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.     

Characterization of infectious laryngotracheitis virus isolates: demonstration of viral subpopulations within vaccine preparations

Infectious laryngotracheitis (ILT) is a severe acute respiratory disease of chickens caused by ILT virus. To better understand the epidemiology of the disease, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay of the glycoprotein E gene has been developed and utilized to characterize vaccine strains and outbreak-related isolates. Enzymes EaeI and DdeI were used to differentiate the tissue culture origin (TCO) vaccine from chicken embryo origin (CEO) vaccines. Two RFLP patterns were observed with enzyme EaeI, one characteristic of the TCO vaccine and a second characteristic of all CEO vaccines. Three RFLP patterns were observed with enzyme DdeI. Patterns A and B were characterized as single patterns, whereas the type C pattern was a combination of patterns A and B. Analysis of vaccine strains showed the presence of patterns A and C. Pattern A was observed for the TCO vaccine and one CEO vaccine, whereas pattern C was observed for five of the six CEO vaccines analyzed. PCR-RFLP analysis of plaque-purified virus from pattern C CEO vaccine preparations demonstrated the presence of two populations (patterns A and B). Identification of molecularly different populations of viruses within currently used ILT vaccine is the first step to develop better molecular epidemiologic tools to track vaccine isolates in the field.     

Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage.

Modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo-origin (CEO), were passaged 20 times in specific-pathogen-free chickens. After serial bird-to-bird passage, increased virulence was observed for CEO virus but not TCO virus. Increased mortality and increased severity and duration of respiratory disease were observed in chickens inoculated with chicken-passaged CEO viruses; only mild respiratory disease (no mortality) occurred in chickens inoculated with chicken-passaged TCO viruses. These findings suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.     

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.     

Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.     

Isolation of very virulent Marek''s disease viruses from vaccinated chickens in Australia

Very virulent Marek's disease viruses (vvMDV), defined as isolates against which the herpesvirus of turkey (HVT) vaccine provide poor protection, have been isolated from poultry flocks in both the United States and Europe. Twenty-one samples from vaccinated Australian flocks, experiencing problems with excessive Marek's disease (MD), were tested for the presence of transmissible MD viruses (MDV). Of the 16 samples which contained a transmissible agent, 14 were pathogenic in chickens, based on the development of MD lesions or depression of the bursa/body weight ratio. Of the pathogenic isolates which have been successfully typed 10 were serotype 1, and one was serotype 2 MDV. Pathogenicity of isolates varied. Several isolates caused tumours in 20-30% of both vaccinated and unvaccinated chickens. Two isolates, MPF6 and MPF23, caused tumours in more than 50% of chickens. When MPF6 and MPF23 were tested in vaccine trials bivalent vaccine gave no better protection against development of MD lesions than a monovalent vaccine. Isolate MPF23 was so pathogenic that lesions were produced in all chickens, regardless of the vaccine protocol used. Therefore vvMDV have been isolated in Australia, and unlike the vaccines tested overseas, bivalent Australian vaccines do not appear to provide greater protection against these vvMDV.     

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.     

Restriction endonuclease analysis of infectious laryngotracheitis viruses: comparison of modified-live vaccine viruses and North Carolina field isolates

Six modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, three reference strains, and 18 field isolates were compared by restriction endonuclease analysis of their DNA. Viral DNA digestion patterns were established for vaccine viruses using restriction endonucleases PstI, BamHI, KpnI, and HindIII. Using these enzymes, five of six ML vaccine viruses had identical restriction endonuclease cleavage patterns. Vaccine viruses had distinct patterns compared with ILT virus reference strains Illinois-N71851, Cover, and NVSL. Restriction endonuclease cleavage patterns of 18 field isolates of ILT virus, obtained from ILT outbreaks in North Carolina, were indistinguishable from vaccine viruses. These results suggest a possible role of vaccine or vaccine-like viruses in recent ILT outbreaks.     

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.     

Epidemiology of recent outbreaks of infectious laryngotracheitis in poultry in Australia

Objective Over the past 3 years, numerous outbreaks of infectious laryngotracheitis (ILT) have occurred in poultry in Australia. The objectives of this study were to identify the viral strains involved in the recent outbreaks and to determine possible epidemiological links between these outbreaks. Procedure A combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of several genes of the ILT virus was used to identify genetic differences in field/vaccine ILT virus isolates. In a previous study, these procedures had demonstrated five classes (1–5) in Australia. Results Analysis of 92 field ILT viruses demonstrated four new classes: 6, 7, 8 and 9. Class 6 was responsible for four outbreaks in one Victorian broiler company and demonstrated to be distinct from other Australian strains of ILT. Class 7 was the Nobilis ILT vaccine (Intervet Pty Ltd). Class 8 was responsible for the majority of the outbreaks in New South Wales and was phylogenetically close to class 7. On one occasion, classes 7 and 8 were identified in an outbreak on a Victorian farm that had used the Nobilis ILT vaccine. Class 9, also phylogenetically close to classes 7 and 8, was found only in New South Wales. The previously identified class 2 was also found to be responsible for a large number of outbreaks, mainly in Victoria. Conclusion The results demonstrate that, epidemiologically, most outbreaks of ILT in New South Wales are unrelated to those in Victoria and suggest a link between classes 8 and 9 and the Nobilis ILT vaccine (class 7).     

