Flow cytofluorometric characterization of bovine blood and milk leukocytes

Flow cytometry and sorting proved to be a rapid method that facilitated the identification of different leukocyte populations in bovine blood and milk. After briefly incubating whole blood and milk samples in a hypotonic phosphate buffer, containing supravital acridine orange, 5 classes of leukocytes were found in the blood (lymphocytes, neutrophils, eosinophils, basophils, and monocytes) and 4 in the milk (lymphocytes, neutrophils, monocytes, and macrophages) by flow cytometry. Cells were morphologically identified by fluorescent microscopy after flow cytometric sorting and by light microscopy after Papanicolaous staining. Udder parenchymal and ductal tissue cells (secretory and epithelial cells) were not found in the milk samples evaluated. Large differences in the total and differential cell counts were found in the different milk secretions.     

Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45

BACKGROUND: In dogs, flow cytometry is used in the phenotyping of immunologic cells and in the diagnosis of hemic neoplasia. However, the paucity of specific antibodies for myeloid cells and B lymphocytes and of labeled antibodies for multicolor techniques limits the ability to detect all leukocyte subpopulations. This is especially true for neoplastic and precursor cells. CD18 and CD45 are expressed on all leukocytes and are involved in cell activation, and together could be useful in helping determine cell lineage. OBJECTIVES: The purpose of this study was to double label canine blood for CD18 and CD45 and to use the differential expression of antigens to identify leukocyte populations in dogs with non-neoplastic and neoplastic hematologic diseases. METHODS: A template was developed using blood samples from 10 clinically healthy dogs and a back-gating technique. Differential leukocyte counts obtained with the template were compared with those obtained by manual and automated methods on blood samples from 17 additional healthy dogs. Blood samples obtained from 9 dogs with non-neoplastic (reactive) hematologic diseases and 27 dogs with hemic neoplasia were double stained for CD18 and CD45 using mouse anticanine CD18 monoclonal antibody (mAb) plus phycoerythrin-conjugated rat anticanine CD45 mAb and fluorescein isothiocyanate-conjugated rabbit antimouse IgG. Hemic neoplasms were diagnosed by cell morphology, and immunophenotypic and cytochemical markers. RESULTS: With the double label, neutrophils, eosinophils, monocytes, and T- and B-lymphocytes were identified. In reactive disorders, a population of activated neutrophils with high CD45 and CD18 expression was detected. In hemic neoplasia, cell lineage was easily determined, even in acute leukemia. CONCLUSIONS: Double labeling for CD18/CD45 may be useful as a screening method to evaluate hematologic diseases and help determine cell lineage, and to aid in the selection of a panel of antibodies that would be useful for further analysis.     

Expression of β2-integrin on monocytes and blood polymorphonuclear leukocytes in the periparturient period in dairy cows

The hypothesis that an altered expression of CD11/CD18 on bovine circulating monocytes, polymorphonuclear leukocytes (PMN), or both, contributes to an increased mastitis susceptibility in periparturient cows was tested. Expression of CD18 and CD11a, -b, -c on bovine monocytes and PMN were assessed in 8 Friesian-Holstein cows by flow cytometry from 2 wk before calving to 5 wk after calving. Minor changes in adhesion molecule expression levels were detected throughout the experimental period. Compared with PMN, monocytes exhibited an expression level that was similar for CD18, higher for CD11a and CD11c, but lower for CD11b. Differences in density may reflect the relative importance of these adhesion molecules on both leukocyte types. In this study, the decreased number of milk resident macrophages and PMN observed during the periparturient period could not be attributed to changes of CD11/CD18 levels on circulating leukocytes.     

Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes

Oxidative stress and abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, current assays for the reduced form of glutathione (GSH) are time-consuming and semi-quantitative and do not allow assessment of GSH concentrations in individual cell populations. Therefore, we developed a flow cytometric assay for rapid determination of intracellular GSH concentrations in feline blood leukocytes. The assay was based on the ability of the non-fluorescent substrate monochlorobimane (mBCl) to form fluorescent adducts with GSH in a reaction catalyzed by the enzyme glutathione-S-transferase. Using flow cytometry, we found that mBCl was sensitive and specific for intracellular detection of the reduced form of GSH in feline leukocytes. Intracellular GSH concentrations were also stable for at least 24h in EDTA preserved whole blood samples stored at 4 degrees C. Neutrophils and monocytes from normal cats had significantly higher intracellular concentrations of GSH than T cells and B cells. The effects of FIV infection on intracellular GSH concentrations in cats were assessed using flow cytometry. We found that neutrophils from FIV-infected cats had significantly increased GSH concentrations, whereas intracellular GSH concentrations were significantly decreased in CD4(+) and CD8(+) lymphocytes from FIV-infected cats, compared to age-matched control animals. We conclude that a flow cytometric assay based on mBCl may be used to accurately and rapidly assess the effects of various disease states and treatments on GSH concentration in cat leukocytes and to help assess intracellular oxidative stress.     

