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MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus)
Gene 526 (2013) 217–222
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MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus) Hui Yu a, Shuwen Tan a,b, Hongjuan Zhao a, Hua Li a,c,⁎ a b c
College of Life Science, Foshan University, Guangdong 528231, China Huanan Normal University, Guangzhou 510631, China Holdone Aquaculture Breeding Limited Company, Foshan, Guangdong 528231, China
a r t i c l e
i n f o
Article history: Accepted 17 May 2013 Available online 30 May 2013 Keywords: Grass carp (Ctenopharyngodon idellus) MH-DAB gene Flavobacterium columnare Polymorphism Expression Disease resistance
a b s t r a c t In this study, the association between MH-DAB gene polymorphism and disease resistance was evaluated by challenging grass carp (Ctenopharyngodon idellus) with Flavobacterium columnare. Eight genotypes and six alleles were found, and named by common nomenclature. The genotypes AA, BB, EE, and DE, and the alleles Ctid-DAB1*0101, Ctid-DAB1*0201 and Ctid-DAB1*0401 were more preponderant in fish. The genotype BB was associated with higher resistance to F. columnare, as well as two alleles Ctid-DAB*0101 and Ctid-DAB*0201. Allele Ctid-DAB*0102 has decreased resistance to F. columnare. The expression of MH-DAB gene was decreased in the liver, kidney, and intestine but not in the spleen, gill, and skin at 2 days post infection (dpi), versus to that in the control group. MH-DAB gene expression was up-regulated in most tissues but remained at normal levels in the intestine at 15 days post infection. Our data suggested that MH-DAB polymorphism can be used as a potential genetic marker for disease resistance breeding of grass carp in the future. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Grass carp (Ctenopharyngodon idellus), which belong to the Cyprinidae family, are widely cultured in China. It is one of the most important freshwater cultured species due to its fast growth rate and good taste. Because of the frequent outbreaks of viral and bacterial diseases in cultivation, the mortality of grass carp is as high as 90%. Flavobacterium columnare, the causative agent of columnaris disease, is a big threat to grass carp, causing severe gill rot and occasional skin ulceration (Darwish et al., 2004). Many vaccine strategies have been used against F. columnare, but with limited success. Gene-assisted selection, which enhances fish resistance to columnaris disease, will be a promising approach to this problem. Disease resistance breeding programmes had been studied in many fish, including Atlantic salmon and rainbow trout (Midtlyng et al., 2002). However, a number of problems still need to be solved, such as the maintenance of the stable heredity of the diseased resistant characteristics and the selection of the target genes. Abbreviations: MHC, major histocompatibility complex; dpi, days post infection; PCR, polymerase chain reaction; PCR-SSCP, PCR-single-strand conformation polymorphism; LD50, lethal dose 50; cfu, colony-forming units; DNA, deoxyribonucleic acid; s, second; min, minute; h, hour; V, volta; RNA, ribonucleic acid; cDNA, complementary deoxyribonucleic acid; BLAST, Basic Local Alignment Search Tool; bp, basic pair; PBR, peptide binding residue; HLA-DR1, human leukocyte antigen-DR1; dN, nonsynonymous substitution rate; dS, synonymous substitution rate; IHNV, infectious hematopoietic necrosis virus. ⁎ Corresponding author at: College of Life Science, Foshan University, No. 1 Xianhu University Road, Nanhai, Foshan, Guangdong, China. Tel.: +86 757 85505797; fax: +86 757 85505013. E-mail address: [email protected] (H. Li). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.05.019
The major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity, characterised with abundant polymorphism. The MHC genes encode cell-surface glycoproteins, which bind and present endogenous and exogenous peptides to T cells, triggering a specific immune response towards the pathogen. MHC class II molecules present mostly exogenous short peptides to CD4+ T helper cells. Generally, MHC class I α and class II genes are highly polymorphic, especially in exon 2 of both genes, which encodes α1 and β1 domains that are principally responsible for peptide binding (Eizaguirre et al., 2012; Langefors et al., 2001). Different allelic MHC molecules bind and present specific peptides, thus the response of an organism towards certain pathogens can be influenced by the different haplotypes of MHC (Rakus et al., 2008). Some pathogens may escape recognition by certain MHC molecules since their peptides are not presentable by MHC, which can lead to increase of the susceptibility to pathogens. Contrarily, resistance to pathogens may be derived through high affinity binding of certain peptides by specific MHC alleles (Kjoglum et al., 2006). It is necessary to detect resistant alleles of MHC in economic species for molecular marker-assisted selective breeding programme, and exon 2 in MHC class II gene was considered to be the responsible candidate. In teleosts, MHC genes are named MH because the MHC I and MHC II genes reside on different chromosomes and segregate independently (Grimholt et al., 2003). MH II genes have been isolated and characterised in fish species including common carp (Hashimoto et al., 1990), cichlid (Murray et al., 2000), and rainbow trout (Grimholt et al., 2000). The expression profiles of these genes have also been analysed (Chen et al., 2006; Shen et al., 2011; Stet et al., 2002). The association between
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polymorphism of MH II genes and disease resistance has been established in several fish species (Glover et al., 2007; Wynne et al., 2007; Zhang et al., 2006). However, there has been no research about the association between the MH-DAB gene polymorphism and diseased resistance in grass carp. In this study, specific genotypes and alleles resistant to F. columnare were detected within MH-DAB gene exon 2 in grass carp by PCR-SSCP, and the induced expressions of MH-DAB in various tissues were examined after infection with F. columnare. The aim was to evaluate the function of MH-DAB gene and select the potential resistant alleles for grass carp disease resistance improvement.
accession number AB190816) was used as a quantitative housekeeping gene (β2mF: 5′-ggctggcagtttcacctcac-3′, β2mR: 5′-ccaccctttgtctggctttg-3′). The qRT-PCR was conducted using an ABI PRISM 7500 Fast Real-time PCR System with SYBR-Green according to the manufacturer's protocol (Takara, Dalian, China). The PCR was performed in three steps using the following condition: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 20 s at 56 °C, and 34 s at 72 °C, and a final extension cycle. Relative gene expression data were calculated using the 2−△△Ct method, and the MH-DAB expression level was normalised to β2m gene expression within each sample.
2. Materials and methods
2.4. Sequence polymorphisms and gene differential expression analysis
2.1. Fish rearing and challenge test
The sequences were compared using the BLAST programme. The sequences and predicted amino acid residues were aligned using BioEdit 7.01 software. Variations of the sequences were analysed by MEGA 4.0. Polymorphisms in MH-DAB exon 2 were analysed using DnaSP 4.0. The statistical analysis was carried out with SAS 8.0 software. Differences in the genotypic and allelic frequencies were calculated using the Chi-square test, while comparisons of the expression levels of MH-DAB after the challenge experiment were calculated using a least squares method. Association between the MH-DAB genotypes and resistance to F. columnare was analysed using Cox proportional hazard regressions (Rakus et al., 2009a).
