Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases.

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.

Comparison of the effect of different opsonins on the phagocytosis of fluorescein-labeled staphylococcal bacteria by chicken heterophils.

Heterophil phagocytosis of fluorescein-labeled staphylococcal bacteria was analyzed by flow cytometry. Opsonization with two types of normal pooled sera and staphylococcal antisera significantly increased bacterial phagocytosis compared to samples without an opsonin. The staphylococcal antisera did not significantly increase bacterial phagocytosis compared to the normal pooled sera. Opsonization appears to increase bacterial phagocytosis but specific antisera may not increase phagocytosis beyond that caused by pooled normal sera.

The effects of haemolysis on serum chemistry measurements in poultry.

The effects of haemolysis on serum chemistry values in broiler and layer chickens, and in turkeys were determined using two types of serum chemistry analysers, a wet reagent analyser and a dry slide reagent analyser. The interfering effects of haemolysis were evaluated using eight levels of haemoglobin in serum analysed by the wet reagent instrument and six levels of haemoglobin in serum analysed by the dry slide reagent instrument. Nine serum chemistry analytical tests were performed on each analyser, including determination of glucose, total protein, albumin, creatine kinase, gamma-glutamyl transferase, aspartate aminotransferase, calcium, phosphorus and uric acid. The interfering effects of haemolysis varied depending on type of analyser, type of bird and the specific test. With the wet reagent chemistry analyser, the gamma-glutamyl transferase, phosphorus and uric acid analytes were most sensitive to haemoglobin interference, and the albumin, total protein and creatine kinase analytes were most resistant. With the dry slide reagent analyser, the gamma-glutamyl transferase, phosphorus, and albumin analytes were most sensitive to haemoglobin interference, and the glucose and aspartate aminotransferase analytes were most resistant. The effects of haemoglobin interference were not consistent from one type of chemistry analyser to another. The dry slide reagent analyser did not appear to resist the effects of haemoglobin interference better than the wet reagent analyser in this study. Our results suggest the need to construct interferographs for each chemistry analyser, species, and type of bird.

Comparison of diagnostic tests for infectious laryngotracheitis.

Diagnostic procedures for detection of infectious laryngotracheitis virus in tracheas of experimentally infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, polymerase chain reaction (PCR), and DNA hybridization, were performed and compared. Using VI as a reference, we calculated the sensitivity and specificity of the tests. The sensitivities of IP, IFAT, histopathology, PCR, and hybridization were 100%, 93%, 7%, 27%, and 0%, respectively, and the specificities of IP, IFAT, histopathology, PCR, and hybridization were 93%, 93%, 100%, 100%, and 100%, respectively. Histopathology, PCR, and hybridization were more specific but lacked sensitivity compared to IP and IFAT. IP and IFAT were equally specific, but IP was more sensitive than IFAT. Based on these results, IP performed better than any other test.

Characterization of monoclonal antibodies against infections laryngotracheitis virus.

Nine monoclonal antibodies (MCAs) produced against two different strains of infectious laryngotracheitis virus (ILTV) were characterized and compared to previously characterized MCA 131-6, produced against a third ILTV strain. In western blotting experiments, MCAs C, E, and 11 resembled MCA 131-6, detecting proteins of 205, 160, 115, and 90 kD as well as several proteins less than 49 kD. The other six MCAs differed from previously described ILTV MCA. MCA D detected a 90-kD protein along with several less than 49 kD. MCAs 4 and 5 each detected proteins of 205, 160, 100, 90, and 70 kD. MCA 9 detected the same proteins detected by MCAs 4 and 5 except the 160-kD protein. MCA 10 detected proteins of 100, 90, and 70 kD and several proteins less than 49 kD. MCAs C, D, and E, like MCA 131-6, failed to react with any ILTV grown in the presence of tunicamycin, suggesting that those MCAs are specific for carbohydrate-based epitopes. MCA 6 reacted with only a 100-kD protein in the presence or absence of tunicamycin. The remaining MCA detected only a 70-kD protein in the presence of tunicamycin except MCA 5, which reacted with proteins of 70 and 90 kD. Only MCA 4 and 6 neutralized ILTV infectivity.

Development of a polymerase chain reaction and a nonradioactive DNA probe for infectious laryngotracheitis virus.

The polymerase chain reaction (PCR) was developed using infectious laryngotracheitis virus (ILTV) primers made from a portion of the ILTV thymidine kinase gene. DNA from various ILTV field isolates, from the USDA challenge strain of ILTV, and from commercial ILTV vaccines was specifically amplified. No amplification occurred using template DNA from uninfected chicken-embryo liver cells (CELC), several nonavian alphaher-pesviruses, Mycoplasma gallisepticum, Mycoplasma synoviae, Pasteurella hemolytica, Escherichia coli, a group I avian adenovirus, fowl poxvirus, or a psittacid herpesvirus. The 647-base pair-amplified ILTV PCR product was labeled to create a nonradioactive, biotinylated DNA probe. Hybridization using the probe detected ILTV DNA. Both PCR and hybridization yielded positive results with ILTV DNA but not with the DNA of other pathogens. Hybridization was specific for ILTV using a stringent salt solution for a 30-min wash step or a somewhat less stringent salt solution for a 60-min wash step. However, slight hybridization occurred with CELC DNA when the less stringent salt solution was used in a 30-min wash step.

Salinomycin toxicosis in male breeder turkeys.

A sudden outbreak of mortality in one house of 600 48-week-old male breeder turkeys on a five-house turkey breeder farm was suspected to be feed-related. The turkeys gasped and became recumbent; 21.7% of affected turkeys died. No significant gross lesions were found at necropsy. Histological lesions, limited to skeletal muscle, consisted of degeneration and necrosis and were judged compatible with ionophore toxicosis. Feed samples from the affected house were analyzed by three techniques and shown to contain 13.4 to 18.4 g of salinomycin per ton of feed. An error at the feed mill was blamed for allowing contamination of the turkey feed with broiler starter feed containing salinomycin.

Chicken heterophil chemotaxis using Staphylococcus-generated chemoattractants.

Heterophil chemotaxis, in response to chemotactic factors generated by three different strains of staphylococcal bacteria, was measured using the modified Boyden-chamber technique. Heterophils were obtained from healthy 6-to-8-week-old broiler chickens. Each bacterial strain generated factors that were chemotactic for chicken heterophils. Factors generated by two pathogenic isolates of Staphylococcus aureus, however, induced significantly greater chemotaxis in chicken heterophils than those generated by a nonpathogenic Staphylococcus isolate.