Isolates of Piscirickettsia salmonis from Scotland and Ireland Show Evidence of Clonal Diversity

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.     

The influence of aquaculture unit proximity on the pattern of Lepeophtheirus salmonis infection of anadromous Salmo trutta populations on the isle of Skye,Scotland

A total of 230 anadromous Salmo trutta (brown trout) were sampled in five sheltered coastal fjords (or sea lochs) on the Isle of Skye, Scotland, U.K., in 2016 at varying distances from active Atlantic salmon Salmo salar farms. Statistical models were developed to investigate potential correlations between salmon lice Lepeophtheirus salmonis burdens on S. trutta hosts and their proximity to S. salar farm cages. Significant correlations were found between lice burdens and fish fork length and proximity to the nearest S. salar farm. The probability of the presence of L. salmonis on fish hosts increased with fish host size and with distance from the nearest S. salar farm, but total lice burdens were highest in fish sampled near S. salar farms and decreased with distance. The proportion of different life‐cycle stages of L. salmonis were also dependent on S. salar farm proximity, with higher juvenile lice numbers recorded at sites near S. salar farm cages. These results highlight the complexity of the relationship between S. trutta and L. salmonis infections on wild fish and emphasize the requirement of further research to quantify these effects to better inform conservation and management strategies, particularly in areas of active S. salar farm facilities.     

Infestations of wild adult Atlantic salmon (Salmo salar L.) by the ectoparasitic copepod sea louse Lepeophtheirus salmonis Krøyer: prevalence,intensity and the spatial distribution of males and [2pt] females on the host fish

The copepod Lepeophtheirus salmonis Krøyer is a specific ectoparasite of North Atlantic and Pacific salmonids in their marine phases. We compared infestations of L. salmonis on wild Atlantic salmon (Salmo salarL.) captured in estuarine (Firth of Tay, east Scotland; 1995, 1996) and marine coastal waters (Strathy Point, north Scotland; 1998, 1999). Host fish from the Tay were caught by sweep netting, whilst those from Strathy Point were trapped in anchored bagnets. Fish capture method and exposure of the parasites to brackish conditions may both have detrimental effects on the retention of L. salmonis by the host, and hence possibly lead to their being under-estimated on returning adult fish. At Strathy Point, we recorded (i) an infestation prevalence of 100%, (ii) mean log abundances of pre-adult + adult L. salmonis at 19 (1998) and 24 (1999) per fish, (iii) 85/93% of all L. salmonis as being adults and (iv) overall 68/69% females. Fish caught in the upper Firth of Tay showed significantly lower prevalences, intensities and abundances of L. salmonis and probably had lost part or all of their lice burdens prior to capture, whereas those sampled from Strathy Point were apparently minimally affected by capture method or brackish water influences. The loss of parasites for the Tay fish was not markedly biased to males or females, or to pre-adult versus adult developmental stages. There were significantly greater abundances of L. salmonis on two sea-winter fish (30 lice per fish) than on one sea-winter fish (17 lice per fish) sampled at Strathy Point in 1998. There are several possible explanations for such age-related patterns of abundance, but the indications are that (i) initial infestation of smolts occurs in coastal waters, (ii) infestation of hosts in the open ocean is a persistent event, and (iii) oceanic reinfestation outweighs mortality losses of L. salmonis. This parasite typically occupies rather few zones on the host fish covering only a small percentage of the total available body surface area. Female predominance appears to be characteristic of L. salmonisinfestations of wild Atlantic salmon; this is in marked contrast to reports of extreme male dominance on farmed stocks. Adult females predominated on the epidermis adjacent to, and posterior of, the insertion of the anal fin and along the posterior dorsal midline between the dorsal and caudal fins. Males predominated on the sides of the head and along the dorsal midline between the head and the dorsal fin. Mate guarding/precopulatory pairs are formed between pre-adult II females and adult males. The significant correlation between the distribution of pre-adult females and adult males may be indicative of pre-adult females actively seeking out adult males, but more likely is due to the (large) adult females competitively ousting all smaller life stages (female and male) from those preferred zones. Given the relatively low fecundity of adult females, and observations of 100% prevalence of L. salmonis, the infective planktonic copepodid stage evidently is extremely efficient at locating and establishing upon its host fish in the pelagic environment.     

The effect of amphotericin B on the ultrastructure of Prototheca species

Prototheca zopfii and Prototheca wickerhamii strains were exposed to subinhibitory concentrations of the antimycotic amphotericin B, and the effect of the treatment on their ultrastructure was assessed. The results revealed ultrastructural changes in the treated cells, expressed by swelling of mitochondria, degradation of cell organelles, accumulation of microbody like structures, lipid droplets and starch granules in the cytoplasm, and changes in the inner layer of the cell wall.     

