Multilobular tumor of bone arising from the palatomaxillary suture line in the skull of a dog.

A 6-year-old neutered male bloodhound dog was presented for surgical evaluation of an intraoral hard palate mass noted during a routine dental cleaning performed by the primary veterinarian. Computed tomography (CT) of the skull revealed a well-defined, multilobular, mineralized mass with a coarse, granular appearance, centered along the palatomaxillary suture extending into both the nasal and oral cavities. Bilateral caudal maxillectomy was performed for curative-intent resection of the tumor. The histopathology of the mass was consistent with a completely excised palatomaxillary multilobular tumor of bone (MLTB). This is apparently the 1st successful report of surgical excision of an MLTB associated with the palatomaxillary suture line. Key clinical message: Our findings emphasized that MLTB should be considered as a differential diagnosis for masses arising in the location of cranial sutures, and a definitive diagnosis can be made postoperatively with histopathology of the mass.

Tumeur osseuse multi-lobulaire résultant de la ligne de suture palato-maxillaire dans le crâne d'un chien. Un chien Saint-Hubert mâle castré de 6 ans a été présenté pour évaluation chirurgicale d'une masse intrabuccale du palais dur notée lors d'un nettoyage dentaire de routine effectué par le vétérinaire initial. La tomodensitométrie (CT) du crâne a révélé une masse minéralisée multi-lobulaire bien définie avec un aspect grossier et granuleux, centrée le long de la suture palato-maxillaire s'étendant dans les cavités nasale et buccale. Une maxillectomie caudale bilatérale a été réalisée pour une résection à visée curative de la tumeur. L'histopathologie de la masse était compatible avec une tumeur osseuse multi-lobulaire palato-maxillaire complètement excisée (MLTB). Il s'agit apparemment du premier rapport réussi d'excision chirurgicale d'une MLTB associé à la ligne de suture palato-maxillaire.Message clinique clé :Nos résultats ont souligné que la MLTB doit être considérée comme un diagnostic différentiel pour les masses apparaissant à l'emplacement des sutures crâniennes, et un diagnostic définitif peut être posé en postopératoire avec l'histopathologie de la masse.(Traduit par Dr Serge Messier).

Immersion challenge of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. did not increase the severity of Neoparamoeba perurans-induced amoebic gill disease (AGD).

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.

Genetic diversity among geographically distant isolates of Neoparamoeba perurans.

The present study explored the use of 2 common genetic markers, the mitochondrial cytochrome oxidase I (COI) and the ribosomal internal transcribed spacer (ITS) to infer the relationship between geographically distant isolates of the protozoan gill parasite Neoparamoeba perurans, the agent responsible for amoebic gill disease in farmed Atlantic salmon worldwide. Present data confirmed that the ITS marker is suitable for Neoparamoeba species discrimination; however, it is not recommended as a population marker due to the presence of multiple copies of ITS within both N. perurans clonal and polycultures. On the other hand, in the partial COI gene analysed here, a low variability was observed, with 8 haplotypes recovered from N. perurans samples collected from Europe (Ireland, Norway, Scotland) and Tasmania (Australia). In Europe, the COI haplotypes which have more recently been detected in aquaculture are different to the haplotypes associated with the original gill disease emergence in Ireland in 1997 and Norway in 2006. The presence of unique COI haplotypes in different continents suggests the presence of multiple distinct reservoirs of the pathogen in both Europe and Tasmania. Isolates from additional geographical locations are required to fully understand the origins and routes for the spread of N. perurans worldwide.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian 'greenlip' abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone 'omics website, http://abalonedb.org.

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD).

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.

Identification of differentially expressed reproductive and metabolic proteins in the female abalone (Haliotis laevigata) gonad following artificial induction of spawning.

