Lymphocytes Of Cattle Research Articles

Studies on haematological changes in GI nematodes in cattle and buffalo in Udaipur, district (Rajasthan)

A study on prevalence of for gastrointestinal nematode in the infected and non-infected cattle and buffalo of Udaipur district (Rajasthan) from Sept. 2020 to January 2021 by haematological parameters such as haemoglobin (Hb), packed cell volume (PCV), erythrocyte sedimentation rate (ESR), total leucocyte Count (TLC) and differential leucocyte count (DLC). The highly significant decrease in haemoglobin concentration innematode infected and non-infected buffalo (8.65±0.2335 and 10.12±0.1077) and (8.63±0.2886 and 09.84±0.1411) in infected and in non-infected cattle, respectively. The packed cell volume showed highly significant decrease in nematode infected and non-infected buffalo (28.98±0.2101 and 32.94±0.2477) and (27.64±0.5337 and 30.65±0.3956) in infected and non-infected cattle, respectively. The highly significant decrease in erythrocyte sedimentation rate in nematode infected and non-infected buffalo (0.80±0.0245 and 1.00±0.0167) and in cattle was found to be in infected (0.63±0.0287) and in non-infected (0.97±0.0240), respectively.Total leukocyte count (TLC) value significant increase for infected and noninfected buffalo and cattle buffalo (8.53±0.2784 and 10.45±0.2538) and (8.36±0.2582 and 10.49±0.1689), respectively. In the infected buffalo neutrophils (32.96±0.3084), eosinophils (5.50±0.2049).Basophils (0), lymphocytes (62.36±0.2948) and monocytes (5.48±0.1831). However, in non-infected buffalo neutrophils (37.21±0.3701), eosinophils (4.60±0.2196), basophils (0), lymphocytes (57.21±0.2802) and monocytes (4.99±0.2094). Whereas, in infected cattle neutrophils (28.02±0.2914), eosinophils (5.74±0.1942) basophils (0), lymphocytes (67.48±0.2979) and monocytes (5.57±0.1547). However, in non-infected cattle neutrophils (32.15±0.3088), eosinophils (3.88±0.1836) basophils (0), lymphocytes (61.59±0.3475) and monocytes (5.23±0.1832).There is reduction in haemoglobin, packed cell volume and ESR in the GI nematode infected cattle and buffalo. In total leukocyte count in buffalo and cattle lymphocytes, eosinophils and monocyte showed increased in infected group.

Использование микроспектрального анализа для оценки морфофункционального статуса иммунокомпетентных клеток при ретровирусных заболеваниях крупного рогатого скота

Abstract. Microspectral analysis is a highly informative way to study the morphofunctional status of cells, including immunocompetent ones. Purpose of the study was an application it for studding of bovine lymphocytes in cattle with enzootic leukemia and viral immunodeficiency, which is important for diagnostics and differential diagnostics of diseases, as well as for analyzing of animal homeostasis and making a prognosis of the disease. Leukemia and immunodeficiency are classified as hematopathological processes. Therefore, we used the hematological method as a comparative method. Spectral analysis of stained with a Leukodif 200 kit agranulocytes was carried out using the universal color analyzer microscope-spectrophotometer LOMO MSFU-K. The value of the light absorption intensity was measured in the visible-light spectrum from 300 to 700 nm. The results of microspectral analysis showed that for cows’ lymphocytes with combined pathology (BLV/BIV), the absorption values in the spectrum of eosin U and azure II were 351.2 ± 17.6 and 751.4 ± 37.6 counts, respectively. For animals with BLV and BIV mono-infection, these indicators were 253.3 ± 12.7; 383.3 ± 19.2 and 371.5 ± 18.5; 500.2 ± 24.9 counts. While in intact cows, these indicators were registered at the level of 210.3 ± 10.5 and 173.6 ± 8.6 counts. In intact animals, the acidic and basic components ratio in the cell was uniformly proportional, the ratio coefficient was 0.83 ± 0.04. For BIV-, BLV- and BLV/BIV-infected animals, this coefficient was 1.34 ± 0.06, 1.51 ± 0.08 and 2.13 ± 0.11, that was in 1.6, 1.8 and 2.6 times higher than in intact animals, which might be an indicator of metabolic acidosis in the cell. The scientific novelty of the research is that the signs of hypoxia and inflammation detected by hematological analysis correlate with the results of microspectral analysis and indicate a change in the homeostasis of the infected animal’s organism, including at the cell level.

