Haplosporosomes, sporoplasmosomes and their putative taxonomic relationships in rhizarians and myxozoans.

This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa - Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.

Haplosporidian host:parasite interactions.

The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma ß-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.

Ultrastructural morphogenesis of a virus associated with lymphocystis-like lesions in parore Girella tricuspidata (Kyphosidae: Perciformes).

The morphogenesis of large icosahedral viruses associated with lymphocystis-like lesions in the skin of parore Girella tricuspidata is described. The electron-lucent perinuclear viromatrix comprised putative DNA with open capsids at the periphery, very large arrays of smooth endoplasmic reticulum (sER), much of it with a reticulated appearance (rsER) or occurring as rows of vesicles. Lysosomes, degenerating mitochondria and virions in various stages of assembly, and paracrystalline arrays were also present. Long electron-dense inclusions (EDIs) with 15 nm repeating units split terminally and curled to form tubular structures internalising the 15 nm repeating structures. These tubular structures appeared to form the virion capsids. Large parallel arrays of sER sometimes alternated with aligned arrays of crinkled cisternae along which passed a uniformly wide (20 nm) thread-like structure. Strings of small vesicles near open capsids may also have been involved in formation of an inner lipid layer. Granules with a fine fibrillar appearance also occurred in the viromatrix, and from the presence of a halo around mature virions it appeared that the fibrils may form a layer around the capsid. The general features of virogenesis of large icosahedral dsDNA viruses, the large amount of ER, particularly rsER and the EDIs, are features of nucleo-cytoplasmic large DNA viruses, rather than features of 1 genus or family.

Legislative and regulatory aspects of molluscan health management.

The world population is growing quickly and there is a need to make sustainable protein available through an integrated approach that includes marine aquaculture. Seafood is already a highly traded commodity but the production from capture fisheries is rarely sustainable, which makes mollusc culture more important. However, an important constraint to its continued expansion is the potential for trade movements to disseminate pathogens that can cause disease problems and loss of production. Therefore, this review considers legislative and regulatory aspects of molluscan health management that have historically attempted to control the spread of mollusc pathogens. It is argued that the legislation has been slow to react to emerging diseases and the appearance of exotic pathogens in new areas. In addition, illegal trade movements are taken into account and possible future developments related to improvements in areas such as data collection and diagnostic techniques, as well as epidemiology, traceability and risk analysis, are outlined.

Ultrastructural comparison of Bonamia spp. (Haplosporidia) infecting ostreid oysters.

The ultrastructure of Bonamia from Ostrea angasi from Australia, Crassostrea ariakensis from the USA, O. puelchana from Argentina and O. edulis from Spain was compared with described Bonamia spp. All appear conspecific with B. exitiosa. The Bonamia sp. from Chile had similarities to the type B. exitiosa from New Zealand (NZ), but less so than the other forms recognized as B. exitiosa. Two groups of ultrastructural features were identified; those associated with metabolism (mitochondrial profiles, lipid droplets and endoplasmic reticulum), and those associated with haplosporogenesis (Golgi, indentations in the nuclear surface, the putative trans-Golgi network, perinuclear granular material and haplosporosome-like bodies). Metabolic features were regarded as having little taxonomic value, and as the process of haplosporogenesis is not understood, only haplosporosome shape and size may be of taxonomic value. However, the uni-nucleate stages of spore-forming haplosporidians are poorly known and may be confused with Bonamia spp. uni-nucleate stages. The many forms of NZ B. exitiosa have not been observed in other hosts, which may indicate that it has a plastic life cycle. Although there are similarities between NZ B. exitiosa and Chilean Bonamia in the development of a larger uni-nucleate stage and the occurrence of cylindrical confronting cisternae, the clarification of the identity of Chilean Bonamia must await molecular studies.

Ultrastructure of Bonamia sp. in Ostrea chilensis in Chile.

