L.) production in European aquaculture. Furthermore, high levels of salmon lice in farms significantly increase challenge pressure against wild salmon populations. Currently, available control methods for salmon louse have limitations, and vaccination appears as an attractive, environmentally sound strategy. In this study, we addressed one of the main limitations for vaccine development, the identification of candidate protective antigens. Based on recent advances in tick vaccine research, herein, we targeted the salmon louse midgut function and blood digestion for the identification of candidate target proteins for the control of ectoparasite infestations. The results of this translational approach resulted in the identification and subsequent evaluation of the new candidate protective antigens, putative Toll-like receptor 6 (P30), and potassium chloride, and amino acid transporter (P33). Vaccination with these antigens provided protection in Atlantic salmon by reducing adult female (P33) or chalimus II (P30) sea lice infestations. These results support the development of vaccines for the control of sea lice infestations.
... As a solution for the industry, a vaccine against the caligus copepod Lepeophteirus salmonis, with the same principle than cattle tick vaccine using a concealed antigen (antigens from a pathogen able to elicit a protective immune response, that are not naturally exposed to the host immune system, for example, a gut protein from a parasite) was made [106,107]. The effort did not stop there, and a vaccine against C. rogercresseyi was proposed [108] using the recombinant protein my32 from C. rogercresseyi. ...
In Chile, the salmon and trout farmed fishing industries have rapidly grown during the last years, becoming one of the most important economic sources for the country. However, infectious diseases caused by bacteria, virus, mycoses and parasites, result in losses of up to 700 million dollars per year for the Chilean aquaculture production with the consequent increase of antibiotic and antiparasitic usage. After 30 years of its first appearance, the main salmon health problem is still the salmonid rickettsial septicaemia (SRS), which together with other disease outbreaks, reveal that vaccines do not provide acceptable levels of long-lasting immune protection in the field. On the other hand, due to the large dependence of the industry on salmonids production, the Chilean government promoted the Aquaculture diversification program by 2009, which includes new species such as Merluccius australis, Cilus gilberti and Genypterus chilensis, however, specific research regarding the immune system and vaccine development are issues that still need to be addressed and must be considered as important as the farm production technologies for new fish species. Based on the experience acquired from the salmonid fish farming, should be mandatory an effort to study the immune system of the new species to develop knowledge for vaccination approaches, aiming to protect these aquaculture species before diseases outbreaks may occur. This review focuses on the current status of the Chilean aquaculture industry, the challenges related to emerging and re-emerging microbial pathogens on salmonid fish farming, and the resulting needs in the development of immune protection by rational designed vaccines. We also discussed about what we have learn from 25 years of salmonid researches and what can be applied to the new Chilean farmed species on immunology and vaccinology.
... This control method was more prevalent in earlier stages of salmon farming; however currently, it is used more in countries that still have treatment-sensitive louse populations (Saksida et al. 2011). Although a vaccine is being developed against sea lice (Raynard et al. 2002), it remains elusive and will not be 100% effective if it eventuates, as few vaccines are. Nevertheless, vaccination resistance is less likely to develop when compared to the rate of resistance to drugs and chemical therapeutants (Kennedy & Read 2017). ...
Host behaviour can prevent infection and moderate the fitness of parasites. Antiparasite behaviours are prevalent in many host–parasite systems and occur over fine or broad scales. With global growth in aquaculture production and the associated proliferation of parasites in farming systems, the behaviour of the fish being farmed has seldom been investigated in relation to parasites. Epidemics and outbreaks of parasites are prevalent in most aquaculture systems, and behaviour could be harnessed in concert with current methods to prevent and control parasites and pathogens. However, this requires a systematic understanding of the behaviours of hosts, their capacity for resistance and their interaction with the environment and the parasite. Herein, we present evidence for how behaviour could be used in aquaculture, and discuss the possibility for behaviour to be used in aquaculture as (i) an indicator of welfare status, (ii) a tool in prevention or control and (iii) to maintain or improve welfare. We apply this framework to a case study of a highly problematic parasite, the salmon louse (Lepeophtheirus salmonis), on farmed Atlantic salmon (Salmo salar). We present the current state of the system, and the drawbacks of current control or prevention methods. We synergise current knowledge on host behaviours and show how behaviour could be incorporated into current and new approaches for prevention and control. Through this first evaluation of the possibilities behaviour presents in disease management, we aim to facilitate a shift in the current disease control paradigm from reactive-based post-infection control to pre-infection prevention approaches.
... These include cleaner fish 10 , thermolicer 11 and alternative production forms limiting the settlement of lice 12 . In addition, the industry is investigating the potential of breeding for increased resistance in host-fish 13,14 , and attempting to develop vaccines 15,16 . Despite this suite of control strategies, the industry is ultimately reliant on a limited number of chemical therapeutants applied as bath or in-feed treatments to control infections 17 . ...
The salmon louse is an ectoparasitic copepod of salmonids in the marine environment, and represents a global challenge to salmon aquaculture. A major issue is the reliance of the industry on a limited number of chemicals to delouse salmonids on farms, and the high levels of resistance that lice have developed to all of these agents. However, for most of these chemicals, resistance and dispersal mechanisms are unknown. We recently demonstrated that the Phe362Tyr mutation is the primary cause of organophosphate resistance in lice collected on Norwegian farms. In the present study, we genotyped >2000 lice collected throughout the entire North Atlantic in the period 1998-2016, using Phe362Tyr and nine tightly linked SNPs. Our results showed that the Phe362Tyr mutation is strongly linked to lice survival following chemical treatment on farms located throughout the North Atlantic, demonstrating for the first time, that this mutation represents the primary mechanism for organophosphate resistance in salmon lice across the North Atlantic. Additionally, we observed multiple and diverse high frequency haplotypes linked with the allele conveying resistance to organophosphate. We, therefore, conclude that Phe362Tyr is not a de novo mutation, but probably existed in salmon lice before the introduction of organophosphates in commercial aquaculture.
... This can be intermittently used to boost the host immune response; for example, when no vaccine is available against the pathogen (Casadei et al. 2013). No commercially available vaccine exists for salmon lice L. salmonis, although efforts towards this goal have been made (Raynard et al. 2002). ...
Salmon lice (Lepeophtheirus salmonis) are important ectoparasites of wild and farmed salmonids and cause major losses to the salmon farming industry throughout the Northern Hemisphere. With the emergence of resistance to several commonly used parasiticides, novel control strategies and integra- tion of multiple treatment options are needed, including host immunostimulation. Here, we investi- gate the effects of a functional feed containing a peptidoglycan and nucleotide formulation on L. salmonis infection of Atlantic salmon (Salmo salar) by characterizing lice infection levels, the expression of several host immune genes, and the parasite transcriptomic response to the immunos- timulated host. Although initial infection intensities were low, the low dose (LD) immunostimulant diet reduced the total lice burden by 50% relative to controls. Immunostimulant fed hosts up- regulated interleukin-1β in the skin and spleen. This gene has been implicated in successful responses of several salmonid species to salmon lice but is typically not observed in Atlantic salmon, suggesting a favorable influence on the immune response. Lice infecting LD immunostimulated salmon overex- pressed genes putatively involved in parasite immunity, including carboxylesterases, and underex- pressed genes putatively involved in feeding (e.g., proteases). These lice response genes further improve the characterization of the transcriptome of the non-model parasite by identifying genes potentially involved in evading host immunity.
... Medical treatment of salmon with parasiticides is widespread; however, where medical treatments are relied on for repeated treatments, the sea lice population will eventually develop resistance or tolerance of the chemicals, as has occurred with other terrestrial parasites. No commercial vaccines for sea lice exist although this has been the focus for research for over 20 years (Raynard et al. 2002 ). ...
One of the main hurdles to sustainable finfish aquaculture in many regions has been the management and control of infectious disease. The most significant diseases of salmonid, carp, catfish, tilapia and marine finfish farming are considered in this chapter by viral, bacterial, parasitic and fungal group. The level of impact caused by disease and methods for control or management are outlined.
... However, salmon immunised with a crude extract of L. salmonis were found to be infected with fewer ovigerous parasites and those present carried fewer developing embryos compared with non-immunised controls following a laboratory exposure . While earlier evidence did not support the development of protective immunity in salmon as a result of a previous infection with L. salmonis or immunization with parasite antigens (Raynard et al. 2002), it is possible that effective vaccination may interfere with parasite reproduction, similar to that seen in the freshwater gourami Helostoma temmincki infected with the copepod Lernea cyprinacea (Woo and Sharif 1990). The recent sequencing of the L. salmonis genome has identified approximately 22,000 genes (Torrissen et al. 2013) and some of these may be shown to encode candidate vaccine antigens. ...
Five species of sea lice (Lepeophtheirus salmonis, L. cuneifer, Caligus elongatus, C. clemensi, C. curtus) are reported from net pen-reared salmonids in Canada. Of these, L. salmonis is the largest and most aggressive species and is the primary focus of management activities and regulations. Sea louse biology is broadly divisible into free-living and parasitic phases. The development and survival of sea lice during both phases of development are regulated by the ambient seawater temperature and salinity. In addition, free-living stages possess numerous adaptations that permit sensing of environmental gradients of light, substances secreted by fish (semiochemicals) and mechanical energy (vibrations derived from fish movements). These adaptations enhance larval survival by optimising host-detection and settlement behaviour. The high fecundity of adult female sea lice further optimises parasite survival. There is a wide range of susceptibilities to L. salmonis among salmonid host species in Canada indicating that sea lice survival is also mediated by host factors. Infestations tend to be of lower intensity and of shorter duration on less susceptible species. There are significant genetic differences between the varieties of L. salmonis that occur on the Pacific and Atlantic coasts of Canada. Further research is required to determine the biological significance of the genetic differences and to better understand the mechanisms responsible for host resistance to sea lice. This Research Document was presented and reviewed as part of the Canadian Science Advisory Secretariat (CSAS) National peer-review meeting, Sea Lice Monitoring and Non-Chemical Measures, held in Ottawa, Ontario, September 25-27, 2012. The objective of this peer-review meeting was to assess the state of knowledge and provide scientific advice on sea lice management measures, monitoring and interactions between cultured and wild fish.
