Bovine Leukocytes Research Articles

Identification of Splice Variants, Targeted MicroRNAs and Functional Single Nucleotide Polymorphisms of the BOLA-DQA2 Gene in Dairy Cattle

Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3'-untranslated regions (UTR) (52 bp+167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. +1283 C>T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3'-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold (p<0.05) in mastitis-infected mammary tissues (n = 5) compared with the healthy mammary gland mammary tissues (n = 5). Except for bta-miR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold (p<0.05), whereas miR-2318 was downregulated in the mastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation.

Antiviral mode of action of bovine dialyzable leukocyte extract against human immunodeficiency virus type 1 infection

BackgroundBovine dialyzable leukocyte extract (bDLE) is derived from immune leukocytes obtained from bovine spleen. DLE has demonstrated to reduce transcription of Human Immunodeficiency Virus Type 1 (HIV-1) and inactivate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Therefore, we decided to clarify the mode of antiviral action of bDLE on the inhibition of HIV-1 infection through a panel of antiviral assays.ResultsThe cytotoxicity, HIV-1 inhibition activity, residual infectivity of bDLE in HIV-1, time of addition experiments, fusion inhibition of bDLE for fusogenic cells and the duration of cell protection even after the removal of bDLE were all assessed in order to discover more about the mode of the antiviral action.HIV-1 infectivity was inhibited by bDLE at doses that were not cytotoxic for HeLa-CD4-LTR-β-gal cells. Pretreatment of HIV-1 with bDLE did not decrease the infectivity of these viral particles. Cell-based fusion assays helped to determine if bDLE could inhibit fusion of Env cells against CD4 cells by membrane fusion and this cell-based fusion was inhibited only when CD4 cells were treated with bDLE. Infection was inhibited in 80% compared with the positive (without EDL) at all viral life cycle stages in the time of addition experiments when bDLE was added at different time points. Finally, a cell-protection assay against HIV-1 infection by bDLE was performed after treating host cells with bDLE for 30 minutes and then removing them from treatment. From 0 to 7 hours after the bDLE was completely removed from the extracellular compartment, HIV-1 was then added to the host cells. The bDLE was found to protect the cells from HIV-1 infection, an effect that was retained for several hours.ConclusionsbDLE acted as an antiviral compound and prevented host cell infection by HIV-1 at all viral life cycle stages. These cell protection effects lingered for hours after the bDLE was removed. Interestingly, bDLE inhibited fusion of fusogenic cells by acting only on CD4 cells. bDLE had no virucidal effect, but could retain its antiviral effect on target cells after it was removed from the extracellular compartment, protecting the cells from infection for hours.bDLE, which has no reported side effects or toxicity in clinical trials, should therefore be further studied to determine its potential use as a therapeutic agent in HIV-1 infection therapy, in combination with known antiretrovirals.

Short communication: Distribution of recessive genetic defect carriers in Chinese Holstein

In dairy cattle, 4 important recessive hereditary diseases exist: complex vertebral malformation (CVM), bovine leukocyte adhesion deficiency (BLAD), citrullinemia (CTLN), and deficiency of uridine monophosphate synthase (DUMPS). Holstein Associations in developed countries have established monitoring systems for such disorders in Holstein bulls for decades. Over the past decades, China has continuously imported Holstein semen and embryos, mainly from North America but also from Europe. The dissemination of such genetic defects was undetermined until now, although efforts were taken to develop molecular techniques and detect carriers for CVM and BLAD in small populations of Chinese dairy cattle. Thus, herein we extensively screened 732 proven bulls participating in artificial insemination programs and 136 young bulls entering progeny test from 15 bull stations in China for CVM, BLAD, CTLN, and DUMPS. The proportion of carriers of the defects was found to be 7.72, 1.38, 0.23, and 0.12%, respectively. Given our findings, early diagnostic and monitoring systems on recessive inherited disorders among proven and young bulls entering the national genetic improvement programs for dairy cattle of China should be established immediately, in which a series of measures will be taken to prevent further spreading of such disorders and gradually eliminate them in the dairy cattle population in China.

