Sheep Lymphocytes Research Articles

Expression of HLA-G1 and G5 Enables Human Mesenchymal Stem Cells To Engraft at High Levels across Xenogeneic Barriers Following Transplantation into Immunocompetent Recipients.

Although mesenchymal stem cells (MSC) have been shown to be fairly non-immunogenic, recent studies demonstrated that MSC are capable of activating and becoming targets of NK-mediated lysis. Furthermore, the ability of IFN-γ to induce MSC expression of HLA-II molecules shows that MSC are not completely immuno-inert. Expression of HLA-G has been associated with the maintenance of fetomaternal tolerance during pregnancy through its inhibition of the cytolytic functions of NK and cytotoxic T cells and of dendritic cell maturation. Here we investigated whether transduction of human MSC with a retroviral vector encoding HLA-G1 (MSC-G1) or G5 (MSC-G5) would render these cells and their differentiated progeny undetectable by the recipient's immune system, and thereby allow us to efficiently transplant these cells into immuno-competent xenogeneic recipients. First, we performed mixed lymphocyte reactions using unmodified MSC (unMSC), MSC-G1 or MSC-G5 as stimulators, and lymphocytes from allogeneic human or sheep donors as responders. While neither unMSC nor MSC-G1 or -G5 elicited a significant proliferative response from human lymphocytes, sheep lymphocytes proliferated 2-6 fold more when cultured with unMSC than with MSC-G1 or MSC-G5. Next, we tested whether HLA-G1 and HLA-G5 expression would enable the engraftment of human MSC in fetal sheep later in gestation when donor cells are normally rejected due to the presence of a competent immune system. To this end, 105 unMSC, MSC-G1, or MSC-G5 were transplanted (Tx) into fetal sheep recipients during the pre-immune period (55 days; n=9), or after immunocompetence was achieved, at 82 days (n=10) or 104 days (n=8) of gestation. Evaluation of the recipients' hematopoietic system at 42 days post-transplant for the presence of human cell engraftment, using a panel of antibodies specific to human blood cells, revealed that Tx at 55 days resulted in similar levels of engraftment for all cell types (MSC:4±0.9; MSC-G1:6±0.3; MSC-G5:5±0.3%). In contrast, while unMSC engrafted at very low levels at 82d and 104d, Tx of MSC-G1 and-G5 at these later time points not only overcame the immune barriers, but resulted in levels of engraftment that were considerably higher than those achieved during the pre-immune period (82d:7±1;18±2.% and 104d:12±2;16±2%). A similar outcome was seen with liver engraftment and hepatic differentiation with MSG-1 and -G5 giving rise to 3–5 times more hepatocytes than unMSC at later time points. However, even at the early Tx time point of 55days, MSC-G1 and -G5 gave rise to, respectively, 5 times and 2 times more donor-derived hepatocytes than their unMSC counterpart. Our studies demonstrate that the forced expression of HLA-G1 or G5 enables MSC to evade a competent recipient immune system and engraft at significant levels at times in gestation when donor cells are normally rejected. These studies may allow the broadening of the use of MSC to diseases in which an underlying MSC defect precludes the use of the patient's own MSC.

Expression and Properties of Recombinant Ovine Uterine Serpin

Ovine uterine serpin (OvUS) is produced in the uterus of sheep under the influence of progesterone. It weakly inhibits pepsin and reduces proliferation of lymphocytes, tumor cell lines, and preimplantation embryos. When purified from uterine fluid, the concentration required for its antiproliferative effect in vitro is approximately 0.25-1 mg/ml. Here we show that recombinant (r) OvUS is a more potent regulator of cell proliferation than native (n) OvUS purified from uterine fluid. To produce rOvUS, RNA was extracted from endometrium from a pregnant ewe and cDNA was amplified by reverse transcription-polymerase chain reaction using gene-specific primers. The purified OvUS cDNA was inserted into the ampicillin-resistant plasmid vector pcDNA3.1/ V5-His-TOPO. The plasmid was introduced into the TOP10 Escherichia coli strain, purified, and used for transfection of Freestyle 293-F cells. Digestion of rOvUS with protein N-glycosidase F confirmed that rOvUS was N-glycosylated. Both rOvUS and nOvUS inhibited proliferation of phytohemagglutin-activated sheep lymphocytes and the P388D1 mouse lymphoma and PC-3 prostate cell lines. Inhibition was greater for rOvUS than for nOvUS, and concentrations as low as 15 microg/ml rOvUS were effective at reducing lymphocyte proliferation. Addition of rOvUS to fertilized bovine embryos reduced the cleavage rate and the percentage of embryos that became blastocysts. Native OvUS did not affect cleavage rate and had a smaller effect on development to the blastocyst stage. Experiments demonstrate that OvUS is a more potent inhibitor of cell proliferation and embryonic development than previously believed and add credence to the putative role for the protein in regulating cell proliferation.

Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester

The cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester (CFSE) is used to quantify cell kinetics. It is particularly important in studies of lymphocyte homeostasis where its labelling of cells irrespective of their stage in the cell cycle makes it preferable to deuterated glucose and BrdU, which only label dividing cells and thus produce unrepresentative results. In the past, experiments have been limited by the need to obtain a clear separation of CFSE peaks forcing scientists to adopt a strategy of in vitro labelling of cells followed by their injection into the host. Here we develop a framework for analysis of in vivo CFSE labelling data. This enables us to estimate the rate of proliferation and death of lymphocytes in situ, and thus represents a considerable advance over current procedures. We illustrate this approach using in vivo CFSE labelling of B lymphocytes in sheep.

Chromosomal aberrations and frequency of micronuclei in sheep subchronically exposed to the fungicide Euparen Multi (tolylfluanid)

We analyzed chromosome aberrations, micronucleus frequency, mitotic index (MI), and nuclear division index (NDI) in peripheral lymphocytes of sheep subchronically exposed to the fungicide Euparen Multi (containing 50% tolylfluanid). Euparen Multi was administered by rumen sonde to group of Merino sheep (seven sheep/group) at 93 mg/kg body weight (1/20 LD 50) daily for 28 days to assess its genotoxic effects. The frequencies of aberrant cells (ABC) in the experimental and control groups were 5.50±1.38% and 2.40±1.14%, respectively, and the increase in ABC in the treated group was significant ( P = 0.003 ). Significantly increased numbers of chromatid breaks (5.67±1.21% against 2.40±1.14%; P = 0.001 ), chromatid gaps (10.33±2.73% against 4.00±1.23%; P = 0.001 ), and chromosome gaps (1.83±0.75% against 0.80±0.45%; P = 0.025 ) and exchanges (3.17±1.94% against 0.20±0.45%; P = 0.009 ) were observed in exposed animals in comparison to control animals. The frequency of micronuclei (MN) was 29.40±5.86 per 1000 binucleated cells in peripheral lymphocytes of sheep in the control group and 49.57±19.12 per 1000 binucleated cells in the treated group. A significant increase in the frequency of MN in peripheral lymphocytes also was observed between the two groups ( P = 0.0477 ). No statistical differences in MI and NDI values were found in the groups ( P = 0.181 and 0.761, respectively). Thus, our results suggest that exposure to Euparen Multi may cause genome damage in somatic cells.

Cloning and characterisation of the primary structure of the sheep lymphocyte function-associated antigen-1 α subunit

The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. The ovine cDNA encoding CD11a, the predominant α subunit of the β 2-integrin family, was sequenced and compared with the human, bovine and murine sequences. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the ovine CD18-encoding cDNA, which is available for a few months, the sequence data provided here will allow the Ovis aries β 2-integrin CD11a/CD18 (LFA-1, α Lβ2) expression in vitro as a tool to examine the specificities of inflammation in the ovine species.

Comparison of modulation of sheep, mouse and buffalo lymphocyte responses by Fasciola hepatica and Fasciola gigantica excretory-secretory products

In order to explain the difference in susceptibility to Fasciola hepatica and F. gigantica between animal species, the activity of their excretory-secretory products (FhESP and FgESP, respectively) on concanavalin A (ConA)-induced proliferation of different animal species (sheep, mouse and buffalo) lymphocytes was compared. At high doses, FhESP inhibited proliferation of lymphocytes of all the animal species tested, and at low doses they inhibited the proliferation of sheep lymphocytes and increased the proliferation of buffalo and mouse lymphocytes. The effects of FgESP were similar but the intensity of FgESP inhibition was less than FhESP. The immunomodulatory effects of FhESP or FgESP could not alone explain the susceptibility level of hosts to Fasciola spp. The immunomodulatory molecules of FhESP and FgESP and their role in the course of Fasciola spp. infection should be further investigated.

