Donald D. Ourth

Secretory IgM, lysozyme and lymphocytes in the skin mucus of the channel catfish, Ictalurus punctatus

Abstract Channel catfish ( Ictalurus punctatus ) skin mucus demonstrated an immunological response. Agglutinating antibody and bactericidal activity to Salmonella paratyphi were found in the mucus after intraperitoneal injection of bacteria. By immunodiffusion in gel, catfish skin mucus gave a precipitin line of identity with catfish 14S serum macroglobulin against rabbit anti-catfish 14S serum indicating the presence of secretory IgM in the mucus. Parenteral immunization (intraperitoneally) can thus yield specific IgM antibody in the mucus. Lysozyme was also demonstrated in the mucus as well as lymphocytes.

Antiviral melanization reaction of Heliothis virescens hemolymph against DNA and RNA viruses in vitro

1. Antiviral activity of Heliothis virescens larval hemolymph was determined using a cytotoxicity/virus inhibition test (TClD50) done in Vero cell tissue cultures. Excellent antiviral activity was found especially against herpes simplex viruses-1 and -2 and also against vesicular stomatitis, parainfluenza-3, coxsackie B3 and sindbis viruses. 2. Prolonged incubation of herpes simplex virus-1 and vesicular stomatitis virus with hemolymph was virucidal and greatly reduced infectivity of the two viruses in tissue culture. 3. Antiviral activity was produced by both normal and immune (vaccinated larvae) cell-free hemolymphs. 4. Antiviral activity against herpes simplex virus-1 could be generated in vitro with hemolymph phenoloxidase or mushroom tyrosinase using four different substrates including tyrosine. 5. Activation of the insect melanization reaction by phenoloxidase was necessary for antiviral activity to occur.

Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae.

Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17965.9+/-5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56-83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted alpha-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic alpha-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240-340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLp-III plays a role in insect immunity.

Bacterial sialic acid modulates activation of the alternative complement pathway of channel catfish (Ictaluruspunctatus)

The alternative complement pathway (ACP) provides the non-immune channel catfish (Ictalurus punctatus) with protection against many Gram-negative bacteria. Very little serum bactericidal activity (0-13%) was found against 8 fish pathogens, but a strong bactericidal response (100%) was found against 7 non-pathogens. MgEGTA chelation of catfish serum did not essentially change the bactericidal results. Catfish serum heated at 56 degrees C and serum adsorbed with zymosan had no bactericidal activity. This demonstrated that the ACP was responsible for the bactericidal response. The molecular nature of the microbial surface determines whether or not the ACP will be activated. A relative lack of surface sialic acid has been found to be important for binding complement Factor B of the ACP by susceptible microbial surfaces. This study therefore examined the 15 Gram-negative bacterial fish pathogens and non-pathogens by determining their sialic acid content and their ability to elicit a bactericidal response by the catfish ACP. It was found that there was very little bactericidal activity against the fish pathogens that contained sialic acid but a very strong bactericidal response (100%) against the non-pathogens that lacked sialic acid (p = .0043). A relative lack of sialic acid or no sialic acid therefore correlated with a strong bactericidal response by the catfish ACP. Neuraminidase treatment of the bacterial fish pathogens to remove sialic acid greatly increased the bactericidal response against them by the catfish ACP when compared with untreated bacteria (p = .0431).

Rhipicephalus sanguinius: localization of vitellogenin synthesis by immunological methods and electron microscopy.

Abstract In ovipositing Rhipicephalus sanguinius (Latrelle), complete immunological identity existed between vitellogenin from the midgut, fat body, and hemolymph and vitellin from eggs. This supported the hypothesis that the same vitellogenin was synthesized by both the midgut and fat body, then released into the hemolymph and transported to the ovary. Vitellogenin was taken up unaltered by the oocytes during vitellogenesis to become vitellin. Antivitellogenin did not react with host (dog) hemoglobin. Transmission electron microscopy showed specialized cells with large amounts of rough endoplasmic reticulum, Golgi complexes, and secretory granules in the midgut and fat body of ovipositing females that were absent in the midgut and fat body of fed males. It is suggested that these cells synthesize vitellogenin.

Alternate pathway of complement and bactericidal response of the channel catfish to Salmonella paratyphi.

