Bovine Leukocytes Research Articles

Prepartal Energy Intake Alters Blood Polymorphonuclear Leukocyte Transcriptome During the Peripartal Period in Holstein Cows.

In the dairy industry, cow health and farmer profits depend on the balance between diet (ie, nutrient composition, daily intake) and metabolism. This is especially true during the transition period, where dramatic physiological changes foster vulnerability to immunosuppression, negative energy balance, and clinical and subclinical disorders. Using an Agilent microarray platform, this study examined changes in the transcriptome of bovine polymorphonuclear leukocytes (PMNLs) due to prepartal dietary intake. Holstein cows were fed a high-straw, control-energy diet (CON; NEL = 1.34 Mcal/kg) or overfed a moderate-energy diet (OVE; NEL = 1.62 Mcal/kg) during the dry period. Blood for PMNL isolation and metabolite analysis was collected at −14 and +7 days relative to parturition. At an analysis of variance false discovery rate <0.05, energy intake (OVE vs CON) influenced 1806 genes. Dynamic Impact Approach bioinformatics analysis classified treatment effects on Kyoto Encyclopedia of Genes and Genomes pathways, including activated oxidative phosphorylation and biosynthesis of unsaturated fatty acids and inhibited RNA polymerase, proteasome, and toll-like receptor signaling pathway. This analysis indicates that processes critical for energy metabolism and cellular and immune function were affected with mixed results. However, overall interpretation of the transcriptome data agreed in part with literature documenting a potentially detrimental, chronic activation of PMNL in response to overfeeding. The widespread, transcriptome-level changes captured here confirm the importance of dietary energy adjustments around calving on the immune system.

Identification of heterozygous cases of Bovine leukocyte adhesion deficiency (BLAD) in Indian Holstein crossbred bulls

BLAD is an Autosomal genetic disorder mainly in Holstein and its crosses are major concern worldwide as heterozygous (carrier) looks normal and therefore, used for breeding programmes. During the last one and half years, blood samples were collected in the heparinized blood collecting tubes from 570 dairy bulls including 11 HF and 81 HF crossbred bulls for investigation of heterozygous/carrier animals for various genetic disorders, including Bovine Leukocyte Adhesion Deficiency Syndrome. The DNA extraction, PCR and RFLP were performed to find out carrier animals. PCR-RFLP techniques exhibited a polymorphism in CD18 gene in two Holstein crossbred bulls, which were confirmed by sequencing. The percentage of recessive allele in Holstein and Holstein crossbred samples was calculated to be 2.17. Rest all animals were found normal.

Genetic variability of the major histocompatibility complex (MHC) class II (DRB3) in South African and Namibian beef cattle breeds

The major histocompatibility complex region has been implicated in explaining some of the variation observed in adaptability and tick susceptibility of cattle. The bovine leukocyte antigen region of 192 cattle representing indigenous, composite and exotic breeds used in commercial beef production in Namibia and South Africa was investigated using four microsatellite markers. Ticks counted under the tail were taken as an indicator of tick susceptibility. Tick scores of all but one population was low (11 to 20 ticks), with only the South African Bonsmara population having an average score of 31 to 40 ticks per animal. The observed variation based on four microsatellite markers ranged from 5.5 alleles in Namibian Afrikaner to 7.7 alleles in South African Nguni and Bonsmara cattle. Unbiased heterozygosity values ranged from 0.66 (Namibian Afrikaner) to 0.76 (South African Bonsmara). Structure analyses grouped the five populations into three indistinct clusters with limited genetic variation between the populations.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata.

SummaryHSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata‐infected counterpart was utilized to test the effects of geldanamycin and the derivative 17‐AAG. T. annulata‐infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17‐AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.

Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells.

