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。     

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．     

Radiographic and immunohistochemical analysis of leukocyte adhesion deficiency in Holstein heifers.

Radiographic and immunohistochemical analyses were performed in two Holstein heifers with leukocyte adhesion deficiency (BLAD). Severe bone resorption, osteolysis and severe progressive periodontitis in submandibula due to dysfunction of leukocytes in heifers affected with BLAD were demonstrated by radiographic examination. Immunohistochemical analysis of lymph nodes using anti-CD18 monoclonal antibody demonstrated that CD18-positive cells were not found on those from a heifer affected with BLAD, whereas CD18-positive cells were clearly present in lymph nodes from a clinically normal heifer. These characteristic findings support the importance of adherence-dependent leukocyte functions in host defense.     

Somatic hypermutations and isotype restricted exceptionally long CDR3H contribute to antibody diversification in cattle

Antibody diversification in IgM and IgG antibodies was analyzed in an 18-month old bovine (Bos taurus) suffering from naturally occurring chronic and recurrent infections due to bovine leukocyte adhesion deficiency (BLAD). The BLAD, involving impaired leukocyte β2 integrin expression on leukocytes, develops due to a single point mutation in conserved region of the CD18 gene resulting in substitution of aspartic acid128 with glycine (D128G). Twenty four VDJCμ and 25 VDJCγ recombinations from randomly constructed cDNA libraries, originating from peripheral blood lymphocytes, were examined for the variable-region structural characteristics in IgM and IgG antibody isotypes. These analyses led to conclude that: (a) expression of exceptionally long CDR3H is isotype restricted to cattle IgM antibody; (b) VDJ recombinations encoding IgM with exceptionally long CDR3H undergo clonal selection and affinity maturation via somatic mutations similar to conventional antibodies; (c) somatic mutations contribute significantly to both IgM and IgG antibody diversification but significant differences exist in the patterns of ‘hot spot’ in the FR1, FR3 and CDR1H and, also, position-dependant amino acid diversity; and (d) transition nucleotide substitutions predominate over transversions in both VDJCμ and VDJCγ recombinations consistent with the evolutionary conservation of somatic mutation machinery. Overall, these studies suggest that both somatic mutations and exceptional CDR3H size generation contribute to IgM and IgG antibody diversification in cattle during the development of immune response to naturally occurring chronic and multiple microbial infections.     

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.     

Bovine leukocyte adhesion deficiency in Holstein cattle.

Two Holstein heifers with persistent and recurrent infections including ulcerative gingivitis, periodontitis, pneumonia, loss of teeth and stunted growth associated with marked neutrophilia were evaluated clinically and for neutrophil function, CD18 expression on neutrophils and CD18 genotype analysis by DNA-polymerase chain reaction (PCR) test. Adherence to nylon fibers and phagocytic activity of neutrophils from affected animals were significantly (p < 0.05) impaired as compared with those of controls. Neutrophils from affected heifers had decreased chemiluminescent (CL) responses when stimulated with opsonized zymosan, compared with those of controls. In contrast, neutrophils from affected heifers produced increased CL responses when stimulated with latex beads and phorbol myristate acetate compared with those of controls. The clinical findings, functional leukocyte abnormalities, deficiency in expression of CD18 on neutrophils, and the D128G mutation detected by DNA-PCR testing of affected heifers demonstrated that these heifers have bovine leukocyte adhesion deficiency (BLAD). Although both animals were confirmed to be homozygotes for BLAD by DNA-PCR test, they had differences in clinical, hematological and neutrophil functional characteristics.     

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.     

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.     

牛白细胞黏附缺陷病液相基因芯片检测方法的建立

从福建某奶牛场200头进口荷斯坦牛采血提取牛血液DNA，根据已知牛染色体上CD18编码基因序列383位基由A变为G而引起牛白细胞黏附缺陷症（BLAD）设计特异性野生型和突变型引物，建立液相基因芯片检测方法用于牛白细胞黏附缺陷症（BLAD）的检测，结果检出2头母牛为BLAD携带者，检出率为0．67％，没有发现患病牛。结果证明建立的液相基因芯片检测方法是一种敏感性和特异性很高的筛选奶牛BLAD有害基因的新方法     

Bovine leucocyte adhesion deficiency: A review of a modern disease and its implications

