Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Fish & Shellfish Immunology 23 (2007) 1152e1160 www.elsevier.com/locate/fsi

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) Donald D. Ourth*, Madhu B. Narra, Bill A. Simco Department of Biology, The University of Memphis, Life Sciences Bldg., Memphis, TN 38152-3560, USA Received 1 January 2007; revised 13 March 2007; accepted 27 March 2007 Available online 6 April 2007

Abstract Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) DþB and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103  blue catfish male DþB) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDSePAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (DþB strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Mannose-binding lectin; Isolation; Comparison; Ictalurus species

1. Introduction Lectins are proteins that recognize specific carbohydrates. Mammalian lectins play an important role in innate immunity and disease resistance [1e4]. Soluble plasma lectins are a first-line of host defense that can initially recognize pathogens as non-self. This carbohydrate recognition leads to phagocytosis by macrophages and enhances complement-mediated cell lysis. Collectins are soluble lectins found in mammals and birds [5] and are C-type (calcium-dependent) lectins composed of multiple subunits [1e4]. The carbohydrate-recognition domains of collectins * Corresponding author. Fax: þ1 901 678 4457. E-mail address: [email protected] (D.D. Ourth). 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.03.014

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recognize specific carbohydrate patterns (which include mannose) on the surfaces of bacteria, viruses and fungi that are not present as surface carbohydrates of higher eukaryotes, and so help to distinguish self from non-self [1e4]. The collectins opsonize non-self microorganisms and also activate the lectin complement pathway, leading to an innate immune response and the killing of many microorganisms. Mannose-binding C-type lectin is an important component of innate immunity in mammals [1e4]. Mannose-binding lectin (MBL), an acute-phase protein produced by liver hepatocytes, increases in response to an infection or inflammatory response. Mannose-binding lectin acts as an opsonin for phagocytosis by macrophages and also activates the mannan-binding lectin complement pathway of the innate immune response [6,7]. Mannose-binding lectin is the initiating lectin for binding to mannose-containing carbohydrates on bacterial and viral surfaces, thus initiating activation of the mammalian mannan-binding lectin complement pathway [1e4]. This leads to the killing of Gramnegative bacteria and enveloped viruses [8]. Mannose-binding lectin therefore acts as a first-line of defense against microbial pathogens. Mannose-binding lectins have been reported in salmon, trout, carp and rohu fish [5,9e11] and now here in several strains of channel catfish and blue catfish. Mannose-binding lectin was purified from channel catfish (Ictalurus punctatus) serum by affinity chromatography. The purification of MBL from channel catfish serum and isolation of MBL from blue catfish (Ictalurus furcatus) serum demonstrates for the first time the existence of the mannan-binding lectin complement pathway in channel catfish and blue catfish and adds a new component of innate immunity and disease resistance in catfish species. The channel catfish  blue catfish hybrid was also included in this study of catfish MBL. Functional binding studies were done to determine the binding activity of channel catfish and blue catfish MBLs in their binding to Edwardsiella ictaluri, an important bacterial pathogen of catfish. 2. Materials and methods 2.1. Maintenance of catfish Channel catfish and blue catfish (2e3-year-old catfish) were maintained in tanks at 27  C in a recirculating waterreuse culture system at the Ecological Research Center, Department of Biology, University of Memphis. Catfish were anaesthetized with tricaine methanesulfonate and bled via the caudal blood sinus [12]. Catfish serum was stored at 80  C. 2.2. Production of guinea pig IgG antibody to mannose-binding lectin Two millilitres of rabbit mannose-binding lectin bound to agarose gel (Pierce Chemical Co., Rockford, IL) were emulsified in 2 ml of Freund’s complete adjuvant [13]. Four guinea pigs were each initially immunized subcutaneously (SC) with 1 ml of the emulsion. Four months later, each guinea pig was again immunized SC with 1 ml of the same vaccine, but this time containing Freund’s incomplete adjuvant. Two months later, the guinea pigs were bled for serum antibody to MBL. A protein A-agarose affinity column (Sigma Chemical Co., St. Louis, MO) was used to isolate guinea pig IgG to MBL according to their procedure. The IgG was eluted with 0.2 M Na2HPO4/0.1 M citric acid, pH 3.5. The IgG antibody to MBL was then used as the primary antibody in the dot-immunoblot ELISA procedure and Western blot analysis. 2.3. Purification of channel catfish mannose-binding lectin by affinity chromatography The following buffers adapted from Nevens et al. [14] were prepared for affinity chromatography: Mixing buffer (20 mM imidazoleeHCl, pH 7.8, 2.5 M NaCl, 40 mM CaCl2); Loading buffer (10 mM imidazoleeHCl, pH 7.8, 1.25 M NaCl, 20 mM CaCl2); Elution buffer (10 mM imidazoleeHCl, pH 7.8, 1.25 M NaCl, 2 mM EDTA). Channel catfish serum was centrifuged at 20,000 rpm for 15 min and then filtered using a 0.45 m filter. One hundred and twenty-five millilitres of channel catfish serum (31 mg ml1 protein; representing a serum pool of 25 two-year old Norris strain channel catfish) were mixed with 125 ml of mixing buffer and stirred for 60 min at 4  C. This mixture was applied to a mannan-agarose affinity column (Sigma Chemical) and then eluted with the elution buffer containing

