Immune responses to infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) is an upper respiratory tract disease in chickens caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. Despite the extensive use of attenuated, and more recently recombinant, vaccines for the control of this disease, ILT continues to affect the intensive poultry industries worldwide. Innate and cell-mediated, rather than humoral immune responses, have been identified as responsible for protection against disease. This review examines the current understandings in innate and adaptive immune responses towards ILTV, as well as the role of ILTV glycoprotein G in modulating the host immune response towards infection. Protective immunity induced by ILT vaccines is also examined. The increasing availability of tools and reagents for the characterisation of avian innate and cell-mediated immune responses are expected to further our understanding of immunity against ILTV and drive the development of new generation vaccines towards enhanced control of this disease.     

Latency and reactivation of infectious laryngotracheitis vaccine virus

Summary Latency and reactivation of a commercial infectious laryngotracheitis virus vaccine were demonstrated in live chickens. Virus was re-isolated at intervals between seven and fourteen weeks post-vaccination and this may be of epizootiological significance.     

Comparison of the replication and transmissibility of an infectious laryngotracheitis virus vaccine delivered via eye-drop or drinking-water

Live attenuated vaccines have been extensively used to control infectious laryngotracheitis (ILT). Most vaccines are registered/recommended for use via eye-drop although vaccination via drinking-water is commonly used in the field. Drinking-water vaccination has been associated with non-uniform protection. Bird-to-bird passage of chick-embryo-origin (CEO) ILT vaccines has been shown to result in reversion to virulence. The purpose of the present study was to examine the replication and transmission of a commercial CEO infectious laryngotracheitis virus (ILTV) vaccine strain following drinking-water or eye-drop inoculation. Two groups of 10 specific-pathogen-free chickens were each vaccinated with Serva ILTV vaccine strain either via eye-drop or drinking-water. Groups of four or five unvaccinated birds were placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days from vaccinated and in-contact birds to assess viral replication and transmission using quantitative polymerase chain reaction. Compared with eye-drop-vaccinated birds, drinking-water-vaccinated birds showed delayed viral replication but had detectable viral DNA for a longer period of time. Transmission to chickens exposed by contact on day 0 of the experiments was similar in both groups. Birds exposed to ILTV by contact with eye-drop vaccinated birds on days 4, 8, 12 and 16 of the experiment had detectable ILTV for up to 8 days post exposure. ILTV was not detected in chickens that were exposed by contact with drinking-water vaccinated birds on day 12 of the experiment or later. Results from this study provide valuable practical information for the use of ILT vaccine.     

Hemagglutination with avian infectious laryngotracheitis virus

Summary Avian infectious laryngotracheitis virus grown in primary chicken kidney cell cultures was tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, 22°C, and 37°C. HA was observed at all temperatures with mouse erythrocytes but not with cattle, sheep, goat, swine, rabbit, guinea pig, chicken, and goose erythrocytes. A strain variation between mice in the agglutinability of their erythrocytes necessitated selection of mice to obtain erythrocytes. The HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were established. HI antibody titers of individual chicken sera had a significant positive correlation with their neutralizing antibody titers.     

Relationship between SMON virus and avian infectious laryngotracheitis virus

Archives of Virology - A new virus, SMON virus, isolated from human cases of subacute myelooptico-neuropathy (S.M.O.N.) was neutralized by anti-avian infectious laryngotracheitis (ILT) virus immune...     