Oyster Ostrea chilensis samples were collected from Quihua Island, Chile, in December 2003 and February 2005, and examined in May 2004, and March, April and July 2005, for an ultrastructural comparison of the Chilean Bonamia sp. with other Bonamia spp. Only uni-nucleate stages were encountered, except in the July sample. The Chilean parasite differs from B. perspora in the apparent lack of a plasmodial stage and of sporulation. It resembles B. ostreae in size, the low number of mitochondrial profiles, and the prevalence and mean number of lipid droplets. It differs from B. ostreae in the greater prevalence of nuclear membrane-bound Golgi (NM-BG), associated haplosporogenesis, and smaller size of haplosporosomes. The Chilean Bonamia sp. resembles B. exitiosa in the number of haplosporosomes, prevalence of lipid droplets, anastomosing endoplasmic reticulum and NM-BG, presence of circles of smooth endoplasmic reticulum (sER), confronting cisternae (CC), and cylindrical CC (CCC). It also appears to have a similar developmental cycle to B. exitiosa with larger forms occurring in winter (August). The circles of sER, CC, and CCC have only been reported from B. exitiosa, and it appears that Chilean Bonamia sp. and B. exitiosa are more closely related than they are to B. perspora or B. ostreae. Similarities in ultrastructure and developmental stages between New Zealand and Chilean parasites suggest that the 2 species are related, and that the Chilean Bonamia sp. is either B. exitiosa, a sub-species of B. exitiosa, or a separate species closely related to B. exitiosa.

Inter-relationships of haplosporidians deduced from ultrastructural studies.

We reviewed papers reporting haplosporidian ultrastructure to compare inter-relationships based on ultrastructure with those based on molecular data, to identify features that may be important in haplosporidian taxonomy, and to consider parasite taxonomy in relation to host taxonomy. There were links between the following: (1) the plasmodia of an abalone parasite, Haplosporidium nelsoni and Urosporidium crescens in the release of haplosporosomes; (2) H. costale and H. armoricanum in haplosporosome shape and presence and shape of Golgi in spores; (3) basal asporous crustacean haplosporidians which form haplosporosomes from formative bodies (FBs) in vegetative stages--H. nelsoni, which forms haplosporosomes from FBs in plasmodial cytoplasm, and H. louisiana, Minchinia spp. and Bonamia perspora, which form haplosporosomes from FBs in spores; (4) crustacean haplosporidians, Bonamia spp. and M. occulta in the predominance of uni- and binucleate stages; and (5) lipid-like vesicles in sporoplasms of H. costale, H. armoricanum, H. lusitanicum, H. pickfordi, H. montforti, and B. perspora. In general, these relationships reflect phylogenies based on molecular studies. As well as spore form and ornamentation, haplosporogenesis in spores appears to be taxonomically important. Parasite and host taxonomy were linked in the infection of lower invertebrates by Urosporidium spp., the infection of oysters by Bonamia spp., and of molluscs by Minchinia spp. Haplosporidium spp. are patently an artificial, paraphyletic group probably comprising many taxa. Consequently, the taxonomy of haplosporidians needs a thorough revision.

Ultrastructure of sporulation in Haplosporidium armoricanum.

An ultrastructural study was carried out on the tissues of an oyster (Ostrea edulis), heavily infected with Haplosporidium armoricanum, that had been fixed in Carson's fixative. The well-fixed tissues revealed details of sporulation and of the spores, which had not been previously reported from H. armoricanum. These include the initial presence of sparse haplosporosomes after thickening of the plasma membrane in early sporonts, division of sporont nuclei by multiple fission, cup-like indentations in the nuclear surface associated with putative nuclear material in both the sporonts and spores, and cytoplasmic multi-vesicular bodies in the cytoplasm of sporonts and spores. The spore wall and operculum were formed from a light matrix that occurred in short cisternae of smooth endoplasmic reticulum in the episporoplasm, and parallel bundles of microfibrils were present in some spores. Spores were rarely bi-nucleate with the nuclei occurring as a diplokaryon, with putative nuclear material at the junction of the 2 nuclei. Nuclear membrane-bound Golgi (NM-BG) cisternae were common in spores, and they appeared to synthesise a light granular material into lysosome-like granules. Dense bodies similar to those reported from H. lusitanicum, H. pickfordi and H. monforti occurred in, or outside, the peripheral endosporoplasm, which was closely apposed to the spore wall. Spore haplosporosomes were frequently axehead-shaped, more like those of H. costale than those previously reported from H. armoricanum, and in some haplosporosomes there was a small round lucent patch with a dark point near the centre of the lucent patch. Overall, H. armoricanum appears to be closely related to H. costale and Bonamia spp. Although the endosporoplasm of H. armoricanum has NM-BG and it resembles the uni-nucleate stage, it appears to be unlikely that they are the same, as the axehead-shaped haplosporosomes of the spore differ considerably from the spherical haplosporosomes of vegetative stages.