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Isolation of Mannose-Binding C-Type Lectin from Channel Catfish 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

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2 mM EDTA (Fig. 1). The EDTA chelates calcium thus releasing MBL from the affinity column. Fractions (2 ml per tube) were collected. The isolation procedure according to Nevens et al. [14] was followed. 2.4. SDSepolyacrylamide gel electrophoresis of catfish mannose-binding lectin SDSePAGE (12% separating gel; 4% stacking gel) was performed under reducing (with 2-ME) conditions [15e 18] to identify the affinity-purified channel catfish MBL (Fig. 1) and resolve MBL from channel catfish, blue catfish and hybrid (channel catfish female  blue catfish male) catfish sera (Figs. 2 and 3). The gel (8  10 cm and 0.75 mm thick) was subjected to electrophoresis at 20 mA for 45 min. Molecular weight (75 kDa, 50 kDa, 37 kDa, 25 kDa) protein standards (Precision Plus, Bio-Rad, Hercules, CA) were used. Western blots were done by electrophoretic transfer to nitrocellulose membrane to identify the catfish MBLs using the guinea pig specific IgG antibody to MBL as the primary antibody.

Fig. 2. SDSepolyacrylamide gel electrophoresis (12%), 2-ME reduction and Western blotting of affinity-purified channel catfish mannose-binding lectin (MBL), blue catfish serum MBL, channel  blue hybrid catfish serum MBL, and channel catfish serum MBL for isolation of mannose-binding lectin. Guinea pig anti-MBL IgG was the primary antibody. Lane 1, reduced affinity-purified channel catfish MBL (Norris strain); lane 2, reduced blue catfish serum MBL (DþB strain); lane 3, reduced hybrid catfish serum MBL (channel catfish female NWAC 103  blue catfish male DþB); lane 4, reduced channel catfish serum MBL (NWAC 103 strain). Molecular weight (kDa) protein standards (Bio-Rad) are shown on the left.

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Fig. 3. SDSepolyacrylamide gel electrophoresis (12%), 2-ME reduction and Western blotting of channel catfish 102 strain mannose-binding lectin (MBL) and Rio Grande blue catfish serum MBL. Guinea pig anti-MBL IgG was the primary antibody. Lane 1, reduced channel catfish 102 strain MBL; lane 2, reduced Rio Grande blue catfish MBL. Molecular weight (kDa) protein standards (Bio-Rad) are shown on the left.

2.5. Enzyme-linked immunosorbent assay (ELISA) for catfish mannose-binding lectin A dot-immunoblot ELISA assay was performed using a dot-blot microfiltration apparatus to determine levels of MBL in the different strains of channel catfish and blue catfish and the affinity-purified MBL (Figs. 4 and 5). The primary antibody used was the guinea pig antirabbit-MBL IgG. The dot-blot apparatus (Bio-Rad, Richmond, CA) was used according to their immunoassay procedure and our previous work using this technique [13,18,19]. Fifty microlitres of a 1:8 dilution of catfish serum or 50 ml of the affinity-purified MBL were spotted on nitrocellulose membrane in the dot-blot apparatus and vacuum applied. The nitrocellulose membrane was removed from the apparatus and put in a dish. Non-specific protein sites were then blocked with 1% purified casein solution in Tris-buffered saline, pH 7.5 (TBS) for 60 min. After washing the membrane three times with TBS, 10 ml of a 1:200 dilution in TBS of the guinea pig antirabbit-MBL IgG was added and incubated for 60 min at room temperature as the primary antibody. After washing the membrane again three times in TBS, a 1:500 dilution in TBS of rabbit anti-guinea pig IgGehorseradish peroxidase conjugate (Sigma Chemical) was added and incubated for 60 min at room temperature as the secondary antibody. This was followed by washing the membrane three times in TBS. A 3,30 -diaminobenzidine solution was then used to develop the brown coloured product for 5 min and the reaction stopped with water.