Comparative in vivo safety and efficacy of a glycoprotein G-deficient candidate vaccine strain of infectious laryngotracheitis virus delivered via eye drop

Infectious laryngotracheitis (ILT) is an acute respiratory disease in poultry that is commonly controlled by vaccination with conventionally attenuated virus strains. Despite the use of these vaccines, ILT remains a threat to the intensive poultry industry. Our laboratory has developed a novel candidate vaccine strain of infectious laryngotracheitis virus (ILTV) lacking glycoprotein G (ΔgG-ILTV). The aim of the present study was to directly compare this candidate vaccine with three currently available commercial vaccines in vivo. Five groups of specific-pathogen-free chickens were eye-drop inoculated with one of the three commercial vaccine strains (SA2-ILTV, A20-ILTV or Serva-ILTV), or ΔgG-ILTV, or sterile medium. Vaccine safety was assessed by examining clinical signs, weight gain and persistence of virus in the trachea. Vaccine efficacy was assessed by scoring clinical signs and conducting post-mortem analyses following challenge with virulent virus. Following vaccination, birds that received ΔgG-ILTV had the highest weight gain among the vaccinated groups and had clinical scores that were significantly lower than birds vaccinated with SA2-ILTV or A20-ILTV, but not significantly different from those of birds vaccinated with Serva-ILTV. Analysis of clinical scores, weight gain, tracheal pathology and virus replication after challenge revealed a comparable level of efficacy for all vaccines. Findings from this study further demonstrate the suitability of ΔgG-ILTV as a vaccine to control ILT.     

Protection of chickens from infectious laryngotracheitis with a recombinant fowlpox virus expressing glycoprotein B of infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) is an economically important disease of chickens caused by a type I gallid herpesvirus, infectious laryngotracheitis virus (ILTV). The vaccines currently available are modified live viruses, which are effective in preventing disease outbreaks. However, they have often been associated with a variety of adverse effects including spread of vaccine virus to non-vaccinates, inadequate attenuation, production of latently infected carriers, and increased virulence as a result of in vivo passage. In this study, a recombinant fowlpox virus expressing glycoprotein B (gB) of ILTV (rFPV-ILTVgB) was constructed. Protection of specific pathogen free (SPF) and commercial chickens from ILT with the rFPV-ILTVgB and commercial ILTV vaccine (Nobilis ILT) were compared after challenge with a lethal dose of virulent ILTV.Both the rFPV-ILTVgB- and the Nobilis ILT-vaccinated SPF chickens were completely protected from death, while 90% of the unvaccinated chickens died after challenge. The immunized commercial chickens were also 100% protected with rFPV-ILTVgB, compared with 85% protected with Nobilis ILT. The protective efficacy was also measured by the antibody response to ILTV gB, isolation of challenge virus and polymerase chain reaction amplification of the ILTV thymidine kinase gene after challenge. The results showed that rFPV-ILTVgB could be a potential safe vaccine to replace current modified live vaccines for preventing ILT.     

Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates.

This study was designed to test local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations. Dextran was chosen as a model polysaccharide antigen and conjugated via adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio)propionate to CTB. Mice were immunized either subcutaneously, intranasally, or perorally three times, and cholera toxin was used as an adjuvant for the mucosal immunizations. Three conjugates with different molecular weights for dextran (40,000 and 76,000) or varying dextran/CTB molar ratios were tested. Peroral immunizations with all conjugates evoked local immunoglobulin A (IgA) antibody responses against dextran in the small intestine, and intranasal immunizations did the same in the lung. Intranasal immunizations also elicited serum antibody titers that were significantly higher than or equal to those after subcutaneous immunizations. Intranasal immunizations evoked serum IgG antidextran titers which were dependent on the dextran/CTB molar ratio and inversely related to the local IgA response, which was not the case for subcutaneous immunizations. This is the first study of local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. The results show that it is possible to evoke a local as well as a systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization.     

MinION sequencing to genotype US strains of infectious laryngotracheitis virus

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing.

RESEARCH HIGHLIGHTS

• A multi-allelic assay identified nine ILTV genotypes circulating in the US

• Single-allele genotyping is congruent with whole genome phylogenetic partitioning

• US ILTV strains can be grouped into five genotypes using the single-allele assay

• The single-allele assay can be done using MinION sequencing of barcoded amplicons

     

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

Summary The 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.     

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.     

