Bovine Leukocytes Research Articles

Mapping of CD4+ T-cell epitopes in bovine leukemia virus from five cattle with differential susceptibilities to bovine leukemia virus disease progression

BackgroundBovine leukemia virus (BLV), which is closely related to human T-cell leukemia virus, is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly prolonged course involving persistent lymphocytosis and B-cell lymphoma. The bovine major histocompatibility complex class II region plays a key role in the subclinical progression of BLV infection. In this study, we aimed to evaluate the roles of CD4+ T-cell epitopes in disease progression in cattle.MethodsWe examined five Japanese Black cattle, including three disease-susceptible animals, one disease-resistant animal, and one normal animal, classified according to genotyping of bovine leukocyte antigen (BoLA)-DRB3 and BoLA-DQA1 alleles using polymerase chain reaction sequence-based typing methods. All cattle were inoculated with BLV-infected blood collected from BLV experimentally infected cattle and then subjected to CD4+ T-cell epitope mapping by cell proliferation assays.ResultsFive Japanese Black cattle were successfully infected with BLV, and CD4+ T-cell epitope mapping was then conducted. Disease-resistant and normal cattle showed low and moderate proviral loads and harbored six or five types of CD4+ T-cell epitopes, respectively. In contrast, the one of three disease-susceptible cattle with the highest proviral load did not harbor CD4+ T-cell epitopes, and two of three other cattle with high proviral loads each had only one epitope. Thus, the CD4+ T-cell epitope repertoire was less frequent in disease-susceptible cattle than in other cattle.ConclusionAlthough only a few cattle were included in this study, our results showed that CD4+ T-cell epitopes may be associated with BoLA-DRB3-DQA1 haplotypes, which conferred differential susceptibilities to BLV proviral loads. These CD4+ T-cell epitopes could be useful for the design of anti-BLV vaccines targeting disease-susceptible Japanese Black cattle. Further studies of CD4+ T-cell epitopes in other breeds and using larger numbers of cattle with differential susceptibilities are required to confirm these findings.

Mutations in the TaPIN1 peptidyl prolyl isomerase gene in Theileria annulata parasites isolated in Sudan

The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.

Technical note: Development and application of KASP assays for rapid screening of 8 genetic defects in Holstein cattle

Specific DNA mutations underlying several genetic defects associated with embryo loss or reduced calf survivability have been identified in dairy cattle, and a convenient and cost-effective platform is required for their routine screening. We developed Kompetitive allele-specific PCR (KASP) assays for discrimination of the wild-type alleles from the associated defective alleles at each of 8 common genetic defects in Holstein cattle, involving 5 SNP [HH1, HH3, HH4, bovine leukocyte adhesion deficiency (BLAD), and complex vertebral malformation (CVM)] and 3 insertion or deletion mutations [HH5, haplotype for cholesterol deficiency (HCD), and brachyspina (BS)]. A total of 390 cows from a Chinese Holstein herd were genotyped and the carriers identified at 7 of these 8 loci (except HH4), with the highest carrier frequencies found for CVM (10.5%) and HH1 (10.0%), followed by HH3 (2.6%), BS (2.1%), HCD (1.3%), HH5 (0.8%), and BLAD (0.5%). Surprisingly, 102 cows (26.2%) carried at least 1 of the 7 defective alleles. Our results demonstrate that these KASP assays are simple, rapid, and reliable for the detection of multiple genetic defects. The high carrier frequency of these genetic defects indicates an urgent need for routine molecular testing to eliminate the deleterious alleles from Chinese Holstein cattle.

Cytotoxic activity of IMMUNEPOTENT CRP against non-small cell lung cancer cell lines.

