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.

Pasteurella anatipestifer-like bacteria associated with respiratory disease in pigeons.

Two cases of respiratory disease in pigeons are described. The first case involved pneumonia and tracheitis, and the second case involved tracheitis. In both cases, unusual gram-negative, non-fermenting, short rod-shaped bacteria were recovered along with other microorganisms. The bacteria produced small, glistening, gray colonies on blood agar, did not grow on MacConkey agar, were unreactive on several biochemical tests, and resembled Pasteurella anatipestifer. Neither pigeon isolate was distinguished from P. anatipestifer by biochemical tests. However, there were morphologic and growth differences between the pigeon isolates and P. anatipestifer. Furthermore, unlike P. anatipestifer, both pigeon isolates were sensitive to aminoglycoside antibiotics and to polymyxin B. Finally, neither isolate was agglutinated by antisera to 15 serotypes of P. anatipestifer. Diagnosticians, especially those who seldom encounter P. anatipestifer, might have difficulty distinguishing the pigeon isolates from P. anatipestifer because of their close resemblance.

Heterophil chemotaxis in chickens with natural Staphylococcal infections.

Heterophil chemotaxis using heterophils isolated from the peripheral blood of five commercial broiler chickens naturally infected with staphylococcal bacteria was compared by the modified Boyden-chamber technique with chemotaxis of heterophils from two chickens from the same flock not infected with Staphylococcus (field controls) and from four healthy laboratory control broiler chickens. The infected chickens had gross and histologic lesions of staphylococcal tenosynovitis and osteomyelitis. Staphylococci were isolated from the lesions. Hematologic parameters and histologic lesions of infected chickens also were examined. Compared with field and laboratory controls, Staphylococcus-infected chickens had heterophilic leukocytosis. The heterophils of Staphylococcus-infected chickens had significantly lower chemotactic activity than both control groups in terms of random movement and directed chemotactic movement in response to stimulus. Toxic changes were observed in heterophils of some of the Staphylococcus-infected broilers.

Intestinal adenocarcinoma of the ileocecal junction in a chicken.

An 89-week-old male chicken was presented with signs of depression, emaciation, and weakness. At necropsy, a stricture was found at the ileocecal junction that resulted in blockage and dilation of the ileum proximal to the stricture. Histologically, neoplastic epithelial cells that contained mucin had invaded the intestinal wall and produced a fibrous connective tissue reaction. The lesion was diagnosed as scirrhous intestinal adenocarcinoma.

Polymerase chain reaction amplification of the genome of infectious bronchitis virus.

The polymerase chain reaction (PCR) was used to amplify a portion of the genome of infectious bronchitis virus (IBV). Two synthetic primers that annealed to segments of the IBV genome in the matrix and nucleocapsid genes facilitated PCR amplification of a 1020-base sequence. Amplification was successful using template DNA from an IBV sequence-containing plasmid and using copy DNA created by reverse transcription of IBV genomic RNA. The PCR product was the expected size and had the expected nucleotide sequence.

Differentiation of vaccine strains and Georgia field isolates of infectious laryngotracheitis virus by their restriction endonuclease fragment patterns.

Seven restriction endonucleases (REs) were used to cleave the DNA from seven vaccine strains of infectious laryngotracheitis (ILT) virus and from six Georgia field isolates of ILT virus. After electrophoresis of the resulting RE fragments, the patterns were compared in order to differentiate strains of ILT virus. The six chicken-embryo-origin (CEO) vaccines were identical with each RE, but the tissue-culture-origin (TCO) vaccine strain differed from the CEO vaccines using five of the REs. Four of the six field isolates were identical by each RE, but two field isolates differed from each other and from the four identical field isolates on the basis of patterns produced by some but not all of the REs. The four identical field isolates could not be differentiated from the CEO vaccine strains by any RE, but the other two field isolates were not identical to either strain of vaccine virus. This work demonstrates that differentiable strains of ILT virus exist in the United States and that viruses other than vaccine viruses are involved in field outbreaks of ILT.

Reproducibility of a virus-neutralization test for infectious laryngotracheitis virus.

A virus-neutralization test for infectious laryngotracheitis virus was performed in microtiter plates using standard techniques. To assess the reproducibility of the test, 11 sera were each titrated repeatedly once a week for 8 weeks, and the results were compared to a standard. The standard used for reproducibility was that the 95% logarithmic confidence intervals of the mean calculated from three titrations of the same serum had to be smaller than the logarithmic distance "within" two microtiter plate wells. For the virus neutralization test to give reproducible results, such confidence intervals had to fall "within" two wells at least 75% of the time. Over the 8 weeks, percent reproducibility varied from 43.5% to 81.5%. The infectious laryngotracheitis virus-neutralization test did not meet our defined standard of reproducibility with positive antisera. Results with negative control sera were reproducible, however. Percent reproducibility varied from 31.8% to 93.8% for different sera tested, but it was not related to the titer of the sera.

Studies of infectious laryngotracheitis vaccines: immunity in broilers.

Broiler chickens were vaccinated at 18 days of age against infectious laryngotracheitis (ILT) using chicken-embryo-origin (CEO) and tissue-culture-origin (TCO) vaccines, each vaccine given either by drinking water, spray, or eyedrop. Controls were not vaccinated. The broilers were challenged 3 weeks later with virulent ILT virus (USDA challenge strain). Serum samples taken before challenge were analyzed by a virus neutralization (VN) test to determine titers due to vaccination. Both vaccines, regardless of route of administration, produced low VN titers, geometric mean titer (GMT) being less than 4.0 in all vaccinated groups. When administered by the same route, the CEO vaccine produced higher titers than the TCO vaccine. Titers following drinking-water or eyedrop administration of vaccines were higher than titers following spray vaccination. There was an inverse relationship between pre-challenge VN titers of groups of birds and the percentage of birds in the groups dying from ILT virus challenge. The drinking-water route of vaccination provided the most protection, while the spray provided the least.

Studies of infectious laryngotracheitis vaccines: immunity in layers.

Ten-week-old layer chickens obtained from a commercial source were eye-drop vaccinated with chicken-embryo-origin (CEO) or tissue-culture-origin (TCO) vaccines for infectious laryngotracheitis (ILT). Controls were not vaccinated. Approximately one-third of the layers were challenged with virulent ILT virus at 21, 40, or 60 weeks of age. Serum samples taken from the layers before challenge were used in a virus neutralization (VN) test to determine vaccination titers at those three ages. Both vaccines induced low VN titers (geometric mean titer [GMT] less than 6). At 21 weeks of age, the titers produced by the two vaccines were not significantly different, but at 40 and 60 weeks of age the VN GMT of the CEO-vaccinated group was significantly greater than that of the TCO-vaccinated group. The VN GMTs did not drop over time in either group and actually rose between 21 and 60 weeks of age in the CEO group. Both vaccines protected layers against severe challenge with virulent ILT virus, neither being significantly better than the other under these experimental conditions. Unvaccinated sentinel chickens were maintained in contact with the vaccinated layers during three intervals between 1 day and 6 weeks post-vaccination. Diagnostic tests performed on the sentinels to detect lateral spread of vaccine virus from vaccinated to unvaccinated chickens showed scattered positive results.