Bovine Lymphocytes Research Articles

A new oxygen modification cyclooctaoxygen binds to nucleic acids as sodium crown complex

BackgroundOxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990. MethodsCyclooctaoxygen sodium was synthesized in vitro from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. Thin-layer chromatographic mobility shift assays were applied on specific nucleic acids and the cyclooctaoxygen sodium complex. ResultsWe report the first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes. The cationic cyclooctaoxygen sodium complex is shown to bind to nucleic acids (RNA and DNA), to associate with single-stranded DNA and spermine phosphate, and to be essentially non-toxic to cultured mammalian cells at 0.1–1.0mM concentration. ConclusionsWe postulate that cyclooctaoxygen is formed in most eukaryotic cells in vivo from dihydrogen peroxide in a catalase reaction catalyzed by cytidine and RNA. A molecular biological model is deduced for a first epigenetic shell of eukaryotic in vivo DNA. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic in vivo DNA. General significanceSince the sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the active regions (2.6%) of bovine lymphocyte interphase genome, and 12.4% of murine enterocyte mitotic chromatin, we propose that the sperminium phosphate/cyclooctaoxygen sodium complex coverage of nucleic acids is essential to eukaryotic gene regulation and promoted proto-eukaryotic evolution.

Analysis of the Cellular Immune Responses to Vaccines.

Flow cytometry, enzyme-linked immunospot (ELISpot) and cellular cytotoxicity assays are powerful tools for studying the cellular immune response towards intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity towards a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific towards cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium ((51)Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included.

The effect of thiacloprid formulation on DNA/chromosome damage and changes in GST activity in bovine peripheral lymphocytes

The potential genotoxic effect of thiacloprid formulation on bovine peripheral lymphocytes was evaluated using the comet assay and the cytogenetic endpoints: chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MNi). Whole blood cultures were treated with the insecticide at concentrations of 30, 60, 120, 240 and 480 μg mL−1 for 24, 48 h and/or 2 h of incubation. A statistically significant increase in the frequency of DNA damage, as well as in unstable chromosome aberrations (% breaks) were found after exposure to the insecticide at concentrations ranging from 120 to 480 μg mL−1 (P < 0.05, P < 0.01, P < 0.001). For the detection of stable structural chromosome aberrations (e.g., translocations) and numerical aberrations by the FISH method, three whole chromosome painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) were used in our experiments. We observed numerical aberrations, but without any statistical significance. Regarding the sister chromatid exchanges, no significant elevation in the SCE frequencies was found after 24-h exposure to the insecticide. A dose-related response in the SCE induction was obtained in bovine cultures after the prolonged time of exposure (48 h) to thiacloprid formulation at concentrations ranging from 120 to 480 μg mL−1 in each donor (P < 0.05, P < 0.01), which was associated with a reduction of the PI (P < 0.05, P < 0.01). The insecticide failed to produce MNi; however, a significant reduction of CBPI was observed. Using real-time PCR, a decrease in the expression of bovine glutathione S-transferase M3 (GSTM3) was detected at the lowest dose. The higher concentrations of thiacloprid formulation caused an increase in the mRNA expression.

Evaluation of DNA damage and cytotoxicity induced by triazole fungicide in cultured bovine lymphocytes

The aim of the present study was to investigate the effect of triazole fungicide Prosaro® (tebuconazole/prothioconazole) on the induction of micronuclei (MNs) and DNA damage in bovine peripheral lymphocytes. Whole blood cultures or isolated lymphocytes were exposed to 1.5, 3, 7.5, 15 and 30 μg ml−1 concentrations of triazole formulation for 24 and 48 h, or 2 h for MN and Comet assays. For 24 and 48 h of incubation with the fungicide no significant elevations in the MN frequencies in bovine lymphocytes were found. However, a clear reduction of cytochalasin-blocked proliferation index (CBPI) was observed in the donors (p < 0.05, p < 0.01, and p < 0.001). After the prolonged time of exposure the highest concentrations resulted in nearly total inhibition of cell proliferation (30 μg ml−1). After the use of Comet assay, only a moderate increase in DNA damage without statistical significance was recorded, except the highest concentration (30 μg ml−1; p < 0.05).

