V. Sivanandan

Isolation of H13N2 influenza A virus from turkeys and surface water

This is the first report of the isolation of H13N2 avian influenza virus (AIV) subtype from domestic turkeys. This subtype was also isolated from nearby surface water. The observation of large numbers of gulls in close association with turkeys on range before the virus isolations suggests that this virus subtype was transmitted from gulls to range turkeys. Turkey flocks infected by this virus subtype did not show any clinical signs of the disease, although seroconversion did occur. The H13N2 isolates were found to be non-pathogenic in chickens.

Biological and molecular characterization of H13N2 influenza type A viruses isolated from turkeys and surface water.

The pathogenicity potential of two H13N2 influenza viruses, one isolated from turkeys and the other isolated from surface water, was evaluated in turkeys, chickens, and mallard ducks (Anas platyrhynchos) after intracranial and oculonasal inoculation. Both isolates replicated in turkey poults, causing depressed weight gain, morbidity and mortality; both also caused histopathological lesions, such as mild to severe pancreatitis, hepatitis, and nephritis in turkeys. These isolates replicated in mallard ducklings but not in chickens. There was depressed weight gain in ducklings given the H13N2 isolate from water. Neither isolate caused morbidity or mortality in ducklings or chicks after inoculation.

Protection and Immunity in Commercial Chicken Layers Administered Mycoplasma gallisepticum Liposomal Bacterins

Six liposomal Mycoplasma gallisepticum (MG) bacterins, differing in charge and size, and two oil-emulsion vaccines (sonicated and non-sonicated) were given to white leghorns in two doses, at 13 weeks and again 1 month later. At 21 weeks of age, all chickens were challenged with a viable 20-hour culture of MG cells (17,800 colony-forming units) intratracheally and with nonviable MG organisms (0.09 mg protein) injected subcutaneously in the wattle center. The three chicken groups that had the lowest tracheal MG-infection rates postchallenge were those given adjuvants of small multilamellar positively charged liposomes (16.67%), large multilamellar negatively charged liposomes (16.67%), and non-sonicated oil-emulsion bacterin (37.5%). These three groups also had significant levels of antibody in sera 4 weeks after the second dose of vaccine. The group given the small multilamellar positively charged liposome also showed significant delayed-type hypersensitivity (wattle swelling) (P less than or equal to 0.05). The group given the large multilamellar negatively charged liposomes had the highest local antibody response (P less than or equal to 0.01) and was the only group that had no microscopic lesions in the trachea.

A lipid-conjugated immunostimulating complex subunit vaccine against Salmonella infection in turkeys.

Immunostaining complexes (ISCOMs) are multimeric particles and have been used successfully for presentation of membrane proteins. In this study, outer-membrane proteins (OMPs) from Salmonella heidelberg were incorporated into lipid-conjugated ISCOM particles and evaluated for their use in a vaccine for turkeys against homologous and heterologous Salmonella challenge. Two types of lipid-conjugated ISCOMs were examined: ISCOM-phospholipid and ISCOM-sphingolipid preparations. The turkeys were challenged with one of the three Salmonella serotypes: S. heidelberg, S. reading, or S. enteritidis. The turkeys were monitored for clinical signs, shedding pattern post-challenge, and clearance of the challenge Salmonella from selected internal organs. Vaccines containing OMP with either lipid-conjugated ISCOM preparation produced significantly greater (P < 0.01) immune response than OMP alone. Cloacal swabs from turkeys given OMP along with ISCOM-phospholipid and challenged with a homologous serotype were completely negative for Salmonella. A certain degree of cross-protection against heterologous Salmonella was afforded by both OMP-ISCOM vaccines. The isolation rate of Salmonella from internal organs was significantly lower (P < 0.0001) in vaccinated turkeys than in unvaccinated controls.

Enhancement of antibody response of turkeys to trivalent avian influenza vaccine by positively charged liposomal avridine adjuvant

Trivalent avian influenza (AIV) antigens (H4N8, H5N2 and H7N3), mixed with positively charged, negatively charged and neutral avridine-containing liposomes, and oil-emulsion were subcutaneously administered to 6-week-old turkeys. Charged liposomal avridine adjuvant, either positive or negative, produced a better antibody response than uncharged liposomal avridine or oil-emulsion adjuvants when used in a trivalent avian influenza vaccine. The antibody response to the different antigens was generally greater to the positively charged adjuvanted vaccine compared with the negatively or neutral charged or oil-emulsion adjuvanted vaccines and these differences were significant (P less than 0.05) with the three antigens. The results suggest that the positively charged liposomal avridine plays a significant role as adjuvant to the AIV antigens.

