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.     

美国发布2000年狂犬病死亡病例报告感染途径值得关注

美国 IAC( Immunization Action Coalition)于 2 0 0 0年 1 2月 2 1日在其“IAC EXPRESS”中发布了2 0 0 0年死于狂犬病的病例报告 ,这是美国自 1 998年 1 2月以来首次发现狂犬病。在 2 0 0 0年中 ,美国共有 5人死于狂犬病 ,其中只有 1人生前曾被未经免疫的幼犬咬伤过拇指和腿 ,其余 4例均与接触蝙蝠有关。据死于 9月 2 0日的 49岁的病人生前报告 ,他没有与蝙蝠接触过 ,但他妻子说 ,在 2 0 0 0年 6月或 7月间 ,一只蝙蝠曾飞进他们居住的房子 ,该病人将其弄了出去 ( remove it)。　　死于 1 0月 1 0日的 2 6岁的病人 ,自 2 0 0 0年 7…     

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.     

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.     

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.     

Analysis of serum cytokine profile in a holstein heifer with leukocyte adhesion deficiency which survived for long period

Serum cytokine levels and their expression of mRNA on neutrophils from a bone marrow (BM) transplanted heifer with leukocyte adhesion deficiency were evaluated. The clinical condition of the affected heifer was relatively stable after BM-transplantation. Persistent hyper gamma-globulinemia and increased serum immunoglobulin G (IgG) concentrations were monitored longitudinally. The concentration of interleukin (IL)-1beta in serum from the affected heifer ranged from 15.8 to 321.7 ng/ml, and maximum concentration occurred at the time which coincided with peak IL-6. Serum levels of IL-6 ranged from 0.32 to 27.9 ng/m l, and they appeared to be associated with the increment of serum IgG in the affected heifer. mRNAs for IL-1beta, IL-6, IL-8 and granulocyte and macrophage colony stimulating factor (GM-CSF) were increased in neutrophils from the affected heifer compared to controls. Persistent hyper gamma-globulinemia of the affected heifer appeared to be associated with enhanced mRNA expression for IL-6 and its serum levels. These findings suggest that humoral immunity of the affected heifer is activated and the production of neutrophils appears to be enhanced under the incapability of beta(2) integrin-mediated functions of phagocytic cells.     

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。     

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.     

重组牛抵抗素对体外培养肝细胞PEPCK活性的影响

为了探讨重组牛抵抗素对体外培养肝细胞PEPCK活性的影响,取72h培养良好的肝细胞培养板(6孔板),每孔分别接种重组牛抵抗素0,10,50,100,300,500ng/L(共6个梯度,每个梯度6个重复),继续培养12h,收集上清,采用乳酸脱氢酶偶联比色法测定PEPCK活性。结果显示,PEPCK酶活性随重组牛抵抗素浓度的升高而增强,10ng/L以上的重组牛抵抗素可明显提高PEPCK活性,这表明牛抵抗素具有生物学活性,能提高PEPCK活性。     

The stress of weaning influences serum levels of acute-phase proteins, iron-binding proteins, inflammatory cytokines, cortisol, and leukocyte subsets in Holstein calves

The purpose of our study was to investigate changes in immunological parameters induced by weaning stress (including milk restriction) in calves. Fifteen Holstein calves were subjected to weaning at 6 weeks of age. Blood samples were collected at -14, -7, -2, 1, 3, and 5 days post-weaning (DPW; 0 DPW = 42 days). Weaning caused significant (p < 0.01) increases in the neutrophil (NE):lymphocyte (LY) ratio at 5 DPW with a significant (p < 0.05) reduction of LYs. The concentration of acute-phase proteins (haptoglobin and serum amyloid A) also increased significantly (p < 0.05) at 3 and 5 DPW compared to -2 DPW. Levels of the iron-binding protein lactoferrin decreased significantly (p < 0.05) after weaning. Serum tumor necrosis factor-α and cortisol levels were elevated (p < 0.05) at 3 DPW, while those of serum interferon-γ decreased (p < 0.05) at 1 and 3 DPW compared to levels observed before weaning. Weaning significantly (p < 0.05) decreased the percentage of CD25⁺ T cells in the peripheral blood. In conclusion, weaning stress affected the NE:LY ratio along with the levels of acute phase proteins, lactoferrin, cortisol, and inflammatory cytokines in the peripheral blood of calves. Weaning stress may induce an acute phase response possibly through the elevation of cortisol production and modulation of inflammatory cytokines.     