Phylogenetic Analysis of Bovine Interferon α

Interferon α is produced by the cells in response to the viral infection and play important role in viral infections. IFN-α coding sequence was amplified from the bovine lymphocytes collected from Bos indicus (Indian cattle breed-Hallikar) and cloned and sequenced. Nucleotide and the deduced amino acid sequence of the bovine IFN-α was compared with the sequences of different animals. The IFN-α nucleotide sequence of B. indicus shows 99.1 % similarity with Bos taurus, with changes at four amino acid positions. Comparison of the nucleotide and deduced amino acid sequence with other species has shown varied structural similarities.

Highly purified mycobacterial phosphatidylinositol mannosides drive cell-mediated responses and activate NKT cells in cattle.

Mycobacterial lipids play an important role in the modulation of the immune response upon contact with the host. Using novel methods, we have isolated highly purified phosphatidylinositol mannoside (PIM) molecules (phosphatidylinositol dimannoside [PIM2], acylphosphatidylinositol dimannoside [AcPIM2], diacyl-phosphatidylinositol dimannoside [Ac2PIM2], acylphosphatidylinositol hexamannoside [AcPIM6], and diacylphosphatidylinositol hexamannoside [Ac2PIM6]) from virulent Mycobacterium tuberculosis to assess their potential to stimulate peripheral blood mononuclear cell (PBMC) responses in Mycobacterium bovis-infected cattle. Of these molecules, one (AcPIM6) induced significant levels of gamma interferon (IFN-γ) in bovine PBMCs. Three PIM molecules (AcPIM6, Ac2PIM2, and Ac2PIM6) were shown to drive significant proliferation in bovine PBMCs. AcPIM6 was subsequently used to phenotype the proliferating cells by flow cytometry. This analysis demonstrated that AcPIM6 was predominantly recognized by CD3(+) CD335(+) NKT cells. In conclusion, we have identified PIM lipid molecules that interact with bovine lymphocyte populations, and these lipids may be useful as future subunit vaccines or diagnostic reagents. Further, these data demonstrate, for the first time, lipid-specific NKT activation in cattle.

Regulatory T Cell Activity and Signs of T Cell Unresponsiveness in Bovine Paratuberculosis.

Johne’s disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a wasting disease of ruminants displaying a long subclinical stage of infection followed by clinical disease characterized by severe diarrhea, wasting, and premature death. Immunologically, subclinical disease is characterized by a Th1 response effective at controlling intracellular infections such as that caused by MAP. In late subclinical disease, the Th1 response subsides and a non-protective Th2 response becomes prominent. One hypothesis for this shift in immune paradigm is that a population of MAP-reactive regulatory T cells (Tregs) develops during subclinical infection, limiting Th1-type responses to MAP antigens. To investigate this, we sought to accomplish the following: (1) determine if CD4+CD25− T cells exposed to MAP-infected macrophages develop a Treg phenotype, (2) develop a method to expand the relative abundance of Tregs in bovine peripheral blood lymphocyte populations, and (3) identify functional activities of expanded Tregs when combined with autologous peripheral blood mononuclear cells (PBMCs) and live MAP. We found that CD4+CD25− T cells exposed to MAP-infected macrophages from cows with Johne’s disease do not show signs of a Treg phenotype and appear unresponsive to MAP antigens. A method for Treg expansion was successfully developed; however, based on results obtained in the subsequent functional studies it appears that these Tregs are not MAP-specific. Overall, it seems that T cell unresponsiveness, rather than Treg activity, is driving the Th1-to-Th2 immune shift observed during Johne’s disease. Further, we have successfully developed a method to enrich non-specific bovine Tregs that exert suppressive effects against Th1 cytokine production.