Haemolymph parasite of the shore crab Carcinus maenas: pathology, ultrastructure and observations on crustacean haplosporidians.

A protozoan parasite with some features of haplosporidians is described from the European shore crab Carcinus maenas. The parasite establishes a systemic infection through the haemal sinuses and connective tissues. Intracellular stages of the parasite were found within reserve inclusion, connective tissue, and muscle cells, while free forms were present in all haemal spaces. A uninucleate stage appeared to develop to a multinucleate plasmodial stage following multiple mitotic divisions of the nucleus. Histopathology also indicated that nuclear division may occur to form multinucleate plasmodia, in connective tissue, reserve inclusion and muscle cells, the multinucleate plasmodium being enclosed in the host-cell plasma membrane. It appears that the multinucleate plasmodium may then undergo internal cleavages which result in plasmodial fragmentation to form many uninucleate stages. Both stages, but particularly the uninucleate stage, contained cytoplasmic, large, ovoid, dense vesicles (DVs), some of which contained an internal membrane separating the medulla from the cortex, as in haplosporosomes. Golgi-like cisternae, closely associated with the nuclear membrane, formed DVs and haplosporosome-like bodies (HLBs), superficially resembling viruses. Infrequently, HLBs may condense to form haplosporosomes. The DVs, as in spores of some Haplosporidium spp. and paramyxeans, may give rise to, and are homologous with, haplosporosomes. Other features, such as the presence of an intranuclear mitotic spindle, lipid droplets, and attachment of DVs and haplosporosomes to the nuclear membrane, indicate that the C. maenas parasite is a haplosporidian. A similar organism reported from the haemolymph of spot prawns Pandalus spp., and haplosporidians reported from prawns Penaeus vannamei and crabs Callinectes sapidus may belong to this group. It is concluded that the well-characterised haplosporidians of molluscs and some other invertebrates may not be characteristic of the whole phylum, and that morphologically and developmentally similar organisms may also be haplosporidians, whether they have haplosporosomes or not.

Mikrocytos roughleyi taxonomic affiliation leads to the genus Bonamia (Haplosporidia).

Microcell-type parasites of oysters are associated with a complex of diseases in different oyster species around the world. The etiological agents are protists of very small size that are very difficult to characterize taxonomically. Associated lesions may vary according to the host species, and their occurrence may be related to variations in tissue structure. Lesion morphology cannot be used to distinguish the different agents involved. Ultrastructural observations on Mikrocytos roughleyi revealed similarities with Bonamia spp., particularly in regard to the presence of electron-dense haplosporosomes and mitochondria, whose absence from M. mackini also indicate that M. roughleyi and M. mackini are not congeneric. A partial small subunit (ssu) rRNA gene sequence of M. roughleyi was determined. This partial sequence, 951 nucleotides in length, has 95.2 and 98.4% sequence similarities with B. ostreae and B. exitiosus ssu rDNA sequences, respectively. Polymorphisms among the ssu rDNA sequences of B. ostreae, B. exitiosus and M. roughleyi allowed identification of restriction enzyme digestion patterns diagnostic for each species. Phylogenetic analysis based on the ssu rDNA data suggested that M. roughleyi belongs in the phylum Haplosporidia and that it is closely related to Bonamia spp. On the basis of ultrastructural and molecular considerations, M. roughleyi should be considered a putative member of the genus Bonamia.

