Prevalence of bovine sperm-bound antisperm antibodies and their association with semen quality.

The objectives of this study were to determine reference intervals (RIs) for sperm-bound immunoglobulins G and A (IgG and IgA), prevalence of antisperm antibodies (ASAs) in satisfactory and nonsatisfactory breeders, and association between ASAs and semen quality in beef bulls. It was hypothesized that ASA binding differed with breeding soundness classification and semen quality. The percentage of IgG- (IgGperc) and IgA-bound (IgAperc) spermatozoa was evaluated in satisfactory (n = 134) and nonsatisfactory (n = 71) breeder beef bulls using flow cytometry. The RI for IgGperc was 0% to 13.5%. The RIs for IgAperc were 0% to 25.8% in yearling Aberdeen Angus bulls and 0% to 12% in all other bulls. The prevalence of IgA-positive samples was higher in nonsatisfactory (14.1%) than that in satisfactory (1.5%) breeders (P = 0.0003). However, the prevalence of IgG-positive samples did not differ. Similarly, IgA binding was higher in nonsatisfactory (median; interquartile range; 2.18; 0.77%-8.57%) than that in satisfactory breeders (median; interquartile range; 1.11; 0.32%-3.16%; P = 0.0035), but IgG binding did not differ. Among ASA-positive bulls, median IgA and IgG binding was 39.7% (range, 18.8%-96.2%) and 24.8% (range, 14.2%-33.1%), respectively. Immunoglobulin A binding correlated with the percentage of total (P < 0.0001; r(2) = -0.345) and progressively motile spermatozoa (P < 0.0001; r(2) = -0.329), morphologically normal spermatozoa (P = 0.0004; r(2) = -0.256), sperm head abnormalities (P = 0.0416; r(2) = 0.149), proximal droplets (P = 0.0227; r(2) = 0.167), and coiled tails (P = 0.0338; r(2) = 0.156). Immunoglobulin G binding correlated with the percentage of total (P < 0.0001; r(2) = -0.373) and progressively motile spermatozoa (P < 0.0001; r(2) = -0.455) and sperm concentration (P = 0.0332; r(2) = -0.195). Reference intervals were established for determination of cutoffs for clinically significant sperm-bound IgA and IgG with flow cytometry. Immunoglobulin A binding was both higher and more prevalent in nonsatisfactory breeder bulls. Although IgG binding did not differ with breeding soundness classification, detection of surface-bound IgG and IgA was associated with changes in semen quality.

Standardization of a method to detect bovine sperm-bound antisperm antibodies by flow cytometry.

The objectives were to standardize some methodological and analytical aspects of a direct technique to detect sperm-bound antisperm antibodies (ASAs) in bovine semen using flow cytometry, including the effects of prefixation of sperm membranes with formalin buffer solution and inclusion of dead cells in the analysis. Fourteen Angus bulls, including ASA-positive (experimentally induced ASAs) and 10 reproductively normal ASA-negative bulls, were used. Fixation of sperm membranes had no significant effect on the percentage of IgG- or IgA-bound spermatozoa detected by flow cytometry. However, including dead cells in the analysis increased the percentage of IgG-bound spermatozoa in fixed (live and dead 18.6 ± 9.7% and live 1.3 ± 0.5%; median ± SEM) and nonfixed samples (live and dead 18.8 ± 9.2%, live 1.5 ± 0.6%; P = 0.0029), as well as IgA-bound spermatozoa in fixed (live and dead 16.3 ± 6.4%, live 0.3 ± 0.5%) and nonfixed samples (live and dead 21.4 ± 4.6%, live 1.0 ± 0.5%; P = 0.0041) in semen from ASA-negative bulls. Intrasample, intra-assay, and interassay coefficients of variation (CV) were 0.8%, 4.6%, and 5.3%, respectively, for determination of sperm-bound IgG, and were 2.8%, 8.4%, and 40.3% for determination of sperm-bound IgA. Despite the high interassay CV for IgA determination, all ASA-positive bulls consistently had high percentages of IgA-bound spermatozoa. Flow cytometry correctly identified ASA-positive bulls. Confocal laser microscopy confirmed binding of ASAs to sperm heads and cytoplasmic droplets, and less frequently to midpieces and principal piece. In conclusion, although fixation was not necessary, dead cells should be excluded from the analysis, because ejaculates with a large proportion of dead cells can yield false-positive results. Flow cytometry was accurate and reliable for detection of sperm-bound IgG and IgA and discrimination between ASA-positive and ASA-negative bulls.