Chromosome damage in peripheral lymphocytes of sheep induced by chlorine in drinking water

The potential of chromosomal damage associated with the effects of chlorine in drinking water was evaluated using chromosome aberrations and micronuclei as cytogenetic endpoints in the lymphocytes of peripheral blood of ewe lambs. The study assessed the in vivo effects of high chlorine doses (1.8 mg l − 1, based on Savo-Super disinfectant) in drinking water on the peripheral lymphocytes of sheep after 30 days. The frequency of aberrant cells (AB.C.) in the experimental and control groups was 31.80 ± 13.68% AB.C. and 4.50 ± 2.07% AB.C. respectively, and the increased AB.C. in the treated group was highly significant (P = < 0.001). In the experimental group chromatid breaks (26.20 ± 10.47%) and gaps (24.20 ± 13.94%) were the dominant types of aberrations, but statistically significant chromosome gaps and exchanges were also present. The frequency of micronuclei in peripheral lymphocytes of sheep in the control group was 21.17 ± 4.36 per 1,000 binucleated cells and 64.20 ± 22.51 per 1,000 binucleated cells in the experimental group. A significant increase in the frequency of micronuclei in peripheral lymphocytes of sheep was observed between the two groups (P = 0.001).

Involvement of glutathione as a mechanism of indirect protection against spontaneous ex vivo apoptosis associated with bovine leukemia virus.

Viruses have developed strategies to counteract the apoptotic response of the infected host cells. Modulation of apoptosis is also thought to be a major component of viral persistence and progression to leukemia induced by retroviruses like human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Here, we analyzed the mechanism of ex vivo apoptosis occurring after isolation of peripheral blood mononuclear cells from BLV-infected sheep. We show that spontaneous apoptosis of ovine B lymphocytes requires at least in part a caspase 8-dependent pathway regardless of viral infection. Cell death is independent of cytotoxic response and does not involve the tumor necrosis factor alpha/NF-kappaB/nitric oxide synthase/cyclooxygenase pathway. In contrast, pharmaceutical depletion of reduced glutathione (namely, gamma-glutamyl-l-cysteinyl-glycine [GSH]) by using ethacrynic acid or 1-pyrrolidinecarbodithioic acid specifically reverts inhibition of spontaneous apoptosis conferred indirectly by protective BLV-conditioned media; inversely, exogenously provided membrane-permeable GSH-monoethyl ester restores cell viability in B lymphocytes of BLV-infected sheep. Most importantly, intracellular GSH levels correlate with virus-associated protection against apoptosis but not with general inhibition of cell death induced by polyclonal activators, such as phorbol esters and ionomycin. Finally, inhibition of apoptosis does not correlate with the activities of GSH peroxidase and GSH reductase. In summary, our data fit into a model in which modulation of the glutathione system is a key event involved in indirect inhibition of apoptosis associated with BLV. These observations could have decisive effects during therapeutic treatment of delta-retroviral pathogenesis.

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory–secretory products

The effect of Fasciola hepatica excretory–secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2 +, CD4 +, CD8 +, γδTCR + or CD21 + cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 μg per well) or suboptimal (0.25 μg per well) doses and with FhESPs at several doses (1.25–20 μg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 μg per well. These cells were incubated with or without monocytes (CD14 + cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 μg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21 + lymphocytes (B lymphocytes), CD14 + cells (monocytes) and γδTCR + cells were not stimulated by ConA. T lymphocyte subsets (CD2 +, CD4 + or CD8 + cells) proliferation was decreased by FhESPs at 5 μg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.

Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cells.

Fasciola hepatica infection has been shown to suppress sheep lymphocyte proliferation in vitro and this is at least partially attributable to excretory/secretory products (ES) released by F. hepatica parasites. We identified a suppressive component in ES by analysing the effect of ES fractions, separated by gel filtration, on the proliferation of sheep T cells in vitro. A major proportion of the suppressive activity in ES was shown to coelute with the cathepsin L proteases: E64, a cysteine protease inhibitor, blocked the suppressive activity of cathepsin L. In order to identify possible mechanisms by which cathepsin L could suppress T cell proliferation, the effect of ES and F. hepatica recombinant cathepsin L (rFhCatL) on the expression of 22 different sheep T cell surface markers was analysed by flow cytometry. Incubation of sheep T cells with ES or two rFhCatL significantly reduced surface CD4 expression and this effect was prevented in the presence of E64. In similar experiments with human T lymphocytes, ES and rFhCatL were shown to down regulate surface CD4 expression. These results show that F. hepatica cathepsin L both suppresses sheep T cell proliferation and reduces surface CD4 expression on both human and ovine T cells.

Changes in behaviour, cortisol and lymphocyte types during isolation and group confinement of sheep.