Preparation, purification and characterization of bovine Peyer''s patch leukocytes.

An enzymatic technique for isolating bovine Peyer's patch leukocytes was developed. Enzymatic dissociation of Peyer's patches yielded a cell population rich in T and B lymphocytes containing 6-10% adherent accessory cells, as defined by morphology and nonspecific esterase staining. Analysis of Peyer's patch lymphocytes, using sheep erythrocyte rosetting and immunofluorescence with conventional antisera and monoclonal antibodies, showed that leukocytes were approximately 45% T cells and 25% Ig-positive B cells. The cell population contained functionally active T and B lymphocytes which proliferated in response to T and B cell mitogens and to exogenous human recombinant interleukin-2. The observed differences in patterns of Peyer's patch leukocyte responsiveness to these mitogens suggest some cellular heterogeneity of the bovine Peyer's patch.     

Identification of feline monocytes and neutrophils as effector cells in antibody-dependent cellular cytotoxicity: sequential analysis, using light microscopy, histochemistry, and scanning electron microscopy

Feline monocytes and neutrophils functioned as effector cells in antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken erythrocytes. Using light microscopy, effector cell populations were identified in effector-target cell interactions, with further characterization of these identical individual effector cells by histochemical evaluations and scanning electron microscopy. Monocytes and neutrophils, but not lymphocytes, were observed attacking target cells. Carbonyl iron depletion of monocytes and neutrophils from peripheral blood leukocytes caused a marked reduction from a mean of 62% to 3.6% lysis in ADCC as measured by a 4-hour 51Cr release assay. Effector cells functioning in the ADCC reaction were visualized, using sequential analysis and light microscopy, histochemistry, and scanning electron microscopy.     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.     

Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.     

Influence of chronic caprine arthritis-encephalitis virus infection on the population of peripheral blood leukocytes

The influence of caprine arthritis-encephalitis (CAE) virus infection on the population of peripheral blood leukocytes in goats was evaluated. For this purpose two groups of adult dairy female goats were formed. The experimental group consisted of 17 goats, which had been naturally infected for many years. The control group comprised 29 non-infected goats, which originated from CAE-free herd. All goats were clinically healthy. Whole blood was collected and tested in hematological analyzer and light microscope to assess the total number of leukocytes and the percentage of four leukocyte populations--neutrophils, eosinophils, monocytes and lymphocytes. Then, flow cytometry with monoclonal antibodies against several surface antigens (namely CD14, CD2, B-B2, CD4, CD8h, TCR-N6, WC1-N2 and WC1-N3) was performed to assess the proportion of lymphocyte subpopulations. Statistically significant differences (alpha < or = 0.01) were observed only in the subpopulations of T lymphocytes--percentage of all subpopulations were significantly higher in the group of seropositive goats. No statistically significant differences were revealed with respect to the total number of blood leukocytes, the average percentage of blood leukocyte populations and proportions of both T and B lymphocytes.     

The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species

We examined the potential of developing a set of species specific and cross reactive monoclonal antibodies (MoAbs) for use in the study of the phylogenetic and functional relation of class I and class II antigens of the major histocompatibility complex (MHC) and leukocyte differentiation antigens in cattle and other species. Comparing immunization strategies demonstrated the number of hybrids producing cross reactive antibodies can be increased by hyperimmunization of mice with lymphoid cells from multiple species. Comparing various methods of assay (antibody-complement mediated cytotoxicity [CT], enzyme linked immunosorbent assay [ELISA] and flow microfluorimetry [FMF]), revealed FMF is the most useful technique for the primary assay of hybridomas producing MoAbs of potential interest. By using dual parameter and dual fluorescence analysis, we could determine whether a given MoAb reacted with mononuclear cells (lymphocytes and monocytes) and/or granulocytes, and also whether any two MoAbs of different isotype and specificity recognized antigens present on identical or separate populations of leukocytes. Comparing the patterns of MoAb reactivity with leukocytes obtained from cows, goats, sheep, pigs, horses and humans, as well as comparing the patterns of reactivity with a panel of lymphoid cell lines derived from cattle (with enzootic bovine leukemia) and humans (with various forms of leukemia), revealed sets of MoAbs reactive with unique antigenic determinants present on BoLA class I (15 MoAbs) and class II (9 MoAbs) antigens, and also MoAbs reactive with determinants present on leukocyte differentiation antigens (36 MoAbs). Dual fluorescence analysis demonstrated the antigens detected by some MoAbs are predominantly expressed on one lineage of leukocytes while others are expressed on two or more lineages of leukocytes. Dual and single fluorescence analysis also demonstrated the PNA receptor(s) is: expressed on T cells, granulocytes and class II antigen monocytes and absent or expressed in low amount on sIgM+ B cells and a newly defined Non T/Non B population of cells. The strategies described for identifying and analyzing the specificity of MoAbs demonstrate the feasibility of developing a set of cross reactive MoAbs for identifying homologous molecules in multiple species and delineating their functional and phylogenetic relation.     

