Bovine Leukocyte Adhesion Deficiency Research Articles

Analysis of surface antigen expression and host defense function in leukocytes from calves heterozygous or homozygous for bovine leukocyte adhesion deficiency

Abstract Objective To analyze surface antigen expression and functional responses of leukocytes from calves heterozygous and homozygous for bovine leukocyte adhesion deficiency (BLAD). Animals 8 clinically normal calves, 4 calves heterozygous for BLAD, and 4 calves homozygous for BLAD. Procedure Surface antigen expression was examined by flow cytometric analysis of leukocytes stained with monoclonal antibodies. Neutrophil function analyses included phagocytosis and killing of Candida albicans and measurement of respiratory burst activity using cytochrome c and dihydrorhodamine 123 assays. Differential leukocyte counts also were performed. Results Leukocytes from heterozygous calves were similar to those of clinically normal calves with respect to surface antigen expression, C albicans phagocytosis and killing, and respiratory burst activity. In contrast, neutrophils from calves homozygous for BLAD had significantly reduced phagocytic and yeast-killing capacity but had higher respiratory burst activity than cells from clinically normal or heterozygous calves. Homozygous calves also had extreme neutrophilia and significantly more immature neutrophils. Conclusions The heterozygous BLAD genotype does not cause detectable functional differences in leukocytes, compared with those of clinically normal calves. In contrast, leukocytes from homozygous calves seem to upregulate alternative host defense capabilities (eg, respiratory burst activity) to partially compensate for the lack of typical adherence-dependent host defense functions. (Am J Vet Res 1999;60:1255–1261)

BLAD genotypes and cow production traits in Hungarian Holsteins

BLAD genotypes and cow production traits in Hungarian Holsteins

Induction of CD18-mediated passage of neutrophils by Pasteurella haemolytica in pulmonary bronchi and bronchioles.

Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs during P. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (beta2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected with P. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P < 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.

Intramammary Infections in Primiparous Holsteins: Heritabilities and Comparisons of Bovine Leukocyte Adhesion Deficiency Carriers and Noncarriers

The objective of this study was to determine the impact of bovine leukocyte adhesion deficiency on intramammary infection (IMI) in Holstein cows at first calving. Quarter milk samples were collected between 3 d prepartum and 4 d postpartum from 756 Holstein cows in first lactation. These samples were frozen and subsequently cultured using National Mastitis Council recommendations. Sixty-eight carriers of bovine leukocyte adhesion deficiency were identified (9.0% of cows) from an additional milk sampling collected in early lactation. Binary variables (infected or uninfected) for each quarter were defined as dependent variables to evaluate IMI incidence from all bacterial species and major species groups: coliforms, coagulase-negative staphylococci, and streptococci other than Streptococcus agalactiae. The model included herd-season of calving, days in milk when samples were collected, age at calving, quarter, cow (random effect), and bovine leukocyte adhesion deficiency. Sire was included as a random effect (instead of cow), and bovine leukocyte adhesion deficiency was dropped from the model to estimate heritabilities. Heritabilities for IMI incidence from the various groups of organisms ranged from 0.02 to 0.66 (0.21 from all bacterial species).No differences were observed between carriers of bovine leukocyte adhesion deficiency and homozygous normal noncarriers for IMI from coliform, coagulase-negative staphylococci, streptococci other than Streptococcus agalactiae, or all bacterial species combined.

Bone marrow transplantation in a holstein heifer withbovine leucocyte adhesion deficiency

Bone marrow transplantation (BMT) was performed in a 9-month-old heifer with bovine leucocyte adhesion deficiency (BLAD). Clinical and haematological findings, selected neutrophil function and CD18 expression of neutrophils in a B2 integrin-deficient heifer were examined. Twelve months after BMT, a small fluorescent region in the CD18-positive area of a flow cytometric profile was demonstrated and estimated to represent 0.3-0.5% of the CD18-positive neutrophils as measured by flow cytometric analysis following immunomagnetic separation. The animal's clinical condition appeared to improve and stabilize over our observation period of 28 months following BMT. Newly expressed CD18 seemed to play an important role in ameliorating the clinical signs of BLAD in this heifer.

