Developmental expression of two spore wall proteins during maturation of the microsporidian Encephalitozoon intestinalis.

Microsporidia are intracellular eukaryotes that infect many animals and cause opportunistic infections in AIDS patients. The disease is transmitted via environmentally resistant spores. Two spore wall constituents from the microsporidian Encephalitozoon intestinalis were characterized. Spore wall protein 1 (SWP1), a 50-kDa glycoprotein recognized by monoclonal antibody (MAb) 11B2, was detected in developing sporonts and at low levels on the surfaces of mature spores. In contrast, SWP2, a 150-kDa glycoprotein recognized by MAb 7G7, was detected on fully formed sporonts and was more abundant on mature spores than SWP1. Nevertheless, the SWPs appeared to be complexed on the surfaces of mature spores. SWP1 and SWP2 are similar at the DNA and protein levels and have 10 conserved cysteines in the N-terminal domain, suggesting similar secondary structures. The C-terminal domain of SWP2 has a unique region containing 50 repeating 12- or 15-amino-acid units that lacks homology to known protein motifs. Antibodies from mice infected with E. intestinalis recognized SWP1 and SWP2. The characterization of two immunogenic SWPs from E. intestinalis will allow the study of exospore structure and function and may lead to the development of useful tools in the diagnosis and treatment of microsporidiosis.

Heavy chain diversity region segments of the channel catfish: structure, organization, expression and phylogenetic implications.

Circular DNA, derived from lymphocytes of juvenile channel catfish, was used to construct lambda libraries that were screened to identify the products of immunoglobulin DH-JH excision events. Clones were characterized that contained DH to JH recombination signal joints. The signal joints represented 23-bp recombination signal sequences (RSS) identical to germline JH segments that were adjacent to DH 12-bp RSS elements. DH flanking regions within the clones were used to probe a genomic library. Three germline DH gene segments containing 11-19 bp coding regions flanked by 12-bp RSS elements with conserved heptamers and nonamers were identified. The DH locus is closely linked to the JH locus, and Southern blots indicate that the DH segments represent different single member gene families. Analysis of H chain cDNA shows that each germline DH segment was expressed in functional VDJ recombination events involving different JH segments and members of different VH families. Several aspects of CDR3 junctional diversity were evident, including deletion of coding region nucleotides, N- and P-region nucleotide additions, alternate DH reading frame utilization, and point mutations. Coding region motifs of catfish DH segments are phylogenetically conserved in some DH segments of higher vertebrates. These studies indicate that the structure, genomic organization, and recombination patterns of DH segments typically associated with higher vertebrates evolved early in vertebrate phylogeny at the level of the bony fish.

Heavy chain joining region segments of the channel catfish. Genomic organization and phylogenetic implications.

The JH locus of the channel catfish has been characterized to determine the organization and structural diversity of JH segments. These analyses indicate that there are a total of nine JH segments tightly clustered within a region spanning about 2.2 kb. The JH locus is closely linked to the CH 1 domain of the expressed catfish H chain; the distance between the CH proximal JH segment (JH9) and the CH 1 domain is about 1.8 kb. Each JH segment has an upstream recombination sequence, which includes a T-rich nonamer, a 22- to 24-bp spacer, and a phylogenetically conserved heptamer. Each JH segment also has an open reading frame that encodes the conserved framework region 4 tryptophan (Trp-103) and terminates with a RNA donor splice site. The catfish JH locus contains an internal repetitive sequence region characterized by a short (183-188 bp) repeat that occurs sequentially five times. Strong sequence homology as well as the unified length of the repeated sequences indicate that JH segments JH3-JH7 probably arose as the result of a series of homologous but unequal crossover events. Sequence alignments of the duplicated JH segments indicates that there is diversity within the 5-11 nucleotides located immediately downstream from the heptamer, an observation which indicates that closely related JH segments can serve to enhance CDR3 diversity in the expressed H chain. Comparisons of the genomic JH sequences with different cDNA clones indicate that each JH segment is probably functional and that junctional diversity serves an important role in the generation of CDR3 diversity. In addition, single base differences observed in comparisons of JH-encoded regions indicate that there is probably somatic mutation or allelic variation of genomic JH segments. These studies suggest that the characteristic structure and organizational pattern of JH segments in higher vertebrates may have evolved early in vertebrate phylogeny at the level of the bony fish.