Pathological and epidemiological observations on rickettsiosis in cultured sea bass (Dicentrarchus labrax L.) from Greece

A systemic infection of a Rickettsia‐like organism (RLO) in cultured sea bass is described for the first time. In hatcheries, clinical signs were lethargy, inappetence and discoloration. Twenty days after transfer to sea cages from hatcheries where the disease existed, fish showed erratic and abnormal swimming behaviour, loss of orientation, and lethargy. Cumulative mortality in colder months of the year reached 30% in hatcheries and 80% in cages. Surviving fish in cages did not show any clinical signs of RLO infection in the subsequent year. Evidence for a systemic distribution of RLO was supported by histolopathological lesions in both infected hatchery and caged fish, where the lesion profile included cranial sensory, central nervous, integumental and alimentary organ systems. Intracranial lesions were primarily characterized by an ascending histiocytic perineuritis and necrotizing congestive meningoencephalitis, with evidence for transfer of infective agents across the blood–brain barrier confirmed by the presence of RLOs within capillary endothelium and histiocytes in inflamed regions of the optic tectum and the cerebellum. In the most severe cases, infection spread to the statoacoustical (semicircular) canal system and the ependymal lining of ventricles, with marked rickettsial‐laden histiocytic infiltration of the canal lumen. Integumental lesions were restricted to the oral submucosa, nares and integumental dermis of the cranium. Alimentary lesions were noted in both the liver parenchyma and mucosa/submucosa of the stomach. In all affected organs the RLOs were found by immunohistochemistry to be related to Piscirickettsia salmonis.     

An Alternative Efficient Procedure for Purification of the Obligate Intracellular Fish Bacterial Pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.     

Rhabdospora thelohani Laguessé, 1895 (Apicomplexa): New Host and Geographic Records with Taxonomic Considerations*

New fish species and geographic records for Rhabdospora thelohani Laguessé, 1895 (rodlet cells) are presented. Additionally, the ultrastructure of R. thelohani in Alburnoides bipunctatus ohridanus Karaman, Borostomias antarcticus (Louml;nnberg), Leuciscus cephalus albus Bonaparte and Rutilus rubilio (Bonaparte) is compared with that reported by other authors and with members of Subphylum Apicomplexa. The ultrastructure of R. thelohani was similar in all the fish species examined: however, the organism was not present in all members of any single species and had intertissue density variations. Rhabdospora thelohani is pyriform. averaging in size 7 × 12 m?, with a basal nucleus. The surface complex is composed of a layer (0.5 m?m diameter) formed by microfilaments (9.3 nm) and an outer trilaminar membrane (9.3 nm). The cytoplasm contains structures identical to rhoptries, micronemes and subpellicular microtubules. Mitochondria. Golgi apparatus, and rough endoplasmic reticulum were not observed, although free ribosomes were present and arranged in a vesicular pattern. The observations suggest that the organism moves between cells of epithelial layers and is either released into a lumen intact or passively or actively discharges its contents into a lumen. Results from this study indicate that R. thelohani should be considered a member of Apicomplexa unless definitive evidence is presented to the contrary.     

Reduced growth in wild juvenile sockeye salmon Oncorhynchus nerka infected with sea lice

Daily growth rings were examined in the otoliths of wild juvenile sockeye salmon Oncorhynchus nerka to determine whether infection by ectoparasitic sea lice Caligus clemensi and Lepeophtheirus salmonis was associated with reduced host body growth, an important determinant of survival. Over 98% of the sea lice proved to be C. clemensi and the fish that were highly infected grew more slowly than uninfected individuals. Larger fish also grew faster than smaller fish. Finally, there was evidence of an interaction between body size and infection status, indicating the potential for parasite‐mediated growth divergence.     

Classification of luminous bacteria from the light organ of the Australian Pinecone fish,Cleidopus gloriamaris

Luminous bacteria isolated from the light organs of the Australian Pinecone fish Cleidopus gloriamaris have been studied. The isolates were from fish from four different geographical estuarine systems on the east coast of Australia. All isolates were found to be strains of Vibrio fischeri, a species not hitherto demonstrated conclusively as forming a symbiotic association. Some ecological considerations are discussed.Non-Standard Abbreviation PHB polyhydroxybutyrate     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.     

Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains

The salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) is an ectoparasitic copepod which represents a major pathogen of wild and farmed salmonid fishes in the marine environment. In order to facilitate research on this ecologically and economically important parasite, a hatchery and culturing system permitting the closure of the life-cycle of L. salmonis in the laboratory was developed. Here, the hatchery system, breeding practices, and selected louse strains that have been maintained in culture in the period 2002–2009 are presented. The hatchery and culture protocol gave rise to predictable hatching of larvae and infections of host fish, permitting the cultivation of specific strains of L. salmonis for 22 generations. Both in- and out-bred lice and mutant colour strains have been established, and some of these strains were characterised by microsatellite DNA markers confirming their pedigree. No evidence of inbreeding depression, fitness or morphological changes was observed in any of the strains cultured. It is suggested that the culturing system, and the strains produced represent a significant resource for future research on this parasite.     