Differentiation induction therapy is an attractive approach in leukemia treatment due to the fact that in blast crisis stage, leukemic cells lose their differentiation capacity. Therefore, it has been proposed as a therapeutic strategy to induce terminal differentiation of leukemic blast cells into a specific lineage, leading to prevention of high proliferation rates. The aim of the present study was to demonstrate the potential of cell differentiation and death induced by bovine dialyzable leukocyte extract (bDLE) in the K562 cell line. For this purpose K562 and MOLT-3 human leukemic cell lines and primary human monocytes and murine peritoneal macrophages were exposed to bDLE, phorbol myristate acetate (PMA) and dimethyl sulfoxide for 96 h, and the viability, proliferation and cell cycle were evaluated. To determine the lineage that led to cell differentiation, Romanowsky staining was performed to observe the morphological changes following the treatments, and the expression of the surface markers cluster of differentiation (CD)14+, CD68+, CD163+ and CD42a+, as well as the phagocytic activity, and the production of nitric oxide (NO) (assessed by colorimetric assay), cytokines [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] and chemokines [chemokine (C-C motif) ligand (CCL)2, CCL5 and chemokine (C-X-C motif) ligand 8] in cell supernatants was assessed by flow cytometry. The results of the present study reveal that high doses of bDLE increase the cell death in K562 and MOLT-3 lines, without affecting the viability of human monocytes and murine peritoneal macrophages. Furthermore, low doses of bDLE induce differentiation in K562 cells towards a monocyte/macrophage lineage with an M2 phenotype, and induced moderately upregulated expression of CD42+, a megakaryocytic marker. Cell cycle arrest in the S and G2/M phases was observed in bDLE-treated K562 cells, which demonstrated similar phagocytic activity, NO levels and cytokine and chemokine production to that of PMA-treated cells. The present study demonstrates that bDLE exhibits an antileukemia effect, suggesting that it may be an effective candidate for leukemia treatment.

Development of a fast and economical genotyping protocol for bovine leukocyte adhesion deficiency (BLAD) in cattle.

Fast and economical means of assaying SNP’s are important in diagnostic assays, especially when a large number of animals have to be screened for a genetic disease. This study was aimed at the development of a fast and economical screening assay for bovine leukocyte adhesion deficiency (BLAD) which is an important genetic disease of cattle industry. Four primers were designed where the outer primers amplify a 354 bp amplicon of CD18 gene carrying the polymorphism responsible for BLAD. The specifically designed inner primers in conjunction with the modified reaction mixture and cyclic conditions ensured amplification of either of wild or mutated alleles. Together with outer primers, the inner primers generated typical banding pattern in agarose gel which discriminated the normal animal against the carrier. We successfully used this protocol in 200 bulls for genotyping the BLAD allele which confirmed by sequencing, showing a cent percentage concordance. With the developed assay the need for restriction digestion or use of costly equipment viz. real time PCR was eliminated. This genotyping assay ensured fast and economical genotyping and could be adopted in every laboratory with a minimum equipment requirement of thermocycler and gel documentation system.

Bovine P-selectin mediates leukocyte adhesion and is highly polymorphic in dairy breeds

Bovine P-selectin (SELP) mediates leukocyte rolling and primes leukocyte adhesion to endothelium, both essential for leukocyte recruitment to an infection site. We investigated SELP-mediated adhesion between bovine peripheral blood leukocytes (PBLs) and cultured bovine aortic endothelial cells pre-activated with lipopolysaccharide (LPS). We examined gene polymorphism for bovine selectins SELP, l-selectin (SELL) and E-selectin (SELE) and compared their SNP frequency between five dairy breeds (Holstein, Friesian, Jersey, Ayrshire and Brown Swiss). LPS treatment caused a rapid (10min) and slower (4h) enhancement of PBL adhesion (P<0.01). Antibody blocking of SELP inhibited LPS induced cell adhesion. SELP was highly polymorphic, with 9 of the 13 SNPs in its exons, whereas only three synonymous SNPs in SELL and one in SELE. The resulting amino acid changes for the three missense SELP SNP were located in the lectin domain and in two consensus repeat (CR) regions, CR2 and CR5. The Val475Met variant locus in the CR4 and CR5 linking region was very close to a predicted N-acetyl-d-glucosamine glycosylation site, which is likely to influence SELP function. The AA genotype was under-represented, only being found in 1% of 373 heifers genotyped from the 5 breeds (P=0.056), suggesting that AA homozygous animals carrying the Val475Met substitution for SELP may have compromised development. Our study thus confirmed that SELP mediates the attachment of PBL to endothelium and provides novel evidence that its high polymorphism is likely to affect biological function. This may potentially influence leukocyte migration and fertility, both key to successful performance in dairy cows.