Fig. 4. Dot-immunoblot ELISA membrane binding assay of different strains of channel catfish and blue catfish sera for levels of mannose-binding lectin. Guinea pig anti-MBL IgG was the primary antibody. Dot 1: DþB blue catfish, Ecological Research Center, University of Memphis, Memphis, TN; Dot 2: DþB blue catfish, Catfish Genetics Research Unit, ARS, USDA, Stoneville, MS; Dot 3: Rio Grande blue catfish, Catfish Genetics Research Unit, ARS, USDA, Stoneville, MS; Dot 4: Northwest Aquaculture Center (NWAC 102), Catfish Genetics Research Unit, ARS, USDA, Stoneville, MS; Dot 5: Northwest Aquaculture Center (NWAC 103), Catfish Genetics Research Unit, ARS, USDA, Stoneville, MS; Dot 6: Northwest Aquaculture Center (NWAC 303), Catfish Genetics Research Unit, ARS, USDA, Stoneville, MS; Dot 7: Hybrid catfish (channel catfish female NWAC 103  blue catfish male DþB), Catfish Genetics Research Unit, ARS, USDA, Stoneville, MS; Dot 8: Stuttgart National Aquaculture Research Center, ARS, USDA, Stuttgart, AR; Dot 9: Ecological Research Center, University of Memphis, Memphis, TN; Dot 10: Tris-buffered saline.

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2.6. Binding of channel catfish and blue catfish mannose-binding lectin to Edwardsiella ictaluri detected by membrane dot-blot ELISA assay The immunoperoxidase method was used to functionally study the binding of channel catfish NWAC 103 strain and blue catfish DþB strain serum MBLs to the surface of Edwardsiella ictaluri (ATCC #33202) on nitrocellulose membrane [13,18,20]. The dot-blot apparatus (Bio-Rad, Richmond, CA) was used for the following immunoblot ELISA method. Bacterial plate cultures of Edwardsiella ictaluri were first removed from plate surfaces and washed six times with sterile saline. To do the binding assay for Edwardsiella ictaluri, 100 ml of a bacterial suspension in sterile saline, containing 850,000 bacteria, was incubated for 30 min with 100 ml of channel catfish or blue catfish serum MBL. After incubation, followed by centrifugation, the bacterial cells were washed three times in Tris-buffered saline, pH 7.5 (TBS) and then reconstituted in 100 ml of TBS. Ten microlitres of the bacterial cells (85,000 bacteria) were applied to a well in the dot-blot apparatus and then vacuum was applied. The nitrocellulose membrane was removed from the apparatus and put in a dish. One percent purified casein in TBS was used as the blocking solution, and the membrane was incubated for 60 min at room temperature After washing the membrane three times with TBS, 10 ml of a 1:200 dilution in TBS of the guinea pig antirabbit-MBL IgG was added and incubated for 60 min at room temperature as the primary antibody. After again washing the membrane three times in TBS, the membrane was incubated for 60 min at room temperature in a dish containing 10 ml of a 1:500 dilution in TBS of rabbit anti-guinea pig IgG conjugated with horseradish peroxidase (Sigma Chemical). The membrane was washed three times with TBS. A 3,30 -diaminobenzidine solution was then used to develop the brown coloured product for 5 min and the reaction stopped with water. The ELISA dot-immunoblots were scanned and compared for inverse colour density using ImageJ software developed by the National Institutes of Health. The binding of channel catfish and blue catfish MBLs by Edwardsiella ictaluri was therefore determined (Fig. 6).

Fig. 6. Binding of channel catfish and blue catfish mannose-binding lectin (MBL) to Edwardsiella ictaluri detected by the dot-immunoblot ELISA method using guinea pig anti-MBL IgG as the primary antibody. Dot 1: Channel catfish (NWAC 103 strain) serum MBL binding to Edwardsiella ictaluri; Dot 2: Blue catfish DþB serum MBL binding to Edwardsiella ictaluri; Dot 3: Tris-buffered saline. A secondary antibody control (no primary antibody) and normal guinea pig IgG used instead of the primary antibody gave inverse density dots nearly equivalent in density to Tris buffered saline.