Haplosporidium sp. (Alveolata: Haplosporidia) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia.

Haplosporidium sp. is described from rock oysters Saccostrea cuccullata Born, 1778 experiencing epizootics on the northwestern coast of Western Australia. All stages were observed as focal infections in the connective tissue of the gills, or as disseminated infections in the mantle and around digestive diverticulae. Haplosporidium sp. occurred between epithelial cells of the gut, in focal lesions in the gills, but not in the epithelium of the digestive diverticulae, and sporulation was confined to the connective tissue. Plasmodia developed into sporonts and sporocysts in a loose syncytium that gave rise to binucleate and uninucleate sporoblasts from which spores developed. Spores were flask-shaped, 5.6-6.7 x 3.3-4.0 microm, with a characteristic operculum, a few filamentous wrappings and rod-like structures in the posterior sporoplasm. Mature spores had a wall comprising inner (90 nm wide), middle (30 nm wide) and outer (130 nm wide) layers, and a surface coat of microtubules giving them a furry appearance. Oysters with empty gonad follicles were most heavily infected, and oyster condition and mortality appeared to be related to degree of infection.

Severe apicomplexan infection in the oyster Ostrea chilensis: a possible predisposing factor in bonamiosis.

Histological examination of 6455 oysters Ostrea chilensis from Foveaux Strait south of New Zealand over a 5 yr period showed >85% contained apicomplexan zoites, irrespective of season. Zoites occurred around the haemolymph sinuses and the digestive diverticulae at all intensities of infection; occurrence in the sub-epithelium, Leydig tissue and gills/mantle increased with increasing intensity of infection. Many (>35%) oysters were heavily infected, and most of them had severely damaged tissues. Heavy infections affected gametogenesis; 1% of lightly infected oysters had empty gonad follicles lacking germinal epithelium compared with 2% of moderately infected oysters and 9% of heavily infected oysters. Of oysters with empty gonad follicles, 75% were heavily infected with zoites. The parasite spread from the haemolymph sinuses and moved between Leydig cells, causing their dissociation and lysis. Some zoites were intracellular in Leydig cells. Lesions contained many haemocytes phagocytosing zoites, leading to haemocyte lysis and causing a haemocytosis. Fibrosis occurred to repair lesions in a few oysters. The zoites had a typical apical complex with 2 polar rings and 84 sub-pellicular microtubules. Prevalence and intensity of concurrent Bonamia exitiosus infection was related to the intensity of zoite infection, with only 3.8% of B. exitiosus infections occurring in the absence of zoites, 20.0% occurring in light zoite infections, 30.9% in moderate zoite infections, and 45.4% when oysters were heavily infected with zoites. The converse was not the case, as 75.3% of zoite infections occurred in the absence of B. exitiosus infection, including 51.1% of moderate to heavy zoite infections. There was a statistically significant association between intensities of B. exitiosus and of zoites (p < 0.0001). Zoites may increase the susceptibility of oysters to B. exitiosus by occupying and destroying haemocytes, and by destroying connective tissue cells and utilising host glycogen reserves. The parasite may be heteroxenous, with other stages in the terebellid polychaete Pseudopista rostrata.

Prokaryote infections in the New Zealand scallops Pecten novaezelandiae and Chlamys delicatula.

Four intracellular prokaryotes are reported from the scallops Pecten novaezelandiae Reeve, 1853 and Chlamys delicatula Hutton, 1873. Elongated (1025 x 110 nm), irregular (390 x 200 nm), or toroidal (410 x 200 nm) mollicute-like organisms (M-LOs) occurred free in the cytoplasm in the digestive diverticular epithelial cells of both scallop species. Those in P. novaezelandiae bore osmiophilic blebs that sometimes connected the organisms together, and some had a rod-like protrusion, both of which resemble the blebs and tip structures of pathogenic mycoplasmas. The M-LOs in C. delicatula had a slightly denser core than periphery. Round M-LOs, 335 x 170 nm, occurred free in the cytoplasm of agranular haemocytes in P. novaezelandiae, without apparent harm to the host cell. In P. novaezelandiae, 2 types of highly prevalent (95 to 100%) basophilic inclusions in the branchial epithelium contained Rickettsia-like organisms (R-LOs). Type 1 inclusions occurred in moderately hypertrophied, intensely basophilic cells, 8 to 10 microm in diameter, containing elongate intracellular R-LOs, 2000 x 500 nm. Type 2 inclusions were elongated and moderately basophilic in markedly hypertrophic branchial epithelial cells, 50 x 20 microm in diameter, containing intracellular organisms 500 x 200 nm in diameter. The possible roles of these organisms in pathogenesis is discussed.