Apoptosis of resident and inflammatory macrophages before and during the inflammatory response of the virgin bovine mammary gland

Background

Macrophages may play a prominent role in defense of the bovine mammary gland, and their functionality is necessary for successful eradication of bacterial pathogens. In contrast to necrosis, however, apoptosis has not yet been studied in macrophages from bovine mammary glands. Therefore, the aim of this study was to confirm the occurrence of apoptosis in macrophages from resting heifer mammary glands and during the inflammatory response.

Methods

Inflammatory response was induced by phosphate buffered saline (PBS) and by lipopolysaccharide (LPS). Resident macrophages (_RESMAC) were obtained before and inflammatory macrophages (_INFMAC) 24, 48, 72 and 168 hours after inducing inflammatory response in mammary glands of unbred heifers. Cell samples were analyzed for differential counts, apoptosis and necrosis using flow cytometry.

Results

Populations of _RESMAC and _INFMAC contained monocyte-like cells and vacuolized cells. Apoptosis was detected differentially in both morphologically different types of _RESMAC and _INFMAC and also during initiation and resolution of the inflammatory response. In the _RESMAC population, approximately one-tenth of monocyte-like cells and one-third of vacuolized cells were apoptotic. In the _INFMAC population obtained 24 h after PBS treatment, approximately one-tenth of monocyte-like cells and almost one-quarter of vacuolized cells were apoptotic. At the same time following LPS, however, we observed a significantly lower percentage of apoptotic cells in the population of monocyte-like _INFMAC and vacuolized _INFMAC. Moreover, a higher percentage of apoptotic cells in _INFMAC was detected during all time points after PBS in contrast to LPS. Comparing _RESMAC and _INFMAC, we observed that vacuolized cells from populations of _RESMAC and _INFMAC underwent apoptosis more intensively than did monocyte-like cells.

Conclusions

We conclude that apoptosis of virgin mammary gland macrophages is involved in regulating their lifespan, and it is involved in the resolution process of the inflammatory response.     

The clinical and pathological findings in two Friesian calves with leukocyte adhesion deficiency

Leukocyte adhesion deficiency is an inherited defect of neutrophil function reported in man, dogs and cattle. In cattle it was first described in 1983 and to date is found only in Holstein Friesians where it is inherited as an autosomal recessive trait. The neu-trophils of affected calves are less able to migrate from blood vessels to sites of inflammation, thus greatly compromising their ability to fight infection.     

Congenital enlargement of the suburethral diverticulum in a Holstein calf

A 3-month-old, female Holstein calf was examined because of marked perineal swelling and tenesmus of 4-days duration. A congenitally enlarged urethral diverticulum was diagnosed using fluoroscopic and ultrasonographic imaging techniques. The urethral diverticulum was surgically resected and the perineal area was reconstructed.     

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.     

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.     

Pancytopenia with bleeding tendency associated with bone marrow aplasia in a Holstein calf

An 11-day-old Holstein calf presented with a high rectal temperature and tachypnea. Treatment with antibiotics and non-steroidal anti-inflammatory drugs did not improve the clinical signs. Bleeding tendency, with several hemorrhage spots on the body surface, appeared five days after admission. Severe pancytopenia was observed in the blood examination. The calf died on the 11th day after admission with severe bleeding from an injection site. Necropsy findings revealed that the pancytopenia had resulted from severe bone marrow aplasia. A congenital disorder was suspected to be the cause of pancytopenia associated with bone marrow aplasia.