Identification of BoLA‐DRB3.2 alleles in Korean native cattle (Hanwoo) and Holstein populations using a next generation sequencer

Bovine leucocyte antigen (encoded by BoLA) has been widely studied to identify the association with many traits related to immunity. Exon2 of BoLA-DRB3 is extremely polymorphic, and more than 100 alleles have been identified. We investigated polymorphisms of BoLA-DRB3.2 in Korean native cattle and Holstein populations using a next generation sequencer of the GS-FLX Titanium system. We found 38 alleles including 11 new alleles (BoLA-DRB3*1303, *4702, *7101, *7501, *7201, *7301, *7601, *1104, *7701, *7401 and *50021) in Hanwoo, and nine alleles including one new allele (BoLA-DRB3*7601) in Holstein. The 454 sequencing method is a promising alternative technology for high throughput genotyping of BoLA-DRB3.2 because of its technical advantages that allow it to overcome the disadvantages of sequence-based typing methods.

BoLA-DRB3 exon 2 mutations associated with paratuberculosis in cattle

A single nucleotide polymorphism at the antigen recognition site of the bovine leucocyte antigen (BoLA) DRB3 gene was assessed in healthy and Mycobacterium avium subsp. paratuberculosis (MAP) - infected cattle, in order to determine if there was a correlation between mutations and altered susceptibility to infection. Of a sample of 200 animals, 19.6% were found to be infected with MAP. PCR - single strand conformational polymorphism analysis of the BoLA DRB3 gene found 19 genotypes (16 in the heterozygous and three in homozygous state, respectively). Four mutations, Val53Glu (OR 453.7), Val53Leu (OR 453.7), Asp57His (OR 1.944) and Arg84Gly (OR 1.458), were linked with increased susceptibility to infection, whereas, Asp57Asn (OR 0) and Phe60Tyr (OR 0.453) were associated with increased resistance. The findings indicate potentially important mutations in the protein-binding site of DRB3, which may be crucial to the activation of an appropriate immune response against MAP.

Vaccine-induced antibodies linked to bovine neonatal pancytopenia (BNP) recognize cattle major histocompatibility complex class I (MHC I).

A mysterious disease affecting calves, named bovine neonatal pancytopenia (BNP), emerged in 2007 in several European countries. Epidemiological studies revealed a connection between BNP and vaccination with an inactivated vaccine against bovine virus diarrhea (BVD). Alloantibodies reacting with blood leukocytes of calves were detected in serum and colostrum of dams, which have given birth to calves affected by BNP. To understand the linkage between vaccination and the development of alloantibodies, we determined the antigens reacting with these alloantibodies. Immunoprecipitation of surface proteins from bovine leukocytes and kidney cells using sera from dams with a confirmed case of BNP in their gestation history reacted with two dominant protein species of 44 and 12 kDa. These proteins were not detected by sera from dams, free of BVDV and not vaccinated against BVD, and from sera of animals vaccinated with a different inactivated BVD vaccine. The 44 kDa protein was identified by mass spectrometry analysis as MHC I, the other as β-2-microglobulin. The presence of major histocompatibility complex class I (MHC I) in the vaccine was confirmed by Western blot using a MHC I specific monoclonal antibody. A model of BNP pathogenesis is proposed.

Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress

BackgroundThe molecular mechanisms by which stress induces the development of pathologies remains unclear, although it is recognised that one of the major factors affecting health as a consequence of stress is the involvement of the neuroendocrine system. In cattle, a number of necessary husbandry practices have been shown to activate the stress response, yet very little is known about the impact these have at the molecular level. The objectives of the study were to characterise, in male and female beef calves, the immune response to weaning stress in bovine leukocytes at the physiological and molecular levels and to assess the difference between calves weaned in the presence of the dam and those weaned and penned away from the dam.ResultsFollowing exposure to weaning stress, total neutrophil number and neutrophil:lymphocyte (N:L) ratio increased (P < 0.01) in calves. Additionally, expression of pro-inflammatory cytokine genes, including IL-1β, IL-8, IFN-γ and TNFα, were up-regulated (P < 0.01). Furthermore, there was increased (P < 0.001) expression of the glucocorticoid receptor, GRα, the pro-apoptotic gene, Fas and the Gram-negative pattern recognition receptor, TLR4. Calves penned away from the dam post-weaning had increased (P < 0.01) neutrophil number and N:L ratio compared with calves penned next to the dam, and female calves had higher (P < 0.05) expression levels of IL-2, IL-8, IFN-γ and TNFα than male calves.ConclusionsWeaning elicits an immediate and somewhat short-lived acute stress response in the calf. The effects serve to enhance, rather than suppress, the immune response by means of a heightened inflammatory response and cellular mobilization. The earlier and more profound increase in neutrophil number and N:L ratio together with reduced lymphocyte number in calves penned away compared with calves penned near their dams post-weaning suggests that the former may be more sensitive to weaning stress. The data also show a clear effect of gender in differential gene expression in response to stress with IFN-γ having increased expression in female calves compared with male calves over the course of the study. Additionally, this study has helped to characterise the inflammatory response to stress in calves and identify a number of novel candidate biomarkers suitable for investigation in future studies of stress.