Analysis of variations, structures, and phylogenic characteristics of bovine leukocyte antigen DRB3 exon2.

Bovine leukocyte antigen (BoLA) DRB3 is a highly polymorphic gene in major histocompatibility complex (MHC) class II that plays a central role in immune responses and production factors. As of yet, molecular and evolutionary characteristics of BoLA-DRB3.2* have not been as fully understood as human and mouse. Therefore, we attempted to analyze variability and phylogeny of BoLA-DRB3.2* and illustrate some novel practical evidence on interspecies diversity, the resistance /susceptibility points in cattle breeding, and vaccine design. Initially, BoLA-DRB3.2* alleles and orthologous exons in the selected livestock were retrieved and checked. In the next step, the secondary/tertiary structure of BoLA-DRB3.2*24 gene product was modeled and validated. Then, hypervariable regions (HVRs) of alleles were identified by hybrid approaches. In the last step, interspecies relationship, allele&amp;rsquo;s phylogeny/grouping, and estimate of average evolutionary divergence were explored. Shannon entropy variation analysis showed eight HVRs and three semi-variable regions in BoLADRB3.2* alleles. These HVRs were present in all the three sub-structures and dominantly existed in alpha helix. In addition, strong relationships and little diversity were noted in phylogenetic trees of cattle, buffaloes, sheep, and goats. Furthermore, there was some evidence on divergence of DRB3 before speciation among the mentioned species and possibility of cross prediction resistance/susceptibility alleles. Finally, DRB3 alleles were grouped into seven clusters, and older and newer alleles were identified. The results show that similar studies should be done in other animals to better understand the nature of the DRB3 attributes.

Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, bovine citrullinaemia and factor XI deficiency in Holstein cattle

Holstein cattle reared in Eskisehir region were monitored with respect to the genetic disorders defined as bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), bovine citrullinaemia (BC) and factor XI deficiency (FXID). To determine the presence of BLAD, DUMPS, BC and FXID genotypes, Holstein cattle (219) were sampled. Three BLAD and 2 FXID carriers were identified among the 219 Holstein cattle examined, while none of the cows carried DUMPS and BC. Current study demonstrated that carriers of BLAD and FXID are present in the Turkish Holstein population in Eskisehir region, although at a low frequency. Since, artificial insemination is widely used in these populations, determination of the carriers of hereditary disorders within the population of breeding sires has become essential. To prevent economic losses resulting from genetic disorders, it is advised to screen breeding sires for these genotypes. The aim of this study was to investigate existence and prevalence of BLAD, DUMPS, BC and FXID alleles in Holstein cattle reared in Eskisehir region by using PCR and PCR-RFLP methods.