Grass carp (40 g on average) were maintained in aerated tap water at 20 °C in 100 L aquaria with Eheim biofilters, and were sampled to ensure no bacterium or virus infection. After a week of acclimatisation, 342 healthy grass carp were intraperitoneally infected with F. columnare (2.5 × 108 cfu, the LD50 in 72 h). F. columnare was re-isolated from infected fish to confirm if the infection was successful. 80 healthy fish in similar aquariums were mock-injected with the same volume of sterilised normal saline. Tissues were collected at different time points post-infection (from 2 days up to 15 days), and stored at −80 °C. 2.2. Detection of MH-DAB gene polymorphisms
3. Results MH-DAB genotyping was performed using PCR-SSCP, and confirmed by clone and sequencing. Genomic DNA was isolated from the liver by phenol–chloroform extraction and purified using ethanol. Specific primers (ex2F: 5′-tctgacataactgtaatgctgc-3′, ex2R: 5′-cagga gagatcagagtcttg-3′) were designed according to the common carp MH-DAB gene sequence (GenBank accession number Z47757) to amplify exon 2 of MH-DAB gene. The PCR profile included an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 55.5 °C, 50 s at 72 °C, and a final extension step at 72 °C for 10 min. The amplified products were assessed by 1% agarose gel electrophoresis. Before PCR-SSCP analysis, the PCR products were denatured at 95 °C for 10 min, immediately cooled on ice for 15 min, and loaded on the 10% polyacrylamide gel (acrylamide/bis: 39/1). Electrophoresis was performed under optimised conditions (300 V for 15 min, then 120 V for 14 to 18 h at 4 °C). The bands were visualised by silver staining. Each PCR product from the different MH-DAB genotypes was ligated into the PMD-18T vector (Takara, Dalian, China), and cloned into Escherichia coli DH5α competent cells. Positive clones were screened via blue–white selection, detected by PCR with M13+/– primers and sequenced.
3.1. MH-DAB polymorphism analysis Eight different MH-DAB genotypes (AA, BB, CC, DD, AB, DE and DF) were detected, and were shown to consist of six different unique alleles, which were named Ctid-DAB1*0101, *0102, *0201, *0202, *0301, and *0401 (Fig. 1. and Table 1) according to accepted nomenclature rules (Klein et al., 1990). In cyprinid fish species, there are at least two paralogous groups of MH class II B genes (DAB1 and DAB3) (Rakus et al., 2009a), and nucleotide sequence analysis revealed that all the sequences belonged to DAB1 group. The complete exon 2 of MH-DAB was 276 bp, in which 273 bp was translated into 91 amino acids (Fig. 2). There were 73 variable sites, and the variation hotspots were located in exon 2 between 14 to 28 bp, 72 to 110 bp, and 24 to 133 bp (Fig. 3). Amino acid sequence analysis revealed 43 variable sites, and showed higher variability in the peptide binding residues (PBRs) compared with that in the HLA-DR1 protein (Brown et al., 1993). The rate of nonsynonymous substitution (dN) was 0.374, and the rate of synonymous substitution (dS) was 0.275 in the PBRs. The rates of dN and dS of the non-PBRs were 0.123 and 0.173, respectively. The dN/dS ratios in the PBRs and non-PBRs were 1.36 and 1.47, respectively, indicating a positive selection pressure in exon 2 of MH-DAB gene.
2.3. Analysis of MH-DAB gene expression Total RNA was extracted from the tested tissues, including the liver, spleen, kidney, gill, intestine, skin, brain and heart, using an RNAiso™ Reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions. The RNA quality was assessed by electrophoresis on a 1% agarose gel, and the total RNA concentration was determined by measuring the absorbance at 260 nm on a spectrophotometer. cDNA was synthesised using a PrimeScript™ RT Reagent Kit (Takara, Dalian, China). The change of MH-DAB expression in different tissues due to F. columnare infection was analysed using qRT-PCR. Quantitative primers (DABF: 5′-cggcttaactaaacccatc-3′, DABR: 5′-ctccctgatttcttcttgt-3′) were designed according to the carp MH-DAB gene sequence (GenBank accession number CCDAB01). The grass carp β2m gene (GenBank
Fig. 1. Different genotypes as characterised by unique SSCP patterns.
H. Yu et al. / Gene 526 (2013) 217–222
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Fig. 2. Alignment of the nucleotide sequences of exon 2 from MH-DAB genes in grass carp. Dots indicate identity with Ctid-DAB*0101.
3.2. Association between MH-DAB gene polymorphisms and resistance to F. columnare Genotypic and allelic frequencies were analysed, and the most frequently occurring genotype was AA, followed by DD, EE and DE, while alleles of Ctid-DAB1*0101, Ctid-DAB1*0102 and Ctid-DAB1*0401 were preponderant alleles (Tables 1 and 2). In Cox proportional hazard regression analysis (Fig. 4), fish with Ctid-DAB1*0101, Ctid-DAB1*0201 showed a statistically significant increase in resistance to F. columnare (P b 0.05), while Ctid-DAB*0102 significantly increased susceptibility to F. columnare (P b 0.05). Genotype BB was significantly reduced in the hazard ratio (HR) (P b 0.05), and fish with this genotype showed a lower mortality than those without BB, suggesting that genotype BB was associated with increased resistance to F. columnare. 3.3. MH-DAB gene expression MH-DAB gene basal expression was investigated in healthy grass carp. The highest expression levels of MH-DAB gene were predominantly observed in the gill and spleen, followed by the brain, kidney, skin, and intestine; there was only faint expression in the heart and liver (Fig. 5). After challenging with F. columnare (Fig. 6), the expression level of MH-DAB in the liver was 2-fold higher in the control group than in the challenged group at 2 dpi (P b 0.05); but the expression had recovered at 15 dpi in the challenged group. A similar expression profile was detected in the kidney. In the intestine, MH-DAB gene expression was decreased by approximately 67% at 2 dpi (P b 0.05) but was increased at 15 dpi (P b 0.05); however, the expression levels at 15 dpi
were still suppressed relative to the pre-infection expression levels. In the spleen, the expression level increased at 2 dpi (P b 0.05), and remained at a high level at 15 dpi (P b 0.05). In the gill and skin, the expression increased and reached a peak at 15 dpi (P b 0.05). 4. Discussion MHC genes are crucial elements of adaptive immunity. It has been shown that polymorphisms in MHC molecules can result in different disease resistance in human (Hendel et al., 1999), chickens (Li et al., 1997; Simonsen et al., 1982), and teleosts (Rakus et al., 2008; Grimholt et al., 2000). Undoubtedly, the polymorphism of MH II genes will provide related gene markers for disease resistance breeding of teleosts. In our study, eight MH-DAB genotypes and six different alleles were detected on grass carp and abundant polymorphisms were demonstrated in the PBRs of the MH-DAB gene exon 2. Many hypotheses have been proposed to explain the vast amount of polymorphisms in MHC genes (Parham and Ohta, 1996). Pathogen-driven selection, which leads to a heterozygote advantage (over-dominance), and frequency-dependent selection are suggested to be the main reasons for the genetic diversity of the MHC genes (Grimholt et al., 2003). Here, the high ratio of dN/dS and the high allelic diversity suggested a strong positive selection, which explained the high polymorphism rate in exon 2 of the MH-DAB gene in grass carp. Different MH-DAB genotypes and alleles indicated the association between the MH-DAB gene and resistance to F. columnare in grass carp. Two alleles (Ctid-DAB1*0101 and Ctid-DAB1*0201) significantly reduced the hazard ratio, suggesting that these alleles could be
Fig. 3. Alignment of the deduced amino acid sequences of exon 2 in MH-DAB. Dots indicate identity with Ctid-DAB*0101(Brown et al., 1993). Asterisks stand for PBRs, and “p” indicates the polymorphic site.