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin

SUMMARY Objective To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils. Animals 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources. Procedure Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils. Results MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P &lt; 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P &lt; 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P &lt; 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P &lt; 0.05) within 10 minutes. Conclusion MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells. (Am J Vet Res 1997;58:1392–1401)

Partial Genomic Structure of the Bovine CD18 Gene and the Refinement of Test for Bovine Leukocyte Adhesion Deficiency

Partial Genomic Structure of the Bovine CD18 Gene and the Refinement of Test for Bovine Leukocyte Adhesion Deficiency

Expression of β2 integrins on blood leukocytes of cows with or without bovine leukocyte adhesion deficiency

Peripheral blood leukocytes of 11 normal cows, 7 cows heterozygous and 2 heifers homozygous for bovine leukocyte adhesion deficiency (BLAD) were analysed by flow cytometry for the intensity of their β2 integrin expression ( LFA-1( CD11a CD18 ) , CR3 ( CD11b CD18 ) and CR4 ( CD11c CD18 )). BLAD-homozygotes revealed no or a very weak expression of the β2 integrins and had a 10-fold and 4- to 5-fold increase in absolute number of neutrophils and monocytes, respectively, whereas the absolute number of lymphocytes remained normal. The mean fluorescence intensity (MFI) of the β2 integrins (CD18) in heterozygous animals was 56 to 90% of this in the normal cows (MFI between 14 and 512). The difference in the expression level was most pronounced for LFA-1 on the small cluster of lymphocytes with the highest MFI for LFA-1. Repeated analysis and phorbol myristate acetate stimulation revealed that the LFA-1 expression on this high-expressing cell population of the peripheral blood allowed a ready identification of BLAD-heterozygotes by flow cytometry.

Enhancement of PCR-RFLP typing of bovine leukocyte adhesion deficiency.

Enhancement of PCR-RFLP typing of bovine leukocyte adhesion deficiency.

Antigen-specific immune responses in cattle with inherited β2-integrin deficiency

The significance of β 2-integrins for the generation of antigen-specific immune responses in vivo was studied employing the bovine model of β 2-integrin deficiency. To that end four cattle with bovine leukocyte adhesion deficiency (BLAD) and healthy age-matched controls were immunized with tetanus toxoid (TT) and rabies virus (RV) vaccines three times in monthly intervals. In addition, two animals with BLAD and three controls received a fourth vaccination 8 months after the start of the study. Proliferative responses of peripheral blood mononuclear cells (PBMC) to the antigens TT and RV as well as specific serum immunoglobulin G (IgG) titers were determined in intervals for up to 10 months after primary vaccination. Proliferative responses of PBMC to TT and RV were substantially lower in cattle with BLAD than in controls, although PBMC from cattle with BLAD were shown to have the capacity to proliferate in the response to the mitogen concanavalin A. Occurrence of antigen-specific IgG titers was delayed and they were considerably lower in cattle with BLAD compared to controls. Finally, treatment of TT- and RV-stimulated PBMC from an immunized control with different concentrations of the anti-CD18 monoclonal antibody R15.7 resulted in a dose-dependent inhibition of lymphocyte proliferation to almost 100%. The results of the present study show that β 2-integrin deficiency leads to delayed and severely impaired immune responsiveness in vivo. The observations that antibody production, although considerably delayed and impaired, does occur and that apparently class-switching takes place in BLAD indicate T-cell reactivity in vivo.

Genetic Association Between Parameters of Innate Immunity and Measures of Mastitis in Periparturient Holstein Cattle

Relationships between genetic measures of mastitis (somatic cell score, score for clinical mastitis, and scores for IMI with major or minor pathogens) and immunological parameters (physiological and molecular markers) were examined for periparturient Holstein cows. Physiological markers included 11 in vitro immunological assays. Molecular markers included the second exon of the DRB3 locus of the bovine major histocompatibility complex, the IgG2 isotype genotype, and the CD18 genotype (the locus responsible for bovine leukocyte adhesion deficiency). A gene substitution model was used to estimate the additive genetic effects of alleles of the three molecular markers on estimated breeding value (EBV) for mastitis measures. Pearson correlation coefficients between EBV for immunological assays and EBV for mastitis measures were computed. Molecular markers explained up to 40% of the variation in EBV for measures of mastitis. The presence of allele DRB3.2*16 was associated with higher EBV for SCS. Allele DRB3.2*8 was associated with increased EBV for clinical mastitis, as was the IgG2b allele and the normal CD18 allele. Alleles DRB3.2*11, *23, IgG2a, and the recessive allele for bovine leukocyte adhesion deficiency were associated with decreased clinical mastitis. A positive genetic association was found between allele DRB3.2*24 and EBV for IMI by major pathogens and between DRB3.2*3 and IMI by minor pathogens. Several correlations between EBV for immunological assays and EBV for mastitis measures were significantly different from 0. Cows with low EBV for SCS tended to have neutrophils that had greater functional ability at maximal immunosuppression, low serum IgG1, and high numbers of circulating mononuclear cells. Immunological parameters, including physiological and molecular markers, are useful aids to understand the genetics of resistance to mastitis.