Fresh channel catfish (Ictalurus punctatus) serum from unimmunized catfish exhibited 100% bactericidal activity against Salmonella paratyphi. Components responsible for bactericidal activity could be absorbed from the fresh catfish serum with S. paratyphi. The bactericidal system of the fresh catfish serum showed a need for magnesium rather than for calcium after EDTA treatment. The addition of salicylaldoxime or ammonium hydroxide to catfish serum indicated the alternate rather than the classical pathway of complement activation to be important in bactericidal activity against S. paratyphi. Bactericidal activity of catfish serum was labile when incubated at 47 degrees C for 30 min., stable for at least 4 mo. at -80 degrees C and could be absorbed with S. paratyphi at 25 degrees C. Very minimal bactericidal activity was present in the descending portion of the first 13.7S peak with most activity being found in the descending portion of the second 7.1S peak and throughout the entire 3.4S peak after Sephadex G-200 catfish serum fractionation.

Opsonic effect of the alternative complement pathway on channel catfish peripheral blood phagocytes

This study determined the effect of the alternative complement pathway (ACP) on neutrophil and monocyte phagocytes from peripheral blood of channel catfish, Ictalurus punctatus. Fluorescent-labeled latex microspheres, Edwardsiella ictaluri and Escherichia coli were used to quantify phagocytic attachment and ingestion. Activation of the ACP enhanced the attachment of bacteria and microspheres to neutrophils and monocytes. Activation of the ACP by serum opsonization of Escherichia coli increased its ingestion by neutrophils in comparison with Edwardsiella ictaluri and microspheres. Inactivation of the ACP and C3b diminished attachment by neutrophils and monocytes, and ingestion by neutrophils of microspheres and bacteria. Ingestion by monocytes was not affected by activation of the ACP. In the present study, we found that the ACP functions in opsonophagocytosis in catfish and that ingestion by neutrophils was especially enhanced.

Spleen Index and Mannose-Binding Lectin Levels in Four Channel Catfish Families Exhibiting Different Susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Electron Microscopy of Bactericidal Effects Produced by the Alternative Complement Pathway of Channel Catfish

Abstract The activation of the alternative complement pathway (ACP) was investigated by incubating serum of channel catfish Ictalurus punctatus with three gram-negative bacteria. Bactericidal activity and ultrastructural damage were not apparent when cells were incubated with serum that had been treated to eliminate complement activity. Proteins of the ACP were responsible for damaging susceptible Escherichia coli and Pseudomonas fuorescens, whereas there was no effect on Aeromonas salmonicida, which was apparently protected by an A-layer. Scanning electron micrographs revealed damaged cells, and transmission electron micrographs showed circular lesions oriented perpendicularly to the cell membrane. These lesions resemble those resulting from ACP activation in sera from frogs, sharks, trout, lizards and mammals. The existence of membrane attack complexes (MACs) in channel catfish, which are similar to MACs seen in other animals, emphasizes the importance of the ACP in host defense against bacterial infect...

Isolation of mannose-binding C-type lectin from sea lamprey (Petromyzon marinus) plasma and binding to Aeromonas salmonicida

The sea lamprey (Petromyzon marinus) is a parasitic cartilaginous fish of the North American Great Lakes and a predator of many bony fish species of commercial importance to the fishing industry. Mannose-binding C-type lectin (MBL) was isolated by mannan-agarose affinity chromatography from sea lamprey plasma. Mannose-binding lectin has not before been identified and quantitated in the plasma of this sea lamprey species. The affinity-purified and 2-ME reduced lamprey MBL showed two bands of 35kDa and 65kDa by SDS-PAGE and Western blotting using guinea pig anti-MBL IgG as the primary antibody. Amino acid composition analysis (mol%) of the purified lamprey MBL found high amounts of histidine, threonine, tyrosine and phenylalanine present when compared with three other vertebrate MBLs. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave XXXTKGCPDA. Lamprey plasma contained 261mug of MBL/ml of plasma. Plasma protein concentration was 40.1mg/ml. Lamprey MBL was present then in plasma at 6.5mug MBL/mg total protein. The sea lamprey MBL also specifically binds to mannose on the surface of the pathogen Aeromonas salmonicida. The presence of MBL in high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. This study describes the presence of MBL in sea lamprey plasma and evidence for a C-type lectin complement pathway of innate immunity.