Denise I. Bounous

Comparison of MTT Colorimetric Assay and Tritiated Thymidine Uptake for Lymphocyte Proliferation Assays Using Chicken Splenocytes

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.

A novel NOD-derived murine model of primary Sjögren's syndrome.

OBJECTIVE The appearance of autoimmune diabetes prior to autoimmune exocrinopathy in the NOD mouse suggests that it is an excellent model of secondary, but not primary, autoimmune sicca complications. Since the unique major histocompatibility complex (MHC) I-A(g7) expression in NOD mice is essential for the development of insulitis and diabetes in these animals, we investigated exocrine gland function in NOD.B10.H2b mice, which have an MHC congenic to NOD, as a potential model for primary Sjögrens syndrome (SS). METHODS Histopathologic manifestations of lymphocytic infiltrates into the pancreas and exocrine tissues were examined by light microscopy. Sera were evaluated for the presence of antinuclear antibodies. Saliva, tears, and gland lysates were evaluated for total volume and protein concentration, the aberrant expression and processing of parotid secretory protein, and cysteine protease activity. RESULTS NOD.B10.H2b mice exhibited the exocrine gland lymphocytic infiltration typical of the SS-like disease and dysfunction observed in NOD mice, but without the insulitis and diabetes. These mice additionally expressed elevated levels of cysteine protease activity (a measure of apoptotic activity) and abnormal expression and cleavage of parotid secretory protein in the submandibular tissues. CONCLUSION The results of this study suggest that the unique NOD MHC I-A(g7) is not essential for exocrine tissue autoimmunity. Furthermore, the findings indicate that sicca syndrome occurs independently of autoimmune diabetes and that the congenic NOD.B10.H2b mouse represents a novel murine model of primary SS.

The effect of dietary aflatoxin on wild turkey poults.

Aflatoxins, toxic metabolites of Aspergillus flavus or Aspergillus parasiticus, cause poor feed utilization, decreased weight gains, depressed immune function, liver dysfunction, coagulation abnormalities, and death in a wide variety of species including humans. Conservationists have become concerned that increasingly popular wildlife feeding or baiting practices could expose wildlife to toxic amounts of aflatoxin-contaminated grains. In particular, the effects of aflatoxins on the wild turkey (Meleagris gallopova silvestris) are of concern because the conspecific domestic turkey is highly susceptible to aflatoxins. To evaluate the effect of dietary aflatoxin on wild turkeys, four groups of 4-mo-old wild turkeys were fed diets containing either 0, 100, 200, or 400 μg aflatoxin/kg feed for 2 wk in September and October 1996. Aflatoxin-fed poults had decreased feed consumption and weight gains as compared with control poults. Decreased liver-to-body weight ratios, liver enzyme alterations, slightly altered blood coagulation patterns, and mild histologic changes indicated low-level liver damage. Compromise of cell-mediated immunity was indicated by decreased lymphoblast transformation. The effects were apparent in all treatment groups to variable levels, but significant differences most often were found at 400 μg aflatoxin/kg feed. This study shows that short-term aflatoxin ingestion by wild turkeys can induce undesirable physiologic changes; therefore, exposure of wild turkeys to feeds containing aflatoxin levels of 100 μg aflatoxin/kg feed or more should be avoided.

Normal hematologic and serum biochemical reference intervals for juvenile wild turkeys.

Blood samples taken from 48 4-mo-old wild turkeys (Meleagris gallopova silvestris) were used to establish reference intervals for hematology and serum chemistry values. The study was conducted during September and October 1996. Packed cell volume, total and differential white cell counts, total protein, albumin, glucose, calcium, uric acid, triglyceride concentrations, as well as aspartate transaminase (AST) and lactate dehydrogenase (LDH) activities were assayed. Reference intervals from wild turkeys are similar to those reported for domestic turkeys.

Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens

N2a and P2a chickens, resistant and susceptible to Mareks disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Mareks disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.

Immunosuppression and Intracellular Calcium Signaling in Splenocytes from Chicks Infected with Chicken Anemia Virus, CL-1 Isolate

Hematocrits, histopathology, concanavalin A-induced lymphocyte proliferation, intracellular calcium signaling, and lymphocyte subpopulations were analyzed over a 6-week period in individual chicks inoculated with the CL-1 isolate of chicken anemia virus. Lymphoid depletion/atrophy was present in the thymus and bone marrow by 11 days post-infection (PI). Anemia was present at 14 days PI. The mean lymphocyte proliferation stimulation index (SI) of the inoculated group was significantly lower than that of the control group at 11 days PI. This response was reversed at 18 days PI, when the SI of the inoculated group was significantly higher than that of the controls; values subsequently returned to baseline. The increase in intracellular calcium levels in CAV-infected chicks and controls paralleled the proliferative response. Percentages of CD3-, CD4-, CD8-, and natural-killer-positive-staining cells decreased significantly at 18 and 25 days PI. The most dramatic decrease occurred in the CD8-positive-staining cell population at 18 and 25 days PI.