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2.7. Protein assay Total protein concentrations of the various catfish sera and the affinity-purified channel catfish MBL were determined using the BCA protein assay (Pierce Chemical). Bovine serum albumin was used as the protein standard.

3. Results The 2-ME reduced Norris strain channel catfish MBL, purified by affinity chromatography (Fig. 1), gave a single electrophoretic band with a molecular mass of 62 kDa by Western blot analysis (Fig. 2). A gradient 7e12% SDS gel was also done with the affinity-purified channel catfish MBL and silver stained by Dr. George Hilliard, Director of the Laboratory for Protein Analysis and Proteomics at the University of Tennessee Health Science Center, Memphis, TN. He obtained a single band of approximately 62 kDa for the purified channel catfish MBL. The channel catfish serum (Norris strain), reduced with 2-ME, also gave a single band of 62 kDa. The 2-ME reduced channel catfish NWAC 103 strain serum and the 2-ME reduced channel  blue hybrid catfish serum both gave single bands of 63 kDa (Fig. 2). Blue catfish serum (DþB strain), reduced with 2-ME, gave a single band of 66 kDa (Fig. 2). The Rio Grande blue catfish strain had a 2-ME reduced serum MBL molecular mass of 65 kDa, and the NWAC 102 strain of channel catfish had a 2-ME reduced serum MBL molecular mass of 63 kDa (Fig. 3). The white catfish, Ameiurus catus, had a 2-ME reduced serum molecular mass of 69 kDa (gel not shown). The guinea pig antirabbit-MBL IgG, used as the primary antibody, was specific for rabbit MBL [13] and insect MBL [18] and is also specific here for the catfish MBLs by Western blot analysis (Figs. 2e4). A standard curve of inverse density vs. mg of purified channel catfish MBL using the primary antibody was done by dot-immunoblot ELISA to corroborate the results obtained by SDSePAGE/Western blot analysis (Fig. 5). Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using the dot-immunoblot ELISA assay for this bacterium on nitrocellulose membrane (Fig. 6). The channel catfish and blue catfish MBLs both bound to Edwardsiella ictaluri with the dot-immunoblot ELISA assay (Fig. 6). The background corrected inverse density by ImageJ analysis was 99 for the channel catfish NWAC 103 strain MBL, and 119 for the DþB blue catfish MBL in their binding to Edwardsiella ictaluri. The blue catfish MBL was therefore 17% greater than channel catfish MBL in the functional binding assay to Edwardsiella ictaluri. In the comparative study of MBL for the different catfish strains and species (Table 1), the Rio Grande strain of blue catfish had four-times the level of MBL when compared with the Norris strain of channel catfish. The DþB strain of blue catfish averaged 2.8 times greater in level of MBL when compared with the Norris strain of channel catfish. Of the different channel catfish strains tested for MBL, the channel catfish NWAC 102 strain had the highest level of MBL, and the channel catfish NWAC 303 strain had the lowest level of MBL (Table 1).

Table 1 Dot-immunoblot ELISA membrane binding assay data of the different strains of channel catfish and blue catfish sera for levels of mannose-binding lectin (from Fig. 4, ELISA immunoblot) Catfish strain

Background corrected inverse density

Number of times greater than Norris

Percent greater than Norris

DþB bluea DþB blueb Rio Grande blueb NWAC 102b NWAC 103b NWAC 303b Channel  blue hybridb Norrisc Norrisa

70 51 88 40 31 20 31 22 22

3.18 2.31 4.00 1.81 1.41 0.91 1.41 1.00 1.00

218 131 300 81 41 0 41 0 0

a b c

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An amino acid composition analysis of the affinity-purified channel catfish MBL was done at the University of Iowa College of Medicine, Molecular Analysis Facility, by Dr. Brian Morrison. A higher serine content (14.4 mol%) was found in the channel catfish MBL when compared with Atlantic salmon MBL (5.5 mol%) [9]. Serum protein concentrations of the six catfish strains used in Western blot analyses (Figs. 2 and 3) were: channel catfish (Norris strain), serum pool of 25 fish, 31 mg ml1; channel catfish (NWAC 103 strain), serum pool of 16 fish, 39.9 mg ml1; blue catfish (DþB strain), serum pool of 20 fish, 29.5 mg ml1; hybrid catfish (channel catfish female NWAC 103  blue catfish male DþB), serum pool of 12 fish, 30.1 mg ml1; Rio Grande blue catfish, serum pool of 5 fish, 43.1 mg ml1; channel catfish (NWAC 102 strain), serum pool of 10 fish, 38.2 mg ml1. Serum protein concentrations of the other three catfish strains were: blue catfish (DþB strain), serum pool of 10 fish, 25.9 mg ml1; channel catfish (NWAC 303 strain), serum pool of 24 fish, 36.1 mg ml1; channel catfish (Norris strain), serum pool of 6 fish, 42.8 mg ml1. After purification of MBL from channel catfish (Norris strain) serum by affinity chromatography (Fig. 1), the yield of MBL was 6 mg ml1 of catfish serum.

