Anatomy and cytology of the thymus in juvenile Australian lungfish, Neoceratodus forsteri.

The anatomy, histology and ultrastructure of the thymus of a dipnoan, the Australian lungfish, Neoceratodus forsteri, was studied by light and transmission electron microscopy. The thymic tissue showed clear demarcation into a cortex and medulla with ample vascularization. Large cells including foamy and giant multinucleated cells with periodic acid Schiff/Alcian blue positive staining properties were localized mainly in the medulla. The major cellular components were epithelial cells and lymphoid cells. The epithelial cells were classified by location and ultrastructure into six sub-populations: capsular cells, cortical and medullary reticular cells, perivascular endothelial cells, intermediate cells, nurse-like cells and Hassall-like corpuscles. Myoid cells were found mainly in the cortico-medullary boundary and medulla. Macrophages and secretory-like cells were also present. These findings will provide a base of knowledge about the cellular immune system of lungfish.

Emerging vesiculo-type virus infections of freshwater fishes in Europe.

Rhabdoviruses were isolated from perch Perca fluviatilis and largemouth bass Micropterus salmoides exhibiting clinical signs of disease. Preliminary studies indicated that these viruses could be neutralised by antisera to perch rhabdovirus (Dorson et al. 1984) and may be similar to those previously isolated from grayling Thymallus thymallus and pike-perch Stizostedion stizostedion. The relationship between these viruses and the previously characterised fish rhabdoviruses, pike fry rhabdovirus (PFRV), spring viraemia of carp virus (SVCV) and lake trout rhabdovirus, was investigated. Viruses were propagated in bluegill fry (BF-2) cells and were characterised using electron microscopy, serum neutralisation tests, immunofluorescence tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nucleotide sequence analysis. The bullet-shaped viral particles appeared to be compact, with spikes visible at the surface, a morphology similar to that of the vesiculovirus group of rhabdoviruses. Serum neutralisation tests showed that the viruses were antigenically closely related to the previously characterised perch rhabdovirus, but were not significantly neutralised by antisera to PFRV, SVCV or viral haemorrhagic septicaemia virus (VHSV). In immunofluorescence tests with perch rhabdovirus antisera, strong specific fluorescence was observed in cell cultures infected with the new rhabdovirus isolates, but no fluorescence was observed with antisera to PFRV, SVCV or VHSV. SDS-PAGE analysis revealed a polypeptide profile typical of vesiculoviruses, but the novel virus isolates had different relative mobilities of their P and M proteins compared to PFRV and SVCV. Nucleotide sequence analysis was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing of a 439 base-pair region of the viral L gene. The novel rhabdovirus isolates had <76% nucleotide sequence identity to PFRV, SVCV and lake trout rhabdovirus and >95% identity to perch rhabdovirus. Phylogenetic analysis using both maximum parsimony and neighbour-joining methods assigned the perch rhaboviruses to a separate group to that of PFRV, SVCV and lake trout rhabdovirus. These data are the initial characterisation of a group of emerging fish vesiculo-type viruses that are biochemically and genetically distinct from the PFRV, SVCV and lake trout rhabdoviruses.

A peptide derived from bovine beta-casein modulates functional properties of bone marrow-derived macrophages from germfree and human flora-associated mice.

The purpose of this study was to evaluate the immunomodulatory activity of a peptide derived from bovine beta-casein (beta-CN), the beta-CN (193-209) peptide, on mouse macrophages that were obtained either from germfree (GF) or from human flora-associated (HF) mice. Macrophages were derived from bone marrow (BMDM) in the presence of recombinant macrophage colony-stimulating factor and exposed to the peptide or lipopolysaccharide (LPS). Membrane marker expression [F4/80, Mac-1, major histocompatibility complex (MHC) class II antigens] and phagocytic activity were assessed by flow cytometry. Production of tumor necrosis factor-alpha and interleukin (IL)-6 was measured by bioassays and production of IL-1alpha, IL-1beta and IL-12 by ELISA. The expression of cytokine mRNA was determined using semi-quantitative reverse transcription-polymerase chain reaction. The beta-CN (193-209) peptide up-regulated MHC class II antigen expression and phagocytic activity of BMDM from GF and HF mice. Its enhancing effect on phagocytosis was greater than that after LPS stimulation (P < 0.01). The peptide induced notable levels of cytokine mRNA in BMDM from GF and HF mice, but it was a significantly weaker inducer of cytokine secretion than LPS. Nevertheless, although flora implantation had no stimulatory influence on basal MHC class II and basal cytokine levels, cells from HF mice were more susceptible than those from GF mice to the peptide effects on these variables. These results indicate that the beta-CN (193-209) peptide could enhance antimicrobial activity of macrophages without proinflammatory effects.

Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph.