LFA-1 expression in cattle affected with Bovine Leukocyte Adhesion Deficiency (BLAD)

LFA-1 expression in cattle affected with Bovine Leukocyte Adhesion Deficiency (BLAD)

LFA-1 expression in cattle affected with Bovine Leukocyte Adhesion Deficiency (BLAD)

LFA-1 expression in cattle affected with Bovine Leukocyte Adhesion Deficiency (BLAD)

Genetic parameters for and Blad effects on beef production traits and disease frequency

Beef production traits measured on 6292 performance tested Danish Friesian bulls from 1973 to 1992 were analysed together with disease records for 4621 bulls performance tested from 1981 to 1992 using a threshold model. Univariate Gaussian and polychotomous and bivariate Gaussian‐binary mixed models with Bovine Leukocyte Adhesion Deficiency (BLAD) test information were used. Danish Friesians were found to have a larger ultrasonic muscle area than North American Holstein‐Friesians and favourable heterosis was observed for daily gain and feed utilization. Carriers of BLAD were observed to have poorer feed utilization than non‐carriers and tended to grow slower and to have a lower incidence of disease treatments. Estimated heritabilities for beef production traits ranged from 0.29 to 0.39. For disease frequency analysed as polychotomous and as binary, the estimated heritabilities ranged from 0.08 to 0.19 and from 0.12 to 0.33, respectively. Simulation showed that heritability estimates tended to be biased with increasing number of pooled categories. Phenotypic and genetic correlations between disease frequency and beef production traits ranged from — 0.32 to 0.10 and from — 0.43 to 0.23, respectively.

Prevalence and Allele Frequency Estimation of Bovine Leukocyte Adhesion Deficiency(BLAD) in Holstein-Friesian Cattle in Japan.

Blood samples from 796 Holstein dairy cows in 20 herds from 6 districts in Japan from June 1994 to August 1995 were examined to determine whether they were BLAD-free, BLAD carriers, or BLAD-affected by use of DNA-polymerase chain reaction (PCR) analysis. The usage of semen of confirmed BLAD-carriers for artificial insemination in the Hokkaido district and two selected dairy farms was examined to estimate the gene frequency of BLAD carriers of sires. BLAD-carrier prevalence in 20 herds (796 cows, over 2.5 years old) ranged from 0 to 23.5%, and the mean BLAD-carrier prevalence was 8.1%. The BLAD-carrier prevalence in 10 herds (363 cows) in which the occurrence of BLAD was not detected by the DNA-PCR test ranged from 0 to 12.5% with a mean of 5.4%. The BLAD-carrier prevalence in 10 herds (433 cows) in which the occurrence of BLAD was confirmed by DNA-PCR analysis ranged from 2.6 to 23.5% with a mean of 10.8%, and these values were significantly (P < 0.05) higher than those of dairy herds in which the occurrence of BLAD was not detected. The age distribution in BLAD carriers in these cows ranged from 2.5 to 11 years. The mean gene frequencies of BLAD among 796 cows from 20 herds and 433 cows from 10 herds in which the occurrence of BLAD was detected were 0.041 and 0.054, respectively. The proportional usage of semen of BLAD carriers for artificial insemination in the Hokkaido district in 1992 was 12.6%, and its gene frequency was 0.058. On two selected farms in which higher BLAD-carrier rates were detected, the prevalences were 35.5% and 25.8%, and their gene frequencies were 0.177 and 0.129, respectively. The occurrence of BLAD-affected in Holstein dairy cattle was estimated to be 0.16-0.31% at birth in Japan without genetic control.

