Prevalence of nine genetic defects in Chinese Holstein cattle.

Worldwide use of elite sires has caused inbreeding accumulation and high frequencies of genetic defects in dairy cattle populations. In recent years, several genetic defect genes or haplotypes have been identified in Holstein cattle. A rapid and reliable microfluidic chip with Kompetitive allele-specific PCR (KASP) assay was developed in our previous study for the detection of heterozygotes at eight genetic defect loci of bovine leukocyte adhesion deficiency (BLAD), Brachyspina syndrome (BS), complex vertebral malformation (CVM), Holstein haplotype 1 (HH1), Holstein haplotype 3 (HH3), Holstein haplotype 4 (HH4), Holstein haplotype 5 (HH5) and haplotype for cholesterol deficiency (HCD). This study aimed to extend that assay to include a newly identified genetic defect of Holstein haplotype 6 (HH6) and to estimate the frequencies of carriers for each of the nine genetic defects in six Chinese Holstein herds. Of the 1633 cows, carrier frequencies of the genetic defects were 6.92%, 5.76%, 4.46%, 4.30%, 3.62%, 2.94%, 1.86% and 0.37% for HH1, HH3, CVM, HH5, HCD, BS, HH6 and BLAD, respectively. No carrier was found for HH4. Notably, 27.43% of cows carried at least one genetic defect, while 2.27% and 0.12% of cows carried double and triple genetic defect alleles, respectively. The existence of genetic defects calls for routine molecular testing and effective management of genetic defects by avoiding carrier-to-carrier mating in production herds and eliminating or at least reducing the frequency of the defective alleles through marker-assisted selection in breeding herds.

Carrier frequency of autosomal recessive disorders (BC, BLAD, FXID and CVM) in Holstein cows in Jalisco, Mexico

The hereditary autosomal recessive disorders bovine citrullinemia (BC), bovine leukocyte adhesion deficiency (BLAD), factor XI deficiency (FXID), and complex vertebral malformation (CVM) have affected dairy cattle breeding significantly around the world. This study examined the carrier frequency of BC, BLAD, FXID, and CVM autosomal recessive disorders in Bos taurus Holstein cows bred in the Altos Norte region of the state of Jalisco, Mexico. We extracted DNA from 408 random samples of peripheral blood, and then used polymerase chain reaction (PCR) to identify insertion mutations for FXID, and PCR with restriction fragment length polymorphism (PCR-RFLP) for CVM, BC and BLAD. We visualized the PCR products using agarose gel electrophoresis stained with GelRed®. We found that 100% of wild-type (N/N) allele homozygous animals for genes CD18, ASS, and FXI were free of the mutations for BLAD, BC and FXID respectively. For gene SLC35A3 we estimated total carrier frequency of 10.3% and allele frequency of 5%.(AU)

Feline leukocyte adhesion (CD18) deficiency caused by a deletion in the integrin ß2 (ITGB2) gene.

BACKGROUND: Leukocyte adhesion deficiency (LAD) or CD18 deficiency is an autosomal recessive immunodeficiency which has been described in people, cattle, dogs, and knockout mice. OBJECTIVES: The study goals were to characterize the clinicopathologic, immunologic, and molecular genetic features of feline LAD (FLAD) in a neutered male adult Domestic Longhair cat with severe leukocytosis and recurrent infections. METHODS: Flow cytometry evaluated surface expression of CD18 on neutrophils. In vitro functional assays assessed CD18-dependent neutrophil adhesion and T-cell proliferation. Genomic DNA and cDNA were used to identify a causative mutation in the coding sequence of the integrin ß2 subunit (ITGB2) gene. RESULTS: The affected cat developed periodontitis during the first months of life followed by recurrent infections poorly responsive to antibiotic therapy, accompanied by extreme neutrophilia. Neutrophils from the proband, compared to feline controls, did not express any CD18 on the cell surface. Adhesion of affected neutrophils was severely impaired with and without phorbol-myristate-acetate activation. The proband's T-cells proliferated weakly to 1 pg but normally to 100 pg staphylococcal enterotoxin A, suggesting a CD18-independent T-cell response at higher doses. Molecular genetic analysis of the ITGB2 gene revealed a 24 bp deletion at the exon 2 to intron 2 boundary (c.46_58 + 11del), predicting premature translational termination due to abnormal splicing of exon 1 to exon 3 or 4. CONCLUSIONS: Feline LAD exhibits features similar to LAD in other species. However, clinical episodes in FLAD appeared milder allowing for an extended life expectancy under long-term antimicrobial therapy, possibly due to an alternative, CD18-independent T-cell proliferation pathway.