An immunoglobulin binding protein (antigen 5) of the stable fly (Diptera: Muscidae) salivary gland stimulates bovine immune responses.

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as ahomolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naive calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with "natural" Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F (ab')2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.

Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules.

Multiparameter flow cytometry analysis and specific cluster differentiation (CD) molecules were used to determine the expression profiles of B- and T-cell antigens on lymph node preparations from 59 dogs with generalized or multisystemic lymphoma. Lymph node samples from 11 healthy dogs were labeled to validate the specificity of antibodies and to formulate guidelines for interpretation of the results obtained from lymphoma samples. In normal lymph nodes, T-lymphocytes expressing CD3, CD4, or CD8 beta represented 59+/-11%, 43+/-8%, or 16+/-5% of the total cells, whereas B-lymphocytes expressing either CD21 or surface IgM (IgM) represented 37+/-9% or 14+/-5%, respectively. Small lymphocytes could be distinguished from large lymphocytes by forward light scatter. Of the patient samples 29 different breeds were represented with Golden and Labrador retriever being the most common. The lymphoma samples segregated into three groups based on CD antigen expression. Thirty cases predominantly expressed one or more combinations of CD79a, IgM, and CD21 representing a B-cell lineage. Three B-cell cases also expressed the stem cell antigen, CD34. Sixteen cases expressed one or more combinations of CD3, CD4, and CD8 consistent with a T-cell lineage and CD3+CD4+CD8--phenotype was the most common. Thirteen cases showed a mixed expression profile for T- and B-cell antigens and in three cases CD14 was highly expressed. Clinical response was poorest for T-cell lymphomas. Leukemic states occurred in all three phenotypes; but mixed cell cases had the greatest proportion. Dual immunofluorescence staining confirmed co-expression of T-cell (CD3) and B-cell antigens (CD79a or CD21) on neoplastic lymphocytes of six mixed cell cases. In one mixed cell case, dual immunostaining identified lymphocyte populations that stained mutually exclusive for CD79a and CD3. Six mixed cell lymphomas tested by PCR showed clonality for rearranged antigen receptor. Four cases that were CD79a+CD3+ had TCRgamma chain gene rearrangements, whereas two cases that were CD3+CD8+CD21+ had Ig heavy chain rearrangement. One case expressing multiple CD molecules (CD3+CD8+CD21+CD14+) was PCR negative for both Ig and TCRgamma gene rearrangement and could not be classified into a B- or T-cell lineage. We show for the first time co-expression of B- and T-cell markers on lymphoma cells that had specific T- or B-cell gene rearrangements. These findings suggest that aberrant CD molecule expression is not an uncommon finding in canine lymphomas and is a useful diagnostic marker for malignancy.

Modulation of bovine lymphocyte response by salivary gland extracts of the stable fly, Stomoxys calcitrans (Diptera: Muscidae).