Plasmid and serological differences between Edwardsiella ictaluri strains.

Several studies have shown that isolates of Edwardsiella ictaluri obtained from infected channel catfish in the southeastern United States harbor two cryptic plasmids, designated pCL1 (5.7 kb) and pCL2 (4.9 kb). These isolates appear to be serologically homogeneous. To extend these studies, we focused our analyses on two isolates of nonictalurid origin. Plasmid analyses of a danio isolate showed that it harbored plasmids which were similar if not identical to pCL1 and pCL2. This strain was also serologically indistinguishable from those isolated from channel catfish. In contrast, a green knife fish (GNF) isolate harbored four plasmids with relative mobilities of 6.0, 5.7, 4.1, and 3.1 kb. Southern blot analyses indicated that only the 5.7- and 4.1-kb plasmids strongly hybridized under high-stringency conditions to probes specific for pCL1 and pCL2, respectively. The GNF isolate showed minimal reactivity when reacted with polyclonal antiserum prepared against a channel catfish isolate. However, polyclonal antiserum to the GNF isolate strongly reacted with the GNF isolate in both surface fluorescence and agglutination reactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of cell lysates showed that the protein banding patterns of the strains compared were similar. However, Western blots of proteinase K-digested cell extracts showed that O antigen of the GNF isolate was antigenically distinct from the O antigen of the other isolates. These studies indicate that there are different serotypes of E. ictaluri and suggest that plasmid and serological analyses of future isolates of E. ictaluri can be used to determine whether structurally distinct strains are emerging in major channel catfish aquaculture areas.

Immunoglobulin in the eggs of the channel catfish (Ictalurus punctatus).

Egg yolk proteins obtained from 2-3-day-old fertilized channel catfish eggs were analyzed to determine if immunoglobulin (Ig) was present. Using both monoclonal and polyclonal antibodies to catfish Ig, egg Ig was isolated and structurally characterized. The egg Ig when analyzed in SDS-gels under nonreducing conditions dissociated into eight distinct subpopulations with relative mobilities identical to that previously described for the serum Ig of the channel catfish. In addition, the component heavy (H) and light (L) chains of the egg Ig had similar relative mobilities as the H and L chains identified in serum Ig. Immunocytological studies using both polyclonal and monoclonal anti-catfish Ig showed that Ig was dispersed throughout the yolk of the egg. In addition these analyses indicated that Ig was localized within the external membranes (eggshell) of the egg.

Activation of complement by different immunoglobulin heavy chain isotypes of the channel catfish (Ictalurus punctatus).

The approximately 750,000 mol. wt tetrameric Ab population from catfish immunized with sheep erythrocytes (E) was purified and shown to sensitize E to the hemolytic activity of catfish serum complement. Catfish complement was heat-labile at 45 degrees C and was hemolytically inactivated by both EGTA and EDTA. Hemolytic activity of EDTA treated serum was restored by the addition of both Ca+ and Mg2+ ions. In comparative assays, the CH50 titers of catfish sera were similar to the CH50 titers of human sera. Catfish complement was hemolytically active at incubation temps ranging from 3 to 40 degrees C, which suggests that complement should be functional throughout the catfish's normal environmental temps. Human complement exhibited considerable cross-reactivity when assayed on catfish Ab sensitized E at 25 degrees C; however, at 3 degrees C, no hemolytic activity was observed. They findings suggest that there may be structural differences between the complement component(s) of these two systems. Mouse mAbs to the recently described H chain isotypes of catfish Ig were used to fractionate catfish anti-sheep E antisera. Each of the purified isotypes contained Ab which sensitized E to the hemolytic activity of catfish complement. Dose-response analysis of the average number of hemolytic sites per cell, as a function of relative Ab concn, predicts that the catfish tetramer conforms to the one-hit theory of immune hemolysis. Purified catfish anti-fluorescein Ab of each isotype was also reacted with fluorescein-labeled sheep E and the level of complement-mediated hemolysis determined. Dose-response analysis indicated that similar numbers of Ab molecules bound to haptenated E resulted in similar levels of complement-mediated lysis. Lastly, the effect of cell surface hapten density of target cells was examined. These studies, similar to those reported with human IgM, showed that the binding of the catfish tetramer to a cell surface hapten may not be sufficient to activate lytic complement.