BackgroundIMMUNEPOTENT-CRP® (I-CRP) is a bovine dialyzable leukocyte extract containing transfer factor. It is a cost-effective, unspecific active immunotherapy that has been used in patients with non-small cell lung cancer (NSCLC) as an adjuvant to reduce the side-effects of chemotherapy and radiotherapy, and has shown cytotoxic activity in vitro on different cancer cell lines. However, its mechanism of action against lung cancer cells has not been assessed. Therefore, the objective of this work was to assess the cytotoxic mechanism of I-CRP on lung cancer cell lines.MethodsWe assessed cell viability through MTT assay on the NSCLC cell lines A549, A427, Calu-1, and INER-51 after treatment with I-CRP. To further understand the mechanisms of cell viability diminution we used fluorescence-activated cell sorting to evaluate cell death (annexin-V and propidium iodide [PI] staining), cell cycle and DNA degradation (PI staining), mitochondrial alterations (TMRE staining), and reactive oxygen species (ROS) production (DCFDA staining). Additionally, we evaluated caspase and ROS dependence of cell death by pretreating the cells with the pan-caspase inhibitor Q-VD-OPH and the antioxidant N-acetylcysteine (NAC), respectively.ResultsOur data shows that I-CRP is cytotoxic to NSCLC cell lines in a dose and time dependent manner, without substantial differences between the four cell lines tested (A549, A427, Calu-1, and INER-51). Cytotoxicity is induced through regulated cell death and cell cycle arrest induction. I-CRP-induced cell death in NSCLC cell lines is characterized by DNA degradation, mitochondrial damage, and ROS production. Moreover, cell death is independent of caspases but relies on ROS production, as it is abrogated with NAC.ConclusionAltogether, these results improve the knowledge about the cytotoxic activity of I-CRP on NSCLC cells, indicating that cell death, cell cycle arrest, DNA degradation and mitochondrial damage are important features, while ROS play the main role for I-CRP mediated cytotoxicity in lung cancer cells.

RNA-Seq Bayesian Network Exploration of Immune System in Bovine.

Background: The stress is one of main factors effects on production system. Several factors (both genetic and environmental elements) regulate immune response to stress.Objectives: In order to determine the major immune system regulatory genes underlying stress responses, a learning Bayesian network approach for those regulatory genes was applied to RNA-Seq data from a bovine leukocyte model system.Material and Methods: The transcriptome dataset GSE37447 was used from GEO and a Bayesian network on differentially expressed genes was learned to investigate the gene regulatory network.Results: Applying the method produced a strongly interconnected network with four genes (TERF2IP, PDCD10, DDX10 and CENPE) acting as nodes, suggesting these genes may be important in the transcriptome regulation program of stress response. Of these genes TERF2IP has been shown previously to regulate gene expression, act as a regulator of the nuclear factor-kappa B (NF-κB) signalling, and to activate expression of NF-κB target genes; PDCD10 encodes a conserved protein associated with cell apoptosis; DDX10 encodes a DEAD box protein and is believed to be associated with cellular growth and division; and CENPE involves unstable spindle microtubule capture at kinetochores. Together these genes are involved in DNA damage of apoptosis, RNA splicing, DNA repairing, and regulating cell division in the bovine genome. The topology of the learned Bayesian gene network indicated that the genes had a minimal interrelationship with each other. This type of structure, using the publically available computational tool, was also observed on human orthologous genes of the differentially expressed genes.Conclusions: Overall, the results might be used in transcriptomic-assisted selection and design of new drug targets to treat stress-related problems in bovines.

A point mutation to the long terminal repeat of bovine leukemia virus related to viral productivity and transmissibility

It is important to establish the molecular basis of the high transmissibility of bovine leukemia virus (BLV) to develop new methods of preventing viral transmission. Hence, the aim of this study was to determine whether some strains had transmission advantages. First, we determined the whole BLV genome sequences of all 34 BLV-infected cows from one farm. Phylogenetic analysis divided strains into 26 major and 8 minor strains. The major strains dominantly spread independent of host factor, bovine leucocyte antigen. Further analysis, with molecular clones, associated transmissibility with viral productivity in vitro. In addition, the two groups could be classified by group-specific mutations. The reverse genetic approach demonstrated that a spontaneous mutation at nucleotide 175 of the BLV genome, which is located in the viral promoter region, could alter viral productivity by changing viral transactivation, suggesting that BLV transmissibility is affected by a spontaneous mutation associated with viral productivity.