Inefficient control of temperate abalone spawning prevents pair-wise breeding and production of abalone with highly marketable traits. Traditionally, abalone farmers have used a combination of UV irradiation and application of temperature gradients to the tank water to artificially induce spawning. Proteins are known to regulate crucial processes such as respiration, muscle contraction, feeding, growth and reproduction. Spawning as a pre-requisite of abalone reproduction is likely to be regulated, in part, by endogenous proteins. A first step in elucidating the mechanisms that regulate spawning is to identify which proteins are directly involved during spawning. The present study examined protein expression following traditional spawning induction in the Haliotis laevigata female. Gonads were collected from abalone in the following physiological states: (1) spawning; (2) post-spawning; and (3) failed-to-spawn. Differential protein abundance was initially assessed using two-dimensional difference in-gel electrophoresis coupled with mass spectrometry for protein identification. A number of reproductive proteins such as vitellogenin, vitelline envelope zona pellucida domain 29 and prohibitin, and metabolic proteins such as thioredoxin peroxidase, superoxide dismutase and heat shock proteins were identified. Differences in protein abundance levels between physiological states were further assessed using scheduled multiple reaction monitoring mass spectrometry. Positive associations were observed between the abundance of specific proteins, such as heat shock cognate 70 and peroxiredoxin 6, and the propensity or failure to spawn in abalone. These findings have contributed to better understand both the effects of oxidative and heat stress over abalone physiology and their influence on abalone spawning.

Expression analysis of sex-determining pathway genes during development in male and female Atlantic salmon (Salmo salar).

We studied the expression of 28 genes that are involved in vertebrate sex-determination or sex-differentiation pathways, in male and female Atlantic salmon (Salmo salar) in 11 stages of development from fertilization to after first feeding. Gene expression was measured in half-sibs that shared the same dam. The sire of family 1 was a sex-reversed female (i.e., genetically female but phenotypically male), and so the progeny of this family are all female. The sire of family 2 was a true male, and so the offspring were 50% male and 50% female. Gene expression levels were compared among three groups: 20 female offspring of the cross between a regular female and the sex-reversed female (family 1, first group), ∼ 10 females from the cross between a regular female and a regular male (family 2, second group) and ∼ 10 males from this same family (family 2, third group). Statistically significant differences in expression levels between males and the two groups of females were observed for two genes, gsdf and amh/mis, in the last four developmental stages examined. SdY, the sex-determining gene in rainbow trout, appeared to be expressed in males from 58 days postfertilization (dpf). Starting at 83 dpf, ovarian aromatase, cyp19a, expression appeared to be greater in both groups of females compared with males, but this difference was not statistically significant. The time course of expression suggests that sdY may be involved in the upregulation of gsdf and amh/mis and the subsequent repression of cyp19a in males via the effect of amh/mis.

Vaccination with recombinant protein (r22C03), a putative attachment factor of Neoparamoeba perurans, against AGD in Atlantic salmon (Salmo salar) and implications of a co-infection with Yersinia ruckeri.

Amoebic gill disease (AGD) affects salmonids during the marine grow-out phase in the Tasmanian industry and in other major salmonid producing countries. During the period post-transfer to seawater, the bacterial condition yersiniosis can also cause high levels of mortality in Atlantic salmon grown in Tasmania, in addition to the hatchery outbreaks. The recombinant protein r22C03, a mannose-binding protein-like (MBP-like) similar to attachment factors of other amoebae, was tested as a vaccine candidate against AGD in a large scale challenge trial. Fish were immunised with r22C03 combined with FCA via intraperitoneal (i.p.) injection, and given a booster five weeks later by either i.p. injection (RP group) or by a dip-immersion (mRP). Fish were then challenged twice with Neoparamoeba perurans: the initial challenge 16 weeks after primary immunisation was terminated due to presence of ulcerative lesions in the skin of salmon; the second challenge was carried out after five weeks of treatment with oxytetracycline. These skin lesions might have been associated with a concurrent infection with Yersinia ruckeri, which was detected by real-time qPCR in serum of a large proportion of moribund and survivor fish after the AGD challenge. Before and during the N. perurans infection, levels of antibodies against r22C03 were measured by ELISA in serum, skin mucus and supernatant from skin and gill explants. For the second challenge, the average size of AGD lesions was recorded from histology sections and survival curves were obtained. Before AGD challenge, r22C03 induced antibody responses in serum and explants with both vaccination strategies. At the end of the challenge, levels of antibodies were lower than before challenge irrespective of treatment. Both vaccinated groups presented increased serum antibody responses, while only mRP presented antibody responses in skin mucus, and no significant antibody responses were measured in the explants. Antibodies did not confer protection to N. perurans infection, as no difference was observed in the survival curves of the vaccinated and control groups, and there was no effect on the gill lesion size. The concurrent yersiniosis infection probably represented more closely infection patterns observed in commercial settings. However, it could have interfered with the survival results and with the ability of the fish to respond to the amoebae infection.