Application of a microspectral analysis for evaluation of the morphofunctional status of immunocompetent cells in cattle with retroviral diseases

Microspectral analysis is a highly informative way to study the morphofunctional status of cells, including immunocompetent ones. Purpose of the study was an application it for studding of lymphocytes in cattle with enzootic leukemia and viral immunodeficiency. It is important for diagnostics and differential diagnostics of diseases, as well as for analyzing of animal homeostasis and making a prognosis of the disease. Spectral analysis of stained with a Leukodif 200 kit agranulocytes was carried out using the universal color analyzer microscope-spectrophotometer LOMO MSFU-K. The value of the light absorption intensity was measured in the spectrum from 300 to 700 nm. The results of microspectral analysis showed that for cows’ lymphocytes with combined pathology (BLV/BIV), the absorption values in the spectrum of Eosin U and Azure II were 351.2 ± 17.6 and 751.4 ± 37.6 counts, respectively. For animals with BLV and BIV mono-infection, these indicators were 253.3 ± 12.7; 383.3 ± 19.2 and 371.5 ± 18.5; 500.2 ± 24.9 counts. While in intact cows, these indicators were registered at the level of 210.3 ± 10.5 and 173.6 ± 8.6 counts. In intact animals, the acidic and basic components ratio in the cell was uniformly proportional, the ratio coefficient was 0.83 ± 0.04. For BIV, BLV and BLV/BIV-infected animals, this coefficient was 1.34 ± 0.06, 1.51 ± 0.08 and 2.13 ± 0.11, that was in 1.6, 1.8 and 2.6 times higher than in intact animals, which might be an indicator of metabolic acidosis in the cell.

Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south-western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci.

Theileria parva is a tick-transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti-transformed and Kiambu 5 stocks of T.parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T.parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T.parva field isolates derived from Ankole (Bos taurus) cattle in south-western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen-encoding genes that are targets of CD8+T-cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T.parva prevalence of 51% confirming endemicity of the parasite in south-western Uganda. Cattle-derived T.parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T.parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.

CD4- and CD8-expressing cells found in the bovine and porcine anterior chamber of the eye.

The aim of the present study was to investigate whether the anterior chamber constitutes part of the normal migratory pathway of CD4+ and CD8+ lymphocytes in cattle and swine. The cells obtained from aqueous humor of cows and pigs were stained for CD4 and CD8 receptors, and subsequently analyzed with flow cytometry. The mean percentage of CD4+CD8-, CD4-CD8+ and CD4+CD8+ cells within the total lymphocyte population of the bovine anterior chamber was, respectively, 17.88, 12.64 and 27.26%. In turn, the mean values of these parameters in pigs were 1.77, 38.48 and 17.45, respectively. Among bovine and porcine CD4+CD8+ cells prevalent were those displaying CD4lowCD8low and CD4lowCD8high phenotypes, respectively. The results suggest that the anterior chamber in cattle and swine is an element in the normal migratory pathway of CD4+, CD8+ and CD4+CD8+ cells. Furthermore, the contribution of these subsets in the anterior chamber lymphocyte population can differ considerably between animal species.

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals

BackgroundTropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane.ResultsIn this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum.ConclusionsThe results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.

Infectious Bovine Rhinotracheitis in a Cattle Farm at Sharkia Governorate with Special Reference to its Effect on T lymphocytes

This study was applied on a cattle farm of Holstein cows at Sharkia Governorate for the isolation and identification of Bovine herpesvirus type-1 (BoHV-1) and to examine its effect on T lymphocytes. The results of clinical examination revealed that there were respiratory disorders in 30 out of 150 (20%) of cattle including elevated body temperature (40 to 42°C), nasal and ocular discharges, some animals developed severe rhinitis, conjunctivitis, corneal opacity, cough and diarrhea. Out of 30 nasal swabs, 15 swabs were positive for virus isolation as indicated by cytopothic effect (CPE) on MDBK cells. Only 11 of the 15 isolates were confirmed by virus neutralization test (VNT) as BoHV-1 isolates. In addition, only 3 out of 4 BoHV-1 isolates were detected by PCR. Peripheral blood T lymphocytes (PBTL) were analyzed using electron microscopy and comet assay to examine the effect of BoHV-1 on lymphocytes. Electron micrographs of T lymphocytes revealed peripheral condensation of chromatin and fragmentation of the nucleus end of the cell leading to the formation of apoptoic bodies. Comet assay denoted fragmentation of cellular DNA. It could be concluded that BoHV-1 can infect T lymphocytes of cattle, causing directly and indirectly apoptosis which subsequently lead to suppression of cell-mediated immunity, enhancing establishment of latency and increasing the probability for secondary bacterial infection.