... Other biological agents such as viruses, fungi, bacteria and hyper-parasites infecting sea lice may become important alternative control agents (Treasurer 2002). Vaccines to control salmon louse may also soon be developed (Raynard, Bricknell, Billingsley, Nisbet, Vigneau & Sommerville 2002 -790, 2000). This limit has been set by the government to protect the wild salmonid stocks (Heuch, Revie, & Gettinby 2003). ...
... These include pest management strategies such as synchronised delousing [30], coordinated fallowing [31] and temporary protected zones [32,33]. They also include more direct control methods such as biological control with cleaner fish [34,35], selective breeding for resistant fish [36,37] and, potentially, vaccine development [38,39]. Nevertheless, despite the availability of a variety of methods, the industry is heavily reliant on anti-parasitic chemicals, applied as bath treatments or orally administered in-feed, to delouse fish in farms [40]. ...
Background
The development of pesticide resistance represents a global challenge to food production. Specifically for the Atlantic salmon aquaculture industry, parasitic sea lice and their developing resistance to delousing chemicals is challenging production. In this study, seventeen full sibling families, established from three strains of Lepeophtheirus salmonis displaying differing backgrounds in emamectin benzoate (EB) tolerance were produced and quantitatively compared under a common-garden experimental design. Lice surviving to the preadult stage were then exposed to EB and finally identified through the application of DNA parentage testing.
Results
With the exception of two families (19 and 29%), survival from the infectious copepod to preadult stage was very similar among families (40-50%). In contrast, very large differences in survival following EB exposure were observed among the families (7.9-74%). Family survival post EB exposure was consistent with the EB tolerance characteristics of the strains from which they were established and no negative effect on infection success were detected in association with increased EB tolerance. Two of the lice families that displayed reduced sensitivity to EB were established from a commercial farm that had previously used this chemical. This demonstrates that resistant alleles were present on this farm even though the farm had not reported treatment failure.
Conclusions
To our knowledge, this represents the first study where families of any multi-cellular parasite have been established and compared in performance under communal rearing conditions in a common-garden experiment. The system performed in a predictable manner and permitted, for the first time, elucidation of quantitative traits among sea lice families. While this experiment concentrated on, and provided a unique insight into EB sensitivity among lice families, the experimental design represents a novel methodology to experimentally address both resistance development and other evolutionary questions in parasitic copepods.
... While there are a range of strategies that can be used in an integrated pest management plan against L. salmonis (Brooks, 2009; Jones et al., 2002; Mordue (Luntz) and Pike, 2002; Rae, 2002; Raynard et al., 2002; Treasurer, 2002), the aquaculture industry is still more or less reliant upon delousing agents to control this parasite on cage-reared salmonids (Grant, 2002; Lees et al., 2008a; Robertson et al., 2009). It is therefore of vital importance to use delousing agents in a controlled manner to inhibit L. salmonis from developing resistance, and also, to inhibit the dispersal of resistance genes among regions. ...
Reduced sensitivity to the avermectin emamectin benzoate (EB) was suspected in salmon lice, collected from five Norwegian salmon farms located in Middle and Western Norway in the period January 2008–February 2009. After being transferred to the laboratory, lice from each farm were compared to an EB sensitive laboratory strain (LsGulen, collected from the field in June 2006) using bioassays. All five samples were thereafter confirmed as displaying reduced sensitivity to EB. A strain (LsAustevoll) based upon lice from one of these farms was subse-quently established in the laboratory. LsAustevoll was compared to the EB sensitive strain, LsGulen, for 4 gener-ations. A hybrid strain (LsHybrid), established by crossing LsGulen and LsAustevoll, was included in the comparisons for 3 generations. Sensitivity to EB was assessed for each generation, and the fitness parameters: egg production, hatching success and copepodid survival were assessed for selected generations. LsAustevoll retained reduced sensitivity to EB throughout all four generations, while LsHybrid displayed intermediate EB sen-sitivity to its parental strains. No fitness costs associated with reduced EB sensitivity were detected for any of the parameters studied.
... The relatively poor response elicited during infection is a likely result of limited exposure of the salmon immune system to copepod antigens, some of which are associated with gut epithelium [37,39], during attachment and feeding on the skin. The available evidence does not indicate that salmon develop protective immunity as a result of a previous infection with L. salmonis [40] and it is not known whether the response elicited by injection with louse homogenates is protective. ...
The purpose of this paper is to review the current knowledge of salmonid defence responses to Lepeophtheirus salmonis. The salmon louse L. salmonis is an important pest of economically valuable salmonids in seawater throughout the northern hemisphere. Treatment of salmon lice on cultured salmon often fails in regions where the parasite has developed resistance to commonly used therapeutants. The development of effcacious vaccines is hampered by limited knowledge of parasite antigens that elicit protective immunity and a poor understanding of defence responses mounted by the salmonid host. Infection kinetics indicate a wide range of susceptibilities to L. salmonis among salmon species: juvenile coho and pink salmon are relatively resistant whereas Atlantic and chum salmon are susceptible. Innate resistance is linked to the speed and intensity of local infammatory reactions at the site of infection. Conversely, susceptibility is related to an absence of these reactions and in Atlantic salmon is mediated in part by hypersecretion by the parasite of prostaglandin E2 and other compounds. Transcriptomic analysis shows that the susceptible salmonid response is characterised by cell stress, tissue remodelling and diminished immunological responsiveness during infection. In contrast, there is evidence of cell motility, somatic growth and immuncompetence among resistant salmon following infection. Future research should apply a combination of genomic, proteomic and immunological studies to better understand defence mechanisms among susceptible and resistant salmonids.
... Vaccination has been proposed as a means of controlling sea lice following advances in the development of vaccines against ectoparasites in mammals. Despite predictions of significant impairment of salmon antibody function in sea lice, due to the similar osmolarity of sea water and hemolymph, no real evidence for this has been provided (Raynard et al. 2002). On the other hand, challenge models with sea lice suffer from a high degree of variability and low reproducibility. ...
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.
... Intensive rearing of fish typically relies on the periodic administration of chemotherapeutic treatments to control parasitic and microbial infestations, although a range of potential alternative methods are under investigation (Jones et al., 2002;Raynard et al., 2002;Treasurer, 2002). Commercial applications to treat sea lice-infested salmon include Salmosan ® , Excis ® and Slice ® . ...
The commercial farming of Atlantic salmon, Salmo salar, typically requires the periodic application of copper-based anti-foulants and chemotherapeutic treatments, including Slice®, Excis®, Salmosan® and Aquatet®/Tetraplex® to reduce the effects of biological pests. Information on the environmental safety of any chemical agent released into the aquatic environment must be obtained before a product can be licensed for use, but such information typically exists only in confidential technical reports which can be difficult to obtain. Furthermore, different test organisms, experimental protocols and reporting procedures make comparison of the various compounds/studies difficult. Here we present a series of 10-day, whole sediment bioassay tests that determined the concentrations of emamectin benzoate (EB), cypermethrin (CP), azamethiphos (AZ) and oxytetracycline hydrochloride (OTC), the active ingredients of the aforementioned chemotherapeutants, and copper, that caused 50% mortality (LC50) in the non-target benthic crustacean, Corophium volutator. Additional whole sediment bioassays of identical design were conducted with the non-target polychaete worm, Hediste diversicolor exposed to copper and EB. C. volutator demonstrated similar sensitivity to EB and AZ, with LC50s of 153 (95% confidence intervals, CI=119–198) and 182 (95% CI=152–217) µg active ingredient [kg wet sediment]−1 respectively. OTC caused 50% mortality of C. volutator at a concentration of 414 (95% CI=233–734) µg OTC [kg wet sediment]−1. The LC50 for H. diversicolor exposed to EB was 1368 (95% CI=744–2516) µg EB [kg wet sediment]−1, an order of magnitude greater than that for C. volutator. Conversely, C. volutator was able to tolerate much higher concentrations of copper than H. diversicolor, with LC50s of 193,326 (95% CI=171,034–218,523) and 74,988 (95% CI=61,192–91,895) µg Cu [kg wet sediment]−1 respectively. CP was the most toxic to C. volutator of all compounds investigated, causing 50% mortality at a concentration of 5 (95% CI=4–6) µg CP [kg wet sediment]−1. These data allow direct inter-comparison of the toxicities of some of the commonly applied treatments used by the global salmonid aquaculture industry.
... Major infestations ultimately cause mortality if not controlled (Pike and Wadsworth, 1999), costing the industry more than US $100 million annually (Johnson et al., 2004). Intensive rearing of fish typically relies on the periodic administration of chemotherapeutic treatments to control parasitic and microbial infestations, although alternative methods including vaccines (Raynard et al., 2002), selective breeding of lice-resistant strains (Jones et al., 2002), and the use of sea lice pathogens and cleaner fish (Treasurer, 2002) are available in some instances. ...