A new method for typing bovine major histocompatibility complex class II DRB3 alleles by combining two established PCR sequence‐based techniques

Recently, two polymerase chain reaction sequence-based typing (PCR-SBT) methods were reported for the genotyping of the bovine leukocyte antigen (BoLA)-DRB3. One technique is a single PCR-SBT (sPCR-SBT) method that generates heterozygous sequences that are subsequently analyzed by the haplofinder program, while the other technique is a nested PCR-SBT (nPCR-SBT) method that allows the analysis of heterozygous sequences using the assign 400ATF software. In this study, these techniques were compared and then integrated to produce an improved genotyping method. The primer set used for sPCR-SBT was more accurate than those used for nPCR-SBT. Combining sPCR-SBT with the assign 400ATF software previously reported for nPCR-SBT enables rapid and accurate genotyping of a large number of DNA samples.

Bovine Neonatal Pancytopenia: Is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies?

Bovine Neonatal Pancytopenia (BNP) is a new emerging disease observed since 2007 in Germany and neighbouring countries. The syndrome affects newborn calves and is characterized by pancytopenia, severe bleeding and high lethality. So far, a causative role of infectious or toxic agents has been ruled out. Instead, the syndrome is induced after ingestion of colostrum, the first milk that supplies the calf with maternal antibodies. In analogy to similar diseases in humans it has therefore been postulated that BNP is caused by alloreactive, maternal antibodies. There is a striking association between BNP and a previous vaccination of the respective dams with a particular vaccine against Bovine Virus Diarrhoea (BVD). This association has led to a suspension of the marketing authorisation for the vaccine, by the European Commission. The current study investigates the role of this vaccine in the pathogenesis of BNP. By flow cytometry we were able to demonstrate that sera of BNP dams (dams that gave birth to a BNP calf) harbour alloreactive antibodies binding to surface antigens on bovine leukocytes. A significantly weaker alloreactivity was observed with sera of non-BNP dams that have been vaccinated with the same vaccine but delivered healthy calves. No binding was seen with non-BVD-vaccinated control cows and animals that were vaccinated with other inactivated BVD vaccines so far not associated with BNP. The binding is functionally relevant, because opsonization of bovine leukocytes with alloantibodies led to an elevated cytophagocytosis by bovine macrophages. To test whether the vaccine induces alloreactive antibodies two strategies were employed: Guinea pigs were vaccinated with a panel of commercially available BVD-vaccines. Only the incriminated vaccine induced antibodies binding surface antigens on bovine leukocytes. Additionally, two calves were repeatedly vaccinated with the suspected vaccine and the development of alloreactivity was monitored. In dependence of the number of booster immunizations the induction of alloreactive antibodies could be observed. Finally, by affinity purification we were able to directly demonstrate that BNP associated alloantibodies cross react with the bovine kidney cell line used for vaccine production. Together this provides strong evidence that this particular BVD vaccine has the potential to induce BNP associated alloantibodies.

Analyses of single nucleotide polymorphisms and haplotypes of BoLA-DRB3 gene in Holstein and Hanwoo

BoLA (bovine leukocyte antigens) have been known as gene complex related with bovine diseases and immunological traits. This study was conducted to find out the characteristics of BoLA-DRB3 gene related to mastitis and BL(bovine leukocyte) from 280 cattle (193 animals of Holstein cattle and 87 animals of Hanwoo). As a result, five PCR-RFLP types (b, d, e, f and g) using HaeⅢ restriction enzyme, three BstYI restriction patterns (b, d and e) and eight RsaI restriction types(b, d, f, I, j, n, o and w) were identified. Moreover, we identified new d' type (197→175/22), having one more cutting site by BstYI enzyme than d type allele and n' type (180→169/11) having one more cutting site by RsaI enzyme than n allele was additionally identified. Next, we identified 53 SNPs in BoLA-DRB3 exon2 from 280 cattle. SNP frequency and heterozygosity of Holstein and Hanwoo were investigated in all the SNP genotype. These results might be based on research for identifying marker associated with bovine diseases.