Influence of PD‐L1 cross‐linking on cell death in PD‐L1‐expressing cell lines and bovine lymphocytes

Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-γ (IFN-γ) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-γ production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.

Nucleotide sequence and polymorphism ofMHCclassIIDQBexon 2 alleles inChinese yakow (Bos grunniens × Bos taurus)

The objective of this study is to describe the gene characteristics of the bovine lymphocyte antigen (BoLA)-DQB exon 2 locus in Chinese yakow (Bos grunniens × Bos taurus) and to compare it with previously reported patterns in other bovidae species to investigate genetic factors for disease resistance. The exon 2 of the MHC class II DQB gene was cloned and sequenced. It was revealed by sequence analyses that there are 36 DQB exon 2 alleles among 44 Chinese yakow. These alleles exhibited a high degree of nucleotide and amino acid polymorphism with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (dS ) and nonsynonymous (dN ) substitution. The Chinese yakow was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (dN = 0.147, P < 0.01). It appears that this variability among Chinese yakow confers the ability to mount immune responses to a wide variety of peptides or pathogens.

Role of the Polymorphic Immunodominant Molecule in Entry of Theileria parva Sporozoites into Bovine Lymphocytes

Theileria parva is a tick-transmitted apicomplexan parasite that infects cattle and African buffalo. In cattle, it causes a fatal lymphoproliferative disease called East Coast fever. The polymorphic immunodominant molecule (PIM) is expressed by two stages of the parasite: the sporozoite, which is inoculated by the tick to infect mammalian lymphocytes, and the schizont, the established intralymphocytic stage. Here, we demonstrate that monoclonal antibodies (MAb) to PIM can reduce the ability of sporozoites to infect bovine lymphocytes in vitro. This reduction appears to be due to blocking of sporozoite attachment by binding of the MAb to several regions of PIM. Interestingly, one MAb, which recognizes an epitope in the central variable region of PIM, did not inhibit sporozoite infectivity. We also demonstrate that PIM antigen, as a recombinant molecule, can also reduce sporozoite infectivity in vitro by blocking both attachment and internalization of sporozoites. Electron microscopic studies showed that PIM is present in microspheres below the sporozoite surface and is transported to the parasite surface soon after contact with bovine lymphocytes. The results suggest that at least two sporozoite molecules, PIM and the previously described p67, are involved in the entry of T. parva into mammalian lymphocytes.

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1 × 10(-4), 1 × 10(-5), 1 × 10(-6), and 1 × 10(-7) mol.L(-1)) for 48 h. The highest concentration of BPA (1 × 10(-4) mol.L(-1)) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50 ± 2.121/1,000 binucleated cells versus 36.0 ± 5.657/1,000 binucleated cells in the DMSO control, P = 0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest concentration.

Effect of zinc treatments on lead exposed periparturient bovine lymphocytes in vitro on their proliferation and superoxide dismutase (SOD) expression

The study was conducted to observe adverse effects of lead (Pb) treatments and protective effect of zinc on lymphocyte proliferation and to quantify the expression levels of superoxide dismutase (Cu/ZnSOD) genes involved in antioxidant’s defenses in periparturient Karan Fries (Tharparker × Holstein-Friesian) cow. Blood samples were collected from peripartum Karan Fries cow at 30 and 15 days prepartum, at day of calving (0 day) and 15 and 30 days postpartum for estimation of lymphocytes proliferation and SOD expression. A fixed number of cells (2×106) were grown in culture for 72 h with different levels of Pb (10–4, 10–5, 10-6 M) and their adverse effects were counteracted by Zn (50, 55, 60 μM) and analysed for the lymphocyte proliferation (MTT assay) and for the expression level of Cu/ZnSOD using the realtime PCR technique with light upon extension (LUX) fluorogenic primers. Pb dosage had no adverse effect and the overall mean proliferation values indicated that 60μM Zn may be optimum for maximizing lymphocyte proliferation. The Cu/ZnSOD mRNA expression in lymphocytes was higher at higher dosage of Pb in comparison to lower dosage in all the 3 (50, 55, 60 μM) Zn treatments. There was no significant difference at different levels of Zn on SOD expression. Cu/ZnSOD mRNA expression decreased from –30 days to 0 days but increased from 0 to 30 days after calving. The results suggested that the zinc may have an ameliorative effect on lead exposed oxidative stress on lymphocytes proliferation and Cu/ZnSOD mRNA expression through modulation of superoxide dismutase expression