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Fig. 4. Kaplan–Meier plot presenting the survival of different MH-DAB genotypes (4a) and alleles (4b) in grass carp challenged with the F. columnare. The mortality was recorded every 4 h during the experiment. The experiment was stopped at 25 days post infection (dpi) and surviving fish were sacrificed.
associated with the resistance to F. columnare in grass carp. The association between MH genetic polymorphism and disease resistance or susceptibility has been successfully demonstrated in teleosts. In Atlantic salmon, UBA*0201 and UBA*0301 were considered to be the major resistant alleles against infectious salmon anaemia (Kjoglum
et al., 2006). In Japanese flounder, a significant association has been detected between resistance to Vibrio anguillarum and the polymorphism of the MH-DAB gene (Xu et al., 2008). In common carp, Cyca-DAB1*05 was the resistance allele to CyHV-3, while Cyca-DAB1*02 and Cyca-DAB1*06 were found to be the susceptible
Table 1 Frequency and hazard ratio of MH-DAB alleles. Allele
Frequency
Hazard ratio (95% CI)
Number of fish
Accession number
A (Ctid-DAB*0101) B (Ctid-DAB*0201) C (Ctid-DAB*0301) D (Ctid-DAB*0102) E (Ctid-DAB*0401) F (Ctid-DAB*0202)
24.56% 13.16% 0.88% 29.39% 31.58% 0.44%
0.768* 0.586* 0.354 1.704* 1.013 2.933
93 54 3 138 144 3
JX305295 JX305297 JX305300 JX305296 JX305301 JX305298
Note: The asterisk (*) indicated the significant Hazard ratio (P b 0.05).
(0.608, 0.972) (0.431, 0.800) (0.088,1.422) (1.397, 2.075) (0.829,1.239) (0.937, 9.174)
H. Yu et al. / Gene 526 (2013) 217–222
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Table 2 Frequency, hazard ration and mortality of MH-DAB genotypes. Genotype
Frequency
Hazard ratio (95% CI)
Mortality
Number of fish
AA (Ctid-DAB*0101/*0101) BB (Ctid-DAB*0201/*0201) CC (Ctid-DAB*0301/*0301) DD (Ctid-DAB*0102/*0102) EE (Ctid-DAB*0401/*0401) AB (Ctid-DAB*0101/*0201) DE (Ctid-DAB*0102/*0401) DF (Ctid-DAB*0102/*0202)
21.93% 10.53% 0.88% 18.42% 21.05% 5.26% 21.05% 0.88%
0.827 0.618* 0.355 1.761 0.889 0.527 1.302 2.950
53.33% 52.78% 33.33% 76.19% 62.5% 44.44% 73.61% 66.67%
75 36 3 63 72 18 72 3
(0.587, (0.385, (0.050, (1.276, (0.637, (0.260, (0.954, (0.937,
1.167) 0.991) 2.531) 2.433) 1.236) 1.067) 1.776) 9.259)
Note: The asterisk (*) indicated the significant Hazard ratio (P b 0.05).
alleles (Rakus et al., 2009b). Moreover, a strong association between Cyca-DAB1 heterozygosity and a lower level of parasitaemia after Trypanoplasma borreli infection was found in common carp (Rakus et al., 2009c). In addition, Scma-DBB1*02 was associated with resistance to Edwardsiella tarda in turbot (Du et al., 2012). The constructive expression of MH II genes was tissue specific in normal teleosts. A previous study showed that the MH II genes were highly expressed in the spleen, intestine, kidney, and liver in uninfected carp, which contained high concentrations of lymphoid and myeloid cells (Rodrigues et al., 1995). In our study, the highest constitutive expression was detected in the gill as formerly observed in Atlantic salmon (Koppang et al., 1998), red sea beam (Chen et al., 2006), and Chinese longsnout catfish (Shen et al., 2011), since the gill is essentially protective barriers to the external environment and infectious pathogens (Caipang et al., 2011; Morris et al., 2000). Challenge of the grass carp with F. columnare resulted in a significant change in MH-DAB gene expression. In the skin and gill, increased expressions were found after the challenge test. The expression in the spleen was also initially up-regulated, and remained at a high level during infection, suggesting that there was interference in the
immune function of the spleen during F. columnare infection. A decrease of the MH-DAB gene expression was detected at 2 dpi in the liver, kidney, and intestine, followed by an increase at 15 dpi. This was similar to the expression profile for the MH-DAB gene in rainbow trout, where a decrease in expression was observed by 72 h in the liver, kidney, and spleen, and then, partial restoration occurred by 192 h after infectious hematopoietic necrosis virus (IHNV) infection (Hansen and La Patra, 2002). The same phenomenon was observed in red sea bream treated with V. anguillarum (Chen et al., 2006). These results suggested that the MH-DAB gene played an important role in the immune response to infection in grass carp, which are responsible for presenting non-self peptides to T cell receptors and promoting a specific immune response towards the foreign pathogens. The expression of MH-DAB gene was significantly changed in liver, kidney, and intestine, suggesting that they might be the important and sensitive organs for MH II protein function. In summary, this study implied that MH-DAB gene polymorphism is associated with grass carp resistance to F. columnare, and unique alleles for disease resistance could be used as potential genetic markers for grass carp resistance breeding. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant Nos. 31072205 and 30972251). References
Fig. 5. The expression of the MH-DAB gene in different tissues.