Construction of a genetic map for Theileria parva: Identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22cMKb−1 per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes.

Screening of Theileria parva apicomplexan antigen homologs for induction of MHC CD4+ and CD8+ T-cell responses.

Theileria parva, an intracellular apicomplexan pathogen transmitted byRhipicephalus appendiculatus ticks, infects and transforms lymphocytes of cattle and African buffalo causing the disease called East Coast fever (ECF). The genome of of T. parva was sequenced in order to facilitate research on parasite biology, assist the identification of schizont antigens for vaccine development and extend comparative apicomplexan genomics. In the present study, eight putative apicomplexan antigens were identified from literature and their homologs purified from a T. parva Muguga schizont cDNA library. These were immunoscreened forMHC CD4+ and CD8+ CTL response. The results showed that of the expressed proteins, only the T. parva T-complex 1 protein zeta subunit ortholog was found to elicit CD4+ response; none elicited CD8+ response. The elicitation of CD4+ T cell response by the T. parva T complex Protein-1 zeta subunit homolog indicates that it is a candidate T-helper cell target antigen. Key words: Theileria parva, apicomplexan antigen, East Coast fever (ECF), major histocompatibility complex (MHC), cytotoxic T lymphocyte.

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis ( M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase ( P < 0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5 dim, CD5 bright, and a minor population that was characterized as CD5 extra bright. A decrease in the CD5 dim B cell population along with a concomitant increase in CD5 bright B cells was observed in infected cows, an effect that was highly significant ( P < 0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5 bright B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5 bright B cells for subclinically infected cows. These results suggest that differential expression of CD5 bright and CD5 dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.

Research on Spontaneously Emerged Chromosomal Aberrations in the Periphery Blood Lymphocytes in Cattle (‘Buša’ Breed)

Knowledge of spontaneous aberrations, namely, of their frequency in non-irradiated cells is of paramount importance not only in cytogenetic research, but also in contemporary animal production. The paper deals with research on spontaneously emerged chromosomal aberrations in the peripheral blood lymphocytes in the cattle of 'Busa' breed. To obtain metaphase chromosomes the conventional method of lymphocyte cultivation was used, albeit slightly modified and adapted to the examined animals and the laboratory conditions. The research findings indicate that a certain percent of spontaneously emerged chromosomal aberrations of chromatid type (gap and break) have been found in the peripheral blood lymphocytes in the cattle of 'Busa' breed.

The micronucleus frequency in cytokinesis-blocked lymphocytes of cattle in the vicinity of a nuclear power plant

Cytogenetic and hematological analyses were performed on the peripheral blood lymphocytes (PBLs) obtained from Korean native cattle bred in the vicinity of three nuclear power plants (Wolsong, Uljin and Yeonggwang) and in a control area. The micronucleus (MN) rates for the cattle from the Wolsong, Uljin and Yeonggwang nuclear power plants and for the control area were 9.87 ± 2.64, 8.90 ± 3.84, 9.20 ± 3.68 and 9.60 ± 3.91 per 1,000 cytokinesis-blocked lymphocytes, respectively. The apparent difference is not statistically significant. The MN frequencies of PBLs from cattle bred in the four areas are within the background variation for this study. The MN frequencies and hematological values were similar regardless of whether the cattle were bred near a nuclear power plant or in the control area.

Identification of Vaccine Candidate Peptides in the NcSRS2 Surface Protein ofNeospora caninumby Using CD4+Cytotoxic T Lymphocytes and Gamma Interferon-Secreting T Lymphocytes of Infected Holstein Cattle

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-gamma) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-gamma secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-gamma enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-gamma ELISPOT and in vitro by measuring T-lymphocyte IFN-gamma production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-gamma-secreting T lymphocytes in cattle with varied MHC genotypes.