Exploiting genomic data to identify proteins involved in abalone reproduction.

Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. BIOLOGICAL SIGNIFICANCE: A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases.

Administration of recombinant attachment protein (r22C03) of Neoparamoeba perurans induces humoral immune response against the parasite in Atlantic salmon (Salmo salar).

This study investigated the use of a recombinant protein of Neoparamoeba perurans, the causative agent of Amoebic gill disease (AGD), as an immunogen to generate systemic and mucosal antibody responses against the parasite. Genes encoding N. perurans homologs of mannose-binding protein (MBP) from Acanthamoeba spp. have been identified. From these, a Neoparamoeba MBP - like EST has been identified and produced as a recombinant fusion protein. Attachment of N. perurans to the gill might be reduced by antibody-mediated interference of this protein, but this is dependent on the presence and level of functional antibodies in the mucus. Fish were immunized with the protein via i.p. injection with Freund's complete adjuvant (FCA); and serum and skin mucus samples were collected before and after immunization. Antibodies (IgM) present in samples were characterized via Western blot and their levels measured with an ELISA. The immunization was able to induce a systemic IgM response 8 weeks after primary exposure and a mucosal response 4 weeks post initial immunization, which were specific to the recombinant protein but not to antigens obtained from crude amoebic preparations. However, adherence of the antibodies to the parasite was observed using immunocytochemistry, and both, serum and skin mucus IgM, were able to bind the surface of formalin-fixed N. perurans. This finding may contribute to further research into the development of a vaccine for AGD.

Development of an in vitro model system for studying bacterially expressed dsRNA-mediated knockdown in Neoparamoeba genus.

RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using ß-actin and EF1-α as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 µg/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, ß-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.

Exploring RNAi as a therapeutic strategy for controlling disease in aquaculture.

Aquatic animal diseases are one of the most significant constraints to the development and management of aquaculture worldwide. As a result, measures to combat diseases of fish and shellfish have assumed a high priority in many aquaculture-producing countries. RNA interference (RNAi), a natural mechanism for post-transcriptional silencing of homologous genes by double-stranded RNA (dsRNA), has emerged as a powerful tool not only to investigate the function of specific genes, but also to suppress infection or replication of many pathogens that cause severe economic losses in aquaculture. However, despite the enormous potential as a novel therapeutical approach, many obstacles must still be overcome before RNAi therapy finds practical application in aquaculture, largely due to the potential for off-target effects and the difficulties in providing safe and effective delivery of RNAi molecules in vivo. In the present review, we discuss the current knowledge of RNAi as an experimental tool, as well as the concerns and challenges ahead for the application of such technology to combat infectious disease of farmed aquatic animals.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways.

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways.

Transcriptome analyses of amoebic gill disease-affected Atlantic salmon (Salmo salar) tissues reveal localized host gene suppression.

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (+/- SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response.

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.).

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.

The opsonising activity of a pentraxin-like protein isolated from snapper (Pagrus auratus, Sparidae) serum.

In mammals the pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are important components of the immune response. Although pentraxins have been isolated from a number of fish species few studies detail their functional immunological role. In this paper we report the establishment of a flow cytometry based assay to measure the phagocytic activity of isolated snapper head kidney leukocytes (HKLs). This assay was then used to examine the ability of a pentraxin-like protein isolated from the serum of snapper (P. auratus) (Sn-PLP) to act as an opsonin. Incubation of snapper head kidney leukocytes (HKL) with FITC-labelled beads resulted in uptake of these particles by approximately 35% of HKLs. Incubation of beads with Sn-PLP enhanced phagocytosis by snapper HKLs in a dose-dependant manner. Enhanced phagocytosis could be inhibited by addition of a rabbit anti-Sn-PLP antibody suggesting that Sn-PLP may act as a ligand for a HKL cell surface receptor. This study provides further evidence toward a functional role for pentraxins in the host defence repertoire of fish.