Intensification of fish farming activity and the amalgamation of farm units has the potential to cause an increased risk of organic and chemical stress on the local environment. Although the amalgamation of fish farm units into a fewer number of larger operations may offer some operators greater financial efficiency, concern exists over whether the discharge of organic matter (waste feed and faeces), sea lice chemical treatments and other synthetic organic compounds from large scale operations may have a greater environmental impact than an equivalent use of such compounds at several smaller farm units. To address this concern, it is necessary to assess how fish farm amalgamation can be implemented in an environmentally sustainable manner. This is an issue facing the entire fin fish marine aquaculture industry to ensure that it can effectively deliver on its obligations to the Strategic Framework for Scottish Aquaculture (Scottish Executive, 2003). Here, we establish the relationship between farm size and zone of impact using a field survey before establishing highly controlled replicated mesocosm studies aimed at establishing the relationship between synthetic organic compounds, including sealice treatment chemicals, and the functioning of the coastal benthos. These experiments avoid the problems associated with confounding and covarying factors in the field and form the basis for numerical simulations that predict how fish farm amalgamation may impact the environment. Should the mechanisms identified in our model systems operate in the real world, the results could be used to constructively inform policymakers on how to design amalgamated fish farms where impact exceeds current practice.
... There is currently very little evidence that Atlantic salmon mount a protective immune response to either L. salmonis infection or immunization with parasite antigens (Grayson et al. 1991; Reilly & Mulcahy 1993; Roper et al. 1995). Furthermore, Atlantic salmon remain susceptible to reinfection following recovery from L. salmonis (Raynard et al. 2002). Thus, the development of a vaccine against L. salmonis in Atlantic salmon remains a long-term goal and may depend on the selection of suitable salmon strains in which natural resistance is already high. ...
Salmon lice, Lepeophtheirus salmonis, are naturally occurring parasites of salmon in sea water. Intensive salmon farming provides better conditions for parasite growth and transmission compared with natural conditions, creating problems for both the salmon farming industry and, under certain conditions , wild salmonids. Salmon lice originating from farms negatively impact wild stocks of salmonids, although the extent of the impact is a matter of debate. Estimates from Ireland and Norway indicate an odds ratio of 1.1:1-1.2:1 for sea lice treated Atlantic salmon smolt to survive sea migration compared to untreated smolts. This is considered to have a moderate population regulatory effect. The development of resistance against drugs most commonly used to treat salmon lice is a serious concern for both wild and farmed fish. Several large initiatives have been taken to encourage the development of new strategies , such as vaccines and novel drugs, for the treatment or removal of salmon lice from farmed fish. The newly sequenced salmon louse genome will be an important tool in this work. The use of cleaner fish has emerged as a robust method for controlling salmon lice, and aquaculture production of wrasse is important towards this aim. Salmon lice have large economic consequences for the salmon industry, both as direct costs for the prevention and treatment, but also indirectly through negative public opinion.
... There is currently very little evidence that Atlantic salmon mount a protective immune response to either L. salmonis infection or immunization with parasite antigens ( Grayson et al. 1991;Reilly & Mulcahy 1993;Roper et al. 1995). Furthermore, Atlantic salmon remain susceptible to reinfection following recovery from L. salmonis ( Raynard et al. 2002). Thus, the development of a vaccine against L. salmonis in Atlantic salmon remains a long-term goal and may depend on the selection of suitable salmon strains in which natural resistance is already high. ...
Salmon lice, Lepeophtheirus salmonis, are naturally occurring parasites of salmon in sea water. Intensive salmon farming provides better conditions for parasite growth and transmission compared with natural conditions, creating problems for both the salmon farming industry and, under certain conditions, wild salmonids. Salmon lice originating from farms negatively impact wild stocks of salmonids, although the extent of the impact is a matter of debate. Estimates from Ireland and Norway indicate an odds ratio of 1.1:1-1.2:1 for sea lice treated Atlantic salmon smolt to survive sea migration compared to untreated smolts. This is considered to have a moderate population regulatory effect. The development of resistance against drugs most commonly used to treat salmon lice is a serious concern for both wild and farmed fish. Several large initiatives have been taken to encourage the development of new strategies, such as vaccines and novel drugs, for the treatment or removal of salmon lice from farmed fish. The newly sequenced salmon louse genome will be an important tool in this work. The use of cleaner fish has emerged as a robust method for controlling salmon lice, and aquaculture production of wrasse is important towards this aim. Salmon lice have large economic consequences for the salmon industry, both as direct costs for the prevention and treatment, but also indirectly through negative public opinion.
... Widespread development of drug resistance in L. salmonis underscores the need for an integrated sea lice management approach to the control of sea lice in salmon farms. This can be achieved through the combined use of drugs, chemicals and non-chemical alternatives including manipulation of host immunity using vaccines and/or immunostimulants (Raynard et al. 2002). Stimulating the innate immunity of fish hosts against pathogen and parasite invasion (Bricknell & Dalmo 2005) is increasingly adopted as part of disease management in aquaculture including sea cage salmon farming (Tacchi et al. 2011). ...
Control of sea lice, Lepeophtheirus salmonis, on farmed Atlantic salmon, Salmo salar, relies heavily on chemotherapeutants. However, reduced efficacy of many treatments and need for integrated sea lice management plans require innovative strategies. Resistance to emamectin benzoate (EMB), a major sea lice parasiticide, has been linked with P-glycoprotein (P-gp) expression. We hypothesized that host immunostimulation would complement EMB treatment outcome. Lepeophtheirus salmonis-infected Atlantic salmon were fed immunostimulatory or control feeds. Sea lice were collected for 24-h EMB bioassays 1 and 2 weeks prior to commencement of EMB treatment of the fish. Two weeks after cessation of immunostimulant-treated feed, EMB was administered at 150 μg kg(-1) fish biomass for 7 days. The bioassay revealed stage, gender and immunostimulant-related differences in EMB EC(50) . Sea lice attached to salmon with a history of immunostimulation exhibited significantly greater survival than those on control feeds, despite similar levels of EMB in host tissues. Lepeophtheirus salmonis from salmon with a history of immunostimulation also exhibited higher P-gp mRNA expression as well as greater survivability compared to controls. Administration of immunostimulants prior to EMB treatment caused increased expression of P-gp mRNA which could have consequently caused decreased efficacy of the parasiticide.
... A vaccine against salmon lice has been described as a 'Holy Grail' [98]. Despite the promise of early efforts to characterize potential candidate L. salmonis antigens [99][100][101] there is no indication that a commercially viable vaccine will be available soon [17]. However, recent applications of microarray technology to understand gene expression [102] and the descriptions of L. salmonis proteins [103,104] suggest genomic and proteomic methods have potential in providing novel approaches for vaccine development. ...
This review documents the extent to which management of salmon lice (Lepeophtheirus salmonis) on farms has a measurable impact on the health of wild salmon. The salmon louse is a persistent and often severe parasite of farmed salmon throughout the Northern Hemisphere. High farm densities in some areas have led to lice levels within adjacent wild salmon populations that are higher than occur naturally. To minimize the additional infection pressure of the parasite on wild salmon, aquaculture industries adopt strategies of integrated pest management that include systematic monitoring, treatment, locating farms to minimize exposure to lice and maintaining optimal stock densities and single year-classes. Often these management activities are coordinated among farms within a region. A number of national or regional programmes collect and publicize salmon louse infection data from farmed and wild salmon. Some but not all management programmes show evidence of reducing infection levels on wild salmon; however, the available data are not yet sufficient to make firm conclusions regarding population effects. The local management and conservation of wild salmon populations should continue to be coordinated and integrated as the abundance of these fish is influenced by climatic, oceanographic and anthropogenic factors, including but not limited to salmon aquaculture.
... If the infestation is serious, then it may impair hypersaline adaptation. Treatment of parasite infestations takes many forms: application of aluminum at a dose that does not affect adult hosts (Pettersen et al. 2006); chemical treatment with emamectin benzoate (Stone et al. 2000); reduction of salinity, which is effective against stenohaline sea louse species (Bricknell et al. 2006); and vaccines under development against sea lice (Raynard et al. 2002). Because of the sensitivity to salinity, we reasoned that a hypersaline salinity stress (i.e., a rise in salinity from 30% to 60%, which is 2.0 Â seawater) may challenge F. heteroclitus without producing mortality in healthy animals, while it may also be lethal to A. funduli. ...
Killifish (Fundulus heteroclitus (L., 1766)) collected in the wild and kept in full-strength seawater were naturally parasitized by the ectoparasite Argulus funduli Kroyer, 1863, a copepod fish louse that creates inflamed skin lesions on the opercular epithelium and host gills. We assessed the damage done by lesions by counting the density of mitochondria-rich cells by fluorescence microscopy and by measuring Cl(-) secretion rate electrophysiologically using control (no lesions) and affected isolated opercular epithelia, often as paired left and right membranes from a single fish. Epithelia with lesions had a significantly reduced Cl- secretion rate, and in the lesions, the density of chloride cells was near zero. Contralateral membranes without lesions from infested fish had transport rates not significantly different from membranes taken from uninfected control animals, indicating no overcompensation on the contralateral membranes. Healthy control and infested animals were transferred to hypersaline conditions (twice seawater). Infested and control animals all survived transfer and had elevated plasma Na(+) and plasma osmolality. Infested animals failed to significantly elevate I(m) to the same level as healthy animals and there was a difference in hematocrit. Happily, the hypersaline challenge also resulted in detachment and death of adult A. funduli. We conclude that Argulus lesions impair salt transport in affected membranes but do not significantly affect survival on hypersaline challenge, and that hypersaline exposure is a successful treatment for A. funduli infestation in these strongly euryhaline teleosts.