Bovine T lymphocyte responses to Brucella abortus

The long-held paradigm of T lymphocyte-mediated activation of mononuclear phagocytes (Mø) as the major mechanism of protection against facultative intracellular pathogens such as Brucella has been modified to include killing of infected Mø by various subsets of T lymphocytes. Remnants of killed infected cells are phagocytosed by immunologically-activated Mø, which are much more efficient at killing such pathogens. Most of the detailed information regarding immunity in general and that of brucellosis specifically has been obtained using murine infection models rather than in cattle. However, there has been considerable definition of cellular phenotypes, cytokines and functional characteristics of T lymphocytes in cattle over the last decade. This was mainly due to development of monoclonal antibodies against cell surface markers and application of molecular cloning and polymerase chain reaction (PCR) for isolation, characterization and detection of genes encoding bovine cytokines. This review discusses cellular and molecular immunity in bovine brucellosis as pertains to T lymphocyte interactions with the Mø. Although current knowledge directly obtained from brucellosis immunity studies in the bovine host is limited and incomplete, the many parallels between the bovine and murine immune systems allow for some extrapolation in the description of bovine host defense mechanisms. Direct information from studies with immunized cattle supports the concepts of coordinate activation of uninfected Mø and killing of Brucella-infected Mø by antigen-specific T lymphocytes as major mechanisms of host defense in bovine brucellosis. There also appears to be a bias in the T lymphocyte compartment towards recognition of particular bacterial stress proteins following immunization with live Brucella vaccines.

Spontaneously proliferating lymphocytes from bovine leukaemia virus-infected, lymphocytotic cattle are not the virus-expressing lymphocytes, as these cells are delayed in G(0)/G(1) of the cell cycle and are spared from apoptosis.

Bovine leukaemia virus (BLV) is in the family of oncogenic retroviruses which includes human T cell leukaemia virus (HTLV). BLV infects B lymphocytes and induces a non-neoplastic persistent lymphocytosis (PL) of B lymphocytes in cattle. A characteristic of BLV- and HTLV-induced disease is spontaneous lymphocyte proliferation of cultured peripheral blood mononuclear cells (PBMC). To investigate the role of virus expression on lymphocyte survival and proliferation, we evaluated cell cycle position, apoptosis and virus expression on a single-cell basis of cultured PBMC from BLV-infected PL cattle, BLV-infected non-PL cattle and uninfected cattle. Results demonstrated that the majority of bovine B lymphocytes spontaneously entered G(2)/M of the cell cycle and died by apoptosis by 24 h post-culture, regardless of BLV infection or PL status. The spontaneous proliferation that characterizes PL cattle was primarily due to a small population of surviving B lymphocytes, but T lymphocytes also contributed. Viral protein expression was detectable in only 5-15% of cultured PBMC from PL cattle and the majority of these lymphocytes were delayed in cell cycle and spared from apoptosis. Unexpectedly, we determined that only 3% of the spontaneously proliferating lymphocytes expressed viral proteins. Previous reports show that spontaneous proliferation decreases when virus expression is suppressed. Together with our results, this suggests that virus expression by one population of B lymphocytes promotes proliferation of another population of B lymphocytes that does not express virus. This may be due to an effect of virus on CD4 T lymphocytes, as depletion of CD4 T lymphocytes significantly decreased spontaneous proliferation.

Analysis of the phenotype and phagocytic activity of monocytes/macrophages from cattle infected with the bovine leukaemia virus

The bovine leukaemia virus (BLV) is a retrovirus that infects mainly B lymphocytes of cattle, but proviral DNA can also be isolated from monocytes/macrophages. This study investigated the effect of BLV infection on surface antigens on freshly isolated peripheral blood monocytes and cultured monocyte-derived macrophages, with and without lipopolysaccharide (LPS) stimulation. The effect of BLV infection on phagocytic activity of CD14 + monocytes was also assessed. The percentage of monocytes expressing the surface antigens CD11b, CD32 (Fc γRII), MHC class II and the surface antigen recognised by mAb DH59B were increased in BLV-positive cattle. In contrast, expression intensity of all markers was low in samples from BLV-positive cattle. CD14 + monocytes from BLV-positive cattle showed less Fc γ-receptor-mediated phagocytosis compared to monocytes from BLV-negative cattle. After 7 days in culture, there was evidence for shedding/downregulation of surface antigens on monocyte-derived macrophages, in particular on cells from BLV-positive cattle. LPS stimulation decreased the percentage of cells expressing the measured markers in monocyte-derived macrophages taken from BLV-negative cattle, but not in cultures derived from BLV-positive cattle. The results provide further evidence for an altered function of monocytes and macrophages in BLV-infected cattle.