Phenotypic and genetic effects of recessive haplotypes on yield, longevity, and fertility

Phenotypes from the August 2015 US national genetic evaluation were used to compute phenotypic effects of 18 recessive haplotypes in Ayrshire (n=1), Brown Swiss (n=5), Holstein (n=10), and Jersey (n=2) cattle on milk, fat, and protein yields, somatic cell score (SCS), single-trait productive life (PL), daughter pregnancy rate (DPR), heifer conception rate (HCR), and cow conception rate (CCR). The haplotypes evaluated were Ayrshire haplotype 1, Brown Swiss haplotypes 1 and 2, spinal dysmyelination, spinal muscular atrophy, Weaver Syndrome, brachyspina, Holstein cholesterol deficiency, Holstein haplotypes 1 to 5, bovine leukocyte adhesion deficiency, complex vertebral malformation, mulefoot (syndactyly), and Jersey haplotypes 1 and 2. When causal variants are unknown and tests are based only on single nucleotide polymorphism haplotypes, it can sometimes be difficult to accurately determine carrier status. For example, 2 Holstein haplotypes for cholesterol deficiency have the same single nucleotide polymorphism genotype, but only one of them carries the causative mutation. Genotyped daughters of carrier bulls included in the analysis ranged from 8 for Weaver Syndrome to 17,869 for Holstein haplotype 3. Lactation records preadjusted for nongenetic factors and direct genomic values (DGV) were used to estimate phenotypic and genetic effects of recessive haplotypes, respectively. We found no phenotypic or genetic differences between carriers and noncarriers of Ayrshire or Brown Swiss defects. Several associations were noted for Holstein haplotypes, including fat and HCR for Holstein haplotype 0 carriers; milk, protein, SCS, PL, and fertility for Holstein haplotype 1; protein, PL, CCR, and HCR for Holstein haplotype 2; milk, protein, and fertility for Holstein haplotype 4; and protein yield and DPR for Holstein haplotype 5. There were no differences among bovine leukocyte adhesion deficiency carriers, but complex vertebral malformation affected fat yield and mulefoot carriers had higher SCS and lower PL DGV. Jersey haplotype 1 was not associated with any phenotypic effects, but we noted significant differences among DGV for fat, protein, PL, DPR, CCR, and HCR. Jersey haplotype 2 was associated only with lower phenotypic CCR. Effects of the recessive haplotypes on other traits studied generally were small even when significant. Almost $11 million of economic losses per year due to reduced fertility and perinatal calf death in the US population can be avoided by selecting mate pairs that will not produce affected embryos. Carrier animals may continue to be selected if the merit of their favorable alleles exceeds the loss from their recessive alleles, but carrier bulls can be generally avoided without reducing the average genetic merit of the sires available for mating.

Quantification methods for human and large animal leukocytes using DNA dyes by flow cytometry.

Ovine and bovine blood is used heavily within the development of blood-handling medical devices, such as heart pumps (left ventricular assist devices, LVADs), for which blood cell damage needs to be monitored during in vitro testing. Hematology analyzers provide cell counts but no information about cell viability. The anthraquinone DNA dyes CyTRAK Orange™ and DRAQ7™ have practical and spectral properties rendering them suitable for multicolor assays. Compared to other DNA dyes such as Vybrant Dyecycle, CyTRAK Orange enables a faster staining protocol and does not require incubation at +37°C. Compared to traditional viability dyes such as propidium iodide and 7AAD, DRAQ7's unique spectral profile of excitation in both blue and red lasers and far-red emission enables identification of dual positive dead cell events and frees up detectors for use with other reagents. CyTRAK Orange and DRAQ7 could be used in combination with absolute counting bead standards to provide cell counts and viability but the combination of these dyes has previously only been used for microscopy on rodent cells. The purpose of this study was to evaluate the use of these dyes in combination in large animal blood samples for flow cytometry. A viability and cell counting protocol for bovine, ovine, and human leukocytes using CyTRAK Orange and DRAQ7 was prepared. Four different counting bead standards were evaluated using the Navios and FACSAria cytometers and compared to counts obtained from hematology analyzers. CyTRAK Orange successfully detected CD45(+) leukocytes in all species. The DRAQ7 single-stained dead cell counts correlated well with the CyTRAK Orange/DRAQ7 double-stained dead cell counts in human and bovine blood, but not in ovine blood, which could be related to the blood source. In conclusion, for human and bovine blood, this method works well for viability counts using different flow cytometers and bead standards. © 2016 International Society for Advancement of Cytometry.

