Characterisation of Saprolegnia sp. isolates from channel catfish.

Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups.

Effects of oleic acid on murine CD4+ T cell death and anti-CD3 or superantigen induced proliferation at low temperature.

Studies were conducted to examine the effects of low, yet physiologically relevant, temperatures on murine T lymphocyte responses. Akin to ectothermic vertebrate responses, lectin-induced murine T cell proliferation was previously shown to be ablated at temperatures 10 degrees C below optimal (i.e. 27 degrees C); responsiveness at 27 degrees C was restored by the addition of oleic acid (18:1). The aim of the present study was to address the mechanism involved in such low temperature suppression. Murine splenic CD4+ T lymphocytes stimulated with either alpha CD3 or SEB exhibited cell death, as opposed to anergy, at 27 degrees C. However proliferation was observed in the presence of 18:1. Thus low temperature-suppression of murine CD4+ T cells is also mediated through TCR and/or CD3 pathways. Additional studies examining the temporal effects of adding 18:1, as well as temperature shifts, indicated that the cell death induced by stimulation at low temperature was preventable by 18:1.

Environmental effects on fish immune mechanisms.

Environmental stress factors which influence fish immune (and likely many other physiological) functions can be divided into two broad, but not mutually exclusive, categories, namely those which occur naturally and those which are artificial. Natural environmental stress factors include season, temperature, salinity and photoperiod as well as social stress factors such as crowding and hierarchy. In general, artificial environmental stress factors are man made, and mainly involve pollutants such as acid rain, heavy metals and organic compounds. The available data indicate that regardless of which immune parameters are assessed, both natural and artificial environmental stress factors appear to suppress immune functions. Of the numerous environmental stress factors considered, pollutants, handling/confinement and low temperature are probably the best studied forms in fish. All three forms of stress factors have been shown to suppress components of both the innate (non-specific) and adaptive arms of the immune system. Since immune responses which protect against invading pathogens frequently involve interactions between both the innate and adaptive arms of the immune system, it seems reasonable to conclude that either acute or chronic exposure to stress factors may predispose fish to infectious diseases. Signalling mechanisms responsible for the effects of these various stress factors on immunity in fish are poorly understood, although elevated serum ACTH and cortisol levels appear to be involved in some cases. A better understanding of the mechanism(s) resulting in immunosuppression should facilitate future in vivo manipulations to reduce susceptibility to disease in aquaculture situations.

Fish immunology: the utility of immortalized lymphoid cells--a mini review.

Long term cell lines can be readily established at high frequency with PBLs from normal channel catfish. Depending upon the mode of stimulation, morphologically and functionally distinct catfish lymphoid cell lines resembling B cells, T cells and monocytes have been developed. These fish cell lines appear unique from their putative mammalian counterparts in that they are immortalized without the need for exogenous factors or overt attempts at transformation.

Identification and characterization of a heat shock protein 70 family member in channel catfish (Ictalurus punctatus).

We have determined the cDNA sequence of a member of the channel catfish heat shock protein 70 (CF Hsp70) family. This protein presumably functions as a molecular chaperone, as is characteristic of this family in other species. Channel catfish peripheral blood leukocytes exhibit a classical heat shock response, in that heat shock (37 degrees C) induces the expression of heat shock genes that are quiescent at normal temperatures (23 degrees C). It was observed that pre-existing synthesis of certain other molecules was suppressed (as evidenced by decreases in actin RNA upon heat shock). Similar trends were observed in mRNA expression for CF Hsp70 in two catfish non-leukocyte cell lines, channel catfish ovary and F59. However, three leukocyte cell lines constitutively expressed high levels of CF Hsp70 mRNA at optimal culture temperature (27 degrees C), whereas heat shock (37 degrees C) elicited only a modest induction of CF Hsp70 expression. Furthermore, continued investigation is warranted to determine whether the apparent upregulation of CF Hsp70 mRNA expression in the catfish long-term leukocyte cell lines is involved in the seemingly immortal phenotype of these cells.

A serum-free medium for human primary T lymphocyte culture.