Elimination of erroneous results related to bovine mononuclear cell immunophenotyping by antibodies binding to Fc receptors

Blocking immunoglobulin G (IgG) binding receptors on leukocytes is an established and highly recommended preventive procedure for immunological assays. Failing to prevent such nonspecific binding can lead to erroneous results. Several studies testing different blocking reagents have been performed in murine or human cells, however, there are no specific studies on bovine cells. Our study aimed to investigate the efficiency of blocking reagents to inhibit the nonspecific binding of mouse monoclonal antibodies (mAbs) to bovine peripheral blood cells. We observed nonspecific interactions of IgG2a and IgG2b negative isotypes with bovine leukocytes, but not IgG1. We found that these nonspecific bindings could be eliminated by blocking with purified mouse IgG, whereas little or no blocking effect was observed when bovine serum or Mouse Seroblock FcR were applied. Moreover, in the absence of an efficient blocking reagent, the percentage of CD335 positive cells was significantly higher than in the group previously blocked with mouse IgG. Based on these results, and due to the lack of specific commercial blocking reagents for bovine cells, our recommendation is to use purified mouse IgG as a blocking reagent for immune assays targeting bovine leukocytes in order to enhance the accuracy of the results.

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

ABSTRACT: 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%.

Screening of Hardhenu Crossbred, Sahiwal and Hariana Bulls for Autosomal Recessive Genetic Disorder Bovine Leukocyte Adhesion Deficiency (BLAD)

The present study involved screening of Hardhenu crossbred, Sahiwal and Hariana bulls for autosomal recessive genetic disorder i.e Bovine Leukocyte Adhesion Deficiency (BLAD) using PCR based techniques. BLAD is a lethal autosomal recessive disease caused due to a point mutation (A→G) at nucleotide 383 in the CD18 gene which results in substitution of aspartic acid with glycine in the adhesion glycoprotein CD18. All animals under study were maintained at Cattle Breeding Farm, LalaLajpat Rai University of Veterinary and Animal Science (LUVAS), Hisar, India. About 5 ml blood was collected aseptically from jugular vein puncture into EDTA containing tubes, transported to the animal genomics laboratory and stored at-20°C until genomic DNA extraction, which was carried out using Phenol Chloroform method. The targeted genomic area of interest was amplified by using simple PCR technique. The amplified PCR products were digested with TaqI restriction enzyme. The targeted amplicon of 357 bp after digestion of the PCR product, the normal allele in unaffected bull produced two fragments of 201 bp and 156 bp. All the animals under study were found to be free from Bovine Leukocyte Adhesion Deficiency. The molecular screening revealed that none of the bulls screened under study was carrier for BLAD. The present study suggests that the PCR and PCR-RFLP based technique could be employed as an efficient technique for screening of various genetic disorders for identification of carriers or affected animals before using them in breeding programs to minimize the spread of defective allele.

Detection of allele and genotype frequencies of bovine leukocyte adhesion deficiency, factor XI deficiency and complex vertebral malformation disease genes in Holstein cattle

Hereditary diseases cause yield and economic loses. It is important to examine hereditary diseases at the molecular level and to remove diseases from the herd. In our study, it was aimed to determine allele frequencies of genes that cause bovine leukocyte adhesion deficiency, factor XI deficiency and complex vertebral malformation diseases in Holstein cattle. Blood samples were randomly taken from 300 Holstein cattle in different dairy farms in Kocaeli, Sakarya and Balıkesir provinces. Deoxyribonucleic acid samples were isolated from blood samples by using the standard ammonium acetate salt-out method. The target regions were amplified by polymerase chain reaction to determine the mutant alleles causing bovine leukocyte adhesion deficiency, factor XI deficiency and complex vertebral malformation. According to the nucleotide chromotograms of the samples subjected to bovine leukocyte adhesion deficiency analysis, it was determined that 4 out of 300 cattle were heterozygous and 296 were homozygous. Polymerase chain reaction procedure for factor XI deficiency disease was sufficient, while samples amplified by polymerase chain reaction for complex vertebral malformation disease were subjected to restriction particle length polymorphism. Factor XI deficiency and complex vertebral malformation disease genes were all homozygous normal.

