Euthanization methods influence cytokine mRNA expression levels in age 0 year Oncorhynchus mykiss.

Significant differences in cytokine transcription were found between Oncorhynchus mykiss euthanized using the pharmacological agents MS-222 v. benzocaine and also when contrasting death induced by carbon dioxide asphyxiation v. physical methods (cervical dislocation). This study highlights the need to consider the potentially confounding effect of euthanization method on gene expression data.

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.

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 beta-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 alpha 1 and alpha 2 domains into different combinations.

Association of plasma IgM with body size, histopathologic changes, and plasma chemistries in adult Pacific herring Clupea pallasi.

Pacific herring Clupea pallasi immunoglobulin is an IgM-like molecule comprised of heavy and light chains with molecular weights of 79 and 25 to 27 kD, respectively. Purified immunoglobulin was used to generate highly specific polyclonal antibodies for development of a sandwich ELISA. The ELISA was used to quantify total plasma IgM in 602 Pacific herring captured in Prince William Sound and Sitka Sound, Alaska, USA. Plasma IgM concentrations ranged from 0.13 to 5.32 mg ml-1. Using multiple stepwise regression analysis, plasma IgM was highly correlated (p < or = 0.01) with body length, Ichthyophonus hoferi infection, plasma albumin, plasma cholesterol, liver macrophage aggregates, and focal skin reddening. I. hoferi was the only organism significantly associated with plasma IgM. Gender, site, and season (spring vs fall) did not contribute to significant differences in plasma IgM. This study contributes to the understanding of the interaction of body size, plasma chemistries, and pathological changes upon circulating immunoglobulins in fish.

A divergent non-classical class I gene conserved in salmonids.

Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UAC32 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 alpha 3 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.

Whirling disease: re-emergence among wild trout.

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.

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.

Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection.

Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizing antibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizing and AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblotting procedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.

Identification of bluetongue virus protein-specific antibody responses in sheep by immunoblotting.

A western immunoblotting procedure was developed for identifying bluetongue virus protein-specific antibody responses in sheep. Assay conditions were optimized and included electrophoretic transfer of viral proteins to a nitrocellulose membrane (NCM), blocking of unbound sites on the NCM, and detection of NCM-bound primary immune complexes. A biotin-avidin-enzyme system was determined to be superior in terms of sensitivity for identification of the NCM-bound virus protein-antibody complexes. The biotin-avidin-enzyme detection system made use of biotinylated rabbit anti-sheep IgG and horseradish peroxidase-conjugated egg white avidin. This system permitted identification of antibodies specific for up to 10 bluetongue virus-associated proteins, 4 of which have not been characterized.

Humoral and cellular immune response of sheep to bluetongue virus.

Plaque cloned strains of the 4 US bluetongue (BT) virus (BTV) serotypes (10, 11, 13 and 17) were pathogenic to sheep and induced mild clinical responses. The clinical responses coincided with the highest titer of viremia reached by day 7 following primary infection. The 4 BTV strains were immunogenic, inducing group-specific (precipitating) and type-specific (neutralizing) antibodies. Primary infection induced an immune response which protected the animals against secondary challenge with the homologous virus. Peripheral blood lymphocytes (PBL) obtained from infected animals responded specifically to in vitro stimulation with pure BT viral antigens. PBL responded to both homologous and heterologous BTV antigens indicating a cross-reactive nature of the lymphocyte response. Perturbations were observed in PBL response to in vitro stimulation with the mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). The response to mitogens was depressed transiently following primary infection and secondary challenge. A significant increase was reached by 3 weeks following primary infection and then gradually leveled off to normal. The specific stimulation of PBL in response to viral antigens and the increase in response to mitogens, as in vitro correlates of cell mediated immunity (CMI), were suggested to play a role in the clearance of BTV infection. Immunoblotting was applied to characterize the specificity of the serologic response to BTV. Sheep antisera to BTV detected 11 specific viral proteins. The maximum response on day 28 post inoculation (PI) detected 11 proteins including both the major and minor protein components.(ABSTRACT TRUNCATED AT 250 WORDS)