Isolation and partial characterization of a pentraxin-like protein with complement-fixing activity from snapper (Pagrus auratus, Sparidae) serum.

Pentraxin-like molecules have been isolated from a number of fish species. However, little is known about the function of these proteins in the teleosts. In this study we report the isolation and characterization of a pentraxin-like molecule from the serum of snapper (Pagrus auratus) that has the ability to activate complement. This pentraxin-like protein was isolated from serum by calcium-dependent binding to agarose. SDS-PAGE analysis demonstrated an oligomeric protein of approximately 200k Da consisting of non-covalently bound subunits of 26 and 23 kDa. Protein sequencing revealed significant (50%) sequence identity with pentraxins from both Atlantic salmon (S. salar) and rainbow trout (O. mykiss). However, polyclonal antibodies raised against snapper pentraxin did not recognise salmon or trout pentraxin in Western blot analysis. Following LPS injection, snapper pentraxin levels increased 2-fold before gradually returning to basal levels. Most significantly, the isolated pentraxin initiated complement-mediated lysis of ligand-coated sheep erythrocytes in a dose-dependent fashion. In view of the similarity between the known fish pentraxins, and their similarity to mammalian serum amyloid P-components we conclude that the isolated protein may be a snapper pentraxin homologue.

Administration of a commercial immunostimulant preparation, EcoActiva as a feed supplement enhances macrophage respiratory burst and the growth rate of snapper (Pagrus auratus, Sparidae (Bloch and Schneider)) in winter.

This study investigated the effects of prolonged administration of a commercial beta-glucan based immunostimulant preparation, EcoActiva, in the form of a feed supplement, on non-specific immune parameters and the growth rate of snapper (Pagrus auratus). Fish held at a temperature representing either summer or winter conditions, were sampled periodically and assayed for head kidney macrophage activity via in vitro superoxide production, and classical and alternative complement activity. Fish were also weighed monthly and the growth rate determined. Fish fed on a diet supplemented with EcoActiva and held at the winter temperature had a significant enhancement of macrophage superoxide anion production upon stimulation with phorbol myristate acetate (PMA), and this increased activity was maintained throughout the trial. Macrophage activity in fish fed the supplemented diet and held at the summer temperature was also increased. However, EcoActiva failed to increase either classical or alternate complement activity. Most significantly EcoActiva resulted in an increase in growth rates of the fish held at the winter temperature as compared to the control fish, although no difference was seen between the groups held at the summer temperature. These results suggest that routine incorporation of beta-glucan preparations like EcoActivaduring winter may enhance macrophage function and growth rates at a time of increased disease susceptibility and little or no growth.

Anti-immunoglobulin binding and activation of snapper (Pagrus auratus) leucocytes.

In order to perform specific immunological assays we have produced and characterised three monoclonal antibodies (MAbs) that bind snapper (Pagrus auratus, Bloch and Schneider) immunoglobulin (Ig). Hybridomas were produced and screened for anti-Ig production using ELISA, Western blot and flow cytometry. All three MAbs (designated 2C5, 4A2 and 1C6) bound specifically to the heavy (H) chain of reduced Ig in Western blot. Furthermore, 1C6 was shown to bind to reduced skin mucus Ig H chain and all three MAbs cross-reacted with the H chain of Atlantic salmon and rainbow trout Ig. In flow cytometric analyses 2C5 and 4A2 bound to B cell populations in the peripheral blood and lymphoid organs. Furthermore, cross-linked 2C5 induced an increase in intracellular protein tyrosine phosphorylation in peripheral blood lymphocytes. Phosphorylated proteins exhibited similar molecular weights to those of mammalian Igalpha and Igbeta and may represent snapper mIg accessory molecule analogues. These data exhibit the potential use of 2C5, 4A2 and 1C6 in both cellular and biochemical analyses of populations of snapper leucocytes.