Ultrastructure of a haplosporidian containing Rickettsiae, associated with mortalities among cultured paua Haliotis iris.

Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand. The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes. Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER. It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network. Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time. The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer. The possible inter-relationships of these features are discussed. The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes. Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster. The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp. and R. massiliae being the closest relatives. The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.

Ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and Ostrea spp. in British Columbia, Canada.

An ultrastructural study was carried out on Mikrocytos mackini, the cause of Denman Island disease in Pacific oysters Crassostrea gigas in western Canada. Three forms were identified, quiescent cells (QC), vesicular cells (VC) and endosomal cells (EC). QC occurred in the vesicular connective tissue (VCT), haemocytes (hyalinocytes), adductor and heart myocytes, and extracellularly. They had a central round to ovoid nucleus, < 7 cisternae of inactive nuclear membrane-bound Golgi, few vesicles and lysosome-like bodies. VC were rarely extracellular and usually occurred in adductor and heart myocytes, in close association with host cell mitochondria. The contents of the host cell mitochondria appeared to pass through a tubular extension into the cytoplasm of the parasite. Cytoplasmic vesicles resembled the tubular structure in appearance and size. EC occurred in the VCT, in haemocytes and extracellularly. They had a dilated nuclear membrane, sometimes containing a looped membranous structure that appeared to derive from the nucleus, and pass into the cytoplasm. A well-developed anastomosing endoplasmic reticulum connected the nuclear and plasma membranes, and endosomes were present in the cytoplasm. QC and EC cells were frequently observed tightly against, or between, the nuclear membranes of the host cell. Few organelles occurred in all forms of M. mackini, especially QC. The lack of organelles found in most eukaryotic cells, including mitochondria or their equivalents, may be due to obligate parasitism and the utilization of host cell organelles reducing the need for parasite organelles. Alternatively, perhaps M. mackini is a primitive eukaryote. Although phylogenetic affinities could not be determined, it is not a haplosporidian. A developmental cycle is proposed from these findings.

Bonamia exitiosus n. sp. (Haplosporidia) infecting flat oysters Ostrea chilensis in New Zealand.

Bonamia sp. is a pathogenic parasite that occurs in the haemocytes of dredge oysters Ostrea chilensis Philippi in New Zealand. Ultrastructurally it resembles other haplosporidians in the possession of haplosporosomes, haplosporogenesis, persistence of mitotic microtubules during interphase and of the nuclear envelope during mitosis, and occurrence of a diplokaryotic or multi-nucleate plasmodial stage. Another stage containing a large vacuole derived from enlargement of 1 or more mitochondria has not previously been described from other haplosporidians. It most closely resembles B. ostreae Pichot et al., 1979, which parasitises and is pathogenic in haemocytes of European flat oysters, O. edulis. However, B. ostreae is smaller and denser, and has fewer lipoid bodies and haplosporosomes. We have nearly completely sequenced the small ribosomal gene of the organism from O. chilensis. Initial comparisons of these sequences with those of other protozoans showed similarities to B. ostreae. Polymorphism within Bonamia sp. was confirmed by restriction fragment length polymorphism analysis. On the basis of ultrastructural and molecular considerations it is proposed that this organism be named Bonamia exitiosus sp. nov.

A survey of some parasites and diseases of several species of bivalve mollusc in northern Western Australia.