4. Discussion The channel catfish is extensively used in aquaculture or fish farming in the Southeast United States and is susceptible to bacterial infections acquired from its pond environment [21e23]. Enteric septicaemia of catfish (ESC), caused by Edwardsiella ictaluri, is the major bacterial disease of channel catfish and can cause high mortality in pond culture [21,22,24e26]. Additional research is needed to understand catfish innate immunity against this important bacterial pathogen. Our discovery of a mannose-binding C-type lectin in channel catfish and blue catfish sera is important in understanding catfish innate immunity and resistance to bacterial pathogens (Figs. 1e6, Table 1). Mannose was found to be a sugar component in the lipopolysaccharide cell wall of Edwardsiella ictaluri [27]. Weete et al. [27] found that 10% of the sugar composition of the lipopolysaccharide cell wall of Edwardsiella ictaluri was a polymer of mannoheptose, a mannose-containing carbohydrate. In functional binding studies, Edwardsiella ictaluri was found to bind both channel catfish and blue catfish MBLs by the dot-immunoblot ELISA method (Fig. 6). Functional binding experiments demonstrated then that catfish serum MBL recognizes mannose on the cell surface of Edwardsiella ictaluri, the most important Gram-negative bacterial pathogen in channel catfish aquaculture. The affinity-purified channel catfish MBL had a 2-ME reduced molecular mass of 62 kDa as determined by SDSe PAGE/Western blot analysis (Figs. 1 and 2). No band was seen to enter the gel with unreduced affinity-purified channel catfish MBL by SDSePAGE and Western blotting, indicating a molecular mass of large molecular weight. The purity of the affinity-isolated catfish MBL was corroborated by gradient SDSePAGE and silver staining in which a single band of the same molecular weight was obtained as that seen by Western blot analysis. The primary antibody reacted with the purified channel catfish MBL in a dose-dependent and quantitative manner (Fig. 5). The dot-immunoblot ELISA standard curve assay for MBL therefore supported the results obtained by SDSePAGE/Western blot analysis (Figs. 2e4). We have molecular mass data for the presence of MBL in channel catfish Norris strain (62 kDa), channel catfish NWAC 103 strain (63 kDa), blue catfish DþB strain (66 kDa) and channel  blue hybrid catfish (63 kDa) (Fig. 2). The hybrid catfish (channel catfish female NWAC 103  blue catfish male DþB) reduced serum MBL had a molecular mass of 63 kDa (Fig. 2). The molecular mass for the reduced hybrid catfish MBL was equivalent then to the molecular mass of the reduced maternal channel catfish NWAC 103 MBL which was also 63 kDa (Fig. 2). The molecular mass of the channel catfish NWAC 102 reduced MBL was also 63 kDa, and the molecular mass of the Rio Grande blue reduced MBL was 65 kDa (Fig. 3). This is evidence that a lectin complement pathway exists in these catfish strains and species (Figs. 1e4), and that catfish MBL has binding activity against the Gramnegative bacterial pathogen Edwardsiella ictaluri (Fig. 6). Ewart et al. [9] found that Atlantic salmon MBL binds to two Gram-negative bacterial pathogens of salmon, Vibrio and Aeromonas. A mannose-binding C-type lectin was purified from channel catfish serum by affinity chromatography and identified by Western blotting using guinea pig anti-MBL IgG as the primary antibody (Figs. 1 and 2). The channel catfish MBL protein concentration was 6 mg ml1 of catfish serum (Fig. 1). This compares with 5 mg ml1 MBL found for Atlantic salmon serum [9]. The amount of serine present (mol%) in the affinity-purified channel catfish MBL was nearly 3 times greater when compared with the amount of serine present in Atlantic salmon MBL [9]. Serine is a polar and hydrophilic amino acid.

