Mark A. Adkison

Whirling disease: re-emergence among wild trout

Summary: Whirling disease of rainbow trout is caused by Myxobolus cerebralis, a myxozoan parasite possessing a life cycle well adapted to the natural environments where salmonid fish are found. Whirling disease was first described in Europe in 1898 among farmed rainbow trout but recent occurrences have been devastating to wild trout in North America. The disease is considered a major threat to survival of wild rainbow trout in the intermountain west of the United States. Difficulties in containing the spread and potentially eliminating the pathogen are tied to features of a complex life cycle involving two hosts, the salmonid fish and an aquatic oligochaete. Details of the morphologic development of the parasite have been described in each host but only now are we beginning to appreciate the breadth of interactions between these developmental forms and the sequential responses of the host. Fundamental mechanisms of the recognition and attachment of the parasite to the hosts, how host immunity is evaded and the unknown influences of environmental factors all contribute to a rather poor understanding of the biology of the parasite. Although the biology and ecology of the salmonid host are better known than for the oligochaete host, our knowledge is inadequate to interpret their complex interactions with the parasite. This uncertainty precludes the development of effective management activities designed to enhance the viability and productivity of wild trout populations in M. cerebralis‐ positive river systems. Improving our understanding of the hosts, the parasite and the environmental factors determining their interaction should provide for more focused and effective control methods for containing the spread and devastating effects whirling disease is causing to our wild trout populations.

Modes of Salmonid MHC Class I and II Evolution Differ from the Primate Paradigm

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15–20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II β-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles α1 and α2 domains into different combinations.

A DIVERGENT NON-CLASSICAL CLASS I GENE CONSERVED IN SALMONIDS

Abstract Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UA-C32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the α3 domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2–6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14–16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene.

Evaluation of Five Diagnostic Methods for the Detection and Quantification of Myxobolus Cerebralis

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin–trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor–I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.

Observations on the Life Stages of Sphaerothecum destruens n. g., n. sp., a Mesomycetozoean Fish Pathogen Formally Referred to as the Rosette Agent

Abstract The rosette agent is an obligate intracellular parasite that causes morbidity and mortality in salmonid fish. In laboratory cultures, the spore stage (2–6 μm diam.) replicates in a salmonid cell line by sequential asexual division, giving rise to daughter cells. If infected cell cultures are transferred to distilled water, the spore stage undergoes internal division to give rise to at least 5 cells each of which develops into a uniflagellated zoospore with a body of approximately 2 μm and a flagellum approximately 10 μm long. Zoosporulation does not occur in cell culture medium alone, artificial seawater, or phosphate-buffered saline. This parasite is currently classified as a member of the Class Mesomycetozoea (formerly Ichthyosporea) based on phylogenetic analyses of the small subunit ribosomal DNA of three different isolates from fish. Given these new morphological observations combined with the available molecular phylogenetic data on other mesomycetozoeans, we propose to classify the rosette agent as Sphaerothecum destruens, n. g., n. sp. This new genus has unique features including (1) intracellular development of spore stages in various organs eliciting a host granulomatous response; and (2) the differentiation of mature spores into multiple, flagellated zoospores. Taken together, these characteristics clearly distinguish it from the closely related genera Dermocystidium and Rhinosporidium.

HUMORAL IMMUNOGLOBULINS OF THE WHITE STURGEON, ACIPENSER TRANSMONTANUS : PARTIAL CHARACTERIZATION OF AND RECOGNITION WITH MONOCLONAL ANTIBODIES