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.     

Expression of CD3 and CD11b antigens on blood and mammary gland leukocytes and bacterial survival in milk of cows with experimentally induced Staphylococcus aureus mastitis.

OBJECTIVES: To differentiate early (1 to 8 days) from late (9 to 14 days) inflammatory phases and assess relationships between leukocyte phenotype and bacterial recovery in cows with Staphylococcus aureus-induced mastitis. ANIMALS: 10 first-lactation Holstein cows. PROCEDURE: Blood and milk samples were collected from 4 or 6 cows before and after intramammary infusion of sterile broth or S. aureus, respectively. Flow cytometric expression of CD3 and CD11b antigens on blood and milk leukocytes, leukocyte differential counts, bacterial counts in milk, and somatic cell counts were determined longitudinally. RESULTS: Density of CD3 molecules decreased on blood lymphocytes and increased on milk lymphocytes after infusion of bacteria. Density of CD11b molecules on lymphocytes and phagocytes and percentage of CD11b+ lymphocytes in milk increased significantly after infusion; maximum values were achieved during the early inflammatory phase. Density of CD3 and CD11b molecules on milk lymphocytes and macrophages, respectively, 1 day after inoculation were negatively correlated with bacterial recovery on day 1 and days 9 to 14, respectively. Density of CD11b molecules on milk macrophages and the ratios of phagocyte to lymphocyte percentages and polymorphonuclear cell to macrophage percentages in milk differentiated the early from the late inflammatory phase. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of bovine mammary gland macrophages and T cells in response to intramammary infusion of S. aureus was associated with an inability to culture this bacterium from milk. Identification of specific inflammatory phases of S. aureus-induced mastitis in cows may allow for the design of more efficacious treatment and control programs.     

Leukocyte profile of cattle persistently infected with bovine viral diarrhea virus

Animals acutely infected with bovine viral diarrhea virus (BVDV) exhibit transient immunosuppression as a result of the virus' predilection for cells that play critical roles in the host immune system. Acute BVDV infections have major effects on thymic and follicular T-lymphocytes, as well as follicular B-lymphocytes, often resulting in severe reduction in circulating numbers of lymphocytes and suppression of functional activities of these cells. Granulocytes and monocytes are equally susceptible to BVDV infections with reduction in numbers and suppression functions. However, there is limited information on the leukocyte profile of cattle persistently infected (PI) with BVDV. This study reports on phagocytic activities of granulocytes and monocytes as well as immunophenotyping by flow cytometric analysis of leukocytes isolated from healthy non-PI (NPI) and PI animals. No significant differences were found between the leukocyte profiles and the phagocytic activities of PI animals when compared to a group of healthy NPI animals.     

Flow cytometric evaluation of canine bone marrow based on intracytoplasmic complexity and CD45 expression