TGF-b2 Induction Regulates Invasiveness of Theileria-Transformed Leukocytes and Disease Susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

BackgroundBovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals.ResultsDegenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression.ConclusionsUsing our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.

The Predominant Infiltration of Neutrophils at the Early Stage of Development into the Cyclic Bovine Corpus Luteum.

In the ovary, ovulation induces a kind of inflammation with infiltration of a large number of macrophages and granulocytes and these leukocytes secrete inflammatory cytokines within the ovulatory/ovulated follicle. After ovulation, the corpus luteum (CL) rapidly develops within one week together with active angiogenesis and progesterone synthesis in the cow. In the life span of CL, the immune cell population presents throughout the estrous cycle. Especially in the regressing CL, T-lymphocytes and macrophages significantly increase, and the luteolytic function of these leukocytes is well illustrated. In the early CL, several types of immune cells including macrophages and eosinophils infiltrate and the primary function of these immune cells is considered to be phagocytosis, but they may also have other critical functions for the CL development. Therefore, we first characterized the distribution of mRNA expression in leukocytes especially for angiogenic factors such as vascular endothelial growth factor (VEGF), nitric oxide (NO) and tumor necrosis factor alpha (TNF). Blood samples were collected from cyclic cows and lymphocytes and granulocytes were purified using Ficoll solution. The mRNA expressions of chemokine receptor CXCR1 and CXCR2 were higher in granulocytes than lymphocytes. The mRNAs of VEGF, VEGF receptor (VEGFR) 1, interleukin-8 (IL8) and TNF were expressed in both lymphocytes and granulocytes, but VEGFR2 and inducible NO synthase mRNAs were expressed only in granulocytes. These results suggest that bovine leukocytes have a potential to act on angiogenesis. In the next, we focused on granulocytes in the early developing CL and investigated the distribution of neutrophils and eosinophils by periodic acid-schiff staining and luna staining, respectively, with related mRNA expressions. CLs were collected at a local slaughterhouse and divided into 4 groups: Days 1-2, 3-4, 5-6 and 7-8 of the estrous cycle (n=5-8 for each luteal phase). The number of neutrophils and eosinophils in the CL were significantly higher on Days 1-2 than Days 5-6 and 7-8. The mRNA expression of IL8, chemoattractant of neutrophils, was highest on Days 1-2 as well as the number of neutrophils in the CL, although mRNA expression of CCL5, major chemoattractant of eosinophils, was not high on Days 1-2. To investigate the function of IL8 as the possible luteal chemotactic mediator, we tested the migration of neutrophils using transmigration assay with the bovine IL8 and supernatants of the CL culture. In transmigration assay, the CL supernatants as well as IL8 (10-100 ng/ml) stimulated neutrophil migration. Although we tested the capacity of migration in neutrophils collected from circulating blood from Day 2, 5 and 10 of the estrous cycle (n=5 for each group), the levels of neutrophil migration were constant in all groups, suggesting that the migration of neutrophils rather depends on the level of IL8 in the CL but not on the stage of estrous cycle or the capacity of neutropils. In conclusion, the bovine granulocytes and lymphocytes express mRNAs for angiogenic factors, and neutrophils and eosinophils exist at the largest number in the early stage of development CL, besides those in regressing CL. Also, the bovine CL is able to active a migration of neutrophils possibly by chemoattractant such as IL8. Further study should be addressed on the possible interactions between the CL and immune cells to enhance luteal development in addition to phagocytosis in the cow. (poster)

The diversity of bovine MHC class II DRB3 and DQA1 alleles in different herds of Japanese Black and Holstein cattle in Japan