Pteriid oysters (Pinctada maxima, Pinctada margaritifera, Pinctada albina, Pteria penguin), rock oysters (Saccostrea glomerata, Saccostrea cuccullata, Saccostrea echinata) and representatives of other taxa (Malleidae, Isognomonidae, Pinnidae, Mytilidae, Spondylidae, Arcidae) from the wild, and 4670 hatchery-reared P. maxima, from northern and Western Australia, were examined for parasites and diseases. Rickettsiales-like inclusions and metacestodes of Tylocephalum occurred in most species. Intranuclear virus-like inclusions occurred in 1/415 wild P. maxima, 1/1254 S. cuccullata, 3/58 Isognomon isognomum, 1/80 Pinna bicolor and 1/45 Pinna deltodes. Perkinsus was histologically observed in 1/4670 P. maxima spat, 2/469 P. albina, 1/933 S. glomerata, 16/20 Malleus meridianus, 12/58 I. isognomum, 1/45 P. deltodes, 5/12 Spondylus sp., 1/16 Septifer bilocularis and 3/6 Barbatia helblingii. One of 1254 S. cuccullata was heavily systematically infected with Perkinsus merozoites, meronts and schizonts, and was patently diseased. Other potentially serious pathogens included Haplosporidium sp. in 6/4670 P. maxima spat, Marteilia sydneyi from 1/933 S. glomerata, and Marteilia sp. (probably M. lengehi) (1/1254) and Haplosporidium sp. (125/1254) from S. cuccullata. The latter were associated with epizootics on offshore islands, with heaviest prevalence (45%) in oysters with empty gonad follicles. Marteilioides sp. infected the oocytes of 9/10 female S. echinata from Darwin Harbour. Details of geographical distribution and pathology are given, and the health of the bivalves examined is discussed.

Effect of Fusobacterium necrophorum leukotoxoid vaccine on susceptibility to experimentally induced liver abscesses in cattle.

The efficacy and the optimum dose of Fusobacterium necrophorum crude leukotoxoid vaccine required to immunize and protect steers against experimentally induced liver abscesses were evaluated. The vaccine consisted of cell-free culture supernatant of a high leukotoxin-producing strain of F. necrophorum, inactivated with formalin and homogenized with an adjuvant. Twenty-five steers were assigned randomly to the following five treatment groups: control; three doses (1.0, 2.0, and 5.0 mL) of the culture supernatant; and 2.25 mL of the concentrated supernatant (equivalent to 5 mL of the original supernatant). Vaccine was injected subcutaneously on d 0 and 21. Blood samples were collected weekly to monitor antileukotoxin antibody titers. Three weeks after the second vaccination (d 42), all steers were injected intraportally with F. necrophorum culture to induce liver abscesses. Three weeks later (d 63), steers were euthanatized and necropsied; livers were examined and protection assessed. Antileukotoxin antibody titers in the control steers generally did not differ from the baseline (wk 0) titers. The titers in the vaccinated groups increased, more so after the second injection, and the increase was generally dose-dependent. Necropsy examination revealed that all steers in the control group had abscesses in the liver. In the vaccinated groups, two of five steers in the 1.0-mL group and one each in the 2.0-, 5.0-, and 2.25-mL (concentrated) groups had liver abscesses. Antileukotoxin antibody titers were higher (P < .05) in steers that did not develop abscesses than in steers that developed abscesses. The difference suggested a protective effect of antileukotoxin antibodies against experimentally induced liver abscesses.

Contamination of fish products: risks and prevention.

The risks of contamination of finfish products with active pathogens largely depend on the type of product concerned and disposal methods of the importing country. Frozen fish used as bait or to feed high-value species present the greatest risk as vehicles of contamination because they are unprocessed. Freezing preserves viral- and some bacterial-pathogens, thus the use of such fish as bait can introduce those pathogens into natural waters. Conversely, processed fish, particularly fillets, which have been heat-treated or cooked, present the lowest risk. If fish are processed after importation, care must be taken to ensure effective waste disposal, with particular attention to the prevention of scavenging by avian vectors and drainage from landfills into natural waters. Liquid waste should be disinfected and disposed of well away from natural waters.