Curcumin induced oxidative stress causes autophagy and apoptosis in bovine leucocytes transformed by Theileria annulata

Bovine tropical theileriosis is a tick-borne disease, caused by Theileria annulata which is a protozoan parasite that resides within the B-cells and macrophages. T. annulata is a unique parasite that can transform bovine leucocytes which leads to the cancer hallmarks in the infected cells. Previously, curcumin has been shown to possess multiple pharmacological activities such as anti-inflammatory and anti-cancer activities. In this study, we demonstrated that curcumin inhibits the proliferation of Theileria-transformed bovine leucocytes by promoting apoptosis and autophagy. The transcriptome analysis of curcumin treated cells showed that the genes involved in cell death and autophagy are also differentially regulated. We further elucidated the mechanism of action of curcumin on Theileria infected bovine cells. We found that curcumin induced the generation of reactive oxygen species (ROS) which activated caspase 8 and destabilized the mitochondrial membrane potential leading to the release of cytochrome c from mitochondria. This subsequently led to the activation of caspase 3 and PARP cleavage, finally leading to apoptosis in the infected cells. Furthermore, curcumin induced the process of autophagy which was characterized by the formation of acidic vesicular organelles, LC3B accumulation with lysosome inhibitor, E64d, and the presence of autophagosomes as visualized by transmission electron microscopy (TEM). Curcumin treatment suppressed the mTOR and increased the expression of autophagy-related proteins. We also found that N- acetylcysteine, an inhibitor of ROS, could rescue the infected cells from curcumin induced apoptosis and autophagy mediated cell death. Intriguingly, curcumin had no effect on uninfected bovine PBMCs. Altogether, these data suggest the therapeutic potential of curcumin against bovine tropical theileriosis.

Prophylactic digoxin treatment reduces IL-17 production in vivo in the neonatal calf and moderates RSV-associated disease.

Respiratory syncytial virus (RSV) is a leading cause of morbidity and mortality in human infants. Bovine RSV infection of neonatal calves is pathologically and immunologically similar to RSV infection in infants, and is therefore a useful preclinical model for testing novel therapeutics. Treatment of severe RSV bronchiolitis relies on supportive care and may include use of bronchodilators and inhaled or systemic corticosteroids. Interleukin-17A (IL-17) is an inflammatory cytokine that plays an important role in neutrophil recruitment and activation. IL-17 is increased in children and rodents with severe RSV infection; and in calves with severe BRSV infection. It is currently unclear if IL-17 and Th17 immunity is beneficial or detrimental to the host during RSV infection. Digoxin was recently identified to selectively inhibit IL-17 production by antagonizing its transcription factor, retinoid-related orphan receptor γ t (RORγt). Digoxin inhibits RORγt binding to IL-17 and Th17 associated genes, and suppresses IL-17 production in vitro in human and murine leukocytes and in vivo in rodent models of autoimmune disease. We demonstrate here that in vitro and in vivo digoxin treatment also inhibits IL-17 production by bovine leukocytes. To determine the role of IL-17 in primary RSV infection, calves were treated prophylactically with digoxin and infected with BRSV. Digoxin treated calves demonstrated reduced signs of clinical illness after BRSV infection, and reduced lung pathology compared to untreated control calves. Digoxin treatment did not adversely affect virus shedding or lung viral burden, but had a significant impact on pulmonary inflammatory cytokine expression on day 10 post infection. Together, our results suggest that exacerbated expression of IL-17 has a negative impact on RSV disease, and that development of specific therapies targeting Th17 immunity may be a promising strategy to improve disease outcome during severe RSV infection.