Fig. 6. The expression changes of the MH-DAB gene across different times.
Brown, J.H., et al., 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364, 33–39. Caipang, C.M.A., Lazado, C.C., Brinchmann, M.F., Rombout, J.H.W.M., Kiron, V., 2011. Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua). Comp. Biochem. Physiol.D. 6, 158–162. Chen, S.L., Zhang, Y.X., Xu, M.Y., Ji, X.S., Yu, G.C., Dong, C.F., 2006. Molecular polymorphism and expression analysis of MHC class II B gene from red sea bream (Chrysophrys major). Dev. Comp. Immunol. 30, 407–418. Darwish, A.M., Ismaiel, A.A., Newton, J.C., Tang, J., 2004. Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae. Mol. Cell. Probes 18, 421–427. Du, M., Chen, S.L., Liu, Y.H., Niu, B.Z., Yang, J.F., Zhang, B., 2012. MHC polymorphism and disease-resistance to Edwardsiella tarda in six turbot (Scophthalmus maximus) families. Chin. Sci. Bull. 57, 3262–3269. Eizaguirre, C., Lenz, T.L., Kalbe, M., Milinski, M., 2012. Rapid and adaptive evolution of MHC genes under parasite selection in experimental vertebrate populations. Nat. Commun. 3, 621. Glover, K.A., Grimholt, U., Bakke, H.G., Nilsen, F., Storset, A., Skaala, O., 2007. Major histocompatibility complex (MHC) variation and susceptibility to the sea louse Lepeophtheirus salmonis in Atlantic salmon Salmo salar. Dis. Aquat. Org. 76, 57–65. Grimholt, U., Getahun, A., Hermsen, T., Stet, R.J., 2000. The major histocompatibility class II alpha chain in salmonid fishes. Dev. Comp. Immunol. 24, 751–763. Grimholt, U., et al., 2003. MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar); facing pathogens with single expressed major histocompatibility class I and class II loci. Immunogenetics 55, 210–219.
222
H. Yu et al. / Gene 526 (2013) 217–222
Hashimoto, K., Nakanishi, T., Kurosawa, Y., 1990. Isolation of carp genes encoding major histocompatibility complex antigens. Proc. Natl. Acad. Sci. U.S.A. 87, 6863–6867. Hendel, H., et al., 1999. New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS. J. Immunol. 162, 6942–6946. Kjoglum, S., Larsen, S., Bakke, H.G., Grimholt, U., 2006. How specific MHC class I and class II combinations affect disease resistance against infectious salmon anaemia in Atlantic salmon (Salmo salar). Fish Shellfish Immunol. 21, 431–441. Klein, J., et al., 1990. Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics 31, 217–219. Koppang, E., Lundin, M., Press, C.M.L., Rønningen, K., Lie, Ø., 1998. Differing levels of Mhc class II [beta] chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 8, 183–196. La Hansen, J.D., Patra, S., 2002. Induction of the rainbow trout MHC class I pathway during acute IHNV infection. Immunogenetics 54, 654–661. Langefors, A., Lohm, J., Von Schantz, T., 2001. Allelic polymorphism in MHC class II B in four populations of Atlantic salmon (Salmo salar). Immunogenetics 53, 329–336. Li, L., Johnson, L.W., Ewald, S.J., 1997. Molecular characterisation of major histocompatibility complex (B) haplotypes in broiler chickens. Anim. Genet. 28, 258–267. Midtlyng, P., Storset, A., Michel, C., Slierendrecht, W., Okamoto, N., 2002. Breeding for disease resistance in fish. Bull. Eur. Assoc. Fish Pathol. 22, 166–172. Morris, D., Adams, A., Richards, R., 2000. In situ hybridisation identifies the gill as a portal of entry for PKX (Phylum Myxozoa), the causative agent of proliferative kidney disease in salmonids. Parasitol. Res. 86, 950–956. Murray, B.W., Shintani, S., Sultmann, H., Klein, J., 2000. Major histocompatibility complex class II A genes in cichlid fishes: identification, expression, linkage relationships, and haplotype variation. Immunogenetics 51, 576–586. Parham, P., Ohta, T., 1996. Population biology of antigen presentation by MHC class I molecules. Science 272, 67–74. Rakus, K.L., Wiegertjes, G.F., Adamek, M., Bekh, V., Stet, R.J., Irnazarow, I., 2008. Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.). Fish Shellfish Immunol. 24, 734–744.
Rakus, K.Ł., Irnazarow, I., Forlenza, M., Stet, R.J.M., Savelkoul, H.F.J., Wiegertjes, G.F., 2009a. Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes. Fish Shellfish Immunol. 26, 352–358. Rakus, K.L., Wiegertjes, G.F., Adamek, M., Siwicki, A.K., Lepa, A., Irnazarow, I., 2009b. Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism. Fish Shellfish Immunol. 26, 737–743. Rakus, K.L., Wiegertjes, G.F., Jurecka, P., Walker, P.D., Pilarczyk, A., Irnazarow, I., 2009c. Major histocompatibility (MH) class II B gene polymorphism influences disease resistance of common carp (Cyprinus carpio L.). Aquaculture 288, 44–50. Rodrigues, P.N., Hermsen, T.T., Rombout, J.H., Egberts, E., Stet, R.J., 1995. Detection of MHC class II transcripts in lymphoid tissues of the common carp (Cyprinus carpio L.). Dev. Comp. Immunol. 19, 483–496. Shen, T., Xu, S., Yang, M., Pang, S., Yang, G., 2011. Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish (Leiocassis longirostris). Vet. Immunol. Immunopathol. 141, 33–45. Simonsen, M., Crone, M., Koch, C., Hala, K., 1982. The MHC haplotypes of the chicken. Immunogenetics 16, 513–532. Stet, R.J., et al., 2002. Unique haplotypes of co-segregating major histocompatibility class II A and class II B alleles in Atlantic salmon (Salmo salar) give rise to diverse class II genotypes. Immunogenetics 54, 320–331. Wynne, J.W., Cook, M.T., Nowak, B.F., Elliott, N.G., 2007. Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 22, 707–717. Xu, T.J., Chen, S.L., Ji, X.S., Tian, Y.S., 2008. MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families. Fish Shellfish Immunol. 25, 213–221. Zhang, Y.X., Chen, S.L., Liu, Y.G., Sha, Z.X., Liu, Z.J., 2006. Major histocompatibility complex class IIB allele polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys olivaceus). Mar. Biotechnol. 8, 600–610.