First isolation and establishment of in vitro culture of Theileria lestoquardi from a naturally infected cow.

Theileria infected cell line was isolated from the prescapular lymph node of an adult crossbred cow. Molecular study confirmed this cell line of bovine lymphocyte has been transformed by the Theileria lestoquardi. This strain of T. lestoquardi designated Ka-6 and sheep were inoculated with this strain didn't show any clinical signs of theileriosis which shows the significance of this cell line to develop a tissue-culture vaccine against malignant ovine theileriosis. Contrary to accepted belief that the T. lestoquardi not capable of causing disease in cattle, the present study describes the first isolation and establishment of in vitro culture of T. lestoquardi-infected cell line from a naturally infected cow with typical singes of acute theileriosis.

Assessment of cytogenetic damage in bovine peripheral lymphocytes exposed to in vitro tebuconazole-based fungicide

The tebuconazole-based fungicide was tested in vitro for its potential genotoxic and cytotoxic effects on cultured bovine peripheral lymphocytes. Following 24h and 48h of incubation, several cytogenetic endpoints were investigated such as: Chromosome Aberrations (CAs); Sister Chromatid Exchanges (SCEs); Micronuclei (MN); Mitotic Index (MI); Proliferation Index (PI); and Cytokinesis Block Proliferation Index (CBPI). The cultured lymphocytes were exposed to the fungicide formulation at concentrations of 3, 6, 15, 30 and 60μgmL−1. Statistical significant increases were seen in the CA assays at concentrations ranging from 6 to 30μgmL−1 for 24h. The higher doses caused a decrease or total inhibition of chromosome damages in comparison to the last active dose, or the control values. The Fluorescence in situ Hybridisation (FISH) technique was also used for the study of stable/unstable structural chromosomal aberrations and numerical aberrations of aneuploidy/polyploidy at the concentrations of 6 and 15μgmL−1. Under conditions of our study, no reciprocal translocations were detected. The more frequent types of aberrations were trisomies and monosomies; both have been identified in association with either bovine chromosome 5 or 7. No statistical significant value was seen in the induced MN; but, the clear, evident reduction of the CBPI was observed. Significant elevations of SCE were observed after the applications of the fungicide formulation at doses from 15 to 60μgmL−1 in each donor for 24h. The highest concentrations also caused a statistical significant decrease in the PI. The treatment for 48h failed to exhibit any genotoxic activity of the fungicide.

Modulation of aryl hydrocarbon receptor target genes in circulating lymphocytes from dairy cows bred in a dioxin-like PCB contaminated area

Animal productions (i.e. fish, eggs, milk and dairy products) represent the major source of exposure to dioxins, furans, and dioxin-like (DL) polychlorobiphenyls for humans. The negative effects of these highly toxic and persistent pollutants are mediated by the activation of the aryl hydrocarbon receptor (AHR) that elicits the transcriptional induction of several genes, including those involved in xenobiotic metabolism. Previously we demonstrated the presence and functioning of the AHR signaling pathway in primary cultures of bovine blood lymphocytes. The aim of the present study was to investigate by real time PCR the expression and the inducibility of selected target genes (i.e. AHR, AHR nuclear translocator (ARNT), AHR repressor, CYP1A1 and CYP1B1) in uncultured cells from dairy cows naturally exposed to DL-compounds. The study was carried out on two groups of animals bred in a highly polluted area and characterized by a different degree of contamination, as assessed by bulk milk TEQ values, and a control group reared in an industry free area. Bovine lymphocytes expressed only AHR, ARNT and CYP1B1 genes to a detectable level; moreover, only CYP1B1 expression appeared to be correlated to TEQ values, being higher in the most contaminated group, and decreasing along with animal decontamination. Finally, lymphocytes from exposed cows displayed a lower inducibility of both CYP1A1 and CYP1B1 after the in vitro treatment with a specific AHR ligand. In conclusion, our results indicate that DL-compound contaminated cows may display significant changes in AHR-target gene expression of circulating lymphocytes.