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The C-type lectin binds mannose in a calcium-dependent manner as the catfish lectin could be eluted from a mannan-agarose affinity column with 2 mM EDTA (Fig. 1). The catfish MBL can be considered then a C-type lectin (Ca-dependent) since it could be eluted from the mannan-agarose affinity column with EDTA. This also means that the catfish C-type lectin has specificity for mannose. Catfish MBL is a member then of the collectin family of Ca-dependent proteins. In mammals and birds, MBL is a collectin composed of subunits containing three identical polypeptide chains of about 30 kDa [5]. The catfish C-type lectin purified in this study is composed of 62 kDae 66 kDa polypeptides. The catfish lectin isolated here may belong then to a different lectin subfamily. A comparative study of MBL was done with the different strains of channel catfish and blue catfish (Fig. 4, Table 1). The highest levels of serum MBL were found in the blue catfish, especially with the Rio Grande strain of blue catfish (Fig. 3, Table 1). Decreasing levels of MBL were observed with NWAC 102, NWAC 103, and NWAC 303 strains of channel catfish (Fig. 4, Table 1). The channel catfish  blue catfish hybrid was equivalent in level of MBL to channel catfish NWAC 103, its maternal cross (Table 1). The channel catfish NWAC 303 strain and the Norris strain of channel catfish had the lowest levels of MBL (Table 1). From the data reported here for levels of MBL, a potential channel  blue hybrid catfish for increased innate resistance could be the channel catfish NWAC 102 strain crossed with the Rio Grande strain of blue catfish (Figs. 3 and 4, Table 1). The present investigation provides new information about the innate immune response of the catfish lectin complement pathway to bacterial pathogens and expands our previous research on channel catfish innate immunity by the alternative complement pathway [12,28e32]. A mannose-containing carbohydrate was previously identified as a cell wall component in Edwardsiella ictaluri [27]. The channel catfish is the major host species for Edwardsiella ictaluri, whereas the blue catfish is generally considered resistant to ESC [33]. The increased serum levels seen for MBL (Fig. 4; Table 1) could be an important factor why the blue catfish and certain strains of channel catfish or the channel  blue catfish hybrid are more resistant to Edwardsiella ictaluri infections when compared with less resistant strains of channel catfish [26,34,35]. The Norris strain of channel catfish is known to be a less resistant catfish and the blue catfish a more resistant catfish to Edwardsiella ictaluri infection [26,34,35]. Bilodeau et al. [36] studied three family groups of 103 strain channel catfish susceptible to ESC and three family groups resistant to ESC. They suggest that a non-specific immune response may be important in ESC resistance which this catfish study of the innate response would support. Chickens are being bred for high levels of MBL to increase their innate resistance to microbial infections [37]. Mannose-binding lectin then may be an innate immune protein that could be used in aquaculture as a molecular resistance marker for genetic selection in the breeding of catfish strains for increased resistance to Edwardsiella ictaluri and other bacterial infections (Fig. 4, Table 1). The different serum levels of MBL found in the different catfish strains and species were compared and studied here (Fig. 4, Table 1). The blue catfish strains had higher levels of MBL then did the channel catfish strains. The guinea pig anti-MBL IgG specific antibody used here could be an important reagent for studying and comparing innate immunity provided by the catfish MBL in the various strains of channel catfish, blue catfish and channel  blue hybrid catfish used in aquaculture production. Acknowledgments The authors thank Dr. Brian Bosworth and Dr. Geoffrey Waldbieser, Catfish Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Stoneville, MS for providing channel catfish, blue catfish, and channel  blue hybrid catfish sera. We also thank Dr. Kenneth Davis, Stuttgart National Aquaculture Research Center, ARS, USDA, Stuttgart, AR for providing channel catfish serum. References [1] [2] [3] [4] [5]

Fujita T. Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol 2002;2:346e53. Jack DL, Turner MW. Anti-microbial activities of mannose-binding lectin. Biochem Soc 2003;31:753e7. Turner MW. The role of mannose-binding lectin in health and disease. Mol Immunol 2003;40:423e9. Gadjeva M, Takahashi K, Thiel S. Mannan-binding lectin-a soluble pattern recognition molecule. Mol Immunol 2004;41:113e21. Vitved L, Holmskov U, Koch C, Teisner B, Hansen S, Skjodt K. The homologue of mannose-binding lectin in the carp family cyprinidae is expressed at high level in spleen, and the deduced primary structure predicts affinity for galactose. Immunogenetics 2000;51:955e64. [6] Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RA. Phylogenetic perspectives in innate immunity. Science 1999;284:1313e8. [7] Suckale J, Sim RB, Dodds AW. Evolution of innate immune systems. Biochem Mol Biol Ed 2005;33:177e83.

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