Comparative Serological Evaluation of Avian Influenza Vaccine in Turkeys

Four- and six-week-old turkeys were vaccinated subcutaneously using avian influenza virus (AIV) A/Duck/613/MN/79 (H4N2) killed oil-emulsion vaccine. Sequential serological tests using agar gel precipitin (AGP), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA) for measuring antibodies to AIV were performed up to 4 weeks postvaccination, when birds were challenged intranasally using A/Turkey/MN/80 (H4N2) live AIV. The ELISA was 25 to 1600 times more sensitive than the HI test and was able to detect antibody production earlier than the HI test. All turkeys with an ELISA titer of greater than or equal to 800 were protected against homologous challenge, as measured by virus recovery 3 days postchallenge. Four turkeys out of 20 serologically negative by AGP and HI tests but ELISA-positive were protected.

Efficacy of Combined Killed-In-Oil Emulsion and Live Newcastle Disease Vaccines in Chickens

Following the introduction of routine vaccination regimes with different types of Newcastle disease (ND) vaccines, the incidence of velogenic viscerotropic Newcastle disease (VVND) in commercial poultry worldwide has declined dramatically. Unfortunately, these vaccination regimes are not feasible in free-range and backyard systems of poultry production practiced in many developing countries. In this study, we sought to develop a single vaccination regime in chickens with ND vaccines to elicit a long-lasting high level of ND virus (NDV) antibodies adequate to protect chickens against ND. The level of antibody response, as measured by the hemagglutination-inhibition (HI) test, and the degree of protection against the virulent strain of NDV were studied in chickens immunized with different vaccines. The vaccines used were: killed-in-oil emulsion (subcutaneous; s.c.) plus live virus (oculanasal; o.n.), given concurrently; experimental vaccine (s.c.) plus live virus (o.n.), given concurrently; killed-in-oil (s.c.); experimental vaccine prepared by homogenizing commercial live vaccine and oil emulsion (s.c.); and live virus (o.n.). The results obtained in this study indicate that concurrent administration of oil emulsion and live NDV vaccines induced the best antibody response, but there was no significant difference in protection among the vaccinated groups.

Effects of Avian Infectious Bronchitis Virus (Arkansas Strain) on Vaccinated Laying Chickens

Twenty-four-week-old white leghorn layers were inoculated subcutaneously with a killed Newcastle-infectious bronchitis (Massachusetts type) virus (MIBV) vaccine. The birds were challenged 194 days later intraocularly with Arkansas strain of infectious bronchitis virus (AIBV). The challenged hens laid significantly (P less than 0.005) fewer eggs than the unchallenged layers, and the eggs laid by the challenged groups weighed significantly less (P less than 0.001) than those laid by the unchallenged groups. Further, the internal quality (Haugh units) and shell quality of eggs laid by the challenged hens were significantly (P less than 0.005) inferior to the quality of eggs from unchallenged hens, and the challenged hens laid more soft-shelled, misshapen, and small-sized eggs than the unchallenged hens. The Arkansas serum hemagglutination-inhibition (AIBV-HI) titers of challenged birds increased continuously through 29 days post-challenge. The MIBV hemagglutination-inhibition (MIBV-HI) titers of killed-MIBV-vaccinated birds decreased during the same period. The study indicates that killed MIBV vaccine offered no protection to birds exposed to AIBV. The same vaccine was quite effective against a homologous (MIBV) virus challenge.

Effect of avian influenza virus infection on the phagocytic function of systemic phagocytes and pulmonary macrophages of turkeys.

The effects of avian influenza virus (AIV) infection on systemic phagocytes and pulmonary macrophages of turkeys were studied. There was a significant increase (P < 0.0001) in oxidative burst in systemic phagocytes of AIV-inoculated turkeys on 2, 4, 6, and 8 days postinoculation (PI), as measured by chemiluminescence. There was also a significant increase (P < 0.02) in oxidative burst in pulmonary macrophages on day 4 PI. The chemiluminescence response was depressed on 6, 8, and 10 days PI in AIV-inoculated turkeys compared with controls. The increase in oxidative response in both systemic phagocytes and pulmonary macrophages correlated with the peak virus titer in the lungs and trachea of AIV-inoculated inoculated turkeys. Bacterial killing by pulmonary macrophages from AIV-inoculated turkeys was reduced on days 6 and 10 PI compared with uninoculated controls. Histopathological changes in trachea were more pronounced on day 6 PI in AIV-inoculated turkeys; no significant changes were detected in the lungs. These data indicate that compromised functional capacity of pulmonary macrophages predisposes turkeys to secondary bacterial infections.

Endotoxin Lipopolysaccharide from Escherichia coli and its Effects on the Phagocytic Function of Systemic and Pulmonary Macrophages in Turkeys

The effect of Escherichia coli lipopolysaccharide (LPS) on the competence of pulmonary macrophages and phagocytic cells from the systemic circulation of turkeys was examined using luminol-enhanced zymosan-stimulated chemiluminescence. The results showed a rapid and accelerated oxidative burst in both systemic and pulmonary macrophages in LPS-treated turkeys that was significantly greater than in untreated controls. However, this increased oxidative metabolism induced by LPS was not associated with enhanced intracellular bacterial killing by pulmonary macrophages. Turkeys treated with LPS showed a highly significant decrease in pulmonary bactericidal activity against Staphylococcus aureus challenge, indicating a defect in pulmonary macrophage function induced by LPS.