Sequence based typing for BoLA-DQA1 alleles in Indian zebu and its crossbred populations

Polymorphism in the major histocompatibility complex of the bovine, which is referred to as bovine leukocyte antigens (BoLAs), was intensively investigated for identifying marker(s) for bovine diseases and immunological traits. This approach requires identifying and documenting the allelic diversity of BoLA among different breeds of cattle using PCR-SBT technique. In this study, we could standardise the most useful approach of unravelling polymorphism in DQA1 exon 2 of cattle using PCR-sequence based typing (SBT). We could identify 16 different DQA1 alleles in 43 zebu and crossbred cattle, out of which 26 were homozygous and 17 were heterozygous. PCR- SBT has proved to be the most useful method for exploring the allelic polymorphism in DQA1 gene in zebu and its crossbred cattle in our study. Further use of this technique is equally appropriate for all kinds of traits i.e. production, reproduction and growth type of traits and beneficial for establishing new breeding objectives.

In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria Box Reveals Novel Compounds with Profound Activities against Theileria annulata Schizonts.

Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leukocytes and thereby cause fatal diseases. The hydroxynaphthoquinone buparvaquone is currently the only option for the treatment of theileriosis, and resistance development has been reported. It is therefore tempting to investigate the repurposing of compounds effective against related apicomplexan parasites, such as Plasmodium Here, we present the results of a screen of 400 compounds included in the open-access Medicines for Malaria Venture (MMV) malaria box on TaC12 cells, a macrophage-derived cell line immortalized by T. annulata schizonts. Using a combination of the classical alamarBlue vitality assay and a recently developed quantitative reverse transcriptase real-time PCR method based on the Theileria TaSP gene, we have identified 5 compounds, characterized their effects on the ultrastructure of TaC12 cells, and investigated whether they easily induce resistance formation. Two compounds, the quinolinols MMV666022 and MMV666054, have 50% inhibitory concentrations (IC50s) of 0.5 and 0.2 μM on TaC12 cells and 5.3 and 5.2 μM on BoMac cells, respectively. Thus, with therapeutic indexes of 11 and 18, they represent promising leads for further development of antitheilerial chemotherapeutics.

Polymorphism and expression of the tumor necrosis factor receptor II gene in cows infected with the bovine leukemia virus

A single T>C nucleotide polymorphism (rs42686850) of bovine tumor necrosis factor receptor type II gene (TNF-RII) is located within a sequence with allele-specific affinity to bind E2F transcription factors, considered pivotal in the regulation of cell cycle and cell proliferation. The objective of the study was to determine the effect of this SNP and BLV infection on the TNF-RII gene expression at the mRNA and protein levels in peripheral blood mononuclear cells (PBMC). We noted that analyzed TNF-RII gene polymorphism influenced the expression of the TNF-RII gene at the mRNA level but only in BLV-positive cows. Concurrently, no statistically significant association was found between gene polymorphism and TNF-RII expression at the protein level. However, we found a significant effect of BLV infection status on the amount of TNF-RII mRNA and the percentage of PBMC expressing TNF-RII. These results show an unclear effect of considered T>C polymorphism on TNF-RII gene expression in bovine leukocytes and they suggest the involvement of BLV in modifying the TNF-RII expression in BLV-infected cows potentially implying the EBL (Enzootic Bovine Leukosis) associated pathogenesis.

In VivoChemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients.

Identification and characterization of common B cell epitope in bovine leukemia virus via high-throughput peptide screening system in infected cattle.