BACKGROUND: Because of the complexity, subjectivity, time, and technical skill required for determination of manual bone marrow differential cell counts, an alternative method is needed. Several flow cytometric methods have been described, but all have limitations. OBJECTIVE: The purpose of this study was to evaluate a technique for bone marrow differential cell counting based on flow cytometric evaluation of CD45 expression and intracellular complexity (CD45 scatter plots). METHODS: Bone marrow was obtained from 15 dogs that were being evaluated for hematologic disorders. In preliminary studies, the location of bone marrow subpopulations in the CD45 scatter plots was evaluated by labeling bone marrow with lineage-specific markers. A template was developed to identify these cell populations. Gates were set to identify granulocytes, myeloblasts, monocyte/macrophages, lymphocytes, and nucleated erythroid populations. RESULTS: The CD45 labeling technique accurately quantified granulocytes, myeloblasts, erythroid precursors, and lymphocytes in canine bone marrow. Correlation coefficients with manual counts for granulocytes, myeloblasts, erythroid cells, lymphocytes, and monocyte/macrophages were 0.90, 0.89, 0.96, 0.91, and 0.54, respectively. CONCLUSIONS: The capacity of the CD45 scatter-plot technique to quantify lymphocytes and myeloblasts is an advantage over previously described techniques. The simplicity of the CD45 labeling method and the ease with which batches of samples can be analyzed makes the technique potentially applicable as a routine test in clinical and research laboratories.     

Glycogen in Leukocytes from Bovine Blood and Milk

Glycogen content was determined quantitatively by the Anthrone reagent method in leukocytes obtained from blood and milk of five cows. Distribution of glycogen in leukocytes was studied by microscopic examination of slides stained by Periodic acid-Schiff (PAS) reaction. Blood glucose concentrations were investigated in these animals by standard procedures. In two of five cows both blood glucose levels and blood leukocyte glycogen levels on the same day were determined for six consecutive days. One hundred and two blood leukocyte samples from five cows had a mean glycogen content of 1.32 ± 0.04 (S.E.) mg/10⁹ WBC, and 6.11 ± 0.17 (S.E.) mg/10⁹ PMNs. Leukocyte preparations from 80 samples of milk comprising 97 to 98% PMNs contained 3.81 ± 0.18 (S.E.) mg glycogen/10⁹ milk leukocytes. In PAS preparations of blood and milk leukocytes glycogen was found almost exclusively in PMNs. Glycogen granules, present frequently in PMNs and occasionally in monocytes and large lymphocytes from blood, were not observed in those from milk. The glycogen level in milk leukocytes was significantly lower (P = <0.01) than that of the blood PMNs in every cow, and the overall mean difference between levels for milk leukocytes and blood PMNs was highly significant (P = <0.001). Mean blood glucose concentration in the five cows was 44.46 ± 0.66 (S.E.) mg%. There was no significant relationship between blood glucose and blood leukocyte glycogen levels in the five corresponding cows; nor between blood glucose and blood PMN glycogen levels on the same day in either of two cows investigated. Leukocyte preparations from milk samples obtained on the second day following intramammary infusion of endotoxin consistently contained markedly less glycogen than the leukocyte preparations from first day post-infusion samples.

These tended to level off and became intermediate between first and second day levels. It is postulated that the poor phagocytic competence of leukocytes from bovine mammary glands compared to their counterparts in blood observed by various workers may be due partially to low energy reserves in these cells.

     

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.     

Leukocytes from heifers at different ages express insulin and insulin-like growth factor-1 (IGF-1) receptors

The objectives of this study were to investigate if insulin receptors (IR) and insulin-like growth factor-1 receptors (IGF-1R) could be detected on bovine leukocytes using fluorescence antibodies and flowcytometry, and use this method to investigate whether the amount of receptors differed among heifers at different ages. Twenty Danish Holstein heifers in the following three age groups were included in the investigation: (1) heifers aged 25-45 days (n = 8), (2) heifers aged 185-205 days (n = 7), and (3) heifers aged 780-900 days (approximately one month prepartum; n = 5). Antibodies against human IR and IGF-1R were used for indirect immunofluorescence staining of cells, and were found to cross-react with the bovine receptors. IR were found on lymphocytes, monocytes, and neutrophils in all animals, while IGF-1R were found only on monocytes and neutrophils. The percentage of IR+ lymphocytes was almost doubled from stage 1 to 3 (34% versus 57%) and IR expression on lymphocytes and monocytes increased significantly (P<0.01) from 6.80 and 13.60 to 8.24 and 17.50 in heifers aged 185-205 days and heifers aged 780-900 days, respectively. No differences were observed for neutrophil IR or IGF-1R expression. In conclusion, surface IR and IGF-1R can be detected on bovine leukocytes by flowcytometry. Interestingly, the highest numbers of IR were found in heifers one month prepartum. The regulation of IR and IGF-1R expression on bovine leukocytes, and their role in host immunity, needs further investigation.     