The effect of salivary gland extract of the stable fly, Stomoxys calcitrans (L), on bovine lymphocyte proliferation was determined, and antibody reactivity to salivary gland proteins was characterized in cattle exposed to stable flies. Salivary glands were dissected from male and female flies (4-8 d after eclosion), and protein extracts were made by freeze-thaw cycles. Salivary gland extract (SGE, 1 and 5 microg) significantly inhibited mitogen-driven proliferation of bovine lymphocytes, compared with 1 and 5 microg of identically prepared midgut extract (ANOVA, P < 0.05). Phytohemagglutinin A (PHA) stimulated lymphocyte responses were suppressed by 61.7 and 79.5% (mean values) with 1 and 5 microg of SCE, whereas concanvalin A (Con A) stimulated responses were suppressed by 62.9 and 77.1% (1 and 5 microg). In contrast, midgut extract (1 and 5 microg) minimally suppressed PHA (12.7% +/- 12.6 and 18.7% +/- 15.5) and Con A-driven responses (13.8% +/- 20.5 and 24.6% +/- 14.9), respectively. Viability studies using propidium iodide and flow cytometry demonstrated that SGE was not cytotoxic. Two-color immunofluorescence studies identified T and B lymphocytes as the nonviable cells in the cultures. Western blot analysis of serum collected from five dairy cows during periods of low and high fly exposure identified an immunodominant 27 kDa protein among the salivary gland proteins. These results indicate that exposure of cattle to stable fly saliva during blood feeding results in an antibody response to salivary proteins and that the saliva has a potential to modulate T lymphocyte function.

Simultaneous flow cytometric analysis of phagocytosis and oxidative burst activity in equine leukocytes.

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.

Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia.

Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9-36.7%, but was eliminated by the use of the F(ab')2 fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs') test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals.

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.

Demodicosis in an American bison.

An 18-month-old, male American bison (Bison bison) was presented with 7- to 9-mm size nodules periorbital, perineal, and on the ventral surface of the tail. Demodex spp. were identified from the exudate by microscopic examination. Examination 6 mo later revealed that the infestation had nearly cleared without treatment.

Clinical and morphologic features of mucopolysaccharidosis type II in a dog: naturally occurring model of Hunter syndrome.

A 5-year-old male Labrador Retriever had progressive incoordination, visual impairment, and exercise intolerance. Coarse facial features, macrodactylia, unilateral corneal dystrophy, generalized osteopenia, progressive neurologic deterioration, and a positive urine spot test for acid mucopolysaccharides suggested mucopolysaccharidosis. Intracytoplasmic vacuoles were most prevalent in epithelial cells, endothelial cells, and histiocytes of liver, kidney, thyroid gland, and spleen. Ultrastructural examination disclosed electron-lucent floccular to lamellar membrane-bound storage material characteristic of mucopolysaccharides. Periodic acid-Schiff-positive intracytoplasmic material was identified in multiple neurons in the medulla, pontine nucleus, cerebellum, and spinal gray matter horns. Biochemical assays identified a deficiency in iduronate-2-sulfatase (IDS) activity in cultured dermal fibroblasts compared with normal dogs. Hair root analysis for IDS showed that the dam was a carrier of X-linked Hunter syndrome and that a phenotypically normal male littermate of the affected dog was normal. This is the first report of Hunter syndrome or mucopolysaccharidosis type II in a dog.

Immunopathology of chronic lentivirus-induced arthritis.

This study evaluated histopathology and mononuclear cell phenotypes in synovial lesions of chronic arthritis induced by experimental infection of Saanen goats with caprine arthritis-encephalitis lentivirus. Histological examination of carpal joint synovium of three infected goats with clinical arthritis revealed progressive lesions consisting of membrane villus hypertrophy with extensive angiogenesis and mononuclear cell infiltration and degenerative changes of membrane villus necrosis associated with loss of vasculature and infiltrates. Changes in synovial tissue of five age-matched infected goats without clinical arthritis were limited to moderate synovial membrane hyperplasia also noted in an age-matched uninfected goat. Immunohistochemistry identified CD45R+ CD5- B lymphocytes as the principal component of most perivascular infiltrates in arthritic synovium. Other mononuclear cells included perivascular CD4+ and CD8+ T lymphocytes and macrophages with a prominent accumulation of CD8+ T lymphocytes at the lining surface of inflamed villi. T lymphocytes and macrophages as well as synovial lining cells were activated with respect to MHC class II but not for interleukin-2 receptors. Inflamed villi also contained lymphoid aggregates comprised of B cell germinal centers and activated T-cell mantles. B cells expressing immunoglobulin occurred around follicles and throughout inflamed villi. These findings indicate that memory immune responses that favor expansion and maturation of B cells and immunoglobulin production contribute to the immunopathology of chronic arthritis.