Leucocyte adhesion deficiency III in a mixed-breed dog.

BACKGROUND: Leucocyte adhesion deficiencies are inherited disorders characterised by immunodeficiency leading to recurrent infections and a marked leucocytosis. We describe the clinical characteristics, diagnosis and management of an Australian mixed- breed dog with leucocyte adhesion deficiency III. CASE REPORT: A 16-month-old male, neutered, German Shepherd × Rottweiler dog was investigated for pyrexia, persistent leucocytosis, marked periodontal disease, lameness, increased mucosal haemorrhages and poor wound healing. Numerous diagnostics were performed including a leucocyte adhesion deficiency III PCR test, which was positive. The patient was managed with topical pressure at bleeding sites, antibiotics, analgesics and dental prophylaxis when required. DISCUSSION: Leucocyte adhesion deficiency III is a rare disorder that manifests because of impaired activation of beta integrins. This results in an absence of neutrophil chemotaxis and adhesion, and platelet dysfunction. Mutations within the KINDLIN3 gene resulting in the absence of the kindlin-3 protein have been identified as the cause of this disease. Leucocyte adhesion deficiency III has previously been reported in humans and a German Shepherd dog. This report describes the first reported case of leucocyte adhesion deficiency III in Australia and the first reported case in a mixed-breed dog worldwide.

Incidence of bovine leukocyte adhesion deficiency, complex vertebral malformation, and deficiency of uridine-5-monophosphate synthase carriers in Brazilian Girolando cattle.

Among the various hereditary diseases that have been widely studied in dairy cattle, bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine-5-monophosphate synthase (DUMPS), and complex vertebral malformation (CVM) are noteworthy because of their high impact on overall herd productivity as a consequence of increased calf mortality. The aim of this study was to verify the frequency of carriers of BLAD, CVM, and DUMPS mutant alleles in cows and bulls from the National Girolando Progeny Test carried out in Brazil by using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR assays. A total of 777 animals were genotyped for BLAD, 783 for CVM, and 122 for DUMPS. The frequencies of carriers for BLAD and CVM were 0.77 and 1.53%, respectively, whereas no carriers of DUMPS were observed.

Leukocyte adhesion deficiency type I in a mixed-breed dog.

A 6-month-old, neutered male, mixed-breed dog was examined for a 2-month persistent fever, nonhealing dermal metacarpal area wound, and leukocytosis (47.0-198.0 × 10(3)/µl). Serum chemistry findings included hypoalbuminemia, hyperglobulinemia, hyperphosphatemia, and hyperphosphatasemia. Complete blood cell count results revealed a moderate microcytic, hypochromic nonregenerative anemia with a profound leukocytosis (198.5 × 10(3)/µl), characterized by neutrophilia with toxicity and hypersegmentation, and significant band cells. Tick-borne disease titers (genera Anaplasma, Ehrlichia, and Borrelia) were negative, as were polymerase chain reaction for other infectious agents (genera Hepatozoon, Mycobacterium, Mycoplasma; and Canine distemper virus). No agents were identified in a deep dermal biopsy (conventional and special histochemical stains) of the chronic draining, metacarpal region lesion. Cytology of the draining tract revealed numerous mixed bacteria and a surprising lack of neutrophils. Chronic occult blood loss with iron deficiency was considered a possible cause of the anemia. Differentials for the leukon were chronic established inflammation (occult infectious agent), chronic neutrophilic leukemia, paraneoplastic leukocytosis (neoplastic source of granulocyte colony-stimulating factor [CSF] or granulocyte-macrophage CSF), and leukocyte adhesion deficiency (LAD). The possibility of a LAD disorder was further investigated because of the noted hypersegmented neutrophils, absence of neutrophils in the cytology sample, the animal's young age, and persistence of clinical and laboratory signs. Flow cytometry of blood neutrophils showed a 60% reduction in surface expression of the ß2-integrin (CD18) subunit, whereas neutrophil function tests (oxidative burst and phagocytosis) were normal. Genetic testing revealed a homozygous missense mutation in the ß2-integrin subunit gene, previously recognized only in purebred Irish Setters, leading to a diagnosis of LAD type 1 disorder in this mixed-breed dog.

