我国研制出检测奶牛隐性有害基因新方法

如何保证种奶牛一代又一代品质优良,拒绝遗传病留给下一代,对奶牛隐性遗传缺陷基因的检测非常关键。因此这一领域成为近年国外奶业发达国家研究的热点。李艳华日前在《遗传》杂志发表“利用单链构象多态性检测牛白细胞黏附缺陷症方法的建立”的文章,报告了她的研究成果：利用自行设计的特异性PCR引物．通过对PCR、PCR—SSCP条件的筛选和优化,并结合DNA序列测定．建立了PCR—SSCP检测牛白细胞黏附缺陷症（BLAD）有害基因的新方法。     

牛白细胞黏附缺陷症(BLAD)的检测与单倍型分析

牛白细胞黏附缺陷症（BLAD）是由常染色体上CD18基因单碱基突变（A→G）引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病从而影响生产性能的表现,严重的影响了奶牛场的经济效益。本研究重点对牛白细胞黏附缺陷症（BLAD）的致病基因CD18基因第2外显子（exon2）进行检测分析,通过对样品进行PCR-SSCP分析,结果发现了4种单倍型：单倍型H1、H2、H3及H4,经测序发现,此扩增片段共存在2个SNPs位点,分别位于CD18基因exon2的C20T和A55G位点,其中C20T突变是首次发现,属于同义突变,而A55G突变即是引起BLAD的错义碱基突变,单倍型为H1、H2和H3的个体是正常牛,单倍型H4是BLAD携带者,从而建立了一种筛选BLAD有害基因的新方法-SSCP法。     

中国荷斯坦牛白细胞粘附缺陷病遗传分析

荷斯坦牛白细胞粘附缺陷病（BLAD）是一种遗传性免疫缺陷病，患病牛出生后，生长发育差，绝大多数在1年内死亡，且不具繁殖和哺育能力。该病的遗传基础为CD18基因编码区383位的A／G点突变导致白细胞表面的β2整合素表达缺陷。目前欧美等奶牛业发达国家都已经建立了完善的BLAD分子检测方法和跟踪监控体系。中国长期从国外进口荷斯坦公牛精液、胚胎或活体，由此可能引进了BLAD基因。本研究采用PCR—RFLP方法对116头可疑中国荷斯坦牛进行了检测．确定了2头BLAD携带者公牛和8头携带者母牛．未发现隐性纯合个体．     

牛白细胞粘附缺陷病携带牛CD18的部分基因序列分析

牛白细胞粘附缺陷病（bovine leukocyte adhesion deficiency ,BLAD）是一种常染色体单基因隐性遗传疾病。目前虽已确定BLAD病因与人的白细胞粘附缺陷病（leukocyte adhesion deficiency, LAD），为白细胞表面的一种称为整合素CD18亚单位表达缺陷所致，但至今关于CD18基因突变情况研究较少。为了深入研究和探讨BLAD发病机制，本研究将在我国黑龙江省东部地区调查发现的6头BLAD携带牛CD18部分基因进行了克隆和序列比较分析。     

北京地区荷斯坦牛白细胞黏附缺陷症的检测分析

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,患病牛只机体免疫力降低、易患病,影响其生产性能的表现。本研究采用限制性片段长度多态性聚合酶链反应方法(PCR-RFLP),检测了北京502头荷斯坦母牛CD18编码基因第二外显子的多态性。结果表明,3个奶牛场中共检测到2种基因型——AA和AG,未检测到GG基因型。检测群体BLAD基因携带率为3.1%,说明北京地区规模化奶牛场使用的公牛冻精中BLAD基因没有得到彻底净化,导致母牛群体中BLAD基因携带率在3.0%以上。在奶牛育种和生产中,应有计划的淘汰携带BLAD基因的个体,净化京郊荷斯坦奶牛群体。     