This paper describes a defined serum-free medium (SFM), designated A-X, which supports in vitro proliferation of human primary T cells stimulated with mitogens, two-way mixed leukocyte reactions, anti-CD3, and a superantigen. A-X is a 1:1 mixture of two commercially available SFM, AIM V and EX-CELL 300. In each assay tested it supported uptake of [3H]thymidine as well as or better than the standard culture medium, namely RPMI 1640 supplemented with 10% fetal calf serum. A-X also allowed the detection of interleukin-2 by ELISA at levels comparable to those produced in serum-supplemented medium. A-X will likely be useful in further studies as it eliminates many of the problems usually associated with the use of serum-supplemented media.

The effects of temperature and oleic acid on murine memory and virgin T cell activation: interleukin-2 secretion and interleukin-2 receptor expression.

Previous studies established that low in vitro temperatures (27 degrees C, termed nonpermissive) suppressed murine primary thymus-dependent (TD), but not primary thymus-independent (TI) or secondary TD, antibody responses. Suppression was rescued by the addition of oleic acid (18:1), recombinant interleukin (rIL)-2 and/or rIL-4, but not rIL-1. These observations suggested that low temperatures suppress the functions of virgin T cells (Tv) but not those of memory T cells (Tm), B cells, or accessory cells and that hypothetically 18:1 may rescue suppressed responses by altering the membranes of Tv cells, allowing them to proliferate and subsequently secrete interleukins. In this study negatively selected Tm and Tv cells were stimulated with various mitogens at 37 or 27 degrees C. The results indicated that proliferation was suppressed at 27 degrees C to all mitogens tested. The subsequent addition of 18:1 induced proliferative responses at 27 degrees C to both concanavalin A (Con A) and a combination of 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore (A23187), but not to phytohemagglutinin-P (PHA). Both Tm and Tv cells showed significant secretion of IL-2 and expression of IL-2 receptor (IL-2R) at 27 degrees C in response to all mitogens, irrespective of proliferation, and the subsequent addition of 18:1 caused little or no change in the levels of IL-2 secretion or IL-2R expression. These findings indicate that suppression of Tm and Tv cell proliferative responses occurs irrespective of IL-2R expression and, unlike antibody production, of IL-2 secretion. Furthermore, it appears that 18:1 can synergistically act with Con A or TPA/A23187, but not PHA, in activating Tm and Tv cells to induce proliferation at 27 degrees C. These findings suggest differences in signal transduction mechanisms between these various mitogens.

Isolation of an acute-phase phosphorylcholine-reactive pentraxin from channel catfish (Ictalurus punctatus).

1. Channel catfish (Ictalurus punctatus) serum contains a protein that precipitates pneumococcal C-polysaccharide (CPS) in a calcium-dependent fashion. 2. The serum titer of this protein follows an acute-phase pattern in catfish injected with turpentine. 3. A non-glycosylated, phosphorylcholine (PC)-reactive protein (PRP) with molecular mass ca 100 kDa, was isolated from channel catfish acute-phase sera by affinity chromatography on PC-Sepharose 4B. 4. Contaminating proteins with molecular masses ca 700 kDa and ca 20 kDa that co-eluted with PRP from PC-Sepharose appear to be aggregated and native low-molecular weight factors (LMFs), respectively. 5. Purified PRP has gamma mobility but in serum samples PRP has gamma-beta mobility. 6. Electron microscopy confirmed that PRP has planar, pentagonal symmetry. 7. The amino terminus of PRP is blocked, but based on comparison of amino-acid compositions channel catfish PRP is clearly similar to human CRP and is most like CRPs from the dogfish (Mustelus canis) and rainbow trout (Oncorhynchus mykiss).

Chelation affects the conformation, lability and aggregation of channel catfish (Ictalurus punctatus) phosphorylcholine-reactive protein (PRP).