Depressed CD4+ T lymphocyte proliferative response and enhanced antibody response to viral antigen in chronic lentivirus-induced arthritis.

The proliferative response of peripheral blood mononuclear cells (PBMC) stimulated with caprine arthritis-encephalitis virus (CAEV) surface glycoprotein (SU) was depressed 4- to 20-fold in Saanen goats with chronic CAEV-induced arthritis compared with asymptomatic goats. Phytohemagglutinin-stimulated responses were not depressed. Complement depletion of PBMC with anti-CD4 or anti-CD8 monoclonal antibodies identified CD4+ T lymphocytes as the antigen-responsive cells in both high-responder asymptomatic goats and low-responder arthritic goats. Serum antibody titers to CAEV SU were 8- to 32-fold higher in goats with chronic arthritis. Increased anti-CAEV SU titers as early as 3 months after infection predicted the eventual development of clinical arthritis. Thus, CAEV-induced arthritis is associated with chronic B cell activation resulting from dominant type 2 immune responses to viral antigen. The clinical outcome of CAEV infection may be determined by differential activation of type 1 or 2 T lymphocyte phenotypes at or near the time of initial exposure to CAEV.

Peripheral blood and synovial fluid mononuclear cell phenotypes in lentivirus induced arthritis.

OBJECTIVE: To identify the phenotypes and activation status of peripheral blood (PB) and synovial fluid (SF) mononuclear cells associated with caprine arthritis encephalitis virus (CAEV) induced arthritis in goats. METHODS: PB and SF mononuclear cells from 8 goats chronically infected with CAEV and 2 mock-infected goats were phenotyped by single immunofluorescence flow cytometry using a panel of monoclonal antibodies (Mab) to caprine leukocyte differentiation molecules. The activation status of mononuclear cell subsets was evaluated by dual immunofluorescence flow cytometry using Mab to major histocompatibility complex (MHC) class II molecules and the interleukin 2 receptor (IL-2R). RESULTS: Three CAEV infected goats had chronic progressive arthritis, clinically evident by periarticular swelling of carpal joints with excessive SF containing inflammatory cells and radiographic changes indicating soft tissue swelling and erosion of articular surfaces. The other 5 infected goats and 2 mock-infected control goats did not exhibit criteria of arthritis. The composition of PB mononuclear cells (11.3 +/- 5.0% monocytes, 21.1 +/- 4.7% B cells, 29.6 +/- 5.4% CD4+ cells, 15.2 +/- 6.1% CD8+ cells, and 9.7 +/- 3.9% gamma delta T cells) was not significantly different between mock-infected and CAEV infected goats. In contrast, cells within the SF of arthritic carpal joints consisted of 71.9 +/- 7.9% CD8+ T cells, 13.3 +/- 5.9% CD4+ T cells and < 2% B cells. The proportions of SF macrophages (7.8 +/- 5.2%) and gamma delta T cells (6.5 +/- 1.3%) were not significantly different from PB. All SF mononuclear cells were activated with respect to class II determinants orthologous to HLA-DR, DP and DQ. IL-2R expression by CD4+ and CD8+ T lymphocytes in SF was reduced compared to PB. CONCLUSION: Lymphocytes infiltrating the SF of arthritic carpal joints of CAEV infected goats consisted of a predominant subset of class II activated CD8+ T lymphocytes and a minority population of CD4+ T lymphocytes, both of which express very little IL-2R. These results parallel previous reports of the phenotypes and activation status of mononuclear cells in rheumatoid SF. A potential connection between T lymphocyte subset reactivity to CAEV and immunopathogenesis of arthritis is suggested.