Identification of a null allele in genetic tests for bovine leukocyte adhesion deficiency in Pakistani Bos indicus × Bos taurus cattle.

Two clinically healthy mature Pakistani Bos indicus × Bos taurus cattle were genotyped as homozygous affected for the lethal immunodeficiency disorder bovine leukocyte adhesion deficiency (BLAD) using previously described PCR-RFLP based DNA tests which was confirmed by sequencing. Sequencing of Bos taurus and B. indicus × B. taurus genomic DNA surrounding the disease causing mutation (c.383A > G) in the ITGB2 gene identified numerous variations in exonic and intronic regions within and between species, including substantial variation in primer annealing sites for three PCR-RFLP tests for one of the B. indicus allelic variants. These variations in the primer annealing sites resulted in a null allele in the DNA tests causing the misdiagnosis of some heterozygous B. taurus × B. indicus cattle to be classified as homozygous affected. New primers were designed and a modified test was developed which simultaneously identified the disease mutation and the Pakistani B. indicus allelic variant associated with the null allele in the previous test.

Gene therapy for canine leukocyte adhesion deficiency with lentiviral vectors using the murine stem cell virus and human phosphoglycerate kinase promoters.

Children with leukocyte adhesion deficiency type 1 (LAD-1) and dogs with canine LAD (CLAD) develop life-threatening bacterial infections due to mutations in the leukocyte integrin CD18. Here, we compared the human phosphoglycerate kinase (hPGK) promoter to the murine stem cell virus (MSCV) promoter/enhancer in a self-inactivating HIV-1-derived lentiviral vector to treat animals with CLAD. Four CLAD dogs were infused with CD34(+) cells transduced with the hPGK vector, and two CLAD dogs received MSCV vector-transduced CD34(+) cells. Infusions were preceded by a nonmyeloablative dose of 200 cGy total body irradiation. Comparable numbers of transduced cells were infused in each group of animals. Only one of four CLAD animals treated with the hPGK-cCD18 vector had reversal of CLAD, whereas both MSCV-cCD18 vector-treated dogs had reversal of the phenotype. Correction of CLAD depends both upon the percentage of CD18(+) myeloid cells and the level of expression of CD18 on individual myeloid cells. In this regard, the hPGK promoter directed low levels of expression of CD18 on neutrophils compared to the MSCV promoter, likely contributing to the suboptimal clinical outcome with the hPGK vector.

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.

Lentiviral vectors incorporating a human elongation factor 1alpha promoter for the treatment of canine leukocyte adhesion deficiency.