This experiment compared changes in complex behaviour patterns, adrenal corticosteroid secretion and the numbers of various types of lymphocytes in sheep that were subjected to the stress of confinement. Grazing Merino ewes (n=80 in five replicated experiments) were confined either in groups of four per pen or in total isolation from other sheep. The percentage of CD4+ lymphocytes increased while the percentage of CD8+ lymphocytes decreased over the experimental period. This result was more pronounced in isolated sheep than in grouped sheep. The increase in CD4:CD8 was greater for isolated sheep than for grouped sheep and greater for 2 week sheep than for 3 week sheep. The percentage of CD5+ cells also increased, less so in isolated than in grouped animals. Interpreting these changes as a recovery of immune competence following introduction of a stressor, it is apparent that isolation impaired immune system recovery more severely than group confinement. Physiological and behavioural adaptation over the period were characterized by a decline in the adrenocortical response, resumption of the normal pattern of flocking behaviour and a reduction in motor activity during the test. These findings add to the evidence pointing to the possible correspondence between critical features of the psychoneural, neuroendocrine and immune systems.

Animals as sentinels of human health hazards of environmental chemicals.

A workshop titled "Using Sentinel Species Data to Address the Potential Human Health Effects of Chemicals in the Environment," sponsored by the U.S. Army Center for Environmental Health Research, the National Center for Environmental Assessment of the EPA, and the Agency for Toxic Substances and Disease Registry, was held to consider the use of sentinel and surrogate animal species data for evaluating the potential human health effects of chemicals in the environment. The workshop took a broad view of the sentinel species concept, and included mammalian and nonmammalian species, companion animals, food animals, fish, amphibians, and other wildlife. Sentinel species data included observations of wild animals in field situations as well as experimental animal data. Workshop participants identified potential applications for sentinel species data derived from monitoring programs or serendipitous observations and explored the potential use of such information in human health hazard and risk assessments and for evaluating causes or mechanisms of effect. Although it is unlikely that sentinel species data will be used as the sole determinative factor in evaluating human health concerns, such data can be useful as for additional weight of evidence in a risk assessment, for providing early warning of situations requiring further study, or for monitoring the course of remedial activities. Attention was given to the factors impeding the application of sentinel species approaches and their acceptance in the scientific and regulatory communities. Workshop participants identified a number of critical research needs and opportunities for interagency collaboration that could help advance the use of sentinel species approaches.

Characterization of immune responses caused by bovine leukemia virus envelope peptides in sheep.

To study the immunomodulative activity caused by bovine leukemia virus envelope (BLV Env) peptide, sheep were immunized with two kinds of Th-epitope peptides, peptide 98 (BLV Env 98-117), and 61 (BLV Env 61-78). Four of eight immunized sheep showed specific proliferative responses against both of the peptide stimulations. To characterize the cells responding to the peptides, peptide-specific cells were established from the responding sheep by the continuous stimulation of peripheral blood mononuclear cells (PBMCs) with either peptide 98 or 61 in vitro. The peptide 98-specific cells consisted of CD4-positive cells, whereas the peptide 61-specific cells consisted of CD8-positive cells and MHC class II-positive cells. In addition, cytokine profile analysis indicated that the peptide 98-stimulated cells expressed IFN-gamma but not IL-10, although the peptide 61-stimulated cells expressed IL-10 but not IFN-gamma. These results show that BLV envelope peptides 98 and 61 can modulate immune responses of sheep lymphocytes in different ways and may contribute to the pathogenesis of BLV infection.

Induction of Micronuclei and Sister Chromatide Exchanges in Sheep Lymphocyte Cultures by Herbicide Chloridazon

Pidova. E .. K. Sivikova: Induction oj Micronuclei and Sister Chromatid Exchanges ill Sheep Lymphocyte Cultures by Herbicide Chloridazoll. Acta vet. Brno 1997.66: 229-234. The effect of herbicide chioridazon was studied using induction of micronuclei (MN) and sister chromatid exchanges (SCE) in vitro. Inductions o(these markers were measured in sheep peripheral lymphocytes with and without of metabolic activation (S-9 mix). Chloridazon affected both MN and SCE at all of the concentrations tested. Significant increases (P<0.05) were achieved at 7 x 1O'~M in both tests. Neither of the lower concentrations (7 x 10·5 M and 7 x 10.6 M) did affect the MN or SCE values. The herbicide was also tested in presence of an S 9 mix activating fraction. in which significant increases of SCE levels were determined for concentrations of 7 x 10'; M and 7 x 1O.~ M (P<0.05 and 0.01. respectively). No induction of the cell cycle delay or reduction of proliferation index (PI) were detected. Chloridazon has. as shown in our assays. a weak influence on induction of tested chromosomal changes. Sheep. peripheral lymphocytes, microlluclei, sister chromatid exchallges. chlorida:oll

