Isolation, characterization and in vitro mitogenic stimulation of peripheral blood lymphocytes from an American buffalo.

A rapid and reproducible method is described for the isolation and characterization of leukocytes from the peripheral blood of an American buffalo (bison). Centrifugation of the buffy coat cells on a Percoll gradient (1.079 g/mL) at 650 x g for 20 min resulted in the separation and high yields of pure viable leukocytes. The sheep erythrocyte-rosetting technique (ER) showed that 59% of the cells were ER+ (T lymphocytes). Fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin and FITC-conjugated concanavalin A revealed 77% and 89% positive cells, respectively. The isolated leukocytes contained adherent accessory cells and functionally active T and B lymphocytes which proliferated in response to both T and B cell mitogens and to exogenous recombinant bovine interleukin-2 in the absence and/or presence of the thiol compound 2-mercaptoethanol.     

Peanut agglutinin (PNA): binding and stimulation of bovine intestinal and peripheral blood leukocytes

The use of peanut agglutinin (PNA) as a reliable marker for bovine T lymphocytes as well as its in vitro lymphoblastogenic capacity were investigated and compared to those of concanavalin-A (ConA). The binding ability of fluorescein isothiocyanate conjugated PNA (FITC-PNA) and FITC-ConA to bovine leukocytes isolated from peripheral blood (PBL) as well as from the intraepithelium (IEL), lamina propria (LPL) and Peyer's patches (PPL) of the small intestinal mucosa of five normal adult cows (n = 5) was analyzed using laser flow cytometry (LFC) and fluorescence microscopy. Monoclonal antibodies (mAb) specific for bovine T cells (B26A), B cells (PIg45A), "null" cells (B7A1) and monocytes/granulocytes (DH59B) were employed to determine the phenotype of the cell lineage(s) expressing PNA surface receptor(s). There were no significant variations (P greater than 0.1) in the proportion of PNA-binding cells in PBL (76%), PPL (77%), IEL (79%) and LPL (81%) even though there were significant differences between the percentages of B26A+ T cells in IEL (26%) and LPL (38%) (P less than 0.001) and in PPL (44%) and PBL (57%) (P less than 0.01). These studies clearly indicate that cells other than T cells bind PNA. Although the proportions of PNA-binding cells in enriched PP-B cells (30%) and enriched PP-plastic adherent cells (44%) were significantly lower (P less than 0.001) than those in enriched PP-T cells (95%), the results indicated that a reasonable number of non-T cells can specifically bind FITC-PNA. Additional support was obtained by similar results observed with the equivalent cell subsets from PBL. Using in vitro lymphoblastogenesis, the PNA stimulating capacity significantly varied between the various cell populations (P less than 0.001 between IEL and PBL; and P less than 0.02 between PPL and PBL). In addition, marked differences were observed between the binding ability and stimulating capacity of PNA on each leukocyte population (P less than 0.01 in PBL to P less than 0.001 in IEL). Concanavalin A which bound to approximately 100% of each cell population, revealed significant variation in its mitogenic activity between IEL and PBL (P less than 0.001) but not between LPL and PPL (P greater than 0.1). The finding that PNA can bind to bovine T cells as well as to some B cells, monocytes/macrophages and possibly some granulocytes and "null" cells disputes its reliability as a specific bovine T cell marker. Furthermore, the binding abilities of PNA and ConA to bovine leukocytes are not necessarily correlated to their in vitro mitogenic capacities.     

Recombinant human interleukin-2-induced mitogenic proliferation of in vitro unstimulated bovine intestinal lymphocytes.

Recombinant human interleukin-2 (rHIL-2) in the absence or presence of additional stimuli, was able to induce and support the proliferation of lymphocytes isolated from the intra-epithelium, lamina propria and Peyer's patches of the small intestine of normal adult cows. Although dose-dependent effects of rHIL-2 were observed with all three cell populations, concentrations as low as 2.5 U/mL were able to induce DNA synthesis as measured by tritiated thymidine incorporation. Furthermore, rHIL-2 as low as 5.0 U/mL was shown to significantly enhance lymphocyte proliferation in response to mitogenic stimulation. These proliferative responses to rHIL-2 were detected within two days of culture and peaked after five days. Although the extent of the blastogenic response was variable in individual animals, the general pattern of time-course and dose-response to rHIL-2 was similar in all animals tested. The response of all three leukocyte populations to rHIL-2 was dependent on the presence of adherent accessory cells and/or 2-mercaptoethanol. Both nylon wool nonadherent (T cells, null cells) and adherent cells (B cells) were shown to be responsive to rHIL-2. These studies demonstrate that bovine lymphocytes isolated from different anatomical locations of the small intestine are capable of proliferation in response to xenogenic IL-2 without in vitro preactivation signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Molecular investigation of foot-and-mouth disease virus in domestic bovids from Gharbia,Egypt

An outbreak of foot-and-mouth disease (FMD) affecting cattle and water buffalo (Bubalus bubalis) occurred in Egypt during 2012/2013. The present study was undertaken to determine the current strains of the FMD virus (FMDV) and the prevalence of FMD among cattle and buffalo in Gharbia, Egypt. The diagnostic sensitivity of two RT-PCR assays for the detection of FMDV was evaluated. The results revealed that SAT2 was the causative agent. The percentage of infected of animals varied with the detection method, ranging from 62.5 % by the untranslated region (UTR) RT-PCR to 75.6 % by SAT2 RT-PCR. The overall prevalence and mortality rates were 100 and 21 %, respectively. The mortality was higher in buffalo (23.3 %) than it was in cattle (17 %). A partial sequence of SAT2 was identical (90–100 %) to Egyptian isolates and was close in similarity to sequences from Sudan and Libya. In conclusion, FMD in Egypt is caused by SAT2. No other serotypes were detected. The results of this study provided the valuable data regarding the epidemiology of SAT2 in cattle and water buffalo from Egypt, which strengthens the need to change the strategies of both control and prevention that help to prevent the spread of the disease.     

A study on the nutritional status of adolescent girls

One hundred and twenty school girls in the age group of 13–15 years from four Government schools of Ludhiana city were selected for the study. On the basis of their family income they were divided into three experimental groups i.e. income group I (IgI), income group II (IgII) and income group III (IgIII). The results showed that the intake was low for all the foods. However, the consumption of fruits, milk and milk-products, sugar and jaggery, fats and oils by the subjects of IgII and IgIII was significantly higher (p<0.05) than IgI. The mean daily intake of energy, protein, iron, calcium, vitamin A & vitamin C was inadequate while the intake of fibre was adequate by the subjects as compared to ICMR recommendations. There was no significant difference in energy, protein and iron intakes among the subjects of three groups. However, the fibre intake by the subjects of IgI was significantly higher (p<0.05) than the subjects of IgIII, whereas the intake of calcium, vitamin A and ascorbic acid by the subjects of IgII & IgIII was significantly higher (p<0.05) than those of IgI. The average body weights and heights of the subjects were normal. The haemoglobin (Hb) level of the subjects ranged from 8.5–12.5 g/dl with a mean value of 10.73+0.07 and reported that only 23 percent of the subjects had acceptable level.