北京地区荷斯坦牛BLAD遗传缺陷基因的分子筛查

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病,从而影响生产性能的表现,可严重影响奶牛场的经济效益。本研究利用PCR-SSCP方法对北京地区246头公牛样本和409头母牛样本进行了检测分析,研究结果表明,在所检测的样本中,荷斯坦种公牛(含后备公牛)和母牛BLAD携带率分别为0.81%和3.91%,BLAD基因频率分别为0.21%和1.95%,并通过系谱追踪发现,BLAD遗传缺陷可追溯到美国一头非常优秀的公牛"Osborndale Ivanhoe"(USA.1189870,BL),其后代在我国有一定的影响。通过剔除BLAD携带者公牛可以有效控制该遗传缺陷的传播,但是,我国部分BLAD携带者公牛冻精依然在商业化使用,所以,有效监控BLAD携带者在奶牛群中的状况对BLAD剔除计划是有益的。     

奶牛白细胞黏附缺陷症焦磷酸测序检测方法的建立及应用

奶牛白细胞黏附缺陷症（bovine leukocyte adhesion deficiency,BLAD）是一种常染色体隐性、致死性、遗传性疾病,严重影响奶牛生产性能和奶牛场的经济效益。为建立快速可靠的BLAD检测方法,本试验根据GenBank中BLAD CD18序列设计引物,PCR扩增后纯化产物,构建A/A、A/G和G/G 3种基因型标准质粒。用标准质粒建立焦磷酸测序检测方法。用建立的方法检测奶牛血液样品,其结果与Sanger测序结果进行比较,分析和验证检测结果的准确性。用建立的方法对300份奶牛血液样品进行检测,结果显示样品中A/A基因型检出294例,占总体比例的98%;A/G基因型检出6例,占总体比例的2%。随机抽取30份已检测样品进行Sanger测序,结果显示与焦磷酸测序法结果一致。本试验结果表明焦磷酸测序法检测BLAD,具有特异、灵敏、快速的特点,其检测结果准确可靠,适合于试剂盒的研发及应用于BLAD的临床诊断。     

中国荷斯坦种公牛BLAD遗传缺陷的分子检测及系谱分析

本试验运用限制性片段长度多态性聚合酶链式反应（RFLP-PCR）方法,检测我国荷斯坦种公牛白细胞粘附缺陷（bovine leukocyte adhesion deficiency,BLAD）基因的携带频率。共检测了来自全国14个公牛站的587头种公牛,发现BLAD携带者8头,携带率为1.36%。对现有公牛系谱信息分析显示,携带者公牛来自美国、加拿大和中国,其中6头携带者公牛可以追溯到共同祖先Osborndale Ivanhoe。此外,本研究还对我国荷斯坦牛遗传缺陷的控制和携带者公牛的利用提出建议。     

牛白细胞黏附缺陷病等位基因频率分析

为了解荷斯坦奶牛群中成母牛白细胞黏附缺陷病情况,抽取1 000头牛血样,采用RT-PCR方法对血样进行分析,并用TaqⅠ内切酶对RT-PCR产物进行消化,再用1.2%琼脂糖凝胶分析Taq Ⅰ限制性位点酶切片段.结果表明:1 000头荷斯坦奶牛中有19头BLAD基因携带者,1头BLAD基因纯合个体;BLAD基因携带者和纯合个体所占的比率分别为1.9%和0.1%;无BLAD和携带BLAD的基因频率分别为0.989 5和0.010 5.     

牛·白·细·胞·粘·附·缺·陷·病·的·诊·断

牛白细胞粘附缺陷病（bovine leukocyte adhesion deficiency,BLAD）是一种常染色体单基因隐性遗传疾病,病因为白细胞表面整合素β（CD18）亚单位的编码383位碱基由A突变为G所致。自1983年报道本病发生以来,BLAD已给奶牛业造成了巨大的经济损失。2004年10月我们在进行本病调查时,在黑龙江省大庆市某奶牛繁殖育种中心发现1头编号为1054的奶牛患有BLAD,报道如下。     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background: Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population. Results: We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM. Conclusions: These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background

Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population.

Results

We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM.