Multiple genotype analysis and sexing of IVF bovine embryos

Twenty-one in vitro-fertilized bovine blastocysts were quartered, lysed and subjected to primer elongation preamplification (PEP) procedure, allowing for the analysis of up to 40 genotypes per quarter embryo. The quarter-embryos were sexed by polymerase chain reaction (PCR) using BRY.1, Bov97M and ZFX/ZFY loci, and then genotyped for k-casein, bovine leukocyte adhesion deficiency (BLAD) and microsatellite D9S1. The mitochondrial cytochrome B locus was used as an internal control with a 95% success rate. The PEP procedure amplified genomic fragments in 93% of all cases. The embryos were identified to be 11 males and 10 females. Sexing accuracy was 87% for BRY.1, 97% for ZFX/ZFY and 100% for Bov97M. False genotyping was due mostly to amplification of BRY.1 in the female embryos and to the nonamplification of the ZFY locus in the male embryos. The results indicate that the combined use of Bov97M and ZFX/ZFY loci is a highly accurate procedure for sexing bovine embryos. Genotyping for κ-casein, D9S1 and BLAD was successful in 94, 99 and 91% of assays, respectively. Sex ratios and allele frequencies of embryos for gk-casein, BLAD and D9S1 were all close to the observed frequencies in the Israeli Holstein population. These results support the conclusion that the genotyping of embryos is as accurate as that of mature animals. Thus, marker-assisted selection can be efficiently applied at the preimplantation embryo level for loci of economic importance.

Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils

Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca 2+([Ca 2+] i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency ( BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca 2+] i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca 2+] i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca 2+] i was almost identical in neutrophils from affected and control heifers. [Ca 2+] i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca 2+] i and the chemiluminescent response of neutrophils. The [Ca 2+] i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca 2+] i signalling and functional responses of B 2-integrin-deficient neutrophils were demonstrated.

Identification of monoclonal antibodies with specificity to α- or β-chains of β2-integrins using peripheral blood leucocytes of normal and Bovine Leucocyte Adhesion Deficient (BLAD) cattle

The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant Leucocyte Antigens with lymphocytes and monocytes of normal and β 2-integrin deficient (Bovine Leucocyte Adhesion Deficiency: BLAD) animals was determined by flow cytometry to investigate potential specificities to α- or β-chains of β 2-integrins. From the 13 monoclonal antibodies that were entered as having specificity for CD11/CD18 antigens, ten were confirmed correct, but three had reactivity with cells from BLAD animals. We conclude that our approach provides an easy way to reliably identify the majority of β 2-integrin specific monoclonal antibodies.

Simultaneous analysis of bovine kappa-casein and BLAD alleles by multiplex PCR followed by parallel digestion with two restriction enzymes.

An improved and simplified method allowing simultaneous genetic typing of kappa-casein and CD 18 (bovine leucocyte adhesion deficiency; BLAD) loci has been developed. The method is based on the simultaneous amplification of fragments of the two groups of alleles by multiplex PCR, and on a concurrent, parallel digestion of the products by two restriction enzymes (PstI and HaeIII) in the same incubation buffer. Digestion with PstI distinguishes kappa-casein A and B alleles and does not cut within any of the BLAD alleles, while digestion with HaeIII allows the differentiation between normal and mutant allele variants of the CD18 locus. All combinations of the known mutants of the two alleles, characterized to the regions amplified and resulting in phenotypic effect, could be detected by electrophoretic separation performed on the same agarose gel owing to the vast differences in the length of the restriction fragments.

An improved DNA test for bovine leucocyte adhesion deficiency

A modified DNA test, based on the polymerase chain reaction, was developed for the monogenic recessive disease bovine leucocyte adhesion deficiency ( BLAD). The test was improved by the selection of new primers which facilitated the interpretation of the results. An easily scorable banding pattern makes the test useful in cattle breeding schemes and for clinical diagnosis. A total of 2381 samples was analysed over a period of three years. The carrier rate among young bulls at artificial insemination ( AI) stations decreased from 11·6 per cent in 1993 to 9·9 per cent in the first five months of 1995. Continuous screening of young bulls before entering AI is still recommended unless both parents are proven to be genetically free of BLAD. The carrier rate among clinically suspect animals was not increased, and carriers are therefore not expected to be immunodeficient. Despite all efforts to eradicate the disease, calves with BLAD were still observed in 1995.