Challenges and recent advancements in infectious laryngotracheitis virus vaccines

Over the past 80 years, biosecurity measures and vaccines have been used to prevent the occurrence of outbreaks of infectious laryngotracheitis (ILT). Despite these control strategies, ILT continues to have an impact on intensive poultry industries. Attenuated vaccines, particularly those derived by passage in chicken embryos, have been associated with a number of side effects, including residual virulence, transmission to naïve birds, establishment of latent infections with subsequent reactivation and shedding of virus, and reversion to virulence after in vivo passage. Most recently, recombination between attenuated ILT vaccines in the field has been shown to be responsible for the emergence of new virulent viruses that have caused widespread disease. To address some of these issues, new-generation virally vectored recombinant vaccines have been developed and recently released in some countries. In addition, recombinant deletion mutants of ILT virus have been proposed as vaccine candidates. In this review, recent advances in the understanding of the epidemiology of traditionally attenuated ILT vaccines as well as in the development and use of new generation vaccines are examined. Next-generation vaccines, along with more appropriate immunological screening strategies, are identified as particularly promising options to enhance ILT control in the future.     

Effects of reticuloendotheliosis virus on the response of chickens to infectious laryngotracheitis virus

Effects of reticuloendotheliosis virus (REV) on the response to infectious laryngotracheitis virus (ILTV) were investigated in young chickens with and without maternally derived antibody (MAb) to REV. In the first experiment a group of 1-day-old chickens without REV MAb were inoculated at 1 day of age with REV whilst another group of similar chickens were left uninoculated. All chickens were vaccinated with ILTV at 7 days of age. There was a significantly higher proportion of infectious laryngotracheitis (ILT) post-vaccinal ophthalmia (p.v.o.) in the group inoculated with REV. In the second experiment chickens with and without MAb to REV were inoculated at 1 day old with REV. These chickens, together with others not inoculated with REV, were vaccinated with ILTV isolate SA-2 8 days later. A virulent ILTV isolate, G, was used to challenge all the chickens 20 days after vaccination. Again the chickens without MAb to REV inoculated with REV showed a higher proportion of ILT p.v.o. and a significantly higher mortality rate due to ILT following vaccination. In the chickens inoculated with REV at 1 day of age and not vaccinated but challenged with ILTV there was a significantly higher mortality and rate of clinical signs due to ILT in those birds without than in those with REV MAb. In both experiments chickens from REV negative parents were found to be free of REV neutralising MAb. However, only 30% of chickens originating from a flock known to be infected with REV had a titre of 1/40 or higher. In spite of this, this group was significantly more resistant than the group without REV MAb to the immunosuppressive effect of inoculation at 1 day old with REV. This was demonstrated by their lower susceptibility (i.e. less p.v.o. and mortality) to the vaccination and challenge with ILTV. Chickens without REV MAb developed neutralising antibodies within 2 weeks of inoculation with REV. Irrespective of the REV MAb status 1-day-old chickens inoculated with REV were viraemic within a week.     

Pathogenicity of latent infectious laryngotracheitis virus in chickens

Groups of specific pathogen-free chickens were inoculated with the same dose of a field strain of infectious laryngotracheitis virus that had either been isolated from tracheal swabs taken from infected birds during acute phase shedding, or that had been isolated during an episode of virus shedding after a latent period of 12 to 17 weeks. Birds in both groups developed characteristic clinical signs of ILT including difficulty in breathing, rales and sneezing. Thus, ILT virus shed after a latent period is capable of causing disease in susceptible birds similar to that seen in the field.     

Epitope-specific antibody responses to virulent and avirulent feline infectious peritonitis virus isolates.