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. In this study, we sequenced alleles of the BoLA class II loci, BoLA-DRB3 and BoLA-DQA1, from 650 Japanese cattle from six herds [three herds (507 animals) of Japanese Black cattle and three herds (143 animals) of Holstein cattle] using polymerase chain reaction-sequence-based typing (PCR-SBT) methods. We identified 26 previously reported distinct DRB3 alleles in the two populations: 22 in Japanese Black and 17 in Holstein. The number of DRB3 alleles detected in each herd ranged from 9 to 20. Next, we identified 15 previously reported distinct DQA1 alleles: 13 in Japanese Black and 10 in Holstein. The number of alleles in each herd ranged from 6 to 10. Thus, allelic divergence is significantly greater for DRB3 than for DQA1. A population tree on the basis of the frequencies of the DRB3 and DQA1 alleles showed that, although the genetic distance differed significantly between the two cattle breeds, it was closely related within the three herds of each breed. In addition, Wu–Kabat variability analysis indicated that the DRB3 gene was more polymorphic than the DQA1 gene in both breeds and in all herds, and that the majority of the hypervariable positions within both loci corresponded to pocket-forming residues. The DRB3 and DQA1 heterozygosity for both breeds within each herd were calculated based on the Hardy–Weinberg equilibrium. Only one Japanese Black herd showed a significant difference between the expected and observed heterozygosity at both loci. This is the first report presenting a detailed study of the allelic distribution of BoLA-DRB3 and -DQA1 genes in Japanese Black and Holstein cattle from different farms in Japan. These results may help to develop improved livestock breeding strategies in the future.

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

BackgroundBovine 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.MethodsGenomic 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.ResultsFourteen 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).ConclusionThis 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.

Mannheimia haemolytica

Mannheimia haemolytica serotype S1 is considered the predominant cause of bovine pneumonic pasteurellosis, or shipping fever. Various virulence factors allow M haemolytica to colonize the lungs and establish infection. These virulence factors include leukotoxin (LKT), lipopolysaccharide, adhesins, capsule, outer membrane proteins, and various proteases. The effects of LKT are species specific for ruminants, which stem from its unique interaction with the bovine β2 integrin receptor present on leukocytes. At low concentration, LKT can activate bovine leukocytes to undergo respiratory burst and degranulation and stimulate cytokine release from macrophages and histamine release from mast cells. At higher concentration, LKT induces formation of transmembrane pores and subsequent oncotic cell necrosis. The interaction of LKT with leukocytes is followed by activation of these leukocytes to undergo oxidative burst and release proinflammatory cytokines such as interleukins 1, 6, and 8 and tumor necrosis factor α. Tumor necrosis factor α and other proinflammatory cytokines contribute to the accumulation of leukocytes in the lung. Formation of transmembrane pores and subsequent cytolysis of activated leukocytes possibly cause leakage of products of respiratory burst and other inflammatory mediators into the surrounding pulmonary parenchyma and so give rise to fibrinous and necrotizing lobar pneumonia. The effects of LKT are enhanced by lipopolysaccharide, which is associated with the release of proinflammatory cytokines from the leukocytes, activation of complement and coagulation cascade, and cell cytolysis. Similarly, adhesins, capsule, outer membrane proteins, and proteases assist in pulmonary colonization, evasion of immune response, and establishment of the infection. This review focuses on the roles of these virulence factors in the pathogenesis of shipping fever.

Binding of the Staphylococcus aureus leucotoxin LukM to its leucocyte targets

The range of leucocytes susceptible to the leucotoxin LukM/F′, a two-component pore-forming toxin of Staphylococcus aureus causing mastitis in ruminants, had not been defined. We used fluorescent-labeled LukM to investigate the binding of this toxin to bovine cells and to identify its cellular targets among bovine, human and murine leucocytes. LukM bound to bovine blood neutrophils from all the individuals tested with similar affinity, with an apparent dissociation constant (Kd) of 1.81 ± 0.14 nM and 13 3100 ± 506 binding sites. The amount of LukM bound to bovine neutrophils did not depend on the presence of the complementary component LukF’, suggesting that the binding of LukM to its ligand does not depend on the formation of pore-forming oligomers, and that the number of bound LukM molecules corresponds to the number of available cell membrane ligands. Other staphylococcal class S components of bipartite leucotoxins (HlgA, HlgC, LukE, LukS-PV) were inefficient competitors of LukM for the binding to bovine neutrophils, indicating that LukM has a distinct ligand on target cells. Bovine blood neutrophils bound slightly more LukM than did milk neutrophils, and much more than did ovine and caprine blood neutrophils. Bovine monocytes and milk macrophages readily bound LukM, whereas blood lymphocytes did not. Human neutrophils bound little LukM and were resistant to LukM/F′ at the highest tested concentration (40 nM). Murine neutrophils bound LukM and were susceptible to the toxicity of LukM/F′, exhibiting flattening and nucleus alteration beginning at 0.3 nM concentration. Among murine peritoneal exudate cells, T lymphocytes (CD3+) and monocytes/macrophages (F4/80+) bound LukM, whereas binding to B lymphocytes (CD19+) was not detected. These results indicate that cells of the myeloid lineage are the main targets of LukM/F′ in dairy ruminants, and that resident or inflammatory migrated phagocytes are susceptible to this toxin.