... Biological control by wrasse is complementary, or in smaller fish also used as a substitute, to medication (Anonymous 2011a;Treasurer 2005). A range of innovative methods to control lice have been investigated, such as water flushing (Nilsen et al. 2010b), vaccines (Raynard et al. 2002), genetic selection of more resistant fish (Kolstad et al. 2005;Gjerde & Saltkjelvik 2009;Gjerde, Ødegård & Thorland 2011) and functional feeds (Anonymous 2009;Refstie et al. 2010). The use of traditional bath agents has also been improved by refining old methods such as bathing in closed tarpaulins or treatment in well boats to ensure appropriate concentration and maximum efficacy (Nilsen et al. 2010a). ...
The aim of this study was to model sea lice levels and the effect on reproduction by a stochastic simulation model and to evaluate the uncertainty of lice estimates based upon counts. Two empirical data sets were examined to parameterize the models. An overall fit of the data to the Poisson distribution was found and thus was used as the base of the stochastic models. In the model, salmon lice reproduction is not linear with the number of adult females and at low lice loads a smaller proportion of the adult female lice will reproduce. Depending on the variance structure, it was estimated that between 40% and 60% of the adult female lice will reproduce at an abundance of 0.5 adult females per fish. Lice counts, especially when examining few fish at low lice loads, are uncertain and at a true abundance of 0.1 one may count between 0 and 5 lice when examining 10 fish. Understanding the dynamics of sea lice reproduction is a key factor in the development of sustainable control strategies.
... According to a recent review ( Raynard et al. 2002), there has been no successful development of a vaccine against sea lice. However, a number of drug companies and academic researchers in both Canada and Norway are continuing research to develop an effective vaccine as we write. ...
... High and very variable unspecific loss of lice has been observed between experimental tanks during development (Bjorn & Finstad 1998, Tucker et al. 2002a) and a significant rate of host transfer has been observed within tanks ( Ritchie 1997, Hull et al. 1998). This complicates experimental design in several fields of L. salmonis research, in particular with respect to long-lasting efficacy assays such as evalu-ation of test vaccines against L. salmonis ( , Roper et al. 1995, Pike & Wadsworth 1999, Raynard et al. 2002. Hosting L. salmonis in tanks with many small fish results in a considerably more unpredictable loss of lice, and female lice are lost at a higher rate than males ( Hamre et al. 2009). ...
In studies of the salmon louse Lepeophtheirus salmonis (Krøyer, 1837), experimental design is complicated by a highly variable and unpredictable lice loss among common experimental tanks and a substantial rate of host transfer within tanks. When fish hosting L. salmonis are maintained in individual tanks, unspecific effects such as host transfer, louse predation by cohabitant hosts and agonistic host interactions are excluded. This study suggests that it is possible to maintain Atlantic salmon Salmo salar infected with L. salmonis in an array of small, single fish tanks and, by doing so, provide an experimental system in which the loss of motile pre-adult and adult stages of L. salmonis is predictable. Here, lice can be collected shortly after detachment for detailed studies or to provide mortality curves of lice from individual fish. This represents an experimental approach improving precision in studies of L. salmonis, such as drug and vaccine efficacy assays, RNA interference (RNAi) studies and host-parasite interactions. The natural loss of pre-adult/adult L. salmonis from the system was higher for males than females. The loss of females appeared to be a process somewhat selective against large individuals. Inherent qualities of the host appeared to be of little significance in explaining the variability in loss of preadult/adult lice.
... Immunization of fish against L. salmonis may be facilitated by an improved understanding of the adaptive immune system and molecules involved therein, particularly how the host responds to parasites. One of the limitations with vaccine development could be the limited exposure of louse to blood and thereby serum antibodies (reviewed in [8] ), and mucosal immunity might play a major role here as L. salmonis are colonizers of cutaneous mucosa of salmonids. Mucosal epithelial cells serve as an initial barrier and, in addition, they are involved in adaptive immunity by Ag presentation and production of Igs along with complement, lectins , CRP, lysozymes, proteolytic enzymes and other effectors484950. ...
The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development.
Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated.
Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses.
Abstract There is tremendous variation in life-history strategies among anadromous salmonids. Species that enter the ocean environment at small sizes ( 90% of all parasites lost by 16 days post-infection (dpi). Rejection was concomitant with host epithelial granulomatous infiltrations that initially targeted the embedded frontal filament (4 dpi) and the entire parasite by 10 dpi. Illumina sequencing, followed by functional enrichment analysis, revealed a concerted defense response in the fin within 1 dpi that included multiple innate and adaptive immunity components. Strikingly, early indications of an allergic-type inflammatory response were associated with chitin sensing pathways orchestrated by early overexpression of the IgE-receptor, fcer1g. Additionally, there was profound overexpression of several classes of c-type lectin receptors, including dectin-2, mincle, and dc-sign at 1 dpi onward. These profiles and upregulation of cellular effector markers were corroborated by histopathological evaluation, revealing the simultaneous presence of mast cell/eosinophilic granular cells, sacciform cells, macrophages/histiocytes, and granulocytes in fin. At 10 dpi and concurrent with parasite expulsion, there was evidence of immunoregulation in addition to tissue remodelling pathways. At 16 dpi, the response was effectively abrogated. Simultaneous profiling of the parasite transcriptome revealed early induction of chitin metabolism and immunomodulation, toxin production and ECM degradation; however, after 7 dpi, these were replaced with overexpression of stress and immune defense genes. These data present the first evidence for Coho salmon demonstrating chitin- and sugar moiety-sensing as key drivers of salmon louse rejection.
Globally, incidences of fish diseases caused by parasites have been on a rise, especially in intensive aquaculture practise, leading to considerable economic losses. The traditional control measures and therapeutics used to manage parasitic infections are associated with numerous limitations as well as risks. Vaccines have emerged as an effective means for control of pathogens; the use of vaccines for bacterial fish diseases has successfully cut down the use of antibiotics in aquaculture. However, development of vaccines for parasitic diseases of fish has seen limited success with availability of only commercial vaccine against sea lice. Nevertheless, significant strides have been made in understanding host-parasite interactions, which provides researchers with the arsenal of information required for identification of vaccine candidates and their development. In this review, we discuss protective responses reported in fish against major group of parasites and various efforts made in the field of vaccine development for important parasite groups of both marine and freshwater fish.
Aim
An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates.
Materials and results
The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4‐7 and SDS‐PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was western blotted to nitrocellulose paper (NCP) and reacted with rohu anti‐A. siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver‐stained gel and subjected to mass spectrometry in MALDI‐TOF/TOF. The MS/MS spectra were analyzed in MASCOT software with taxonomy ‘other metazoa’ and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in ‘molecular function’ and belonging to metabolic processes in ‘biologic process’ categories. The possibility of these proteins as vaccine candidates against A. siamensis is discussed in the paper.
Conclusion
Three of the identified proteins namely, bromodomain‐containing protein, anaphase‐promoting complex subunit 5, and elongation factor‐2 could possibly serve as vaccine candidates against argulosis in carps.
Infection with parasitic copepod salmon louse Lepeophtheirus salmonis, represents one of the most important limitations to sustainable Atlantic salmon (Salmo salar L.) farming today in the North Atlantic region. The parasite exerts negative impact on health, growth and welfare of farmed fish as well as impact on wild salmonid populations. It is therefore central to ensure continuous low level of salmon lice with the least possible handling of the salmon and drug use. To address this, vaccination is a cost-effective and environmentally friendly control approach. In this study, efficacy of a vaccine candidate, containing a peptide derived from ribosomal protein P0, was validated post infestation with L. salmonis, at the lab-scale. The sampling results showed good potential of the vaccine candidate when administered intraperitoneally in the host, in reducing the ectoparasite load, through reduction of adult female lice counts and fecundity and with greater presumptive effect in F1 lice generation. The sampling results correlated well with the differential modulation of pro-inflammatory, Th1, Th2 and T regulatory mediators at the transcript level at different lice stages. Overall, the results supports approximately 56% efficacy when administered by intraperitoneal injection. However, additional validation is necessary under large-scale laboratory trial for further application under field conditions.
The Atlantic salmon aquaculture industry still struggles with ectoparasitic sea lice despite decades of research and development invested into louse removal methods. In contrast, methods to prevent infestations before they occur have received relatively little research effort, yet may offer key benefits over treatment-focused methods. Here, we summarise the range of potential and existing preventative methods, conduct a meta-analysis of studies trialling the efficacy of existing preventative methods, and discuss the rationale for a shift to the prevention-focused louse management paradigm. Barrier technologies that minimise host-parasite encounter rates provide the greatest protection against lice, with a weighted median 76% reduction in infestation density in cages with plankton mesh ‘snorkels’ or ‘skirts’, and up to a 100% reduction for fully enclosed cages. Other methods such as geographic spatiotemporal management, manipulation of swimming depth, functional feeds, repellents, and host cue masking can drive smaller reductions that may be additive when used in combination with barrier technologies. Finally, ongoing development of louse-resistant salmon lineages may lead to long term improvements if genetic gain is maintained, while the development of an effective vaccine remains a key target. Preventative methods emphasise host resistance traits while simultaneously reducing host-parasite encounters. Effective implementation has the potential to dramatically reduce the need for delousing and thus improve fish welfare, productivity and sustainability in louse-prone salmon farming regions.