1. Phosphorylcholine-reactive protein (PRP) affinity-purified from channel catfish (Ictalurus punctatus) serum on phosphorylcholine-Sepharose, eluted from Bio-Gel A-5M as a 94.6 +/- 2.4 kDa protein when the gel filtration column buffer (Tris-saline) contained 25mM ethylenediaminetetraacetic acid (EDTA). 2. PRP chelated with EDTA immediately after affinity purification and gel-filtered in Tris-saline-EDTA, eluted as a 75.5 +/- 2.67 kDa protein referred to as fast-PRP (F-PRP). 3. PRP and F-PRP were identical on SDS-PAGE. Both resolved as a broad band of protein (ca 86-100 kDa) on non-reducing gels or as a ca 100 kDa protein after reduction with 2-mercaptoethanol (2-ME). 4. After gel-filtration in Tris-saline-EDTA, nearly complete reduction of 100 kDa PRP was achieved on SDS-PAGE. However, the protein regained its resistance to reduction upon storage at -60 degrees C. 5. SDS-PAGE and native PAGE also revealed that during storage, PRP and F-PRP combined to form 3 different aggregates referred to as aggregated-PRP (aggPRP). These aggregates are readily dissociated in the presence of 2-ME, suggesting a covalent interaction between adjacent pentamers comprising decameric aggPRPs. 6. PRP, F-PRP, and aggPRP have similar amino acid compositions.

Temperature-mediated processes in teleost immunity: in vitro immunosuppression induced by in vivo low temperature in channel catfish.

In an attempt to understand the interrelationships between environmental temperature and immune competence, channel catfish in the laboratory were subjected to a rapid change in water temperature in order to mimic conditions which might be encountered in commercial ponds during the winter months and subsequently examined for a variety of immune parameters. The results indicated that lowering the water temperature from 23 to 11 degrees C over a 24 h period suppressed both B and T cell functions for 3-5 weeks as assessed by in vitro responses. Furthermore, this form of suppression was not a typical stress-induced response, i.e. blood serum chemistry and lymphocyte and neutrophil compositions did not change in a manner reminiscent of transport-induced stress. Collectively these results indicate that channel catfish are probably immunocompromised during the winter months and consequently it seems plausible that many of the fish losses associated with the syndrome termed "winter kill" may be attributable, at least in part, to a low temperature-induced immuno-deficient state.

Pristane induced changes in rat lymphocyte membrane fluidity.

The ability of pristane (2,6,10,14-tetramethylpentadecane) to act as a membrane perturbant was examined. Data obtained from rats treated with pristane by either intraperitoneal injection or the diet indicated there were significant increases over normal in the amount of pristane in lymphoid cells; 50-89% was incorporated into the plasma membranes. Fluorescence polarization analyses, using 1,6-diphenyl-1,3,5-hexatriene, of normal plasma membrane isolates demonstrated that splenic and Peyer's patch lymphocytic membranes were more viscous than those of the thymus, mesenteric lymph nodes or peripheral blood. Studies to assess the effects of pristane on membrane viscosity demonstrated that there were significant differences in the viscosities of plasma membrane isolates from lymphocytes of normal versus pristane treated rats. The observed changes were dependent on route of administration, length of exposure and the lymphoid organ examined.

A monoclonal antibody specific for neutrophils in normal and stressed channel catfish.

A murine monoclonal antibody, designated mAb 13C5, was found to react specifically with channel catfish neutrophils based upon its ability to identify a subpopulation of leukocytes that are phagocytic and stain positive with both Sudan Black B and nitro-blue tetrazolium. Cytofluorographic analysis with mAb 13C5 was used to assess trafficking of neutrophils in various tissues of catfish subjected to transport stress. Since no prestress neutrophil reservoir was apparent, it seems likely that stress-induced neutrophilia in catfish, as in endotherms, may result from demargination of a pool of capillary-bound neutrophils. MAb 13C5 was also used to successfully "pan" for neutrophil-enriched and depleted populations of peripheral blood leukocytes from transport stressed channel catfish. "Panning" indicated that the continued physical presence of elevated numbers of neutrophils is not responsible for the suppression of T and B cell in vitro proliferation responses to mitogens observed in stressed fish.

Differential effects of temperature and exogenous fatty acids on mitogen-induced proliferation in channel catfish T and B lymphocytes.