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Gene journal homepage: www.elsevier.com/locate/gene
MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus) Hui Yu a, Shuwen Tan a,b, Hongjuan Zhao a, Hua Li a,c,⁎ a b c
College of Life Science, Foshan University, Guangdong 528231, China Huanan Normal University, Guangzhou 510631, China Holdone Aquaculture Breeding Limited Company, Foshan, Guangdong 528231, China
a r t i c l e
i n f o
Article history: Accepted 17 May 2013 Available online 30 May 2013 Keywords: Grass carp (Ctenopharyngodon idellus) MH-DAB gene Flavobacterium columnare Polymorphism Expression Disease resistance
a b s t r a c t In this study, the association between MH-DAB gene polymorphism and disease resistance was evaluated by challenging grass carp (Ctenopharyngodon idellus) with Flavobacterium columnare. Eight genotypes and six alleles were found, and named by common nomenclature. The genotypes AA, BB, EE, and DE, and the alleles Ctid-DAB1*0101, Ctid-DAB1*0201 and Ctid-DAB1*0401 were more preponderant in fish. The genotype BB was associated with higher resistance to F. columnare, as well as two alleles Ctid-DAB*0101 and Ctid-DAB*0201. Allele Ctid-DAB*0102 has decreased resistance to F. columnare. The expression of MH-DAB gene was decreased in the liver, kidney, and intestine but not in the spleen, gill, and skin at 2 days post infection (dpi), versus to that in the control group. MH-DAB gene expression was up-regulated in most tissues but remained at normal levels in the intestine at 15 days post infection. Our data suggested that MH-DAB polymorphism can be used as a potential genetic marker for disease resistance breeding of grass carp in the future. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Grass carp (Ctenopharyngodon idellus), which belong to the Cyprinidae family, are widely cultured in China. It is one of the most important freshwater cultured species due to its fast growth rate and good taste. Because of the frequent outbreaks of viral and bacterial diseases in cultivation, the mortality of grass carp is as high as 90%. Flavobacterium columnare, the causative agent of columnaris disease, is a big threat to grass carp, causing severe gill rot and occasional skin ulceration (Darwish et al., 2004). Many vaccine strategies have been used against F. columnare, but with limited success. Gene-assisted selection, which enhances fish resistance to columnaris disease, will be a promising approach to this problem. Disease resistance breeding programmes had been studied in many fish, including Atlantic salmon and rainbow trout (Midtlyng et al., 2002). However, a number of problems still need to be solved, such as the maintenance of the stable heredity of the diseased resistant characteristics and the selection of the target genes. Abbreviations: MHC, major histocompatibility complex; dpi, days post infection; PCR, polymerase chain reaction; PCR-SSCP, PCR-single-strand conformation polymorphism; LD50, lethal dose 50; cfu, colony-forming units; DNA, deoxyribonucleic acid; s, second; min, minute; h, hour; V, volta; RNA, ribonucleic acid; cDNA, complementary deoxyribonucleic acid; BLAST, Basic Local Alignment Search Tool; bp, basic pair; PBR, peptide binding residue; HLA-DR1, human leukocyte antigen-DR1; dN, nonsynonymous substitution rate; dS, synonymous substitution rate; IHNV, infectious hematopoietic necrosis virus. ⁎ Corresponding author at: College of Life Science, Foshan University, No. 1 Xianhu University Road, Nanhai, Foshan, Guangdong, China. Tel.: +86 757 85505797; fax: +86 757 85505013. E-mail address: [email protected] (H. Li). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.05.019
The major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity, characterised with abundant polymorphism. The MHC genes encode cell-surface glycoproteins, which bind and present endogenous and exogenous peptides to T cells, triggering a specific immune response towards the pathogen. MHC class II molecules present mostly exogenous short peptides to CD4+ T helper cells. Generally, MHC class I α and class II genes are highly polymorphic, especially in exon 2 of both genes, which encodes α1 and β1 domains that are principally responsible for peptide binding (Eizaguirre et al., 2012; Langefors et al., 2001). Different allelic MHC molecules bind and present specific peptides, thus the response of an organism towards certain pathogens can be influenced by the different haplotypes of MHC (Rakus et al., 2008). Some pathogens may escape recognition by certain MHC molecules since their peptides are not presentable by MHC, which can lead to increase of the susceptibility to pathogens. Contrarily, resistance to pathogens may be derived through high affinity binding of certain peptides by specific MHC alleles (Kjoglum et al., 2006). It is necessary to detect resistant alleles of MHC in economic species for molecular marker-assisted selective breeding programme, and exon 2 in MHC class II gene was considered to be the responsible candidate. In teleosts, MHC genes are named MH because the MHC I and MHC II genes reside on different chromosomes and segregate independently (Grimholt et al., 2003). MH II genes have been isolated and characterised in fish species including common carp (Hashimoto et al., 1990), cichlid (Murray et al., 2000), and rainbow trout (Grimholt et al., 2000). The expression profiles of these genes have also been analysed (Chen et al., 2006; Shen et al., 2011; Stet et al., 2002). The association between
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polymorphism of MH II genes and disease resistance has been established in several fish species (Glover et al., 2007; Wynne et al., 2007; Zhang et al., 2006). However, there has been no research about the association between the MH-DAB gene polymorphism and diseased resistance in grass carp. In this study, specific genotypes and alleles resistant to F. columnare were detected within MH-DAB gene exon 2 in grass carp by PCR-SSCP, and the induced expressions of MH-DAB in various tissues were examined after infection with F. columnare. The aim was to evaluate the function of MH-DAB gene and select the potential resistant alleles for grass carp disease resistance improvement.
accession number AB190816) was used as a quantitative housekeeping gene (β2mF: 5′-ggctggcagtttcacctcac-3′, β2mR: 5′-ccaccctttgtctggctttg-3′). The qRT-PCR was conducted using an ABI PRISM 7500 Fast Real-time PCR System with SYBR-Green according to the manufacturer's protocol (Takara, Dalian, China). The PCR was performed in three steps using the following condition: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, 20 s at 56 °C, and 34 s at 72 °C, and a final extension cycle. Relative gene expression data were calculated using the 2−△△Ct method, and the MH-DAB expression level was normalised to β2m gene expression within each sample.