The morphology and functions of haemocytes from the haemolymph of the European oyster, Ostrea edulis, were analysed by flow cytometry on the basis of cellular structures and incorporation of fluorescent markers. O. edulis circulating haemocytes appear to be composed of one to three cell populations based on cell size and granularity, with many individual variations. Analysis of haemocytes after propidium iodide staining indicated that the majority of oyster haemocytes are alive after sampling. The phagocytic activity level of haemocytes was analysed using fluorescent beads and this cell activity varied greatly depending on the oysters. The use of 3,3'dihexyloxacarbocyanine iodide (DIOC6) allowed the demonstration of several cell populations on the basis of labelled intensity. One to three cell sub-populations can be observed depending on the oysters. The haemocytes characterised by high granularity showed a strong fluorescence intensity related to high mitochondrial activity.

Comparative study of in-situ cell death induced by the viruses of viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN) in rainbow trout.

The causative viruses of two diseases of rainbow trout, viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN), exert much of their cytopathogenic effect in cell culture through the induction of apoptosis. In the present study, the TUNEL procedure was used to investigate the presence of apoptotic cells in different organs of rainbow trout infected with the viruses of VHS and IPN. VHS viral infection resulted in massive apoptosis in renal lymphoid tissue, where viral antigens were also detected. Large numbers of viral particles were observed in close proximity to apoptotic cells. Apoptosis was not detected in excretory cells of the renal tubules or in infected muscle cells. IPN virus did not induce apoptosis in the pancreas. However, the DNA degradation associated with apoptotic nuclei was observed in muscle lesions. Taken together, these results indicated that induction of apoptosis in vivo was critically influenced by the species of virus and the cell type. Moreover, it would seem likely that apoptosis contributed to the nature of the two diseases and to mortality.

Flow cytometric analysis of European flat oyster, Ostrea edulis, haemocytes using a monoclonal antibody specific for granulocytes.

The importance of haemocytes in mollusc defence mechanisms can be inferred from their functions. They participate in pathogen elimination by phagocytosis (Cheng, 1981; Fisher, 1986). Hydrolytic enzymes and cytotoxic molecules produced by haemocytes contribute to the destruction of pathogenic organisms (Cheng, 1983; Leippe & Renwrantz, 1988; Charlet et al., 1996; Hubert et al., 1996; Roch et al., 1996). Haemocytes may also be involved in immunity modulation by the production of cytokines and neuropeptides (Hughes et al., 1990; Stefano et al., 1991; Ottaviani et al., 1996). As a result, the literature dealing with bivalve haemocyte studies has increased during the last two decades. Most of these publications use microscopy for morphological analysis (Seiler & Morse, 1988; Auffret, 1989; Hine & Wesney, 1994; Giamberini et al., 1996; Carballal et al., 1997; Lopez et al., 1997; Nakayama et al., 1997), and functional analysis (e.g. phagocytosis) (Hinsch & Hunte, 1990; Tripp, 1992; Mourton et al., 1992; Fryer & Bayne, 1996; Mortensen & Glette, 1996). Flow cytometry represents a rapid technique applicable to both morphological and functional studies of cells in suspension. While the measurements based on autofluorescence provide information on cell morphology, the analyses with fluorescent markers including labelled antibodies, offer data on phenotyping and cell functions. As a result, its application has greatly contributed to the investigation of immunocyte functions and differentiation in vertebrates (Stewart et al., 1986; Rothe & Valet, 1988; Ashmore et al., 1989; Koumans-van Diepen et al., 1994; Rombout et al., 1996; Caruso et al., 1997). Some authors studied oyster haemocyte populations by flow cytometry based on cellular autofluorescence (Friedl et al., 1988; Fisher & Ford, 1988; Ford et al., 1994). However, no analysis using specific monoclonal antibodies has been reported to date. In this study, a protocol for studying European flat oyster, Ostrea edulis, haemocytes by flow cytometry using a monoclonal antibody specific for granulocytes and an indirect immunofluorescence technique have been developed. European flat oysters, Ostrea edulis, 7-9 cm in shell length were obtained from shellfish farms in Marenne Oléron bay (Charente Maritime, France) on the French Atlantic coast. All individuals were purchased just before each experiment and processed without any previous treatment.

Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species.

BACKGROUND: The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the (51)Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species. METHODS: This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18((3)) (3, 3'-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations. RESULTS: A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the (51)Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus. CONCLUSIONS: We provide evidence that the flow cytometric assay using DIOC18((3)) is highly reproducible and is suitable to measure different types of cell cytotoxicity.

Rainbow trout sleeping disease virus is an atypical alphavirus.

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10 degrees C, while little virus was produced at 14 degrees C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.

Fish rhabdovirus cell entry is mediated by fibronectin.

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family.

Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway.

In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-kappaB, a transcription factor known to be activated by oxidative stress, was examined. NF-kappaB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-kappaB activation is not critical for TGEV-induced apoptosis.

Comparative development of two microsporidian species: Enterocytozoon bieneusi and Enterocytozoon salmonis, reported in AIDS patients and salmonid fish, respectively.