Feline infectious peritonitis virus (FIPV) has been isolated several times from infected cats. Some of these isolates vary markedly in their ability to cause disease. Specific-pathogen-free cats were inoculated with the avirulent FIPV-UCD-2 isolate or the extremely virulent FIPV-79-1146 isolate or both. After 1 month, cats which had received FIPV-79-1146 were either dead or showed clinical signs of FIP. All cats which received only FIPV-UCD-2 remained healthy up to 6 months after inoculation. Antibody-mediated immune enhancement of disease was not observed in cats which received FIPV-UCD-2 before inoculation with FIPV-79-1146. Monoclonal antibodies which recognized type-specific epitopes on each of the structural polypeptides of these two viruses were used in competitive-inhibition enzyme-linked immunosorbent assays to analyze the humoral immune responses of the cats. All cats produced antibodies to epitopes found on the homologous virus. In addition, cats inoculated with FIPV-79-1146 also produced antibodies which inhibited the binding of the anti-FIPV-UCD-2 E1 monoclonal antibody. One cat inoculated twice with FIPV-UCD-2 produced antibodies which inhibited the binding of the anti-FIPV-79-1146 N- and E1-specific monoclonal antibodies. Competitive enzyme-linked immunosorbent assays may prove useful in distinguishing cats which are infected with virulent FIPV isolates from cats infected with avirulent feline coronaviruses.     

Local and systemic antibody responses in high-risk adults given live-attenuated and inactivated influenza A virus vaccines.

Forty seropositive older adults with chronic diseases were vaccinated intranasally with either influenza A/California/10/78 (H1N1) (CR37) or influenza A/Washington/897/80 (H3N2) (CR48) virus. No clinically significant decrements in pulmonary function occurred postvaccination. Eight (62%) recipients of CR37 virus and 16 (59%) recipients of CR48 virus became infected with vaccine virus, as indicated by a fourfold rise in nasal wash immunoglobulin G (IgG) or IgA antibody titer, a fourfold rise in serum antibody titer, isolation of vaccine virus from nasal washings, or all of these. Within 2 years after cold-recombinant virus vaccination, 29 vaccinees received trivalent inactivated influenza virus vaccine parenterally. After inactivated virus vaccination, 23 (79%) vaccinees developed a fourfold rise in nasal wash or serum antibody titer to H1 antigen and 24 (83%) developed a fourfold rise in nasal wash or serum antibody titer to H3 antigen. Significantly more cold-recombinant virus vaccinees developed a fourfold rise in nasal wash IgA antibody to H1 or H3 hemagglutinin compared with inactivated virus vaccinees (17 [43%] versus 9 [17%], P = 0.01). We conclude that these cold-recombinant virus vaccines are safe and immunogenic in seropositive older high-risk adults and more often induced a nasal wash IgA antibody response than the inactivated virus vaccine.     

Comparison of serological tests for detection of antibodies to infectious laryngotracheitis virus

Four serological tests i.e. ELISA, serum neutralisation (SN), fluorescent antibody (FA), and agar gel immunodiffusion (AGID) were compared for sensitivity using several criteria, for detection and titration of infectious laryngotracheitis (ILT) virus antibodies in chicken sera. In the ELISA test, sera were tested in parallel on virus positive and negative control antigens with results expressed as positive-negative difference. Non-specific binding was not a problem in this test. SN tests were performed in microtitre plates using chick embryo liver cells, while sera for FA tests were titrated on multispot slides on which were fixed ILT virus-infected chick embryo liver cell cultures. The AGID test was the standard test still widely used for serological diagnosis of ILT. The four tests were compared using (1) sera from experimentally inoculated birds bled regularly at intervals from 4 to 35 days post-inoculation, (2) convalescent sera from a natural outbreak of ILT, and (3) serial dilutions of an ILT positive serum. In all experiments the ELISA test was of slightly greater sensitivity than SN and was comparable to the FA test. In the experimentally infected birds ELISA and FA test detected sero-conversion in more birds at 7 days than SN. In tests with the serially diluted hyperimmune ILT serum, ELISA, FA and SN tests were comparable. SN however was the most useful test for quantification of ILT antibodies. ILT-SN titres in birds were never high, the highest titre recorded in experimental birds and in convalescent sera was 1/48. AGID was found to be less sensitive than ELISA, FA or SN test but was considered useful for detection of antibodies on a flock basis, since, with the convalescent sera AGID detected a significant proportion of positives.