Canine leukocyte adhesion deficiency (CLAD) provides a unique large animal model for testing new therapeutic approaches for the treatment of children with leukocyte adhesion deficiency (LAD). In our CLAD model, we examined two different fragments of the human elongation factor 1alpha (EF1alpha) promoter (EF1alphaL, 1189 bp and EF1alphaS, 233 bp) driving the expression of canine CD18 in a self-inactivating (SIN) lentiviral vector. The EF1alphaS vector resulted in the highest levels of canine CD18 expression in CLAD CD34(+) cells in vitro. Subsequently, autologous CD34(+) bone marrow cells from four CLAD pups were transduced with the EF1alphaS vector and infused following a non-myeloablative dose of 200 cGy total-body irradiation. None of the CLAD pups achieved levels of circulating CD18(+) neutrophils sufficient to reverse the CLAD phenotype, and all four animals were euthanized because of infections within 9 weeks of treatment. These results indicate that the EF1alphaS promoter-driven CD18 expression in the context of a RRLSIN lentiviral vector does not lead to sufficient numbers of CD18(+) neutrophils in vivo to reverse the CLAD phenotype when used in a non-myeloablative transplant regimen in dogs.

Morphologic and cytokine profile characterization of Salmonella enterica serovar typhimurium infection in calves with bovine leukocyte adhesion deficiency.

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18-), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18- calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18- calves--including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration--were similar to the ones reported in the literature for CD18- calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18- calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Detection and screening of bovine leukocyte adhesion deficiency in Pakistan using molecular methods.

Bovine leukocyte adhesion deficiency (BLAD) is an autosomal recessive disease. Affected animals die because of extreme susceptibility to infections caused by the lack of a membrane glycoprotein called the leukocyte integrin beta-2 subunit of CD18. The present study was planned to standardize a technique for the diagnosis of BLAD and to get an estimation of BLAD allele in the Pakistani cattle population. The study was performed on 700 animals including Holstein-Friesian (HF) (n=280), Friesian-Sahiwal (FS) (n=120) Sahiwal (n=100) cows and HF calves (n=59) from Government as well as private farms. Similarly 141 bulls of Sahiwal (n=100), HF (n=18) and FS (n=23) from the Semen Production Unit Qadirabad and Kherimorat were also sampled. The identification of normal, carrier and affected animals were made by the PCR-RFLP method. No animal was found homozygous for BLAD while 10 animals including HF calve (n=1), FS bull (n=1), HF (n=6) and FS (n=2) cows were BLAD carrier. The Hardy-Weinberg frequency of the mutant allele in HF and FS population in Pakistan was calculated to 0.01. Thus there is a need of regular screening of the bulls used for artificial insemination to avoid the risk of spreading BLAD in the cattle population of Pakistan.

Identification of novel allelic variants of integrin beta 2 (ITGB2) gene and screening for Bubaline leukocyte adhesion deficiency syndrome in Indian water buffaloes (Bubalus bubalis).

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).

Canine neutrophil dysfunction caused by downregulation of beta2-integrin expression without mutation.

Canine leukocyte adhesion deficiency (CLAD) in Irish setters is caused by genetic defects of leukocyte integrin CD18 leading to recurrent bacterial infections. We report clinical features and analysis of neutrophil function from two mixed-breed canine littermates (one female and one male dog) similar to CLAD. The symptoms of pyogenic infection were first recognized at 3 months of age and since then the patients suffered from recurrent bacterial infections. These clinical findings were strongly suggestive of genetic phagocyte dysfunction. Neutrophil function tests revealed a marked reduction of serum-opsonized zymosan-mediated superoxide production in the two littermates. Neutrophils of the male dog revealed impaired integrin-mediated adherence and phagocytic activity, whereas ability of serum opsonization was normal. There was also a profound decrease of surface expression of CD11b/CD18 and beta2-integrin transcript level, detected by real-time RT-PCR without missense mutations unlike CLAD. Immunoblot analysis indicated that protein expression of cytochrome b(558) component gp91(phox), the cytosolic components p47(phox) and p67(phox) of NADPH oxidase components increased profoundly in the male. Our study suggests that decreased transcriptional levels of beta2-integrin without mutations, lead to downregulation of surface expression, resulting in multiple defects in adhesion-related neutrophil functions and consequently, recurrent bacterial infections from puppyhood.

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 beta2 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 VDJCmu and 25 VDJCgamma 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 VDJCmu and VDJCgamma 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.

Frequency of BLAD and CVM alleles in sires and elite heifers of Czech Holstein cattle.