Characterization of bovine neutrophil β2‐adrenergic receptor function

This study compares bovine leukocyte beta-adrenergic receptor densities to that of the rat, demonstrates for the first time a functional beta(2)-adrenergic receptor signaling pathway in steer neutrophils, and investigates the effect of an inflammatory stimulus on that signaling pathway. The beta(1)-/beta(2)-adrenergic antagonist ([3H])CGP-12177 demonstrated that rat lymphocyte specific binding-site density was highest, followed by steer and dairy cow lymphocytes, and lastly steer and dairy cow neutrophils. The beta(2)-adrenergic agonist terbutaline stimulated steer neutrophil adenosine 3,5-cyclic monophosphate (cAMP) production, an effect increased by inclusion of > or = 1 x 10(-8) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Both terbutaline and the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) independently decreased steer neutrophil superoxide anion production in a concentration-dependent manner, with 1 x 10(-4) M IBMX enhancing both the potency and efficacy of the terbutaline effect (up to 74% reduction in superoxide anion production). Superoxide anion production was also reduced by the synthetic cAMP analog 8-bromo-cAMP, which increased the potency of the IBMX effect on superoxide anion production. Taken together, these data demonstrate the presence of a beta(2)-adrenergic receptor signaling pathway in bovine neutrophils much like that described in other animal species, as well as the potential for an inflammatory stimulus to alter its function.

Biological markers of neonatal calf performance: The relationship of insulin-like growth factor-I, zinc, and copper to poor neonatal growth1

Raising a heifer calf to reproductive age represents an enormous cost to the producer. Poor neonatal growth exacerbates the costs incurred for rearing, and use of blood variables that may be associated with poorly growing calves may offer predictive value for growth and performance. Thus, the principal objective of the present study was to describe changes in serum IGF-I, zinc, and copper from birth to 90 d in Holstein calves, while accounting for sex and twin status, in poorly growing calves and calves growing well. A second objective was to test the hypothesis that an association exists between these serum variables and morphometric indicators of growth. Measurements of BW, length, and height were recorded at birth and at 30, 60, and 90 d of age. Jugular blood (12 mL) was collected from each calf on d 1 to determine serum total protein, serum IgG, packed cell volume, serum zinc, serum copper, serum IGF-I, and CD18 genotype for bovine leukocyte adhesion deficiency; serum zinc, serum copper, and serum IGF-I (predictor variables) were also determined for each calf on d 2 through 10 and on d 30, 60, and 90. Stepwise multiple regression and logistic regression analyses were used to examine the relationships between the predictor variables and the dependent variables (BW, height, and length at d 30, 60, and 90 of life). Birth weight, sex, serum IGF-I (at all ages), serum copper, and the serum copper-to-zinc ratio were associated, to varying degrees, with the dependent growth variables. Birth weight was consistently the dominant predictor. In conclusion, these results suggest that lighter birth weight, reduced serum IGF-I, and inflammation may be important causes of poor growth in neonatal Holstein dairy calves.

Cytoplasmic sequestration of p53 promotes survival in leukocytes transformed by Theileria

The function of the p53 protein as the central effector molecule of the p53 apoptotic pathway was investigated in a reversible model of epigenetic transformation. The infection of bovine leukocytes by the intracellular protozoan parasite Theileria annulata results in parasite-dependent transformation and proliferation of the host cells. We found p53 to be largely localized in the host cell cytoplasm and associated with the parasite membrane of isolated schizonts. Curing infected cells of the parasite with the theilericidal drug buparvaquone resulted in a time-dependent translocation of p53 into the host cell nucleus and the upregulation of the proapoptotic Bax and Apaf-1 and the downregulation of the anti-apoptotic Bcl-2 proteins. Although buparvaquone treatment led to apoptosis of the host cell, inhibition of either p53 or Bax significantly reduced buparvaquone-induced apoptosis of the transformed cells. Thus, the p53 apoptotic pathway of host cells is not induced by infection and transformation with Theileria by a mechanism involving cytoplasmic sequestration of p53. The close association of host cell p53 with the parasite membrane implies that the parasite either interacts directly with p53 or mediates cytoplasmic sequestration of p53 by interacting with other host cell proteins regulating p53 localization.