2. Materials and methods
2.4. Sequence polymorphisms and gene differential expression analysis
2.1. Fish rearing and challenge test
The sequences were compared using the BLAST programme. The sequences and predicted amino acid residues were aligned using BioEdit 7.01 software. Variations of the sequences were analysed by MEGA 4.0. Polymorphisms in MH-DAB exon 2 were analysed using DnaSP 4.0. The statistical analysis was carried out with SAS 8.0 software. Differences in the genotypic and allelic frequencies were calculated using the Chi-square test, while comparisons of the expression levels of MH-DAB after the challenge experiment were calculated using a least squares method. Association between the MH-DAB genotypes and resistance to F. columnare was analysed using Cox proportional hazard regressions (Rakus et al., 2009a).
Grass carp (40 g on average) were maintained in aerated tap water at 20 °C in 100 L aquaria with Eheim biofilters, and were sampled to ensure no bacterium or virus infection. After a week of acclimatisation, 342 healthy grass carp were intraperitoneally infected with F. columnare (2.5 × 108 cfu, the LD50 in 72 h). F. columnare was re-isolated from infected fish to confirm if the infection was successful. 80 healthy fish in similar aquariums were mock-injected with the same volume of sterilised normal saline. Tissues were collected at different time points post-infection (from 2 days up to 15 days), and stored at −80 °C. 2.2. Detection of MH-DAB gene polymorphisms
3. Results MH-DAB genotyping was performed using PCR-SSCP, and confirmed by clone and sequencing. Genomic DNA was isolated from the liver by phenol–chloroform extraction and purified using ethanol. Specific primers (ex2F: 5′-tctgacataactgtaatgctgc-3′, ex2R: 5′-cagga gagatcagagtcttg-3′) were designed according to the common carp MH-DAB gene sequence (GenBank accession number Z47757) to amplify exon 2 of MH-DAB gene. The PCR profile included an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 55.5 °C, 50 s at 72 °C, and a final extension step at 72 °C for 10 min. The amplified products were assessed by 1% agarose gel electrophoresis. Before PCR-SSCP analysis, the PCR products were denatured at 95 °C for 10 min, immediately cooled on ice for 15 min, and loaded on the 10% polyacrylamide gel (acrylamide/bis: 39/1). Electrophoresis was performed under optimised conditions (300 V for 15 min, then 120 V for 14 to 18 h at 4 °C). The bands were visualised by silver staining. Each PCR product from the different MH-DAB genotypes was ligated into the PMD-18T vector (Takara, Dalian, China), and cloned into Escherichia coli DH5α competent cells. Positive clones were screened via blue–white selection, detected by PCR with M13+/– primers and sequenced.
3.1. MH-DAB polymorphism analysis Eight different MH-DAB genotypes (AA, BB, CC, DD, AB, DE and DF) were detected, and were shown to consist of six different unique alleles, which were named Ctid-DAB1*0101, *0102, *0201, *0202, *0301, and *0401 (Fig. 1. and Table 1) according to accepted nomenclature rules (Klein et al., 1990). In cyprinid fish species, there are at least two paralogous groups of MH class II B genes (DAB1 and DAB3) (Rakus et al., 2009a), and nucleotide sequence analysis revealed that all the sequences belonged to DAB1 group. The complete exon 2 of MH-DAB was 276 bp, in which 273 bp was translated into 91 amino acids (Fig. 2). There were 73 variable sites, and the variation hotspots were located in exon 2 between 14 to 28 bp, 72 to 110 bp, and 24 to 133 bp (Fig. 3). Amino acid sequence analysis revealed 43 variable sites, and showed higher variability in the peptide binding residues (PBRs) compared with that in the HLA-DR1 protein (Brown et al., 1993). The rate of nonsynonymous substitution (dN) was 0.374, and the rate of synonymous substitution (dS) was 0.275 in the PBRs. The rates of dN and dS of the non-PBRs were 0.123 and 0.173, respectively. The dN/dS ratios in the PBRs and non-PBRs were 1.36 and 1.47, respectively, indicating a positive selection pressure in exon 2 of MH-DAB gene.
2.3. Analysis of MH-DAB gene expression Total RNA was extracted from the tested tissues, including the liver, spleen, kidney, gill, intestine, skin, brain and heart, using an RNAiso™ Reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions. The RNA quality was assessed by electrophoresis on a 1% agarose gel, and the total RNA concentration was determined by measuring the absorbance at 260 nm on a spectrophotometer. cDNA was synthesised using a PrimeScript™ RT Reagent Kit (Takara, Dalian, China). The change of MH-DAB expression in different tissues due to F. columnare infection was analysed using qRT-PCR. Quantitative primers (DABF: 5′-cggcttaactaaacccatc-3′, DABR: 5′-ctccctgatttcttcttgt-3′) were designed according to the carp MH-DAB gene sequence (GenBank accession number CCDAB01). The grass carp β2m gene (GenBank
Fig. 1. Different genotypes as characterised by unique SSCP patterns.
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Fig. 2. Alignment of the nucleotide sequences of exon 2 from MH-DAB genes in grass carp. Dots indicate identity with Ctid-DAB*0101.
3.2. Association between MH-DAB gene polymorphisms and resistance to F. columnare Genotypic and allelic frequencies were analysed, and the most frequently occurring genotype was AA, followed by DD, EE and DE, while alleles of Ctid-DAB1*0101, Ctid-DAB1*0102 and Ctid-DAB1*0401 were preponderant alleles (Tables 1 and 2). In Cox proportional hazard regression analysis (Fig. 4), fish with Ctid-DAB1*0101, Ctid-DAB1*0201 showed a statistically significant increase in resistance to F. columnare (P b 0.05), while Ctid-DAB*0102 significantly increased susceptibility to F. columnare (P b 0.05). Genotype BB was significantly reduced in the hazard ratio (HR) (P b 0.05), and fish with this genotype showed a lower mortality than those without BB, suggesting that genotype BB was associated with increased resistance to F. columnare. 3.3. MH-DAB gene expression MH-DAB gene basal expression was investigated in healthy grass carp. The highest expression levels of MH-DAB gene were predominantly observed in the gill and spleen, followed by the brain, kidney, skin, and intestine; there was only faint expression in the heart and liver (Fig. 5). After challenging with F. columnare (Fig. 6), the expression level of MH-DAB in the liver was 2-fold higher in the control group than in the challenged group at 2 dpi (P b 0.05); but the expression had recovered at 15 dpi in the challenged group. A similar expression profile was detected in the kidney. In the intestine, MH-DAB gene expression was decreased by approximately 67% at 2 dpi (P b 0.05) but was increased at 15 dpi (P b 0.05); however, the expression levels at 15 dpi
were still suppressed relative to the pre-infection expression levels. In the spleen, the expression level increased at 2 dpi (P b 0.05), and remained at a high level at 15 dpi (P b 0.05). In the gill and skin, the expression increased and reached a peak at 15 dpi (P b 0.05). 4. Discussion MHC genes are crucial elements of adaptive immunity. It has been shown that polymorphisms in MHC molecules can result in different disease resistance in human (Hendel et al., 1999), chickens (Li et al., 1997; Simonsen et al., 1982), and teleosts (Rakus et al., 2008; Grimholt et al., 2000). Undoubtedly, the polymorphism of MH II genes will provide related gene markers for disease resistance breeding of teleosts. In our study, eight MH-DAB genotypes and six different alleles were detected on grass carp and abundant polymorphisms were demonstrated in the PBRs of the MH-DAB gene exon 2. Many hypotheses have been proposed to explain the vast amount of polymorphisms in MHC genes (Parham and Ohta, 1996). Pathogen-driven selection, which leads to a heterozygote advantage (over-dominance), and frequency-dependent selection are suggested to be the main reasons for the genetic diversity of the MHC genes (Grimholt et al., 2003). Here, the high ratio of dN/dS and the high allelic diversity suggested a strong positive selection, which explained the high polymorphism rate in exon 2 of the MH-DAB gene in grass carp. Different MH-DAB genotypes and alleles indicated the association between the MH-DAB gene and resistance to F. columnare in grass carp. Two alleles (Ctid-DAB1*0101 and Ctid-DAB1*0201) significantly reduced the hazard ratio, suggesting that these alleles could be
Fig. 3. Alignment of the deduced amino acid sequences of exon 2 in MH-DAB. Dots indicate identity with Ctid-DAB*0101(Brown et al., 1993). Asterisks stand for PBRs, and “p” indicates the polymorphic site.