Immune cell populations residing in mesenteric adipose depots and mesenteric lymph nodes of lean dairy cows

Inconsistent evidence of inflammatory immune cell infiltrates in adipose tissues with extensive triglyceride mobilization raises the possibility that regulatory or anti-inflammatory immune cell populations reside within the mesenteric adipose tissue (MAT) and mesenteric lymph nodes (MLN). These resident immune cell populations may be involved in attenuating the inflammatory response. We explored the immune cell population of MAT and MLN collected from lean, lactating Holstein cows without apparent disease in an abattoir (n = 42). Lean cows had a body condition score of 2.6 ± 0.1 (mean ± SD) with a greater frequency of adipocyte area occurring in small rather than large adipocytes. Cells were labeled with monoclonal antibodies specific to bovine leukocyte antigens for enumeration by flow cytometry. Within both lymph node and adipose tissues, relatively large subpopulations of cells expressed the β2 integrins CD11b and CD11c, class II major histocompatibility antigens (MHCII), and the SIIRP-1α receptor (CD172a) typical of dendritic cells and macrophages. Macrophage/dendritic cell heterogeneity was marked by β2 integrin expression alone or in conjunction with CD172a or MHCII across subpopulations from both tissues; CD209, the DC-SIGN c-type lectin receptor of dendritic cells, was not detected by fluorescence-activated cell sorting in either tissue. Lymphocytes comprised 74.1 ± 3.7% and 13.7 ± 3.7% of the MLN and MAT cell populations, respectively, and CD3+CD4+ lymphocytes accounted for 49.8 ± 9.9% of the MLN and 6.13 ± 1.23% of the MAT cells. Fox P3+ regulatory lymphocytes comprised 15.3 ± 1.1% and 6.73 ± 0.52% of the MLN and MAT cells, whereas γδ+ lymphocytes accounted for 6.65 ± 0.74% and 3.91 ± 0.43% of the MLN and MAT cells, respectively. Subpopulations of CD3+CD8+ cytotoxic T cells and CD3+CD11c+ innate lymphocytes were present in MLN but not MAT. These results show that subpopulations of resident tissue macrophages, dendritic cells, T helper lymphocytes, regulatory T lymphocytes (Tregs), and γδ lymphocytes reside in mesenteric lymph nodes and adipose tissues. Balance in the innate and adaptive immune functions embedded in these tissues could support metabolic health.

The new bovine reference genome assembly provides new insight into genomic organization of the bovine major histocompatibility complex

The reference genome sequence represents a key resource for genetic studies of the target species. In 2009, two reference assemblies of the cattle (Bos taurus) genome, were published (Btau 4.0 and UMD 2.0). Both assemblies were upgraded several times since then. Highly polymorphic major histocompatibility complex (MHC) encodes proteins crucial for immune recognition and regulation of immune response in vertebrates. It is characterised by extensive nucleotide diversity, copy number variation of paralogous genes, and long repetitive sequences. In cattle, MHC is designated as BoLA (bovine leucocyte antigen), located on the chromosome 23. Its organisation differs from typical mammalian MHCs. The structural complexity makes it difficult to assemble a reliable reference sequence of this genomic region. Therefore, this region represents a good genomic model region to compare the accuracy of different assembly strategies. Recent advances in long-read sequence technology, combined with new scaffolding technologies, enabled issuing of the new bovine reference genome assembly build ARS-UCD 1.2, which is significantly improved over previous bovine genome assembly releases. In the current study the software tool Mauve for multiple alignment of conserved genomic sequences with rearrangements was used to identify the differences of genomic organization in the BoLA region assembled in three bovine reference genomes, Btau 5.0.1, UMD 3.1.1, and ARS-UCD 1.2. Multiple alignment of the bovine chromosome 23 sequences extracted from three genome assemblies revealed differences in the structure of the BoLA region. Segments encoding genes BOLA-DMA and BOLA-DQB are rearranged and inverted in the new assembly relative to the previous builds.

