Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines.

A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the DeltaC(t) values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5'-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines.

Pathogenicity and growth characteristics of selected infectious laryngotracheitis virus strains from the United States.

In a recent study, several US infectious laryngotracheitis virus (ILTV) strains and field isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) into nine different genotypes. All of the commercial poultry isolates were identified within genotypes IV, V, and VI. Based on the PCR-RFLP, Group IV isolates were characterized as genetically identical to the chicken embryo origin (CEO) vaccines, Group V as genetically closely related to the CEO vaccines, and Group VI as genetically different to the vaccine strains. The objective of this study was to determine the pathogenicity and growth characteristics of six ILTV commercial poultry isolates as compared with the CEO vaccine. Two isolates representative of PCR-RFLP Groups IV, V, and VI were selected. Differences in disease severity, viral tissue distribution in chickens, and plaque formation ability in cell culture were observed among viral genotypes IV, V, and VI, and between V-A and V-B isolates. Mild respiratory clinical signs were produced by IV-A, IV-B and the CEO vaccine, while VI-A and VI-B isolates produced severe respiratory signs and severe depression, and during the peak of clinical signs both isolates were re-isolated from the conjunctiva, sinus, trachea and thymus. Similarly to Group VI isolates, V-A and V-B produced severe respiratory signs, depression, and were re-isolated from conjunctiva, sinus, and trachea; on cell culture, both isolates produced significant larger plaques than any of the other isolates analysed. Overall, differences in pathogenicity and growth characteristics were observed among genetically closely related US ILTV isolates; however, complete genomes will be necessary to identify molecular determinants linked to the pathogenic viral phenotypes.

Evaluation of the protection elicited by direct and indirect exposure to live attenuated infectious laryngotracheitis virus vaccines against a recent challenge strain from the United States.

In a recent study (Oldoni & García, 2007), some field strains of infectious laryngotracheitis viruses (ILTV) were characterized as genotypically different (group VI) from ILT vaccine strains. The objective of this study was to evaluate the protection elicited by one chicken embryo origin (CEO) and one tissue culture origin (TCO) vaccine against a field isolate from group VI after direct and indirect exposure to ILTV live attenuated vaccines. In phase 1 of the experiment, non-vaccinated chickens were placed into contact with the eye drop vaccinates for a period of four weeks after vaccination. Transmission of the vaccine virus to these in-contact birds was demonstrated by real time PCR and antibody production, although the in-contact birds did not become protected against disease when subsequently challenged in phase 2 of the experiment. This emphasized the importance of uniform vaccination to obtain adequate protection, both to avoid the occurrence of susceptible chickens, and to minimize the potential for reversion to virulence of live-attenuated vaccines. In phase 2, protection against challenge with a group VI field virus was assessed four weeks after vaccination by scoring clinical signs and mortality, and quantifying weight gain. Sentinel birds were added to the groups one day after challenge to assess shedding of challenge virus, using real time PCR and virus isolation, during the period 2 to 12 days post challenge. The results showed that the CEO and TCO eye drop-vaccinated chickens were protected against challenge with the group VI virus, even though it was genetically different from the vaccine strains, and that challenge virus was not transmitted from these protected birds to the sentinels.

Characterization of infectious laryngotracheitis virus (ILTV) isolates from commercial poultry by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).

Infectious laryngotracheitis (ILT) is a highly contagious, acute respiratory disease of chickens, of worldwide distribution, that affects growth and egg production and leads to significant economic losses during periodic outbreaks of the disease. Live attenuated vaccines (chicken embryo origin [CEO] and tissue-culture origin [TCO]) have been widely used to control the disease in the United States. It is believed that most of the outbreaks in the United States are caused by vaccine-related isolates that persist in the field and spill over into naïve poultry populations. The objective of this study was to utilize the previously developed polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis to genotype recent ILT virus (ILTV) isolates from commercial poultry. Forty-six samples were collected during January 2006 to April 2007 from five poultry production regions of the United States and were characterized within PCR-RFLP groups III-VI. Sixty-three percent of the samples analyzed were categorized as closely related to the vaccine strains (groups III-V), whereas 33% were categorized as group VI viruses that differed in six and nine PCR-RFLP patterns from the CEO and TCO vaccines; a mixture of group IV and V viruses was detected in two samples (4%). In general, groups V and VI were the most prevalent viruses, found in 52% and 33% of the samples tested respectively. Both types of viruses were detected in vaccinated and nonvaccinated flocks. Although genetically different, both viruses produced severe disease in the field.

Replication and transmission of live attenuated infectious laryngotracheitis virus (ILTV) vaccines.

The aim of this study was to evaluate the replication of live attenuated infectious laryngotracheitis virus vaccines in selected tissues and their ability to transmit to contact-exposed birds. Four-week-old specific-pathogen-free chickens were eye drop-inoculated with tissue culture origin (TCO) and chicken embryo origin (CEO) vaccines. Contact-exposed chickens were housed in direct contact with eye drop-inoculated chickens from the first day postinoculation. Virus isolation and real-time polymerase chain reaction were used to detect the presence of live virus and viral DNA, respectively, in the trachea, trigeminal ganglia, eye conjunctiva, cecal tonsils, and cloaca from eye drop-inoculated and contact-exposed birds at days 2, 4, 5 to 10, 14, 18, 21, 24, and 28 postinoculation. No differences were observed in the ability of the TCO and CEO vaccines to replicate in the examined tissues. Both vaccines presented a localized replication in the eye conjunctiva and the trachea. Both vaccines were capable of transmitting to contact-exposed birds, attaining peaks of viral DNA as elevated as those observed in inoculated birds. The CEO vaccine replicated faster and reached higher viral genome copy number than the TCO vaccine in the conjunctiva and trachea of eye drop-inoculated and contact-exposed birds. The viral DNA from both vaccines migrated to the trigeminal ganglia during early stages of infection. Although the CEO and TCO vaccines were not recovered from the cecal tonsils and the cloaca, low levels of viral DNA were detected at these sites during the peak of viral replication in the upper respiratory tract.