Background Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. BLV is closely related to human T cell leukemia virus. B cell epitopes are important for the use of antibodies as therapeutic agents, the epitope-driven vaccine design, and immunological assays. A common B cell epitope for BLV has not yet been found due to individual differences in disease susceptibility.ResultsWe used a peptide microarray with 156 synthetic 15-mer peptides covering the envelope glycoprotein gp51 and the Gag proteins p15, p24, and p12 to map B cell epitope and one B cell epitope, gp51p16, was recognized by all four cattle experimentally infected with BLV. A newly developed high-throughput peptide ELISA system revealed 590 (91.2 %) of 647 cattle naturally infected with BLV, carrying 25 different bovine leukocyte antigen class II DRB3 (BoLA-DRB3) alleles, responded to a 20-mer gp51p16-C peptide containing a C-terminal cysteine and gp51p16. Alanine mutation and comparison of the sequences at 17 amino acid positions within gp51p16-C revealed that R7, R9, F10, V16, and Y18 were the common binding sites to BLV antibodies, and two of these sites were found to be highly conserved. Transient expression in the cells of five infectious molecular clones of BLV with a single alanine mutation at five common antibody binding sites had no effect syncytia formation of the gp51 protein. In addition, the mutant proteins, R7A and R9A had no effect on the expression of gp51 protein; the gp51 protein expressions of F10A, V16A and Y18A were lower than that of the wild type protein.ConclusionsThis is the first report to identify a common B cell epitope in BLV by comprehensive screening of BLV-infected cattle with varied genetic backgrounds in BoLA-DRB3. Our results have important implications for disease control and diagnosis.

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules.

Major histocompatibility complex (MHC) class Imolecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network-based predictions (NetMHCpan). The updated NetMHCpan server was used to predict BoLA-I binding peptides within the P1 structural polyprotein sequence of FMDV (strain A24 Cruzeiro) for Bo-LA-1*01901, BoLA-2*00801, BoLA-2*01201, and BoLA-4*02401. Peptide binding affinity and stability were determined for these BoLA-I molecules using the luminescent oxygen channeling immunoassay (LOCI) and scintillation proximity assay (SPA). The functional diversity of known BoLA alleles was predicted using theMHCcluster tool, and functional predictions for peptide motifs were compared to observed data from this and prior studies. The results of these analyses showed that BoLA alleles cluster into three distinct groups with the potential to define BBoLA supertypes.^ This streamlined approach identifies potential T cell epitopes from pathogens, such as FMDV, and provides insight into T cell immunity following infection or vaccination.

Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum

The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25×107 live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7×107 NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3+, CD4+, γδ-T cells, CD8+ cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P<0.001). The results from this study showed that the strongest cellular immune responses were observed in the placentomes of animals that were immunized with inactivated vaccines (groups B and C) and in the placentomes of animals that were sc-sham-inoculated (groups D and E). On the other hand, animals that were immunized with live tachyzoites showed a milder immune cell infiltration to the placenta possibly due to the existence of a protective systemic maternal immune response that helped to minimize N. caninum infection at the maternal-fetal interface.

Reference ranges of hematology and lymphocyte subsets in healthy Korean native cattle (Hanwoo) and Holstein dairy cattle.

There are no accurate reference ranges for hematology parameters and lymphocyte subsets in Korean native beef cattle (Hanwoo). This study was performed to establish reliable reference ranges of hematology and lymphocyte subsets using a large number of Hanwoo cattle (n = 350) and to compare differences between Hanwoo and Holstein dairy cattle (n = 334). Additionally, age-related changes in lymphocyte subsets were studied. Bovine leukocyte subpopulation analysis was performed using mono or dual color flow cytometry. The leukocyte subpopulations investigated in healthy cattle included: CD2(+) cells, sIgM(+) cells, MHC class II(+) cells, CD3(+) CD4(+) cells, CD3(+) CD8(+) cells, and WC1(+) cells. Although Hanwoo and Holstein cattle are the same species, results showed several differences in hematology and lymphocyte subsets between Hanwoo and Holstein cattle. This study is the first report to establish reference ranges of hematology and lymphocyte subsets in adult Hanwoo cattle.

Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins.

Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF’ on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF’, shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0243-7) contains supplementary material, which is available to authorized users.