Effect of BoLA-DRB3 exon2 polymorphisms on lameness of Chinese Holstein cows

The involvement of bovine lymphocyte antigen (BoLA) in immune system and its role in susceptibility/resistance to infectious diseases has been extensively studied. However, few studies have been conducted to investigate the association between BoLA and gait scores. Our objective was to investigate whether polymorphisms in BoLA gene are associated with susceptibility of lameness in 435 Chinese Holstein cows. Genotyping of the BoLA-DRB3.2 gene was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with three restriction endonucleases (BstUI, BstYI and HaeIII). The relationship between the polymorphisms in BoLA-DRB3.2 gene and gait scores was analyzed by least-squares linear model. The gait score was non-significant among all five BstUI-RFLP and BstYI-RFLP genotypes. However, analysis of seven HaeIII-RFLP genotypes revealed a significantly higher gait score for AB genotype than others. In conclusion, BoLA-DRB3.2 may be a candidate gene for lameness susceptibility in Chinese Holstein cows.

Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

BackgroundGene expression of lymphocytes was found to be influenced by histone methylation in mammals and trimethylation of lysine 27 on histone H3 (H3K27me3) normally represses genes expressions. Peripheral blood lymphocytes are the main source of somatic cells in the milk of dairy cows that vary frequently in response to the infection or injury of mammary gland and number of parities.MethodsThe genome-wide status of H3K27me3 modifications on blood lymphocytes in lactating Holsteins was performed via ChIP-Seq approach. Combined with digital gene expression (DGE) technique, the regulation effects of H3K27me3 on genes expressions were analyzed.ResultsThe ChIP-seq results showed that the peaks of H3K27me3 in cows lymphocytes were mainly enriched in the regions of up20K (∼50%), down20K (∼30%) and intron (∼28%) of the genes. Only ∼3% peaks were enriched in exon regions. Moreover, the highest H3K27me3 modification levels were mainly around the 2 Kb upstream of transcriptional start sites (TSS) of the genes. Using conjoint analysis with DGE data, we found that H3K27me3 marks tended to repress target genes expressions throughout whole gene regions especially acting on the promoter region. A total of 53 differential expressed genes were detected in third parity cows compared to first parity, and the 25 down-regulated genes (PSEN2 etc.) were negatively correlated with H3K27me3 levels on up2Kb to up1Kb of the genes, while the up-regulated genes were not showed in this relationship.ConclusionsThe first blueprint of bovine H3K27me3 marks that mediates gene silencing was generated. H3K27me3 plays its repressed role mainly in the regulatory region in bovine lymphocytes. The up2Kb to up1Kb region of the down-regulated genes in third parity cows could be potential target of H3K27me3 regulation. Further studies are warranted to understand the regulation mechanisms of H3K27me3 on somatic cell count increases and milk losses in latter parities of cows.

Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors

Luteal cell-induced proliferation of T lymphocytes devoid of the nuclear progesterone receptor (PGR) is inhibited by progesterone. Functional effects of progesterone on bovine lymphocytes and the expression of membrane progesterone receptors (mPRs) alpha (PAQR7), beta (PAQR8), gamma (PAQR5), and progesterone receptor membrane component 1 (PGRMC1) mRNA were analyzed in corpus luteum (CL) and lymphocytes. Progesterone and a cell-impermeable progesterone conjugate caused a dose-dependent decrease in IL2 receptor α-subunit (IL2RA) mRNA and an increase in interleukin 2 (IL2) mRNA concentrations in cultured PBMCs. In luteal tissues, concentrations of PAQR7 and PAQR8 mRNA were lower in CL collected on day 11 compared with day 18, whereas PGRMC1 and PGR mRNA were greater on day 11 than on day 18. The mRNA of all three PAQRs and PGRMC1 were detected in bovine T lymphocytes, but not in B cells/monocytes. Progesterone increased intracellular Ca++ and reduced the phosphorylation of zeta-chain-associated protein kinase 70 (Zap70). A specific, saturable, and single progesterone binding site with a steroid specificity characteristic of mPRs was demonstrated by saturation and competitive binding assays using T lymphocyte membranes, and PAQR7 receptors were localized on the plasma membranes by immunofluorescence. Thus, progesterone induces specific and rapid functional effects on T lymphocytes in the absence of PGR. The mPRs are potential intermediaries of the cell-surface actions of progesterone because they are expressed in lymphocytes, the actions of progesterone are mimicked by a cell-impermeable form of progesterone, and specific, saturable progesterone binding, which is characteristic of mPRs, is present on lymphocyte membranes.

Association between BoLA-DRB3 and somatic cell count in Holstein cattle from Argentina

Different studies have proved that the resistance/susceptibility to mastitis is genetically determined. The major histocompatibility complex in cows is known as bovine lymphocyte antigen (BoLA). Genes from the BoLA have been associated with the occurrence of infectious diseases such as mastitis and leukosis, especially the BoLA-DRB gene. The object of the present study was to detect associations between BoLA-DRB3 alleles and somatic cell count (SCC), as an indicator of resistance/susceptibility to mastitis in Holstein cattle (N = 123) from La Pampa, Argentina. Fisher's exact test and Woolf-Haldane odds ratio were applied to study the association between SCC and BoLA-DRB3 allele frequencies. Significant association was noted between BoLA-DRB3.2*23 and *27 alleles (p < 0.05) and protective or susceptibility effects, respectively. In addition, alleles BoLA-DRB3.2*20 and *25 exhibit suggestive association with high SCC (p < 0.1). These results were partially in agreement with data reported from Japanese Holstein cattle, but differed from those published by other authors. A possible explanation for the contrasting results could be that the mastitis is a multifactor disease caused by different pathogens. Moreover, most of the studies were carried out using PCR-RFLP method, which has less resolution than PCR-SBT because PCR-RFLP defined alleles included more than one sequenced alleles.

Polymorphism in The Region of Bovine Prion Protein Gene and Selected Haematological Indices in Cows Naturally Infected with Bovine Leukaemia Virus

Abstract The objective of this study was to determine whether insertion-deletion (indel) 23 bp polymorphism of the bovine prion protein (PRNP) gene differentiates the total number of leukocytes and lymphocytes and the number of virus-infected lymphocytes. The experimental materials comprised 119 Black-and-White Polish Holstein-Friesian cows. Bovine leukosis was diagnosed by an indirect immunofluorescence based on the detection of viral protein p24 in bovine lymphocytes infected with leukaemia virus (BLV). Indel23 polymorphism was determined by PCR. Blood haematological parameters (total leukocyte counts, total lymphocyte counts, and their percentages) were determined at a specialist haematological laboratory. The examined indices were analysed in three replications, at one-month intervals. It was found that indel23 polymorphism significantly differentiated blood leukocyte counts and the total number and percentage of lymphocytes. Cows with the 23 bp del/del genotype showed significantly higher leukocyte and lymphocyte counts than animals with the remaining two genotypes. Higher values of the analysed haematological parameters noted in homozygotes with 23 bp deletion are similar to the values reported in cows affected by persistent lymphocytosis, thus pointing to an adverse effect of this genotype on the haemopoiesis process. The variations between indel23 genotypes and the number and percentage of BLV-infected lymphocytes are less obvious and more difficult to interpret.