In this paper, we analyse the occurrence of BLAD and CVM heterozygous animals in Holstein cattle in the Czech Republic in 1993-2005. The occurrence of BLAD heterozygous sires and heifers (BL) during the period 1993-1998 in Czech Holsteins was 13.9% and 10.7%. Radical measures have been taken to restore the population. Evidently, the measures have been efficient, in 2005 one BLAD heterozygous sire of 101 was found. Continuous testing is necessary, because in commercial herds, the eradication process is not short-term. The found occurrence ofCVM heterozygous sires (CV) decreased from 20% in 2001 to 8% (7 positive of 85) in 2005.This is still quite a high frequency. The occurrence in CV females of 20% remains higher. Therefore, the use of CV sires should be restricted thoroughly. Identification of the molecular basis for inherited diseases, should lead to control measures which would enable the quick recovery of the population.

Implication of complex vertebral malformation and bovine leukocyte adhesion deficiency DNA-based testing on disease frequency in the Holstein population.

Two inherited lethal disorders, bovine leukocyte adhesion deficiency (BLAD) and complex vertebral malformation (CVM), play a major role in breeding of Holstein cattle. Both inherited diseases are based on single nucleotide polymorphisms that have been known for 12 and 7 yr, respectively. A total of 25,753 cattle were genotyped for BLAD (18,200 tests) and CVM (14,493 tests) in our laboratory since the beginning of the genotyping programs for these diseases. Based on founder effects, the CVM mutation is thought to be linked to milk production. The BLAD was genotyped using RFLP until 2001; then a fluorescence resonance energy transfer assay on a LightCycler was used, as for CVM genotyping. By using single nucleotide polymorphism-aided breeding, the allelic frequency of the BLAD and CVM mutations in the active sire population was reduced from 9.4% in 1997 to 0.3% in 2007 (BLAD) and from 8.3% in 2002 to 2.3% in 2007 (CVM), with calculated half-life of the mutant allele of 2.1 yr for BLAD and 3.6 yr for CVM. An observed increase of BLAD frequency in 1999 could be attributed to the massive use of a BLAD-positive sire tested falsely negative in another laboratory. These data show that marker-assisted selection is capable of substantially reducing the frequency of a mutation within a period of not more than 5 yr. The different selection strategies against the lethal recessive allele in CVM and BLAD are reflected in the different reduction rates of the specific allele frequencies.

Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors.

Recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy. However, the use of gammaretroviral vectors in these trials led to insertional activation of nearby oncogenes and leukemias in some study subjects, prompting studies of modified or alternative vector systems. Here we describe the use of foamy virus vectors to treat canine leukocyte adhesion deficiency (CLAD). Four of five dogs with CLAD that received nonmyeloablative conditioning and infusion of autologous, CD34+ hematopoietic stem cells transduced by a foamy virus vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained more than 2 years after infusion. In vitro assays showed correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis showed polyclonality of transduced cells and a decreased risk of integration near oncogenes as compared to gammaretroviral vectors. These results represent the first successful use of a foamy virus vector to treat a genetic disease, to our knowledge, and suggest that foamy virus vectors will be effective in treating human hematopoietic diseases.

[A method for detecting bovine leukocyte adhesion deficiency in Holstein calves using polymerase chain reaction-single strand conformation polymorphism analysis].

Bovine leukocyte adhesion deficiency (BLAD) is an autosomal recessive disease caused by A-->G mutation in the CD18 gene. The disease is characterized by greatly reduced expression of the heterodimeric beta2 integrin adhesion molecules on leukocytes, resulting in multiple defects in leukocyte function and significant deficits on performance traits in Holstein cattle. In this study, primers were designed to amplify the CD18 gene fragment and BLAD was detected by PCR-SSCP in a simple, highly accurate and high throughput manner. Results were confirmed by DNA sequencing. This study provides a more reliable and useful method for extensive screening of BLAD and also offers a theoretical basis for molecular diagnosis in Holstein calves.