White sturgeon (Acipenser transmontanus) immunoglobulin (Ig) was purified from serum by two methods, ion-exchange chromatography and gel filtration and precipitation of the euglobulin fraction. The purity of these immunoglobulin preparations was confirmed by gel electrophoresis. Sequence analysis of the N-terminal amino acids confirmed that the purified protein was immunoglobulin. The major portion of the immunoglobulin preparation consisted of two proteins with estimated molecular weights (m.w.) of 870 and 170 kDa. The m.w. of the H- and L-chains of the purified Ig were 73 and 27-30 kDa, respectively, as determined by SDS-PAGE. Ion-exchange purified Ig was used to immunize mice for the production of monoclonal antibodies. This resulted in the production of six stable hybrids that recognized sturgeon Ig, two specific for heavy chain and four specific for light chain. The two anti-H-chain mabs were highly specific for white sturgeon Ig while all four anti-L-chain mabs cross reacted with Ig from green sturgeon (A. medirostris), Atlantic sturgeon (A. oxyrhynchus oxyrhynchus), shovelnose sturgeon (Scaphirhynchus platorynchus), and paddlefish (Polyodon spathula), (all Chondrosteans), but not with channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss) or striped bass (Morone saxatilis). The mabs were used to enumerate the percentage of sIg+ lymphocytes in the peripheral blood of white sturgeon by flow cytometry. The percentage of cells positively stained with the mabs ranged from 12 to 28%. In a comparison of mabs with polyclonal rabbit anti-sturgeon Ig serum by ELISA the mabs produced a larger signal and less background than the polyclonal serum.

Humoral immune responses to phocine herpesvirus-1 in Pacific harbor seals (Phoca vitulina richardsii) during an outbreak of clinical disease.

Infection with phocine herpesvirus type-1 (PHV-1) has been associated with morbidity and high mortality in neonatal harbor seals (Phoca vitulina). A PHV-1 specific indirect enzyme linked immunosorbent assay (ELISA) was developed to sequentially measure the serological status of 106 harbor seal neonates admitted to a Pacific coast rehabilitation center (total number of sera tested was 371). Early in the season (February-April), the majority of pups had low serum levels of PHV-1 specific antibody. A dramatic increase in PHV-1 specific antibody, involving the majority of hospitalized pups, was observed during a 4-week period in May. This coincided with a high incidence of PHV-1 associated adrenal lesions and mortality. Although there was overall agreement between the timing of seroconversion to PHV-1 and histological evidence of PHV-1 infection, 82.4% of individual pups with adrenalitis had no evidence of a humoral response to PHV-1 at the time of their death. This suggests either a rapid disease course, or an inability to develop a humoral response in some neonatal seals.

Invasion and initial replication of ultraviolet irradiated waterborne infective stages of Myxobolus cerebralis results in immunity to whirling disease in rainbow trout.

Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80 mJ/cm(2) either prevented (20-80 mJ/cm(2)) or significantly inhibited (10 mJ/cm(2)) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80 mJ/cm(2)) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80 mJ/cm(2) dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10 days and continuing thereafter until the end of the study at 46 days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20 mJ/cm(2)) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49 days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20 mJ/cm(2)) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5 months post parasite exposure to fully infective triactinomyxons.

Viral nervous necrosis (VNN) in white seabass, Atractoscion nobilis, cultured in Southern California, and implications for marine fish aquaculture

The organism responsible for causing viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) has been demonstrated to belong to the group Nodaviridae [K. Mori, et al., 1992] [M. Comps, et al., 1994]. Since its initial identification in 1990 by Yoshikoshi and Inoue (1990), in Japanese parrotfish, its distribution and/or identification has become global. Methods of identification developed and results from outbreaks of VNN in white seabass between 1992 and 1999 have been published in Curtis et al. (2001). The white seabass cultured at the Hubbs-SeaWorld Research Institutes hatchery in Carlsbad California (CBD), have experienced VNN, of varying severity, since 1992. Disease in 1992 was thought to be due to a picrona-like virus but the indirect fluorescent antibody technique (IFAT) performed in 1999 on samples from 1992, indicated the same nodavirus was responsible. Confirmation of the nodavirus in white seabass was done using IFAT, reverse transcription-polymerase chain reaction (RT-PCR), electron microscopy and virus isolation via cell culture. The strain of nodavirus isolated from white seabass was shown to be similar to that recovered from redspotted grouper in Japan. The most serious and extensive outbreak experienced in white seabass at CBD extended from August of 2002 to May 2003. Since 1999 a concerted effort has been made to further the understanding of VNN epidemiology in white seabass. Recently, enzyme linked immunosorbant assays (ELISA) performed on sera samples from wild fish have demonstrated VNN exposure in wild juvenile white seabass. With the apparent lack of species specificity of VNN and its global distribution, continued devastating impacts on new marine species development are a distinct possibility. Along with cooperative measures for identification and epidemiology, there needs to be a drive towards development of preventative husbandry protocols and methods of prophylaxis.