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Fig. 4. Kaplan–Meier plot presenting the survival of different MH-DAB genotypes (4a) and alleles (4b) in grass carp challenged with the F. columnare. The mortality was recorded every 4 h during the experiment. The experiment was stopped at 25 days post infection (dpi) and surviving fish were sacrificed.
associated with the resistance to F. columnare in grass carp. The association between MH genetic polymorphism and disease resistance or susceptibility has been successfully demonstrated in teleosts. In Atlantic salmon, UBA*0201 and UBA*0301 were considered to be the major resistant alleles against infectious salmon anaemia (Kjoglum
et al., 2006). In Japanese flounder, a significant association has been detected between resistance to Vibrio anguillarum and the polymorphism of the MH-DAB gene (Xu et al., 2008). In common carp, Cyca-DAB1*05 was the resistance allele to CyHV-3, while Cyca-DAB1*02 and Cyca-DAB1*06 were found to be the susceptible
Table 1 Frequency and hazard ratio of MH-DAB alleles. Allele
Frequency
Hazard ratio (95% CI)
Number of fish
Accession number
A (Ctid-DAB*0101) B (Ctid-DAB*0201) C (Ctid-DAB*0301) D (Ctid-DAB*0102) E (Ctid-DAB*0401) F (Ctid-DAB*0202)
24.56% 13.16% 0.88% 29.39% 31.58% 0.44%
0.768* 0.586* 0.354 1.704* 1.013 2.933
93 54 3 138 144 3
JX305295 JX305297 JX305300 JX305296 JX305301 JX305298
Note: The asterisk (*) indicated the significant Hazard ratio (P b 0.05).
(0.608, 0.972) (0.431, 0.800) (0.088,1.422) (1.397, 2.075) (0.829,1.239) (0.937, 9.174)
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Table 2 Frequency, hazard ration and mortality of MH-DAB genotypes. Genotype
Frequency
Hazard ratio (95% CI)
Mortality
Number of fish
AA (Ctid-DAB*0101/*0101) BB (Ctid-DAB*0201/*0201) CC (Ctid-DAB*0301/*0301) DD (Ctid-DAB*0102/*0102) EE (Ctid-DAB*0401/*0401) AB (Ctid-DAB*0101/*0201) DE (Ctid-DAB*0102/*0401) DF (Ctid-DAB*0102/*0202)
21.93% 10.53% 0.88% 18.42% 21.05% 5.26% 21.05% 0.88%
0.827 0.618* 0.355 1.761 0.889 0.527 1.302 2.950
53.33% 52.78% 33.33% 76.19% 62.5% 44.44% 73.61% 66.67%
75 36 3 63 72 18 72 3
(0.587, (0.385, (0.050, (1.276, (0.637, (0.260, (0.954, (0.937,
1.167) 0.991) 2.531) 2.433) 1.236) 1.067) 1.776) 9.259)
Note: The asterisk (*) indicated the significant Hazard ratio (P b 0.05).
alleles (Rakus et al., 2009b). Moreover, a strong association between Cyca-DAB1 heterozygosity and a lower level of parasitaemia after Trypanoplasma borreli infection was found in common carp (Rakus et al., 2009c). In addition, Scma-DBB1*02 was associated with resistance to Edwardsiella tarda in turbot (Du et al., 2012). The constructive expression of MH II genes was tissue specific in normal teleosts. A previous study showed that the MH II genes were highly expressed in the spleen, intestine, kidney, and liver in uninfected carp, which contained high concentrations of lymphoid and myeloid cells (Rodrigues et al., 1995). In our study, the highest constitutive expression was detected in the gill as formerly observed in Atlantic salmon (Koppang et al., 1998), red sea beam (Chen et al., 2006), and Chinese longsnout catfish (Shen et al., 2011), since the gill is essentially protective barriers to the external environment and infectious pathogens (Caipang et al., 2011; Morris et al., 2000). Challenge of the grass carp with F. columnare resulted in a significant change in MH-DAB gene expression. In the skin and gill, increased expressions were found after the challenge test. The expression in the spleen was also initially up-regulated, and remained at a high level during infection, suggesting that there was interference in the
immune function of the spleen during F. columnare infection. A decrease of the MH-DAB gene expression was detected at 2 dpi in the liver, kidney, and intestine, followed by an increase at 15 dpi. This was similar to the expression profile for the MH-DAB gene in rainbow trout, where a decrease in expression was observed by 72 h in the liver, kidney, and spleen, and then, partial restoration occurred by 192 h after infectious hematopoietic necrosis virus (IHNV) infection (Hansen and La Patra, 2002). The same phenomenon was observed in red sea bream treated with V. anguillarum (Chen et al., 2006). These results suggested that the MH-DAB gene played an important role in the immune response to infection in grass carp, which are responsible for presenting non-self peptides to T cell receptors and promoting a specific immune response towards the foreign pathogens. The expression of MH-DAB gene was significantly changed in liver, kidney, and intestine, suggesting that they might be the important and sensitive organs for MH II protein function. In summary, this study implied that MH-DAB gene polymorphism is associated with grass carp resistance to F. columnare, and unique alleles for disease resistance could be used as potential genetic markers for grass carp resistance breeding. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant Nos. 31072205 and 30972251). References
Fig. 5. The expression of the MH-DAB gene in different tissues.