Lysozyme Content in Organs and Blood Serum in Various Species in the Volga River

The results of study of lysozyme content in kidney, liver, spleen, and blood serum of 12 fish species in the Volga River, which belong to six different orders, are presented. It has been established that the lysozyme level in the organs and blood serum is different in representatives of different taxons. The highest enzyme content was observed in sterlet (order Acipenseriformes), while the lowest content, in burbot (order Gadiformes). The enzyme content and its distribution in visceral organs depended on fish phylogeny, but not on the type of feeding (vegetarian or predatory) and the place of the constant habitation (fresh river or salted sea water). There were seasonal and yearly variations of the lysozyme concentration in fish organs and tissues. The many-year studies have established the regularity in enzyme distribution in sturgeon fish: kidney > spleen > liver > blood serum, this regularity being independent of the sturgeon species and constant during intra- and inter-year variations.     

Ultrastructure of the Peritrich Ciliate Ambiphrya ameiuri and Its Attachment to the Gills of the Catfish Ictalurus punctatus1

Ambiphrya ameiuri is an ectocommensal peritrich that attaches to the gills of warm-water fishes and filters bacteria from the water. The ultrastructure of this protozoon, its attachment to the fish gills, and its effect on the gill tissue were investigated by scanning and transmission electron microscopy. The peritrich attached to the gills by fibers extending from the scopula. A microtubular array, apparently a barren kinetosome, was present in each lobular projection, but no scopular cilia were observed. At low densities Ambiphrya had no apparent harmful effects on the fish; however, at high densities respiration may be impeded. Ultrastructural studies indicate that this organism receives no nourishment from the host tissue.     

First case of human protothecosis in Canada: Laboratory aspects

A case of bursitis due to Prototheca wickerhamii is briefly reported. In histological sections the organism stained well with fungal stains, grey with silver methanamine and red with periodic acid Schiff reagent. This unicellular achlorophyllous alga was studied on common laboratory media. The characterization of the Prototheca sp. depends largely on wet mount microscopic examination from broth or agar cultures which ensures the observation of endosporulation and a consistent absence of budding. Otherwise the growth rate and the pasty white colonies may lead to an erroneous identification, most likely as a Cryptococcus sp. P. wickerhamii lends itself very well to standard physiological tests used for the identification of yeasts. The strain was found insensitive to 5-fluorocytosine. The MIC of amphotericin B was 0.15 g/ml.     

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Saprolegnia salmonis sp. nov. isolated from sockeye salmon,Onchrhynchus nerka

A saprolegniasis occurred in cultured sockeye salmon,Onchorhynchus nerka, raised in Hokkaido, Japan. The lesions were mainly observed in the head, peduncle region and the caudal fin. All strains isolated were morphologically classified in the genusSaprolegnia. They were identified as a new species in the genus from the characteristics of the sexual organs, and namedSaprolegnia salmonis.     

Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from teat cup rubbers during milking (n = 2); water (n = 6); soil (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the teat cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.This revised version was published online in October 2005 with corrections to the Cover Date.     

Stable isotopes reveal contrasting trophic dynamics between host–parasite relationships: A case study of Atlantic salmon (Salmo salar) and parasitic lice (Lepeophtheirus salmonis and Argulus foliaceus)

Across existing fish host–parasite literature, endoparasites were depleted in δ¹⁵N compared to their hosts, while ectoparasitic values demonstrated enrichment, depletion and equivalence relative to their hosts. δ¹³C enrichment varied extensively for both endo- and ectoparasites across taxa and host tissues. In our case study, sea lice (Lepeophtheirus salmonis) were enriched in δ¹⁵N relative to their farmed Atlantic salmon (Salmo salar) hosts, although the value contradicted the average that is currently assumed across the animal kingdom. Common fish lice (Argulus foliaceus) did not show a consistent trend in δ¹⁵N compared to their wild S. salar hosts. Both parasitic species had a range of δ¹³C enrichment patterns relative to their hosts. Farmed and wild S. salar had contrasting δ¹³C and δ¹⁵N, and signals varied across muscle, fin and skin within both groups. L. salmonis and A. foliaceus subsequently had unique δ¹³C and δ¹⁵N, and L. salmonis from opposite US coasts differed in δ¹⁵N. Given the range of enrichment patterns that were exhibited across the literature and in our study system, trophic dynamics from host to parasite do not conform to traditional prey to predator standards. Furthermore, there does not appear to be a universal enrichment pathway for δ¹³C nor δ¹⁵N in parasitic relationships, which emphasizes the need to investigate host–parasite linkages across species.