Conclusions

These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

Bovine leukocyte adhesion deficiency (BLAD): a review

Bovine leukocyte adhesion deficiency (BLAD) in Holstein cattle is an autosomal recessive congenital disease characterized by recurrent bacterial infections, delayed wound healing and stunted growth, and is also associated with persistent marked neutrophilia. The molecular basis of BLAD is a single point mutation (adenine to guanine) at position 383 of the CD18 gene, which caused an aspartic acid to glycine substitution at amino acid 128 (D128G) in the adhesion molecule CD18. Neutrophils from BLAD cattle have impaired expression of the beta2 integrin (CD11a,b,c/CD18) of the leukocyte adhesion molecule. Abnormalities in a wide spectrum of adherence dependent functions of leukocytes have been fully characterized. Cattle affected with BLAD have severe ulcers on oral mucous membranes, severe periodontitis, loss of teeth, chronic pneumonia and recurrent or chronic diarrhea. Affected cattle die at an early age due to the infectious complications. Holstein bulls, including carrier sires that had a mutant BLAD gene in heterozygote were controlled from dairy cattle for a decade. The control of BLAD in Holstein cattle by publishing the genotypes and avoiding the mating between BLAD carriers was found to be successful. This paper provides an overview of the genetic disease BLAD with reference to the disease in Holstein cattle.     

Screening for bovine leukocyte adhesion deficiency,deficiency of uridine monophosphate synthase,complex vertebral malformation,bovine citrullinaemia,and factor XI deficiency in Holstein cows reared in Turkey

Background

Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins.

Methods

Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not.

Results

Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID).

Conclusion

This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.     

Detection of bovine leukocyte adhesion deficiency (BLAD) in Turkish native and Holstein cattle

The purpose of this work was to study whether the bovine leukocyte adhesion deficiency (BLAD) allele is present in native cattle breeds and the Holstein breed in Turkey. Blood samples were obtained from 120 Holstein, 20 Brown Swiss, 20 Anatolian Black, 20 Turkish Grey, 20 South Anatolian Red and 20 East Anatolian Red cattle. The isolated DNA materials were multiplied in PCR using the primer developed by Kriegesmann et al. (1997). In order to determine the area of mutation in PCR products, the PCR products were digested with TaqI endonuclease enzyme. The resulting fragments were analysed on 2% agarose gel for the absence of a TaqI restriction site. It was found that two of the Holstein cattle (a bull and a cow) were heterozygote BLAD carriers. There was no homozygote BLAD animal. The BLAD allele was not found in the other breeds used in the study. The mutant BLAD allele frequency in the 120 Holstein cattle calculations was 0.0084.     

奶牛白细胞黏附缺陷症(BLAD)检测方法的研究进展

奶牛白细胞黏附缺陷症是近年新发现的一种常染色体单基因隐性遗传病,由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病从而影响生产性能,严重影响了奶牛场的经济效益。文章综述了该病检测方法的研究进展。     

In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non-BLAD calves.     

Bovine leukocyte adhesion deficiency: in vitro assessment of neutrophil function and leukocyte integrin expression.

Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders.     

中国荷斯坦奶牛RPL23A、ACACB基因3个SNPs检测及其与产奶性状的关联分析

旨在开展奶牛群体高乳成分功能基因的验证与筛选,利用微流控芯片自主选育技术分析北京地区母牛群体高乳蛋白、高乳脂基因频率,同时分析基因多态性及其与产奶性状的相关性。本研究对北京地区8个大型奶牛场1 596头中国荷斯坦奶牛RPL23A、ACACB基因的多态性进行了检测,所有个体均为3胎以内的泌乳牛,收集每头牛所有测定日的产奶量、乳脂率、乳蛋白率等数据,同时对3个多态位点不同基因型与产奶性状进行了关联分析。RPL23A基因的SNP位点g.20146771G>A,在第一泌乳期,与产奶量、乳脂量、乳脂率和乳蛋白量均达到极显著关联（P<0.01）,第二泌乳期,g.20146771G>A与5个产奶性状均呈极显著关联（P<0.01）。ACACB基因的SNP位点g.63962768C>T,在第一泌乳期,与产奶量、乳脂量、乳脂率和乳蛋白量均呈极显著关联（P<0.01）,在第二泌乳期,与产奶量、乳脂量、乳脂率和乳蛋白率呈极显著关联（P<0.01）。结果表明,RPL23A、ACACB基因可以作为影响中国荷斯坦奶牛产奶性状的候选基因用于标记辅助选择,以上基因位点可能通过直接或间接的途径影响奶牛的乳脂或乳蛋白性状,对产奶性状起到重要调控作用。本研究为荷斯坦奶牛后续的标记辅助选择奠定了良好基础。