The salmon louse Lepeophtheirus salmonis (Lsal) is an ectoparasitic copepod that exerts immunomodulatory and physiological effects on its host Atlantic salmon. Over 30 years of research on louse biology, control, host responses and the host‐parasite relationship has provided a plethora of information on the intricacies of host resistance and parasite adaptation. Atlantic salmon exhibit temporal and spatial impairment of the immune system and wound healing ability during infection. This immunosuppression may render Atlantic salmon less tolerant to stress and other confounders associated with current management strategies. Contrasting susceptibility of salmonid hosts exists and early pro‐inflammatory Th1 type responses are associated with resistance. Rapid cellular responses to larvae appear to tip the balance of the host‐parasite relationship in favour of the host, preventing severe immune‐physiological impacts of the more invasive adults. Immunological, transcriptomic, genomic and proteomic evidence suggests pathological impacts occur in susceptible hosts through modulation of host immunity and physiology via pharmacologically active molecules. Co‐evolutionary and farming selection pressures may have incurred preference of Atlantic salmon as a host for Lsal reflected in their interactome. Here we review host‐parasite interactions at the primary attachment/feeding site, and the complex life‐stage dependent molecular mechanisms employed to subvert host physiology and immune responses.
Infection with parasitic copepod salmon louse Lepeophtheirus salmonis, represents one of the most important limitations to sustainable Atlantic salmon (Salmo salar L.) farming today in the North Atlantic region. The parasite exerts negative impact on health, growth and welfare of farmed fish as well as impact on wild salmonid populations. It is therefore central to ensure continuous low level of salmon lice with the least possible handling of the salmon and drug use. To address this, vaccination is a cost-effective and environmentally friendly control approach. In this study, efficacy of a vaccine candidate, containing a peptide derived from ribosomal protein P0, was validated post infestation with L. salmonis, at the lab-scale. The sampling results showed good potential of the vaccine candidate when administered intraperitoneally in the host, in reducing the ectoparasite load, through reduction of adult female lice counts and fecundity and with greater presumptive effect in F1 lice generation. The sampling results correlated well with the differential modulation of pro-inflammatory, Th1, Th2 and T regulatory mediators at the transcript level at different lice stages. Overall, the results supports the effectiveness of the vaccine candidate in controlling salmon lice infestation load. However, further validation is necessary under field conditions.
The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg⁻¹ MOS, 2 g kg⁻¹ cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1β (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1β IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1β whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.
This paper describes the sea lice control methods that are currently applicable to salmon farming in Canada. Three commercial chemical products are approved for use against sea lice in Canada: Slice®, Interox® Paramove® 50, and Calicide®. Physical control methods include photoperiod and cage depth manipulations, desiccation of farm equipment and the use of electric fences. Biological control measures include the polyculture of salmon with wrasse or mussel. Nutraceuticals and immunostimulents shown to reduce sea lice infestations in salmon include unmethylated DNA, yeast fermentation extract-Brewer’s yeast compound, AllBrew and NuPro, MacroGard® and Optimûn®. Salmon farmers are recommended to use site-specific integrated approaches, by properly adapting a combination of chemical, physical, biological and nutritional measures to the environmental and financial characteristics of their farm. In the future, development of vaccines against sea lice and of salmon lines resistant to sea lice will play a pivotal role in fighting against this parasite.
The expanding global aquaculture industry is urgently in need of more effective methods for the control of parasitic diseases. Advances in our understanding of parasite biology, host parasite interactions and improved diagnostic methods using new technologies which contribute to better management and control are highlighted in this chapter. There is an increasing volume of research pointing to effective control without the use of chemical interventions. The processes leading to increasing regulation and cost of drugs and chemicals are described. Their integration into a more sustainable pest management strategy is discussed and the imperative for the inclusion of resistance management principles in any strategy is emphasised.
Four novel trypsin-like S1A peptidase transcripts (LsTryp2–5) from the marine parasitic copepod Lepeophtheirus salmonis were characterised based on analyses of 1918 expressed sequence tags from two adult female libraries. In addition, one previously described salmon louse trypsin, LsTryp1, has been further characterised. The five peptidases possessed all residues typically found in trypsins in correct sequence contexts. Interestingly, two cysteine residues, possibly involved in a disulphide bridge not previously reported in trypsins are conserved in all louse trypsin sequences. Phylogenetic analyses showed that the five louse peptidases form a monophyletic group with other crustacean trypsins (Brachyurin Ts). Quantitative PCR analyses demonstrated increased transcript levels from planktonic to early host-attached stages and from preadult to sexually mature adult stages. Furthermore, sex-specific differences in transcription regulation were found. In situ hybridisation demonstrated that all five trypsin-like peptidases are transcribed throughout the undifferentiated midgut, indicating a digestive function. The sequence characteristics, histological localisation and transcript regulation suggest that LsTryp1–4 encode typical digestive trypsins. LsTryp5, however, showed some sequence and regulatory peculiarities that rendered its function less clear. Our findings support earlier suggestions for the function of the midgut cells and suggest the existence of an additional undifferentiated cell-type.
The outbreak, persistence, and eradication of infectious diseases often depend on the density of hosts. In coastal seas, many fisheries are fully or over-exploited; meanwhile, farmed populations are increasing rapidly with aquaculture growth. Marine aquaculture facilities are typically open to the surrounding ecosystem and, therefore, wild and farmed populations are connected by their shared parasites. At the core of epidemiological theory are host density thresholds, above which diseases can persist or invade and below which diseases can be eradicated. Host density thresholds in aquaculture-fishery interactions likely function at regional scales that encompass multiple farms, which are connected by pathogen dispersal and the movement of wild hosts. Sudden outbreaks of parasitic copepods in wild-farmed salmon systems may be linked to aquaculture growth exceeding host density thresholds. Abiotic (e.g. temperature and salinity), management (e.g. husbandry and farm siting), and biotic factors (e.g. migrations of wild hosts) likely affect threshold values. A connected wild-farmed host population can exceed a host density threshold due to an influx of wild hosts via migration, increases in aquaculture production, or environmental change such as climate warming. Coastal management and policy should heed the disease implications of climate warming, aquaculture growth, and fisheries restoration that suggest increasing host densities and decreasing threshold values.
The 3 post-marsupial juvenile stages of the gnathiid isopod, Paragnathia formica, are haematophagous ectoparasites of fishes that may, in heavy infestations, cause host mortality. Protein digestion in fed stage 3 juveniles is accomplished by cysteine proteinases, but what bioactive compounds attenuate host haemostatic, inflammatory and immunological responses during feeding is unknown. Trypsin inhibitory activity and anticoagulant activity were detected in crude extracts of unfed P. formica stage 1 juveniles; fractionation of stage 1 crude extracts by ion exchange chromatography resulted in 3 preparations each displaying these bioactivities. Further characterization revealed anti-thrombin activity in 2 of these preparations, whilst the third displayed the strongest anticoagulant activity that targeted a factor of the intrinsic coagulation pathway. Three trypsin inhibitors (18 kDa, 21 kDa, and 22 kDa) were also detected using reverse zymography. In parallel, homogenates of fed stage 2 and 3 juveniles were used to identify their fish hosts by amplifying the 16S mitochondrial rDNA and 18S genomic rDNA vertebrate gene regions. Blood from at least 4 fish families had been ingested by separate individuals during feeding. This study demonstrates that trypsin inhibitors and anticoagulants are present in P. formica juveniles which could suppress host haemostatic, inflammatory and immunological responses during feeding, and that juveniles are not host specific.
Introduction: This section provides a brief overview of aspects of sea lice biology and simplified methods to identify the difference species found on salmonids in British Columbia. The term sea louse (pl. sea lice) is the common name used for several species of marine ectoparasitic copepods of the family Caligidae (Order Copepoda: Suborder Siphonostomatoida) that infect fish. In B.C. these species include Caligus clemensi, Lepeophtheirus salmonis, and Lepeophtheirus cuneifer that have been reported from salmonids and nonsalmonid hosts. In addition to these species there are another nine species of Lepeophtheirus and one species of Caligus reported from numerous non-salmonid hosts (Margolis and Arthur 1979, Kabata 1988; Johnson and Albright 1991a; McDonald and Margolis 1995). Emphasis here is only on C. clemensi, L. salmonis, and L. cuneifer that use wild and farmed salmonids in British Columbia waters as hosts. For more detailed reviews of their sea lice biology readers should consult Pike and Wadsworth (1999), Tully and Nolan (2002), Johnson and Fast (2004) and Johnson et al. (2004). An identification key for the adults of species of sea lice found in British Columbia is given in Kabata (1988). yes published online
The effects of hydrocortisol implants on the susceptibility of naive coho salmon Oncorhynchus kisutch to infection with the economically important marine ectoparasitic copepod Lepeophtheirus salmonis was investigated under laboratory conditions. Cortisol-implanted coho salmon were more susceptible to infection than the control coho salmon. Copepods were lost from the gills of the control coho salmon by 10 d post-infection, and only a few remained on the body and fins at 20 d post-infection. Copepods were retained on the gills, fins and body of the cortisol-implanted coho salmon over the 20 d studied. Histological sections of control coho gills and fins revealed well developed epithelial hyperplasias and inflammatory responses to the presence of L. salmonis. The magnitude of the inflammatory response and the development of epithelial hyperplasia was suppressed in the cortisol-implanted coho. These data support the hypothesis that non-specific host defence mechanisms are important in resistance of coho salmon to infection with L. salmonis.