Fig. 6. The expression changes of the MH-DAB gene across different times.
Brown, J.H., et al., 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364, 33–39. Caipang, C.M.A., Lazado, C.C., Brinchmann, M.F., Rombout, J.H.W.M., Kiron, V., 2011. Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua). Comp. Biochem. Physiol.D. 6, 158–162. Chen, S.L., Zhang, Y.X., Xu, M.Y., Ji, X.S., Yu, G.C., Dong, C.F., 2006. Molecular polymorphism and expression analysis of MHC class II B gene from red sea bream (Chrysophrys major). Dev. Comp. Immunol. 30, 407–418. Darwish, A.M., Ismaiel, A.A., Newton, J.C., Tang, J., 2004. Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae. Mol. Cell. Probes 18, 421–427. Du, M., Chen, S.L., Liu, Y.H., Niu, B.Z., Yang, J.F., Zhang, B., 2012. MHC polymorphism and disease-resistance to Edwardsiella tarda in six turbot (Scophthalmus maximus) families. Chin. Sci. Bull. 57, 3262–3269. Eizaguirre, C., Lenz, T.L., Kalbe, M., Milinski, M., 2012. Rapid and adaptive evolution of MHC genes under parasite selection in experimental vertebrate populations. Nat. Commun. 3, 621. Glover, K.A., Grimholt, U., Bakke, H.G., Nilsen, F., Storset, A., Skaala, O., 2007. Major histocompatibility complex (MHC) variation and susceptibility to the sea louse Lepeophtheirus salmonis in Atlantic salmon Salmo salar. Dis. Aquat. Org. 76, 57–65. Grimholt, U., Getahun, A., Hermsen, T., Stet, R.J., 2000. The major histocompatibility class II alpha chain in salmonid fishes. Dev. Comp. Immunol. 24, 751–763. Grimholt, U., et al., 2003. MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar); facing pathogens with single expressed major histocompatibility class I and class II loci. Immunogenetics 55, 210–219.
222
H. Yu et al. / Gene 526 (2013) 217–222
Hashimoto, K., Nakanishi, T., Kurosawa, Y., 1990. Isolation of carp genes encoding major histocompatibility complex antigens. Proc. Natl. Acad. Sci. U.S.A. 87, 6863–6867. Hendel, H., et al., 1999. New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS. J. Immunol. 162, 6942–6946. Kjoglum, S., Larsen, S., Bakke, H.G., Grimholt, U., 2006. How specific MHC class I and class II combinations affect disease resistance against infectious salmon anaemia in Atlantic salmon (Salmo salar). Fish Shellfish Immunol. 21, 431–441. Klein, J., et al., 1990. Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics 31, 217–219. Koppang, E., Lundin, M., Press, C.M.L., Rønningen, K., Lie, Ø., 1998. Differing levels of Mhc class II [beta] chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 8, 183–196. La Hansen, J.D., Patra, S., 2002. Induction of the rainbow trout MHC class I pathway during acute IHNV infection. Immunogenetics 54, 654–661. Langefors, A., Lohm, J., Von Schantz, T., 2001. Allelic polymorphism in MHC class II B in four populations of Atlantic salmon (Salmo salar). Immunogenetics 53, 329–336. Li, L., Johnson, L.W., Ewald, S.J., 1997. Molecular characterisation of major histocompatibility complex (B) haplotypes in broiler chickens. Anim. Genet. 28, 258–267. Midtlyng, P., Storset, A., Michel, C., Slierendrecht, W., Okamoto, N., 2002. Breeding for disease resistance in fish. Bull. Eur. Assoc. Fish Pathol. 22, 166–172. Morris, D., Adams, A., Richards, R., 2000. In situ hybridisation identifies the gill as a portal of entry for PKX (Phylum Myxozoa), the causative agent of proliferative kidney disease in salmonids. Parasitol. Res. 86, 950–956. Murray, B.W., Shintani, S., Sultmann, H., Klein, J., 2000. Major histocompatibility complex class II A genes in cichlid fishes: identification, expression, linkage relationships, and haplotype variation. Immunogenetics 51, 576–586. Parham, P., Ohta, T., 1996. Population biology of antigen presentation by MHC class I molecules. Science 272, 67–74. Rakus, K.L., Wiegertjes, G.F., Adamek, M., Bekh, V., Stet, R.J., Irnazarow, I., 2008. Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.). Fish Shellfish Immunol. 24, 734–744.
Rakus, K.Ł., Irnazarow, I., Forlenza, M., Stet, R.J.M., Savelkoul, H.F.J., Wiegertjes, G.F., 2009a. Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes. Fish Shellfish Immunol. 26, 352–358. Rakus, K.L., Wiegertjes, G.F., Adamek, M., Siwicki, A.K., Lepa, A., Irnazarow, I., 2009b. Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism. Fish Shellfish Immunol. 26, 737–743. Rakus, K.L., Wiegertjes, G.F., Jurecka, P., Walker, P.D., Pilarczyk, A., Irnazarow, I., 2009c. Major histocompatibility (MH) class II B gene polymorphism influences disease resistance of common carp (Cyprinus carpio L.). Aquaculture 288, 44–50. Rodrigues, P.N., Hermsen, T.T., Rombout, J.H., Egberts, E., Stet, R.J., 1995. Detection of MHC class II transcripts in lymphoid tissues of the common carp (Cyprinus carpio L.). Dev. Comp. Immunol. 19, 483–496. Shen, T., Xu, S., Yang, M., Pang, S., Yang, G., 2011. Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish (Leiocassis longirostris). Vet. Immunol. Immunopathol. 141, 33–45. Simonsen, M., Crone, M., Koch, C., Hala, K., 1982. The MHC haplotypes of the chicken. Immunogenetics 16, 513–532. Stet, R.J., et al., 2002. Unique haplotypes of co-segregating major histocompatibility class II A and class II B alleles in Atlantic salmon (Salmo salar) give rise to diverse class II genotypes. Immunogenetics 54, 320–331. Wynne, J.W., Cook, M.T., Nowak, B.F., Elliott, N.G., 2007. Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 22, 707–717. Xu, T.J., Chen, S.L., Ji, X.S., Tian, Y.S., 2008. MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families. Fish Shellfish Immunol. 25, 213–221. Zhang, Y.X., Chen, S.L., Liu, Y.G., Sha, Z.X., Liu, Z.J., 2006. Major histocompatibility complex class IIB allele polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys olivaceus). Mar. Biotechnol. 8, 600–610.