Mimicry of a 21.5 kDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TLC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide ( P < 0.001) and to a lesser extent sheep MBP ( P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogensis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Immunomodulation of Sheep and Human Lymphocytes by Fasciola hepatica Excretory-secretory Products

Immunomodulation of Sheep and Human Lymphocytes by Fasciola hepatica Excretory-secretory Products

Immunomodulation of sheep and human lymphocytes by Fasciola hepatica excretory-secretory products

Jefferies J. R., Barrett J. & Turner R. J. 1996. Immunomodulation of sheep and human lymphocytes by Fasciola hepatica excretory-secretory products. International Journal for Parasitology 26: 1119–1121. Fasciola hepatica excretory-secretory (ES) products (30 μg ml −1 total ES protein upwards) significantly decreased tritiated thymidine ( 3HTh) uptake in phytolectin-stimulated sheep lymphocytes. Lower doses of ES products, however, caused an increase in 3HTh uptake in sheep cells stimulated with phytohaemagglutinin (PHA) and Concanavalin A (Con A) and, indeed, ES products were mitogenic on their own. In contrast, 3HTh uptake by human lymphocyte cultures in the presence of phytolectins was progressively inhibited by increasing doses of ES products, and ES products were not mitogenic on their own. The observed inhibitory effects on 3HTh incorporation were not due to toxicity of ES products.

The effect of small doses of mercury on the level of selected lysosomal enzymes in the plasma and lymphocytes of sheep

1. 1. The 12-month-old ewes, Slovak Merinos breed, were given 40 mg of mercury daily for 28 days in the form of HgCl 2. 2. 2. Administration of mercury had a significant influence on the activity of the investigated lyso-somal enzymes in the plasma and lymphocytes of sheep.

Investigation of sheep-associated malignant catarrhal fever virus infection in ruminants by PCR and competitive inhibition enzyme-linked immunosorbent assay

Development of control measures for the gammaherpesviral disease of cattle known as sheep-associated malignant catarrhal fever (SA-MCF) has been hampered by a lack of accurate diagnostic tests either for the causative virus or for antibody against that virus. A recently developed competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA) for the detection of antibody to malignant catarrhal fever (MCF) virus (MCFV) in ruminants based on a monoclonal antibody to a widely conserved epitope of MCFV (H. Li, D. T. Shen, D. P. Knowles, J. R. Gorham, and T. B. Crawford, J. Clin. Microbiol. 32:1674-1679, 1994) and a PCR assay based on previously reported primers (S. I. F. Baxter, I. Pow, A. Bridgen, and H. W. Reid, Arch. Virol. 132:145-159, 1993) were used to detect anti-MCFV antibody and SA-MCFV DNA in sheep and other ruminants. The PCR amplified a specific 238-bp SA-MCFV genomic DNA fragment from peripheral blood lymphocytes of adult sheep and other ruminants with clinical MCF. Of 144 samples from randomly selected healthy adult sheep, 143 (99%) were positive by PCR and 136 (94%) were positive by CI-ELISA. The agreement between the two assays exceeded 95%. Of nine samples collected from cattle and deer with clinical MCF of apparent sheep origin, seven were CI-ELISA positive and all 9 were PCR positive. Among 59 serum samples from presuckling lambs, none contained antibody detectable by CI-ELISA. After suckling, maternal anti-MCFV antibody was detectable for about 10 +/- 3 weeks. Although all colostrum and milk samples from infected ewes were strongly PCR positive, the appearance of detectable SA-MCFV DNA in lambs was correlated generally with antibody patterns, which suggests that the natural infection event in sheep may not occur during the perinatal period but occurs sometime later in life.

The effect of storage on immunophenotyping of sheep peripheral blood lymphocytes by flow cytometry

We have shown that when a whole blood method of cell staining is used for flow cytometric analysis of sheep lymphocytes (i.e. red cell lysis after addition of antibody), staining may be delayed for up to 48 h after blood collection without significant effect on expression of CD4, CD5, CD8 or B cell markers. If cells were stained immediately after blood collection, using the same whole blood method, and then fixed with paraformaldehyde, cell samples could be stored for 24 h without change in marker expression. However, by 7 days there was a significant decrease in the percentage of cells expressing CD8, T19 and B cell markers. Cryopreservation prior to staining was found to markedly affect the expression of all cell surface markers investigated. These results indicate that storage of sheep blood prior to flow cytometric analysis is feasible but may affect the results obtained. Thus is it important to standardise the handling of samples, especially when comparative studies are being undertaken.