The comparative susceptibility of naive Atlantic Salmo salar, chinook Oncorhynchus tshawytscha and coho Oncorhynchus kisutch salmon to infection with the economically important marine ectoparasitic copepod Lepeophtheirus salmonis was investigated under laboratory conditions. Coho salmon were the most resistant to infection followed by chinook then Atlantic salmon. Copepods were lost from the gills of coho salmon by 10 d post-infection and only a few remained on the fins at 20 d post-infection. Although their abundance declined significantly, copepods were retained on both the gills and fins of chinook and Atlantic salmon over the 20 d studied. Rejection of L. salmonis on all 3 host species appears to be due to non-specific host responses. Histological sections of coho fins and gills revealed well-developed epithelial hyperplasias and inflammatory responses to the presence of L. salmonis. Gill and fin tissue responses of chinook salmon to L. salmonis appeared to be intermediate in intensity between those of coho and Atlantic salmon. Only minor gill and fin tissue response to the presence of L. salmonis was observed in Atlantic salmon.
Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence of several epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of beta-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen.
Cattle can be vaccinated against the tick Boophilus microplus by inducing an immunologic reaction against Ag in the tick gut. The uptake of antibody during feeding leads to severe damage to the parasite. One of the responsible tick gut Ag has now been purified and characterized: the Bm86 Ag. It is a membrane-bound glycoprotein present in very low abundance in extracts of partially engorged adult female ticks. It has an apparent m.w. of 89,000, an isoelectric point of 5.1 to 5.6 and an affinity for wheat germ lectin. Microgram amounts of this Ag are able to induce effective protection in cattle against the parasite, as shown by the decreased survival of ticks on vaccinated cattle and a reduction in engorgement weights and egg laying capacity of the survivors. Antisera to the Ag react with the surface of digest cells in the tick gut. As a result of the reaction with antibody, the endocytotic activity of these cells, which is a critical step in bloodmeal digestion in this tick, is strongly and rapidly inhibited. A number of peptides from this Ag, produced by digestion of the reduced and alkylated protein with endoproteinase lys-C, have been sequenced. One peptide has significant amino acid sequence homology with the epidermal growth factor precursor and a second peptide has homology with a putative protective antigen from Plasmodium falciparum.
The sheep blowfly, Lucilia cuprina, is responsible for over 80%
of cases of blowfly strike in Australia and the tosses in production and sheep deaths due to flystrike exceed S200 million per annum. Traditional methods of control are becoming less effective because of the blowfly's resistance to insecticides and thus other methods of control are necessary. In general, sheep develop very little immunity to flystrike even after repeated infestation, however vaccination against L. cuprina has shown considerable potential for controlling flystrike. The most successful sources of antigens have been the larvae's secreted proteases and several extracts from gut or peritrophic membrane. Immunization with these antigens results in retardation of larval growth and in some cases larval mortality. On sheep immunized with peritrophic membrane extracts, the growth retardation appears to be caused by a blocking of the peritrophic membrane which results in the larvae being starved of nutrients. The prospects for vaccine development and the remaining barriers to be overcome are discussed.
The changes in the activities of mucus hydrolytic enzymes and plasma cortisol levels were examined following infection of Atlantic salmon Salmo salar with the salmon louse Lepeophtheirus salmonis and these changes were compared with those resulting from elevated plasma cortisol. Salmon were infected at high (Trial 1; 178 +/- 67) and low (Trial 2; 20 +/- 13) numbers of lice per fish and the activities of proteases, alkaline phosphatase, esterase and lysozyme in the mucus, as well as plasma cortisol levels were determined. At both levels of infection, there were significant increases of protease activity over time (1-way K-WANOVA; Trial 1, p = 0.004; Trial 2, p < 0.001). On several sampling days, generally on later days in the infections, the mucus protease activities of infected fish were significantly higher than control fish (Student's t-tests; p < 0.05). In addition, zymography experiments demonstrated bands of proteases at 17 to 22 kDa in the mucus of infected salmon that were absent in the mucus from non-infected fish and absent in the plasma of salmon. The intensity of these protease bands increased in the mucus over the course of both infections. However, plasma cortisol levels were elevated only in the heavily infected fish from the first trial. At high infection levels (Trial 1), alkaline phosphatase activity was higher in the mucus of infected fish at all days (t-test, p < 0.05). However, at the lower infection level (Trial 2), the mucus alkaline phosphatase activity did not differ significantly between infected and non-infected fish. Esterase and lysozyme activities were very low and did not change with time nor between non-infected and infected salmon in either challenge. Mucus enzyme activities of cortisol-implanted salmon did not change over time, nor were there any differences in activities between cortisol-implanted and control salmon. The present study demonstrates biochemical changes resulting from sea lice infection of Atlantic salmon occurring at the site of host-pathogen interaction, the mucus layer. However, the origin of these enzymes, whether host or pathogen, remains to be determined.
As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.
For many parasites, the interaction between the immunogenicity of the parasite and the immunological response of the host is a dynamic equilibrium that allows both to survive, albeit often with severe consequences for the host. Vaccines, if intended as a means of parasite control, are unlikely to be generally successful if they do no more than mimic an immunological equilibrium that would be reached after natural exposure to the parasites. The situation must be tipped in favour of the host. It has been difficult to find ways around this impasse. One approach has been receiving practical attention over recent years, an approach that Peter Willadsen, Craig Eisemann and Ross Tellam have called vaccination against 'concealed' antigens.
The architecture and ultrastructure of the alimentary canal in Lepeophtheirus salmonis is described and compared with that of other copepods. The midgut of L. salmonis cannot be divided into different zones, and, hence, differs from most other copepods in this respect. The midgut cells seem to be involved in secretion/excretion, absorption, and intracellular digestion. Three different distinct cell types are identified in the midgut epithelium. Two of these cells could represent early and late stages of the same cell type. Bacteria were found in association with the midgut, both in the midgut lumen and in the midgut cells. Large colonies of bacteria were also found on the surface of L. salmonis. It is suggested that L. salmonis may function as a vector in the spread of pathogenic diseases in salmonids.
The midgut diverticulum of the marine calanoid copepod C. helgolandicus consists of a columnar epithelial layer, a myoepithelial layer, and between these a well-developed basement membrane. The apical region of the epithelial cell is thrown into tightly packed microvilli which showed an alcian blue reaction indicating the presence of acid mucopolysaccharides. The apical half of the cell contains numerous microvesicles and mitochondria as well as tiny Golgi-like bodies. The plasma membrane of the basal region of the epithelium is extremely digitated. The digitations contain numerous mitochondria and the whole structure resembles mitochondrial pumps. The epithelial cells contain a large centrally situated oval nucleus with its single nucleolus. The myoepithelial cell is squamous and contains a flattened nucleus as well as very well-developed circularly and longitudinally arranged myofibrils. It is suggested that the midgut diverticulum of Calanus is probably analogous to the mammalian stomach in that food is mechanically churned. However, it does not appear to be involved in the secretion of digestive juices but only mucopolysaccharides; it is probably involved in the absorption of amino acids which are probably actively transported, by the "mitochondrial pump" in the basal region of the epithelial cells, into the haemocoel.
The piscine immune system is well developed and is normally quite efficient in protecting healthy free ranging fish from parasitic diseases. However, when fish are cultured in high numbers and are stressed by adverse environmental factors (e.g. heavy metal pollution, low dissolved oxygen, nutritional deficiencies, and/or overcrowding), parasites may have the advantage and the risk of disease outbreak increases in the fish population. We know very little about innate immunity against parasites. Hence it has not been considered a viable strategy to protect fish from diseases. The alternative pathway of complement activation is the protective mechanism in certain fish species against hemoflagellates (Cryptobia salmositica and Cryptobia catostomi). This mechanism also operates in some resistant individuals (in a susceptible fish species) against C. salmositica. Complement is also involved in innate parasiticidal activities against adult intestinal tapeworms (Acanthobothrium quadripartitum) and encysting larval stages of digeneans (Diplostomum spathaceum and Cryptocotyle lingua). It is suggested here that more studies be conducted to elucidate the mechanism(s) of innate immunity in nonsusceptible host species, and also to follow the heredity of parasite resistant factor(s) in individuals that belong to an otherwise susceptible host fish species.
FORSTER1 has recently examined the peritrophic membrane in Caridea (Crustacea, Decapoda). He found a membrane in the mid-gut of all the Caridea examined and was able to show that it was chitinous. A similar membrane can easily be shown, to surround the fæcal pellets of calanoid copepods, where it often forms a `tail', occasionally of considerable length, at each end of the pellet. That the pellet is indeed enclosed in a membrane can easily be demonstrated by compressing it carefully under a coverslip on a microscope slide. As pressure is applied the pellet first flattens more or less evenly throughout its length ; then the membrane suddenly ruptures at one point only and its contents can be seen streaming through the point of rupture. The membranes can also be demonstrated by careful heating of a pellet in a concentrated potassium hydroxide solution: the alkali dissolves most of the fæcal matter, leaving the membrane intact. No positive results were obtained in tests for chitin but this may well be because of the small size of the pellets (c. 1.5 × 0.15 mm.) and the delicacy of the membrane.
A humoral antibody response was demonstrated by Western blotting and Crossover immunoelectrophoresis, in Atlantic salmon (Salmo salar L.) immunised with crude extracts derived from the copepod ectoparasites (sea lice) Caligus elongatus (Nordmann) and Lepeophtheirus salmonis (Kroyer). Antibodies were not detected in control fish. Many cross-reacting antigens were observed between both sea lice species and their various life stages. The antigenic components most clearly resolved on blots probed with salmon sera were in excess of approximately 75 kDa, although equal numbers of components of lower molecular weight were identified. The response from Atlantic salmon was compared to that from rabbits immunised with similar sea lice extracts. On blots probed with rabbit antisera a greater number of antigenic components were identified than from salmon immunoblots. Antigenic components detected with rabbit antisera were more clearly stained with little distinction between components of high or low molecular weights.
