Jennifer J. York

Development of T cell immune responsiveness in the chicken

Chickens are highly susceptible to infection by opportunistic pathogens during the first few days after hatching. This observation has generally been attributed to an immaturity of the immune system; however, the mechanisms responsible are not known.

Laryngotracheitis herpesvirus infection in the chicken: The role of humoral antibody in immunity to a graded challenge infection

The clinical responses of SPF White Leghorn chickens to graded levels of infection with virulent (wild-strain) infectious laryngotracheitis (ILT) herpesvirus, administered by the tracheal route, were investigated in chickens from 1 day to 8 weeks of age. In 1-day-old chickens 40 plaque forming units (PFU) of ILT virus caused 55% mortality within 8 days. At least 500 PFU was needed to achieve comparable mortality at 3 weeks of age and this increased to 4,500 PFU of ILT virus by 6 to 8 weeks of age. A combination of surgical bursectomy at 1 day old and cyclophosphamide treatment ablated the ability of chickens to generate humoral antibody to ILT virus, but did not impair the level of protection induced by commercial ILT vaccine. Further, the passive transfer to ILT antibody-positive serum to 2-day-old or 4-week-old chickens did not significantly alter their susceptibility to tracheal challenge with virulent virus. Serum antibody was therefore discounted as a major immune mechanism in resistance to ILT virus infection. An experimental inactivated vaccine to ILT virus was also investigated. One intramuscular injection induced low serological responses, but no significant protection to ILT. A second injection of inactivated vaccine only marginally increased the titre of humoral antibody, but seemed to reduce the degree of respiratory distress. However, the levels of protection afforded by the inactivated vaccine were not significant compared with a live commercial ILT vaccine.

The role of mucosal antibody in immunity to infectious laryngotracheitis virus in chickens

The role of mucosal antibody in recovery from a primary infection and resistance to reinfection with infectious laryngotracheitis (ILT) herpesvirus was studied in bursectomized chickens, which were unable to synthesize specific antibodies. Viral antigen in the infected trachea was assessed by indirect immunofluorescence on tissue sections and by ELISA. The ability of bursectomized chickens to resolve primary infections as effectively as intact chickens and of vaccinated-bursectomized chickens to prevent the replication of challenge virus without the participation of mucosal antibody, is evidence for the importance of local cell-mediated rather than humoral immune mechanisms in the outcome of infection with ILT virus.

Laryngotracheitis herpesvirus infection in the chicken. II. The adoptive transfer of resistance with immune spleen cells

Protection against virulent infectious laryngotracheitis (ILT) virus was successfully transferred between inbred white leghorn chickens with spleen cells or peripheral blood leukocytes from immune cock birds. Resistance to infection could be demonstrated 7 to 8 days, but not 2 days after cell-transfer. Both hyperimmune spleen cells and memory spleen cells conferred resistance to infection, while the transfer of non-immune spleen cells failed to protect the chickens. Thymocytes or bursal cells from immune donors also failed to confer protection. The majority of cyclophosphamide pretreated recipients of immune spleen cells survived the challenge with ILT virus without synthesising detectable levels of humoral antibody. These findings indicate that cell-mediated immune mechanisms are involved in vaccine-induced immunity to acute ILT infections.

Diagnosis of infectious laryngotracheitis using a monoclonal antibody ELISA

An ELISA has been developed which uses a selected monoclonal antibody specific for ILT virus. The ELISA proved to be as accurate as, yet faster than, virus isolation, more accurate than the fluorescent antibody test and more accurate and rapid than the relatively simple agar gel precipitin test. The ELISA clearly differentiated between chickens from commercial flocks infected with ILT virus and non-infected chickens, or chickens infected with other respiratory pathogens.

Immunogenic glycoproteins of infectious laryngotracheitis herpesvirus

The viral glycoproteins produced in cells infected with either vaccine strain or virulent isolates of infectious laryngotracheitis virus, an avian herpesvirus, were identified by in vitro labeling using [14C]glucosamine and [14C]mannose. Chicken antisera to the vaccine strain and to a virulent isolate, and rabbit antisera to the vaccine strain, immunoprecipitated four major viral glycoproteins of 205, 115, 90, and 60K mol wt. Additional glycoprotein bands were recognized by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus-infected cells. Monoclonal antibodies to the immunogenic glycoproteins were produced and characterized by immunoprecipitation and Western blotting. One group of monoclonal antibodies reacted only with the 60K glycoprotein, by both techniques, while a second group reacted with the 205, 115, and 90K glycoproteins in immunoprecipitation and with additional bands of 85 and 160K in Western blotting.