Kinetics of expression and subset distribution of the TNF superfamily members CD40 ligand and Fas ligand on T lymphocytes in cattle

CD40 and Fas are members of the tumor necrosis factor receptor (TNFR) superfamily. CD40 and Fas play key roles in T cell–B cell interactions. Cross linkage of these molecules induces cell activation and cell death, respectively. The interaction of CD40 with its ligand (CD40L), which is expressed on activated T cells, plays a pivotal role in the generation of the T-dependent (TD) immune response, and FasL-bearing T cells, which have been shown to be predominantly of either the T H0 or T H1 type, have the potential to induce the apoptotic death of Fas expressing B cells. We investigated bovine CD40L mRNA expression in established T cell clones by RT-PCR and Southern blotting. T cells analyzed included CD4 + T H0 and T H1 cell subpopulations, CD8 +, and γ/ δ T cells stimulated with either specific antigen or Con A. All CD4 + clones but not all CD8 + or γ/ δ T cell receptor (TCR)-bearing clones expressed mRNA for CD40L. To determine the activation requirements for CD40L expression in cattle, we examined the kinetics and induction requirements for CD40L transcription in peripheral blood T cells using a phorbol ester and/or ionomycin, immobilized mouse anti-bovine CD3, or Con A. Our results demonstrate that CD40L mRNA appears relatively early after activation (1 h) and peaks at 2–4 h poststimulation. A rise in intracellular calcium concentration mediated by ionomycin treatment alone was sufficient to induce CD40L mRNA expression at relatively high levels. Ionomycin treatment in combination with other agonists (anti-CD3, PMA) did not enhance CD40L mRNA expression above levels obtained with ionomycin alone. The bovine Fas ligand gene was partially cloned and mRNA expression determined by RT-PCR in a panel of T cell clones. Our results demonstrate that T H0 and T H1 bovine T cell clones expressed Fas ligand transcripts although only one γ/ δ T cell clone did. This expression was upregulated within 3 h after mitogen stimulation and reduced by 24 h.

Quantification of Cytokine Gene Expression in Lamina Propria Lymphocytes of Cattle Following Infection with Ostertagia ostertagi

Changes in cell surface markers and cytokine transcription were analyzed in lamina propria lymphocytes from control animals (noninfected calves) and calves after a single high but nonprotective primary infection with Ostertagia ostertagi. Flow cytometry of cells recovered from the lamina propria showed an increase in the percentages of IgM+, WC1+, and IL-2R+-bearing cells 10 days after infection; however, 2 mo after infection, cell staining was comparable to preinfection levels. Transcription levels of interleukins IL-2, IL-4, IL-10, and interferon (IFN)-gamma mRNA were measured using a competitive reverse transcription-polymerase chain reaction. Results indicated elevated levels of IL-4 and IFN-gamma in the infected animals at 10 days and at 60 days after infection. Transcription of IL-10 also increased; however, this change was not observed until 60 days postinfection.

Immunoanalysis of IgG and IgM molecules on the surface of BLV infected cattle lymphocytes

The BLV-infected animals were divided into lymphocytotic (9 animals: BLV+PL+) and non lymphocytotic (11 animals: BLV+PL -). BLV-free animals formed a control group (7 animals: BLV-PL-). Flow cytometry immunofluorescence (FACS) and scanning electron microscopy (SEM) were used to evaluate the expression of IgG and IgM molecules on the surface of the lymphocytes of cattle infected with the bovine leukaemia virus (BLV). The percentage of IgM-bearing cells was significantly higher in the BLV+PL+ (75% and 74%) than in BLV+PL (43% and 50%) and BLV-PL (39% and 44%) groups by FACS and SEM respectively. The percentage of IgG-bearing cells was higher in BLV+PL+ (6%) and BLV+PL (8%) compared to BLV-PL (0.7%) determined by FACS and significantly higher in BLV+PL+ (62%) in relation to BLV+PL (17%) and BLV-PL (8%) groups ascertained by SEM. A significantly higher intensity of IgM expression was observed in the BLV+PL+ group by SEM and FACS; a higher intensity of IgG expression in BLV+PL+ group was detected only by SEM. The results suggest that FACS and SEM are complementary. The finding of significant differences in the expression of IgG molecules in BLV+ groups compared with BLV - animals confirms the sensitivity of the SEM method.