The interrelationship between plasma cortisol levels, iodine-iodide nutritional supplementation, plasma thyroid hormone levels (tri- and tetra-iodothyronine, T3 and T4 respectively), and infection intensity with the sea louse Caligus elongatus were investigated in Atlantic salmon, Salmo salar L., and Arctic char, Salvelinus alpinus (L.). Cortisol-implanted Atlantic salmon had significantly higher mean intensities of sea lice than salmon not implanted with cortisol. Untreated Arctic char had significantly higher plasma cortisol levels and sea lice infections than untreated salmon. Cortisol-implanted salmon had lower plasma levels of T3 and T4. Salmon and char treated with iodized feed and iodinated water had higher plasma T3 and T4 levels, higher plasma T3/T4 ratios, and lower plasma cortisol levels than controls. Sea lice infections were significantly reduced on salmon treated with either iodized feed (P < 0.05) or iodinated water (P < 0.05). Char treated with iodized feed also had significantly reduced infections (P < 0.05). In general, iodized feed had a greater effect than iodinated water in reducing sea lice numbers. This study indicates that elevated plasma cortisol levels in salmon and char result in lower plasma levels of thyroid hormone and an increased susceptibility to sea lice infections. It is concluded that providing the fish with sufficient iodine-iodide increases thyroid hormone levels, reduces plasma cortisol levels, and reduces susceptibility to sea lice infections.
Atlantic salmon (Salmo salar), naturally infected with Caligus elongatus in seapen cages in Brandy Cove, St. Andrews, N.B., were examined histologically and ultrastructurally for signs of pathology associated with the attached chalimus larvae. Naturally -infected fish and fish that had never been exposed to sea lice infections were examined serologically for altered white blood cell counts and for any antibody response to the adult parasite. The chalimus larva feeds on the epidermal cells of the scale to which it is attached and denudes the epidermis down to the basement membrane. Ruptured and pycnotic cells line the hole created by the larva, and there is some evidence of hyperplasia around stage IV chalimus larvae. Little other damage or host reaction is evident. There is no seral antibody response to the patent infection as assayed by electroimmuno transfer blot, and white blood cell counts from infected and control groups of fish are statistically the same.
In 1939 Lepeophtheirus salmonis infected salmon entering Moser river, Nova Scotia. Some fish had much of skin in occipital region removed and died some time after entering fresh water. It is suggested that these copepods cause the condition on the top of the salmon's head known as "white spot."
Synopsis
The general biology and pathology of Lepeophtheirus salmonis and Caligus elongatus and the prevention and treatment of such ‘sea-lice’ infestations on farmed salmonids are described from the literature and original observations. The life-cycle of L. salmonis and probably also that of C. elongatus comprises the egg and 10 stages separated by moults, namely, two nauplius, one infective copepodite, four chalimus, two pre-adult and the adult (male and female) stages. Water temperature greatly affects the rate of development, especially for early larval stages. Heavy infestations of wild fish seem rare, and lice are lost fairly rapidly in freshwater. In Scotland at least L. salmonis shows a succession of generations on farmed salmonids; generation time is about six weeks at 9–12 C. Post-chalimus stages of C. elongatus may exchange between farmed salmonids and wild fish (especially gadoids). Epizootics (particularly with L. salmonis) cause great damage to salmonids in Norwegian and Scottish farms largely through feeding on host skin. The dermis is oedematous and haemorrhaged where lice feed, and blood seeps between scales; deaths probably result from osmoregulatory failure. Whilst prevention of infestation is difficult, a bath treatment for 1 h with 1 ppm of the organophosphorus compound Dichlorvos is effective against post-chalimus stages of L. salmonis on caged salmonids. Side effects are minimal and clearance rates from fish tissues satisfactory, but treatment may be required every 3–4 weeks.
The immune responses of rainbow trout and Atlantic salmon naturally infected by the copepod parasite, Lepeophtheirus salmonis, were compared, by ELISA, Western blotting and immunohistochemistry with the responses of rabbits and rainbow trout immunised with whole adult lice homogenates. At least 31 antigens of adult lice were identified by serum from immunised rabbits and a comparable number by serum from immunised trout. Compared with immunised animals, the serum responses of naturally infected salmon were at a very low level recognising only five antigens in samples reduced with 2-mercaptoethanol and one major antigen in unreduced samples, which were shown by immunohistochemistry to be exclusively associated with the gut of the parasite. No serum response was observed in either naturally infected or control trout or in control salmon. Antigenic differences between chalimus and adult stages of the parasite were examined by probing with rabbit and trout antisera. The results demon-strate that the trout immune system is capable of producing antibodies to many lice components and that some antigenic differences exist between adult and chalimus stages of L. salmonis.
The functional morphology of the alimentary canal of copepodite and chalimus stages of Lepeophtheirus salmonis (Krøyer, 1837) is described and compared with that found in other copepods studied to date.
The buccal cavity passes into a gut comprising three major regions: foregut (oesophagus), midgut and hindgut. The foregut and hindgut both posscss a cuticular lining whereas the midgut is lined with specialized epithelial cells. The midgut is divided into three recognizable zones, namely anterior midgut caecum, anterior midgut and posterior midgut. Three main types of epithelial cell are recognizable in the midgut: vesicular cells, microvillous cells and basal cells which correspond to the cell types normally described in other parasitic and free-living copepod species.
Digestion is thought to occur in the midgut and be mediated by the epithelial cells that line it. Although several glands appear to discharge into the area of the buccal cavity, none was seen to interface to any other area of the gut. There was no evidence for the involvement of commensal gut bacteria in food digestion.
Injection of extracts derived from adult caligid copepods induced a partial immunity to Lepeophtheirus salmonis in Atlantic salmon. Antigens were derived from the supernatant of adult lice extracts and were partially purified by Con A affinity chromatography. The antigens were also present in the pellet derived from adult lice extracts, Immunohistochemistry showed that antibodies present in the serum of rats immunized with the Con A purified extract bound predominantly to the gut of L. salmonis. Components of apparent sizes > 205 000, 165 000, 133 000, 130 000, 125 000, 114 000, 110 000, 96 000, 82 000, 78 000, 65 000, 46 000, 35 000, 31 000 and < 29 000 were present in the extract. Although the numbers of attached lice were initially higher on those fish in the immunized group, throughout the course of the challenge experiment the total numbers of lice did not vary greatly between the immunized or control groups of fish. Initially, an overall average of about 17 attached stages were recorded from each fish sampled and by the end of the experiment this figure had fallen to about five adult lice per fish. However, compared with control fish fewer gravid female lice were present on immunized fish and furthermore these lice possessed fewer eggs (P<0.01). No major differences in egg hatching success were recorded. Further exploitation of this would require the isolation and purification of the antigens responsible for the observed effects. The possibilities for the development of a salmon louse vaccine are promising.
The osmotic changes in haemolymph and body tissues of the ectoparasitic salmon louse,Lepeophtheirus salmonis, have been studied upon transfer from sea water (SW) to dilute sea water (37% SW), and then to fresh water (FW). The parasite shows osmoconformity in SW but hyperosmotic regulation in 37% SW regardless of whether it is attached to the salmon host or free swimming in the water. The same conclusion is reached by haemolymph Cl− measurements. In FW, the osmotic tolerance and response of attached and free swimming parasites differ: Attached animals maintain steady haemolymph osmolality and Cl− concentration and survive for at least 1 week, while free swimming parasites quickly become diluted and start to die within 8 h.
Acclimation to 37% SW is accompanied by changes in body tissue water content and in the content of ninhydrin positive substances and specific amino acids which suggest the presence of cell volume regulation. Glycine is the dominating free amino acid in the cephalothorax tissues but alanine, proline and taurine also occur in high amounts. Lysine is found to increase significantly during FW acclimation of attached parasites. A breakdown of cell volume regulation is suggested to limit the survival of attached salmon louse in fresh water.
Faecal pellet formation within the gut of Stage V and adult females of the copepod Calanus helgolandicus Claus involves (1) cyclical processes of digestion and (2) the contribution of parts of the gut epithelium to the pellets. During an experimental regime in which dim lighting was restricted to day-time and feeding to night-time (17.00 to 09.00 hrs), the copepods responded with cyclical changes in both the quantity of pellets they produced and the fine structure of the contents. During the feeding period, the contents showed changes in the relative amounts of materials originating from disintegrated cells of the digestive epithelium and those derived directly from the ingested food. The vacuolar B-cells of the gut contribute to the content of the pellets and the distal, necrotic N-cells appear to be involved in forming the peritrophic membrane which encloses each pellet. Cells of the gut epithelium which are broken down during feeding are all replaced during the non-feeding period. Other individuals were taken directly from the sea and in these, also, the cells of the gut broke down during feeding and contributed to the faecal pellets. The supply of epithelial cells may limit the duration of the feeding period.
Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well characterized antigen from the tick gut. Several factors explain how cattle could produce antibody to such antigens, which one would expect to be 'concealed' from the host's immune system, during natural infestation. It has been shown that the carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and that the reaction of bovine antibodies with intrinsic membrane glycoprotein is at least partially blocked by low molecular weight carbohydrate. Further, antisera from cattle exposed to ticks react with a glycosylated, native Bm86 but not with a non-glycosylated, recombinant Bm86. Thus the reaction of concealed antigens with antibodies produced as a result of tick infestation appears to be due to a relatively non-specific reaction with carbohydrate determinants on tick glycoprotein. Evidence is also presented that antibodies directed against carbohydrate determinants of Bm86 are not protective. Care must therefore be exercised in interpreting the results of antibody reaction with glycoproteins in such complex organisms.