Lack of Correlation Between Microscopic Lesion Scores and Gross Lesion Scores in Commercially Grown Broilers Examined for Small Intestinal Eimeria spp. Coccidiosis

Comparisons were made between microscopic lesion scores (MLSs) and gross lesion scores (GLSs) in sections from small intestine of broilers during three routine coccidiosis screenings. The duodenal and jejunal GLS were determined and recorded by different evaluators. During each screening, 2-cm sections of duodenum and jejunum were collected, and sections of intestine were then scored using a microscopic lesion scoring system. No correlation between MLS and GLS was observed in duodenum in two out of three coccidiosis screenings, and no correlation was observed between MLS and GLS in jejunum in three out of three screenings. Our findings demonstrate that, if used alone in coccidiosis screening, GLSs can underestimate infections and may not provide a true representation of the magnitude of Eimeria maxima infection within broiler flocks.

Detection of equine and bovine T- and B-lymphocytes in formalin-fixed paraffin-embedded tissues

Formalin-fixed paraffin-embedded sections of equine and bovine lymph nodes, spleen, thymus, and Peyers patches were incubated with monoclonal antibodies to B-lymphocyte markers BLA.36, B29, and mb-1 and T-lymphocyte markers CD3 and CD5. The monoclonal antibody BLA.36 reacted with 80-90% of lymphocytes in the germinal centers and mantle zones of follicles in lymph nodes, spleen, and Peyers patches. In addition, 90% of lymphocytes in the marginal zone of the spleen, and variable numbers of lymphocytes within lymph node medullary cords were immunopositive for BLA.36. Antibodies to B29 and mb-1 produced similar staining patterns as BLA.36 with fewer positive cells in the germinal centers and medullary cords. BLA.36, B29, and mb-1 reacted with 30-50% of lymphocytes in the medulla of the thymus and with 5-10% of lymphocytes in the cortex. CD3 and CD5 reacted with 90% of lymphocytes in the paracortex and parafollicular zones of lymph nodes, spleen, and Peyers patches; 40-50% of lymphocytes in the medullary cords of lymph nodes, and scattered positive cells within follicles. Anti-CD3 antibody reacted with 95% of lymphocytes in the splenic red pulp, but antibodies directed against CD5 reacted only faintly with approximately 5-10% of lymphocytes in the red pulp. CD3 and CD5 reacted with 50-60% of cells in the medulla of the thymus and with 40-80% of lymphocytes in the thymic cortex. The biochemical characterization of the antibodies by Western blotting against lysates of equine and bovine peripheral blood mononuclear cells confirmed that antibodies to BLA.36, mb-1, B29, CD3, and CD5 detected molecules of the same approximate molecular mass as found on lymphoid cells of human beings and rats.

Infiltrating Lymphocyte Populations and Cytokine Production in the Salivary and Lacrimal Glands of Autoimmune NOD Mice

It is only in the last five years that significant interest has developed in detailing the autoimmune destruction of the salivary and lacrimal tissues in the non-obese diabetic (NOD) mouse. Pioneering studies have demonstrated that the lymphocytic infiltration of the salivary and lacrimal glands correlates with a functional decline in saliva flow and tear production, respectively, that is not attributable to the loss of blood glucose regulation in NOD mice.1,2 Together, these studies indicate that the NOD mouse represents the first-described animal model for the spontaneous, autoimmune-induced loss of both tear production and saliva flow, and is, therefore, emerging as an excellent animal model for the study of human Sjogren’s syndrome.

Viral proventriculitis in chickens

The objectives of the present study were to examine proventriculi of broiler chicks for lesions, and to classify and record the incidence of these lesions. Deep non-purulent necrotizing proventriculitis (accompanied by adenoepithelial hypertrophy and hyperplasia) was the most common (109/220 = 49.5%) light microscopic diagnosis in the proventriculi examined. Degenerating and necrotic alveolar secretory cells had amorphous, granular or vacuolated cytoplasm. Nuclei usually were either pyknotic, karyorrhectic or karyolytic; however, fewer attached or sloughed cells had swollen nuclei with marginated chromatin and clear centres. Basophilic inclusion bodies were never seen. In five cases examined ultrastructurally, hexagonal virus particles were found in intact nuclei (average size = 68.9 nm, n = 89), associated with unbound condensed chromatin, and within vacuolated spaces in the cytoplasm (average size = 62.3 nm, n = 109). DNA in situ hybridization failed to detect adenovirus or polyomavirus nucleic acids. The presence of intralesional virus suggests that a causal relationship might exist between the virus and the proventricular lesions.