Bovine leucocyte adhesion deficiency (BLAD) is a genetic disease of cattle which affects the hematopoietic system. In the last decade BLAD has become a disease of economic importance in the dairy industry. This review describes the chronological developments and thinking that led to the elucidation of BLAD as a disease distinct from previous models in canine and human populations. All species affected show signs of chronic and recurrent infections. Necrotic and/or gangrenous infections of soft tissues are prevalent, in addition to secondary infections with bacteria or fungi. Low birthweight and unthriftiness are key signs in all species affected by leucocyte adhesion deficiency (LAD). Dermatomycoses and impaired pus formation are also common findings. The physiological basis for BLAD is a deficiency in the chemotactic and phagocytic properties of leucocytes and particularly neutrophils. The inhibition of diapedesis in the inflammatory response prevents normal immune reactions to invading pathogens. Chronic infections are a consequence of the faulty immune mechanisms. The biochemical aetiology of BLAD involves cell surface glycoprotein molecules known as integrins. These are responsible for the cell-cell interactions necessary for neutrophils to adhere to vascular endothelium in a normal individual. Experiments with monoclonal antibodies to block LFA-1, Mac-1, and p150,95 (three integrins vital for cell-cell interactions) mimic BLAD symptomatology and have led to the discovery of the reciprocal intercellular adhesion molecule (ICAM). Through pedigree analysis and biochemical detection with restrictive endonucleases, BLAD has been isolated genetically to a single gene locus. The economic significance and prophylaxis of the disease are briefly discussed. In addition, the beneficial aspects of the study of BLAD are considered. There are advantages in producing a BLAD-like state for preventing transplant rejection, ischaemia-reperfusion injury and other problems arising from the deleterious effects of the inflammatory response.     

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。     

Canine leukocyte adhesion deficiency colony for investigation of novel hematopoietic therapies

The genetic immunodeficiency disease canine leukocyte adhesion deficiency (CLAD) was originally described in juvenile Irish Setters with severe, recurrent bacterial infections. CLAD was subsequently shown to result from a mutation in the leukocyte integrin CD18 subunit which prevents leukocyte surface expression of the CD11/CD18 complex. We describe the development of a mixed-breed CLAD colony with clinical features that closely parallel those described in Irish Setters. We demonstrate that the early identification of CLAD heterozygotes and CLAD-affected dogs by a combination of flow cytometry and DNA sequencing allows the CLAD-affected animals to receive life-saving antibiotic therapy. The distinct clinical phenotype in CLAD, the ability to detect CD18 on the leukocyte surface by flow cytometry, and the history of the canine model in marrow transplantation, enable CLAD to serve as an attractive large-animal model for the investigation of novel hematopoietic stem cell and gene therapy strategies.     

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.     

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 Danish Holstein-Friesian Cattle: I. PCR Screening and Allele Frequency Estimation

Jergensen, C. B., J. S. Agerholm, J. Pedersen and P. D. Thomsen: Bovine leukocyte adhesion deficiency in Danish Holstein-Friesian Cattle I. PCR screening and allele frequency estimation. Acta vet. Scand. 1993,34,231-236.–A screening program for bovine leukocyte adhesion deficiency (BLAD) in Danish Holstein-Friesian cattle has been initiated. During the first months 1611 animals were tested by a PCR based assay. Of these animals 1256, 346, and 8 were assigned normal, BLAD carriers, and BLAD affected animals, respectively One bull, born as a co-twin, showed weak reaction for the BLAD allele on DNA isolated from leukocytes, but a normal genotype on DNA isolated from semen. Chromosome analysis showed that this bull was a blood chimaera. Estimation of the BLAD allele frequency upon the PCR test results showed that around 450 Danish calves born in 1991 might have been affected with the recessive disorder.     

Cytokine profile of a Holstein calf with bovine leukocyte adhesion deficiency during the acute-phase inflammatory response

Changes in interleukin (IL)-1beta, IL-6 and IL-8 in serum, and their mRNA expression on neutrophils from a 4.6-month old Holstein young calf with bovine leukocyte adhesion deficiency (BLAD) during the acute phase were evaluated. IL-1beta concentrations in the serum of the calf with BLAD at age 143-162 days ranged from 8.7 to 16.6 ng/ml, whereas the values were less than 2.7 ng/ml in control calves. Serum IL-6 (0.04 ng/ml) was only detected on the 1st day when the animal was diagnosed with the BLAD. IL-1beta and IL-8 mRNA expression on neutrophils from the affected calf appeared to be similar to those of controls. Serum cytokine levels and their mRNA expression on neutrophils from the calf with BLAD appeared to be little affected by the deficient expression of beta(2)-integrin on leukocytes, and are considered to be modulated by the inflammatory stimuli.     

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。