Humoral and cell-mediated immune responses to the glycoproteins of infectious laryngotracheitis herpesvirus.

SummaryThe viral glycoproteins of infectious laryngotracheitis virus, an alphaherpesvirus, were the dominant antigens recognised by immune chickens. Glycoproteins with molecular weights of 205, 160, 115, 90, 67, 60, and 52 k reacted strongly in Western blotting studies with a majority of chicken antisera. Viral glycoproteins immunoprecipitated using monoclonal antibodies were also able to elicit a delayed-type hypersensitivity reaction in chickens previously vaccinated with a live vaccine. The 60 k glycoprotein alone and the antigenically related family of higher molecular weight glycoproteins (205, 160, 115, 90, and 85 k) both elicited significant increased in the thickness of the wattles of immune cockerels. Because the glycoproteins induce both antibody and cell-mediated immune responses they may prove to be important protective immunogens in a subunit vaccine.

The appearance of viral antigen and antibody in the trachea of naive and vaccinated chickens infected with infectious laryngotracheitis virus

In chickens vaccinated with SA-2 infectious laryngotracheitis (ILT) virus, viral antigen could no longer be detected in tracheal washings from day 7 post infection (pi). Total specific antibody was detected in tracheal washings from day 5 pi, IgA antibody appeared at day 6 pi, but neutralising antibody could not be detected until day 14. In the serum of vaccinated chickens, total antibody appeared on day 5 pi and neutralising antibody on day 7. However, no IgA antibody could be detected in serum. There was a substantial increase in the numbers of IgA- and IgG-synthesising cells in the trachea by day 3 pi, with a marked increase in the numbers of IgA-positive cells at day 7 pi. Following challenge with virulent CSW-1 ILT virus, no virus could be detected in the trachea of vaccinated chickens. There was also no evidence of an anamnestic antibody response in the trachea or in serum up to day 10 post challenge, and there was no significant change in the numbers of IgA- or IgG-synthesising cells in the tracheas of vaccinated chickens up to day 7 post challenge.

Vaccination with affinity‐purified glycoproteins protects chickens against infectious laryngotracheitis herpesvirus

Glycoproteins of infectious laryngotracheitis virus (ILTV) were purified from detergent extracts of virus-infected cells by lectin affinity chromatography. In these preparations, glycoproteins of 205, 160, 115, 90, 85, 74, 60 and 50 kDa were recognized by immune chicken serum in Western blotting. Vaccination of chickens with these ILTV glycoproteins protected up to 83% of chickens against replication of the challenge virus as demonstrated by the absence of viral antigen in the trachea. Both neutralizing antibody and delayed-type hypersensitivity responses were induced by vaccination with glycoprotein preparations, but neither correlated with protection.

Antigens of infectious laryngotracheitis herpesvirus defined by monoclonal antibodies

SummaryMonoclonal antibodies to glycoprotein and protein antigens of infectious laryngotracheitis virus (ILTV) were divided into five groups on the basis of their reactivity in immunofluorescence and Western blotting. Group I antibodies recognised a single band of 60 k and Group II antibodies recognised bands of 205, 160, 115, 90 and 85 k in Western blotting. In immunofluorescence both these groups of antibodies reacted with antigens located in the cytoplasm of fixed virus-infected cells and they also reacted with unfixed cells, suggesting that these antigens are on the surface of virus-infected cells. While Group I monoclonal antibodies did not react with extracts of tunicamycin-treated cells, some Group II antibodies recognised bands of decreased molecular weight compared to those present in untreated cells. The reactivity of the Group II antibodies with extracts of tunicamycin-treated cells suggested that they recognised at least three different epitopes which was confirmed by ELISA additivity assays. Monoclonal antibodies of Group III, Group IV and Group V recognised several low molecular weight proteins from 45 to 24 k. Immunofluorescence studies showed that these were nuclear and cytoplasmic antigens that were not present on the surface of virus-infected cells.