1. Exogenously supplied, BSA complexed saturated and unsaturated fatty acids were compared for their effects on mitogen-induced DNA synthesis in channel catfish T and B lymphocytes. 2. At "permissive" in vitro temperatures (27 degrees C), high concentrations (greater than or equal to 240 microM) of all the fatty acids used were inhibitory. However, at lower concentrations (80-160 microM), differences were noted in the ability of some fatty acids to modulate mitogen responses. While palmitic acid (16:0) and linoleic acid (18:2) had little effect on LPS-induced B cell- or Con A-induced T cell proliferation, stearic acid (18:0) suppressed while oleic acid (18:1) enhanced T cell responses only. 3. Adding equimolar amounts of 18:0 and 18:1 obviated the effects of singularly added fatty acids on T cell mitogenesis. 4. 18:1 was used to successfully "rescue" approximately 60% of the Con A-induced T cell proliferation normally inhibited at "nonpermissive" in vitro temperatures (17 degrees C). 5. While B cells readily appear to desaturate 18:0 and synthesize unsaturated fatty acids, T cells accumulate comparatively large amounts of 18:0 in membrane associated phospholipids. 6. It is proposed that 18:1 enhances T cell responses at permissive high temperatures and rescues suppressed T cell responses at nonpermissive low temperatures by increasing membrane fluidity.

Channel catfish as an unconventional model for immunological studies.

The channel catfish, Ictalurus punctatus, is an economically important species which is readily available, acclimates well to the laboratory setting, and is amenable to considerable experimental manipulation. Although the channel catfish is still a relatively circumscribed species in terms of comprehensive physiologic and/or endocrinologic studies, our current understanding of the basic immunobiology and immunochemistry of the channel catfish is significantly further advanced than for any other teleost species. In this respect the channel catfish is not only proving useful in the general areas of comparative immunology but it is also showing considerable promise as a model system for definitive studies on problems which bridge the fields of immunology and endocrinology, i.e., understanding the effects of environmental temperature and stress on the immune system.

Phylogeny of lymphocyte heterogeneity: the thymus of the channel catfish.

The number of thymocytes (approximately 3 x 10(7)) that were recoverable from fingerling channel catfish remained constant from about 3 to 10 months of age, i.e. from September to April following hatching the previous June. Between 11 and 12 months, i.e. May and June, the thymus dramatically increased in size with 3 x 10(9) thymocytes being recoverable from the tissue of individual fish. The thymus remained enlarged for several months (throughout the summer) but at about 15 months (in September) began to involute such that by 17 months (November) no thymus tissue could be seen macroscopically. This natural involution could be accelerated by subjecting the fish to handling and transport stress. Thymocytes of channel catfish aged 4 to 16 months exhibited reactivity with monoclonal antibodies against peripheral T cells but not B cells. Thymocytes responded to the mitogen Concanavalin A only in the presence of added accessory cells (peripheral blood monocytes) or a monocyte-derived supernatant (presumably containing IL-1) at permissive temperatures (27 degrees C). Thymocytes could also be induced to divide at nonpermissive temperatures (17 degrees C) when incubated in the presence of the following combinations of stimulants, a) the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the calcium ionophore A23187, b) TPA and ConA, or c) A23187 and ConA. In those cases where TPA or A23187 were used, accessory cells or their products were not needed. Collectively, these results support the notion that channel catfish thymocytes functionally mimic those lymphocytes in the peripheral blood previously designated as T cells.

Temperature mediated processes in teleost immunity: differential abilities of channel catfish T and B lymphocytes to cap membrane antigen.

1. The effect of both in vivo acclimation temperature and in vitro assay temperatures on channel catfish T and B lymphocyte membrane antigen (mAg) capping were investigated to determine if capping might be the temperature sensitive step involved in the low temperature immunosuppression of channel catfish T cell responses. 2. Flow cytometry was used to monitor the kinetics of capping induced by a mouse monoclonal antibody (mAb 11G3) specific for a common antigenic determinant present on channel catfish T and B cells. Results indicated that the kinetics of mAg capping were dependent on in vitro assay and in vivo acclimation temperatures and the length of time of in vivo acclimation. 3. T cells from fish appropriately acclimated to 27 degrees C cap mAg more efficiently at low assay temperatures than do B cells. 4. Activation energies were 32 and 47 kcal/mol for B and T cells, respectively, from fish acclimated to 17 degrees C for 3 weeks, but were significantly lower (14 and 22 kcal/mol, respectively) after acclimation for 5 weeks. 5. In summary, it appears that after appropriate in vivo acclimation, channel catfish T cells are better able to cap mAg at low assay temperatures than are B cells. These results suggest that mAg capping is not the low temperature sensitive step involved in T cell immunosuppression in channel catfish.

Temperature-mediated processes in teleost immunity: homeoviscous adaptation by channel catfish peripheral blood cells.