Zygotes and ookinetes of the rodent malaria Plasmodium berghei can be enriched 50-fold, from whole blood cultures by ammonium chloride lysis. Three monoclonal antibodies (MoAbs) raised against such enriched preparations specifically bind to a determinant of Mr 21 kD as assessed by 125I-labelled goat anti-mouse IgG probed immunoblots of Western transfers of SDS-PAGE gels. Indirect immunofluorescence indicates that the 21 kD determinant bound by specific MoAbs, whilst not detectable on gametocytes or gametes, appears on the parasite surface within 2 h of exflagellation/fertilization and increases thereafter. The three MoAbs specifically binding the 21 kD determinant block oocyst development in mosquitoes by at least 90%, as assessed either by in-vitro membrane feeds or by live feeds on passively immunized mice. These MoAbs reduce ookinete formation in vitro by between 52 and 100%. Possible mechanisms of action of these MoAbs are discussed.
Sheep were immunised with ovalbumin and then infected with the sheep blowfly, Lucilia cuprina in order to study immunoglobulin and specific antibody degradation at the wound site. Serum and wound exudates were collected over the infection period and the dry weight and protein content of the exudates were determined. Exudates were analysed by SDS-PAGE and immunoblotting for IgG degradation. Levels of IgG and specific anti-ovalbumin antibodies in the exudates were measured by ELISA. The total weight of exudates increased over the whole period of the infection, while protein content increased in the first 24 h and then remained relatively constant. Immunoglobulin was present 6 h after infection and levels increased with protein content. However, the levels of IgG measured were quite different depending on the secondary antibody used in the ELISA. A monoclonal antibody measured mainly intact IgG while a polyclonal anti-IgG measured intact and degraded IgG. This allowed an estimation that approximately 60% of the IgG in exudates was degraded from 6 h after infection. Assays in vitro showed that L. cuprina larval enzymes degraded sheep antibody. However, measurement of specific anti-ovalbumin levels in exudates suggested that although high levels of antibody were degraded this did not necessarily decrease the level of antigen binding. As a result, IgG degradation may assist and not hinder vaccine development by allowing antibody fragments to penetrate the peritrophic membrane and access gut cell antigens.
Increasingly, there is need for methods to control cattle tick (Boophilus microplus) infestations by the use of non-chemical technology. This need is brought about by a mixture of market forces and the failure or inadequacy of existing technology. A recombinant vaccine has now been developed against the tick. This vaccine relies on the uptake with the blood meal of antibody directed against a critical protein in the tick gut. The isolation of the vaccine antigen, Bm86, and its production as a recombinant protein is briefly described. The vaccine has been tested in the field, has been taken through the full registration process and is now in commercial use in Australia. A related development has occurred in Cuba. The potential for improvement of the current vaccine and for the development of similar vaccines against other haematophagous parasites is discussed.
The tick-host-pathogen interface is characterised by complex immunological interactions. Host immune responses to tick infestation and infection with tick-borne pathogens involve cytokines, antibodies, complement and T lymphocyte regulatory and effector pathways. A successful host-parasite relationship is a balance between limiting the parasite by host defenses and the ability of the parasite to modulate, evade or restrict the host response. Hosts acquire immunological based resistance to tick infestation, which reduces engorgement, production of ova and viability. Salivary glands of ixodid ticks produce a complex array of immunogens and pharmacologically active molecules. Tick salivary gland derived material can modulate host cytokine, antibody and cell mediated immune responses. Both immunoregulatory and immune effector pathways of the host are suppressed. Tick feeding impairs the ability to develop a primary immune response to a thymic dependent immunogen. Lymphocytes obtained from tick infested hosts are reduced in their ability to proliferate in vitro to T lymphocyte mitogens, while responses to B lymphocyte polyclonal activators are unaltered. Normal macrophages and lymphocytes were exposed to female tick salivary gland extracts prepared daily during the course of engorgement. All extracts reduced lymphocyte responses to T cell mitogens and enhanced in vitro proliferation in the presence of a B lymphocyte mitogen. Macrophage elaboration of tumour necrosis factor alpha and interleukin-1 are significantly reduced in a differential manner. Production of interleukin-2 and interferon-gamma by T lymphocytes is reduced. Tick modulation of the host immune response could enhance the ability of the arthropod to obtain a blood meal and facilitate pathogen transmission to an immunocompetent host.
A preembedding immunogold technique was used to locate Bm86, an antigen from the gut digest cells of the cattle tick Boophilus microplus. Gut from partially engorged female ticks was everted to expose the cells, lightly fixed in 4% paraformaldehyde, and then incubated in rabbit antisera against a recombinant form of Bm86. Following incubation in a secondary antibody conjugated to 1-nm colloidal gold, Bm86 antigenic sites were visualized for both light and electron microscopy using silver enhancement. Bm86 was shown to be located predominantly on the microvilli of digest cells. Antiserum against a nonglycosylated Escherichia coli recombinant form of Bm86 was used to avoid cross-reactivity with carbohydrate epitopes of other digest cell proteins.
The quantity of specific antibody ingested by larvae of Lucilia cuprina and its fate after ingestion were studied in larvae grown on sheep and on an artificial diet. Larvae grown to late first or early second instar on sheep vaccinated with horse myoglobin contained 66% less specific antibody detected by enzyme linked immunosorbent assay than larvae grown to a similar stage on an artificial diet containing 75% serum from the same sheep. A similar result was obtained when larvae were grown to mid-third instar. Larvae grown on sheep to first or second instar contained approximately the same quantity of specific antibody per unit weight of larvae as those grown to third instar. Larvae grown on diet to third instar contained 22% less specific antibody per unit weight than those grown to first or second instar. In larvae grown on diet to late third instar, ingested diet retained 91 +/- 12% of its original specific antibody activity in the crop, 50 +/- 11% in the anterior midgut, 8 +/- 2% in the posterior midgut and 13 +/- 6% in the hindgut. The mean concentration of total immunoglobulin detectable in the haemolymph of individual third instar larvae grown on diet was 1.7 +/- 2.8 micrograms/ml. Assays of specific antibody in the haemolymph of similarly reared larvae indicated that all or most of this immunoglobulin remained functional. The implications of the quantities and distribution of ingested functional antibody found in feeding larvae of L.cuprina are discussed in relation to the possibility of vaccinating sheep against these larvae and the selection of likely internal targets as sources of potential protective antigens.
Novel vaccines against ectoparasites have the potential to be cost-effective new technology for pest control that avoids some of the real and perceived problems with insecticide and acaricide usage. Nevertheless, their development is in its infancy. A vaccine against the cattle tick Boophilus microplus, the world's first vaccine against an ectoparasite, is in field use in Australia. Considerable effort had gone into the development of a vaccine against the sheep blowfly Lucilia cuprina, while other vaccines are at an earlier stage of development. The identification of critical antigens and their production as effective recombinant proteins remains the greatest hurdle. Characteristics of the few known antigens and the mode of action of the protective immune response are discussed. Development of further vaccines will depend on recognition of likely antigenic targets. The efficacy of such vaccines will depend on the characteristics of the target species, in particular its digestive biology and the way in which the novel vaccine impacts on the parasite population.
Parasitologists have not, in the past, exploited the immune system to protect fish against parasitic diseases. In the past few years, however, there has been an increased interest in adopting this strategy, and we have made steady and promising progress against a few parasites which are of economic importance. Amyloodinium ocellatum is an ectoparasitic dinoflagellate on brackish and marine fishes, which may also cause problems to aquarium fishes. Antiserum from fish inoculated intraperitoneally (i.p.) with living dinospores of the parasite immobilizes and agglutinates living dinospores; it also reduces parasite infectivity in cell culture. Cryptobia salmositica is a pathogenic haemoflagellate of salmonids on the Pacific coast of North America, causing mortality in semi-natural and intensive salmon culture facilities. A live attenuated vaccine inoculated i.p. protects susceptible juvenile and adult fish for at least 24 months. The protection involves production of complement fixing antibodies, phagocytosis, and antibody-dependent and antibody-independent T-cell cytotoxicity. A monoclonal antibody against a surface membrane glycoprotein (199-200 kDa is therapeutic in that it significantly reduces parasitaemias when inoculated into fish with acute disease. Ichthyophthirius multifiliis is an ectoparasitic ciliate of freshwater fishes with world wide distribution, usually causing disease when fish are stressed and/or when environmental conditions are favourable for parasite multiplication. Live theronts injected into the body cavity protect fish, and monoclonal antibodies with immobilizing activity upon parasites have been developed. There is some evidence of passive transfer of protective immunity from immune to naive fish, and to eggs. Diplostomum spathaceum is an intestinal parasite of gulls; the metacercaria stage of the parasite encyst and causes disease and mortality in numerous species of freshwater fish in Europe and in North America. Fish injected i.p. with sonicated/killed cercariae or metacercariae have fewer metacercariae in the eyes and survives longer. Lepeophtheirus salmonis and Caligus elongatus are parasitic copepods (sea lice), and they are important parasites of Atlantic salmon in cage cultures. A vaccine against fish lice is plausible, and the efficacy of about 20 candidate antigens in protecting fish is being tested.