HIGH- RESOLUTION MELTING (HRM) CURVE ANALYSIS: NEW APPROACH USED TO DETECT BLAD AND DUMPS IN URUGUAYAN HOLSTEIN BREED

The widespread use of artificial insemination has allowed the expansion of genetic progress. However, it also brought consequences such as the expansion of lethal hereditary diseases and the increase in inbreeding. The object of this study was to establish a fast and sensitive molecular assay to detect bovine leukocyte adhesion deficiency (BLAD) and deficiency of uridine monophosphate synthase (DUMPS) carriers in Uruguayan Holstein cattle by means of high resolution melting (HRM) curve analysis. By testing previously confirmed carrier and non-carrier animals, we set up a rapid, simple, and inexpensive diagnostic test using PCR followed by HRM curve analysis. The PCR-HRM genotyping method was effective for the discrimination of BLAD and DUMPS homozygous genotypes, and the BLAD heterozygous genotype. We conclude that the PCR-HRM assay is a robust, reliable, and economical tool for the detection of these mutations in the Holstein breed, which may be implemented in genetic selection programs.

PRODUCTION OF INTERLEUKIN 1BETA AND INTERFERON GAMMA BY BOVINE MAMMARY GLAND LEUKOCYTES STIMULATED BY LIPOPOLYSACCHARIDE

The aim of this trial was to investigate the production of the pro-inflammatory cytokines interleukin 1beta (IL-1β) and interferon gamma (IFN-γ) produced by bovine mammary gland leukocytes following lipopolysaccharide (LPS) challenge (5 µg LPS of Escherichia coli in 20 ml of PBS) or PBS (20 ml) as a control. The leukocytes were obtained by mammary gland lavage 1, 2, 3, and 7 days following the stimulation. The concentration levels of these cytokines were analyzed by sandwich ELISA. Eight clinically healthy crossbred heifers (Holstein x Czech Pied) were selected for this study. The heifers were group housed in a tie-stall barn and fed a total mixed diet. Higher average number of leukocytes (P

Allele frequency estimation of BLAD (Bovine Leukocyte Adhesion Deficiency) in dairy cattle in Enrekang regency South Sulawesi Indonesia

Bovine leukocyte adhesion deficiency (BLAD) is a genetic disorder in dairy cows that are generally lethal or premature in cattle that can have an impact on productivity and result in losses for farmers. The purpose of this study is to identify the distribution of the BLAD recessive allele in dairy cows raised in Enrekang dairy cattle development centers. Identification of recessive BLAD allele using the PCR-RFLP method. A total of 80 DNA samples in a collection of blood samples from FH dairy cows from Enrekang regency (50 heads from Cendana and 30 heads from Anggeraja district) were extracted and amplified by PCR, then the PCR product was cut with the Taq1 restriction enzymes. The identification of the BLAD carrier alleles is calculated according to the frequency of genotypes and alleles. The results obtained from this study is (+/-) heterozygous cows (BLAD carrier) found 1 head (with a frequency of the allele (-) (BLAD recessive) of 0.01) in the Cendana district of 50 samples, while the Bovine population in the district Anggeraja did not found any BLAD recessive allele. The results of this study concluded that there is a normal heterozygous dairy cows (BLAD carrier) in the dairy cows population in the Enrekang regency, although the proportion / frequency of alleles is very low (0.625%). This research can be used as a reference in selecting dairy cattle, especially those related to the spread of lethal alleles in dairy cattle development areas in South Sulawesi.

The protective effect of chlorogenic acid on bovine mammary epithelial cells and neutrophil function