1. Channel catfish peripheral blood erythrocyte, thrombocyte, T cell and B cell membranes were assayed by fluorescence depolarization using the fluorescent probe 1,6-diphenyl-1,3,5 hexatriene (DPH) to determine the effects of in vivo temperature acclimation on membrane viscosity and the kinetics of homeoviscous adaptation. 2. Erythrocyte membranes did not undergo homeoviscous adaptation during the 8 week time period studied and were more rigid compared with those of the other cell types. 3. The kinetics of homeoviscous adaptation exhibited by membranes from T cells, B cells and thrombocytes differed: B cells required 1-3 weeks while T cells and thrombocytes each required 3-5 weeks. Membranes from T cells, B cells and thrombocytes from fish acclimated for relatively short times (less than or equal to 3 weeks) exhibited similar membrane fluidities. 4. T cells from channel catfish appeared not only to be sensitive to temperature but also to a factor(s) independent of temperature but correlated to long term in vivo acclimation, i.e. T cell membranes underwent additional decreases in membrane viscosity between 3 and 5 weeks. 5. In conclusion, it appears that low temperature-mediated immunosuppression of T cell functions in channel catfish is probably not due to an inherent non-adaptability or rigid nature of the T cell membranes.

Temperature-mediated processes in teleost immunity: the effects of temperature on membrane immunoglobulin capping on channel catfish B lymphocytes.

1. In order to better understand ligand-induced redistribution of membrane receptors and lymphocyte activation in ectothermic vertebrates, flow cytometry was used to monitor the effects of both in vivo acclimation temperature and in vitro assay temperatures on the kinetics of monoclonal antibody-induced membrane immunoglobulin (mIg) capping on channel catfish lymphocytes. 2. It was observed that the kinetics of mIg capping were dependent on in vitro assay temperatures, in vivo acclimation temperatures, and the length of time of in vivo acclimation. In the latter situation in vivo acclimation of fish to 27, 22 and 17 degrees C was considered complete after 3 weeks, while acclimation to 12 degrees C required a minimum of 5 weeks. 3. The energies of activation required for mIg capping ranged from 33 to 24 kcal/mol; lower energies of activation were observed with lower temperature acclimation. 4. It was also noted that the lower energies of activation were associated with concomitant decreases in cellular phospholipid saturated/unsaturated fatty acid ratios. 5. It appears that channel catfish B cell mIg capping, presumably a requisite for immune function, can be significantly affected by environmental temperatures; most likely such effects are attributable to changes in plasma membrane viscosities.

Phylogeny of lymphocyte heterogeneity: identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies.

The purpose of this study was to identify monoclonal antibodies reactive with channel catfish T cells. Since a variety of commercially available anti-human and mouse T cell reagents failed to react with channel catfish leucocytes, the development of anti-fish T cell monoclonal antibodies was undertaken. One of these reagents, designated mAb 13C10, reacted with channel catfish surface immunoglobulin negative, but not positive, lymphocytes. This reagent, when used in "panning" protocols, was able to isolate those channel catfish lymphocytes which provide helper activity for antibody synthesis to a thymus dependent antigen. In addition, this antibody was observed to react with most thymocytes, neutrophils, and thrombocytes, few hepatocytes, and with some brain cells (possibly neurons). It was concluded that this antibody reacts with relatively high molecular weight antigens on channel catfish T cells and that it may be a pan anti-T cell reagent (possibly akin to Thy-2) for this species.

Temperature-mediated processes in teleost immunity: binding and mitogenic properties of concanavalin A with channel catfish lymphocytes.

Low (non-permissive) temperatures inhibit the proliferation responses of channel catfish T cells to stimulation with Con A. The study reported here was undertaken to ascertain if failure to bind Con A at non-permissive temperatures could explain the observed suppression at such temperatures. The influence of temperature on the binding of fluorescein-labelled succinyl Con A to channel catfish T and B cells was studied by cytofluorography. The results indicated that Con A bound equally well at both mitogenically permissive (22 degrees, 27 degrees and 32 degrees) and non-permissive (12 degrees and 17 degrees) temperatures. Hence, temperature effects on mitogen-binding to cell surface receptors cannot readily explain the observed suppression of channel catfish T-cell proliferative responses at non-permissive temperatures.