Enterocytozoon bieneusi and Enterocytozoon salmonis are reported in HIV-infected patients and in salmonid fish, respectively. Both species share the early development of the extrusion apparatus of the spores, which is completed prior to fission of the sporogonic syncytium into sporoblasts, and the early synthesis of polar tube constituents, but they differ in other developmental and sporogenetic processes. Enterocytozoon bieneusi develops in direct contact with the cytoplasm of epithelial cells whereas E. salmonis occurs only in the nucleus of leucocytes and epithelioid cells. Sporogonic nuclei, which are scattered throughout the sporont in E. bieneusi, are located in the periphery in E. salmonis. The multilamellar structures associated with the nuclear envelopes and the endoplasmic reticulum cisternae are specific for E. bieneusi. Additionally, the evolution of the polar tube precursors proceeds differently in the two parasites. In E. bieneusi, they transform into electron-dense bodies associated with a reticulum and polar tubes derive from these structures according to a process similar to that reported in other microsporidia. In E. salmonis, polar tube precursors fuse directly at their ends and the polar tubes appear to be formed by the assemblage of these fused precursors with a material previously synthesized in the vicinity of nuclei. In conclusion, both species appear to be less closely related than was supposed in earlier descriptions.

Different peptides from hemorrhagic septicemia rhabdoviral proteins stimulate leucocyte proliferation with individual fish variation.

Trout leucocytes from most of the survivors of viral hemorrhagic septicemia virus (VHSV) infections were capable of in vitro proliferation (T-like response) when cultured in the presence of short synthetic peptides designed from the G and the N cDNA-derived protein sequences of VHSV, a virus with substantial economic impact in trout farms. In contrast, no significant proliferative responses were obtained for the above-mentioned peptides from leucocytes obtained from either noninfected or genetically VHSV-resistant trout. However, since the anamnestic recognition of particular peptides (epitopes) of the G and the N protein by trout leucocytes varies largely within the outbred trout population, larger VHSV protein fragments were also tested. The finding that recombinant G and N fragments carrying multiple epitopes are recognized by the majority of the individual trout surviving VHSV infections and with higher stimulation indexes suggests that the recombinant viral proteins could be used as vaccines given the outbred nature of the fish.

Pathogenesis of viral haemorrhagic septicaemia virus: cellular aspects.

Leucocyte populations from rainbow trout subjected to experimental viral haemorrhagic septicaemia virus (VHSV) infections under in vivo and in vitro conditions were analysed by flow cytometry. Quantitative analysis shows that only a low percentage of the leucocytes support viral replication. Lymphocytes, compared with monocytes granulocytes and macrophages, are the least susceptible sub-population. A decrease in the amount of the phagocytic activity was observed after the cells were infected with VHSV. Obvious modifications were observed through dot plot profiles in the composition of the cell sub-populations and in the cell morphology. In addition to a direct effect of VHSV on the macrophages, a systemic effect is produced. This could also be related to the stress induced by the experimental infection process.

Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish.

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.

Structure and development of Sphaerospora elegans Thélohan 1892 (Myxozoa: Myxosporea) in the sticklebacks Gasterosteus aculeatus L. and Pungitius pungitius L. (Gasterosteidae).

The structure and development of Sphaerospora elegans Thélohan 1892 was studied by light and electron microscopy. Specimens of Gasterosteus aculeatus and Pungitius pungitius were examined from localities in England, Scotland and France. The kidney was found to be the main site of infection in both species, with a frequent co-infection with Myxobilatus gasterostei (Parisi, 1912) Davis, 1944 present in several populations of G. aculeatus. Possible extrasporogonic stages were located in the interstitial haematopoietic tissue of the kidney and in the choroidal rete of the eye. Within renal tubules sporogonic events could be followed and were seen to result in the formation of mature sub-spherical spores 10.2 × 10.1 µm in size, containing 2 almost spherical polar capsules of equal size. Only minor pathological changes were found, even in heavy infections. Relationships with other Sphaerospora spp. and PKX cells are discussed.

Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus.

The nucleotide sequence of the gene that encodes for the structural viral protein VP1 of bovine rotavirus (RF strain) has been determined. The sequence data indicate that segment 1 contains 3302 bp and is A + T rich (65.3%). The positive strand of segment 1 contains a single open reading frame that extends 1088 codons and possesses 5'- and 3'-terminal untranslated regions of 18 and 20 bp, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 124,847, very close to the apparent molecular weight of VP1 (M.W. 125,000). The deduced amino acid sequence presents significant similarities with RNA-dependent RNA polymerase of several RNA viruses. VP1 was also synthesized in baculovirus using two transfer vecors: pAC461 and pVL941. Following infection of Sf9 cells with a recombinant baculovirus, a full-length nonfusion protein was synthesised which shares properties with authentic VP1 made in monkey kidney cells. The level of VP1 synthesis was about 10-fold higher when the baculovirus recombinant was derived from the pVL941 transfer vector. In that case, VP1 was expressed in yields approximately equivalent to 10% of the cellular protein. The recombinant protein was immunoprecipitated by hyperimmune serum raised against purified rotavirus. It also was immunogenic; a hyperimmune serum made in guinea pigs reacted with VP1 using immunoprecipitation and Western blot. This serum did not possess neutralization activity.