Chlorogenic acid (CGA) is the ester of caffeic acid and quinic acid and plays an important role in antibacterial activity and anti-inflammatory properties. The objective of this study was to examine the effects of CGA on the growth of Staphylococcus aureus and the mRNA levels of the genes encoding the inflammatory response cytokines, κ-casein, and neutrophil function in bovine mammary epithelial cells (BMEC) exposed to S. aureus. Chlorogenic acid has important antibacterial, antioxidant, and anti-inflammatory functions; however, the effect of CGA on BMEC and neutrophils exposed to S. aureus has not been investigated previously. Our results demonstrated that 10, 20, and 30 μg/mL CGA had no cytotoxic effects on BMEC in culture, and that 20 μg/mL CGA enhanced the viability of BMEC exposed to S. aureus, whereas 30 μg/mL CGA reduced S. aureus growth after 9 h compared with controls. The rate of S. aureus invasion into BMEC was also attenuated by 30 μg/mL CGA compared with controls, whereas this treatment led to reduced abundance of IL6, IL8, and TLR2 mRNA in S. aureus-exposed BMEC. Migration of bovine polymorphonuclear leukocytes was significantly decreased in S. aureus-exposed BMEC with 10 and 20 μg/mL CGA treatment when compared with S. aureus treatment alone. In addition, incubation with 20 or 30 μg/mL CGA enhanced the phagocytic ability of polymorphonuclear leukocytes compared with the control group. Importantly, levels of κ-casein were enhanced by treatment of S. aureus-exposed BMEC with CGA. Our results suggest that the use of CGA may be a potent therapeutic tool against bovine mastitis caused by S. aureus.

Bovine Neonatal Pancytopenia-Associated Alloantibodies Recognize Individual Bovine Leukocyte Antigen 1 Alleles.

Bovine neonatal pancytopenia (BNP) was a vaccine-induced alloimmune disease observed in young calves and characterized by hemorrhages, pancytopenia, and severe destruction of the hematopoietic tissues. BNP was induced by alloreactive maternal antibodies present in the colostrum of certain cows vaccinated with a highly adjuvanted vaccine against bovine viral diarrhea. Bioprocess impurities, originating from the production cell line of the vaccine, are likely to have induced these alloreactive antibodies. One prominent alloantigen recognized by vaccine-induced alloantibodies is highly polymorphic bovine major histocompatibility complex class I antigen (bovine leukocyte antigen 1—BoLA I). Aim of this study was to define the fine specificity of BNP-associated anti-BoLA I alloantibodies. In total, eight different BoLA I alleles from the production cell line were identified. All genes were cloned and recombinantly expressed in murine cell lines. Using these cells in a flow cytometric assay, the presence of BoLA I specific alloantibodies in BNP dam sera was proven. Three BoLA I variants were identified that accounted for the majority of vaccine-induced BoLA I reactivity. By comparing the sequence of immunogenic to non-immunogenic BoLA I variants probable minimal epitopes on BoLA I were identified. In general, dams of BNP calves displayed high levels of BoLA I reactive alloantibodies, while vaccinated cows delivering healthy calves had significantly lower alloantibody titers. We identified a subgroup of vaccinated cows with healthy calves displaying very high alloantibody titers. Between these cows and BNP dams no principle difference in the BoLA I reactivity pattern was observed. However, with a limited set of dam-calf pairs it could be demonstrated that serum from these cows did not bind to BoLA I expressing leukocytes of their offspring. By contrast, when testing cells from surviving BNP calves with the corresponding dam’s serum there was significant binding. We therefore conclude that predominantly highly alloreactive cows are at risk to induce BNP and it depends on the paternally inherited BoLA I whether or not the calf develops BNP.

Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis (EBL), which causes enormous economic losses in the livestock industry worldwide. To reduce the economic loss caused by BLV infection, it is important to clarify the characters associated with BLV transmissibility and pathogenesis in cattle. In this study, we focused on viral characters and examined spontaneous mutations in the virus and viral properties by analyses of whole genome sequences and BLV molecular clones derived from cows with and without EBL. Genomic analysis indicated that all 28 strains harbored limited genetic variations but no deletion mutations that allowed classification into three groups (A, B, and C), except for one strain. Some nucleotide/amino acid substitutions were specific to a particular group. On the other hand, these genetic variations were not associated with the host bovine leukocyte antigen-DRB3 allele, which is known to be related to BLV pathogenesis. The viral replication activity in vitro was high, moderate, and low in groups A, B, and C, respectively. In addition, the proviral load, which is related to BLV transmissibility and pathogenesis, was high in cows infected with group A strains and low in those infected with group B/C strains. Therefore, these results suggest that limited genetic variations could affect viral properties relating to BLV transmissibility and pathogenesis.