Canine differential leukocyte counting with the CellaVision DM96Vision, Sysmex XT-2000iV, and Advia 2120 hematology analyzers and a manual method

Background: For differential leukocyte counts, automated blood smear evaluation systems have been too slow or inaccurate to replace or supplement the manual differential count. The CellaVision DM96Vision (DM96V), a new instrument, is an automated image analysis system that is rapid and accurate enough to be used for enumerating human leukocytes and may be useful for analysis of canine blood. Objectives: The aims of this study were to evaluate the performance of the DM96V in differential counting of canine leukocytes, to compare its performance with that of other methods, and to analyze interoperator variability. Methods: Four methods of determining the leukocyte differential count of 108 canine blood samples were compared based on agreement, precision, and errors as well as relative performance. Differential counts were obtained using the DM96V, the manual method, and automated methods performed by the Advia 2120 and Sysmex XT‐2000iV. Results: All leukocyte types were detected by the DM96V and the manual method, and all 4 methods had similar mean and median results in most cases. The automated methods were more precise than either the DM96V or manual method when comparing identification of a single type of leukocyte, especially neutrophils and lymphocytes. However, precision of the automated methods was only fair for monocytes, and the Advia and Sysmex failed to identify basophils. The Advia reported fewer monocytes and eosinophils than did the other methods. Significantly fewer lymphocytes were identified by the manual method than by the Sysmex, Advia, and DM96V. The DM96V occasionally presented duplicate images of the same neutrophils. Conclusions: The CellaVision DM96V is a satisfactory system for facilitating canine differential leukocyte counting. The DM96V differential count was more similar to the manual count than to automated counts, which were more precise but had errors and omissions in detecting some types of leukocytes.     

Use of flow cytometry for determination of differential leukocyte counts in bovine blood

A flow cytometric method was developed to perform differential leukocyte counts on bovine blood. Blood specimens from 50 healthy Holstein cows were analyzed by use of a flow cytometer. The method entailed diluting blood with phosphate-buffered, hypotonic saline solution containing acridine orange, and performing a step-wise, 3-parameter analysis on the bases of cell size, cellular granularity, and granulocyte fluorescence. Initially, proportions of monocytes, granulocytes, and lymphocytes were determined by creating appropriate windows on dot plots of cell size (determined by forward light scatter) vs cellular granularity (determined by the logarithm of side light scatter). Eosinophils were resolved by analysis of granulocytes as dot plots of logarithms of green vs red fluorescence ascribed to acridine orange. Proportions of eosinophils and neutrophils were computed from data so generated. Microclumps of platelets spuriously affected counts of some granulocytes, particularly eosinophils. Differential leukocyte counts determined by flow cytometry generally compared favorably with those obtained by use of the conventional microscopic method, using Wright-stained blood films. Mean neutrophil and eosinophil counts determined by the 2 methods did not differ significantly, but lymphocyte counts determined by flow cytometry were significantly higher than those determined by microscopy (P less than 0.01). Correlation coefficients for counts of neutrophils, eosinophils, and lymphocytes determined by the 2 methods ranged from 0.519 to 0.833. Correlation between monocyte counts was low (r = 0.147), although mean monocyte counts determined by the 2 methods did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of the green turtle’s leukocyte subpopulations by flow cytometry and evaluation of their phagocytic activity

Phagocytosis is a fundamental aspect of innate immunity that is conserved across many species making it a potentially useful health-assessment tool for wildlife. In non-mammalian vertebrates, heterophils, monocytes, macrophages, melanomacrophages, and thrombocytes all have phagocytic properties. Recently, B lymphocytes from fish, amphibians, and aquatic turtles have also showed phagocytic capacity. Phagocytes can be studied by flow cytometry; however, the use of this tool is complicated in reptiles partly because nucleated erythrocytes complicate the procedure. We separated green turtle leukocytes by density gradient centrifugation and identified subpopulations by flow cytometry and confocal microscopy. Additionally, we assessed their ability to phagocytize Fluorspheres and Ovoalbumin-Alexa. We found that heterophils and lymphocytes but not monocytes could be easily identified by flow cytometry. While heterophils from adults and juvenile turtles were equally able to phagocytize fluorspheres, adults had significantly more phagocytic ability for OVA-Alexa. Lymphocytes had a mild phagocytic activity with fluorospheres (27–38 %; 39–45 %) and OVA-Alexa (35–46 %; 14–22 %) in juvenile and adult green turtles, respectively. Confocal microscopy confirmed phagocytosis of fluorospheres in both heterophils and lymphocytes. This provides the first evidence that green turtle lymphocytes have phagocytic activity and that this assay could potentially be useful to measure one aspect of innate immunity in this species.