Protection induced by infectious laryngotracheitis virus vaccines alone and combined with Newcastle disease virus and/or infectious bronchitis virus vaccines

Two types of live attenuated vaccines have been used worldwide for the control of infectious laryngotracheitis virus (ILTV): 1) chicken embryo origin (CEO) vaccines; and 2) tissue culture origin vaccines (TCO). However, the disease persists in spite of extensive use of vaccination, particularly in areas of intense broiler production. Among the factors that may influence the efficiency of ILTV live attenuated vaccines is a possible interference of Newcastle Disease virus (NDV) and infectious bronchitis virus (IBV) vaccines with the protection induced by ILTV vaccines. The protection induced by CEO and TCO vaccines was evaluated when administered at 14 days of age alone or in combination with the B1 type strain of NDV (B1) and/or the Arkansas (ARK) and Massachusetts (MASS) serotypes of IBV vaccines. Two weeks after vaccination (28 days of age), the chickens were challenged with a virulent ILTV field strain (63140 isolate, group V genotype). Protection was evaluated at 5 and 7 days postchallenge by scoring clinical signs and quantifying the challenge virus load in the trachea using real-time PCR (qPCR). In addition, the viral load of the vaccine viruses (ILTV, NDV, and IBV) was quantified 3 and 5 days postvaccination also using qPCR. The results of this study indicate that the NDV (B1) and IBV (ARK) vaccines and a multivalent vaccine constituted by NDV (B1) and IBV (ARK and MASS) did not interfere with the protection induced by the CEO ILTV vaccine. However, the NDV (BI) and the multivalent (B1/MASS/ARK) vaccines interfered with the protection induced by the TCO vaccine (P < 0.05). Either in combination or by themselves, the NDV and IBV vaccines decreased the tracheal replication of the TCO vaccine and the protection induced by this vaccine, since the ILTV-vaccinated and -challenged chickens displayed significantly more severe clinical signs and ILTV load (P < 0.05) than chickens vaccinated with the TCO vaccine alone. Although NDV and IBV challenges were not performed, the antibody responses elicited by NDV and/or the IBV vaccinations were significantly reduced (P < 0.05) when applied in combination with the CEO vaccine.     

Sequence analysis of related low-pathogenic and highly pathogenic H5N2 avian influenza isolates from United States live bird markets and poultry farms from 1983 to 1989

The last highly pathogenic outbreak of avian influenza in the United States was caused by an H5N2 influenza virus in Pennsylvania and New Jersey in 1983-84. Through a combined federal and state eradication effort, the outbreak was controlled. However, in 1986-89, multiple H5N2 viruses were isolated from poultry farms and the live bird markets (LBMs) in the United States. To determine the epidemiologic relationships of these viruses, the complete coding sequence of the nonstructural gene and the hemagglutinin protein subunit 1 of the hemagglutinin gene was determined for 11 H5N2 viruses and compared with previously available influenza sequences. The H5N2 isolates from 1986-89 were all closely related to the isolates from the 1983-84 Pennsylvania outbreak by nucleotide and amino acid sequence analysis for both genes, providing additional evidence that the Pennsylvania/83 (PA/83) virus lineage was not completely eradicated. The PA/83 lineage also had a large number of unique amino acid changes not found in other avian influenza viruses, which was suggestive that this lineage of virus had been circulating in poultry for an extended period of time before the first isolation of virus in 1983. High substitution and evolutionary rates were measured by examining the number of nucleotide or amino acid substitutions over time as compared with the index case, CK/PA/21525/83. These rates, however, were similar to other outbreaks of avian influenza in poultry. This study provides another example of the long-term maintenance and evolution of influenza viruses in the U.S. LBMs and provides further evidence of the connection of the LBMs and the Pennsylvania 1983 H5N2 outbreak.     

Protection against infectious laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant vaccines

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines. The chicken embryo origin (CEO) vaccines, although proven to be effective in experimental settings, have limited efficacy in controlling the disease in dense broiler production sites due to unrestricted use and poor mass vaccination coverage. These factors allowed CEO vaccines to regain virulence, causing long lasting and, consequently, severe outbreaks of the disease. A new generation of viral vector fowl poxvirus (FPV) and herpesvirus of turkey (HVT) vaccines carrying ILTV genes has been developed and such vaccines are commercially available. These vaccines are characterized by their lack of transmission, lack of ILTV-associated latent infections, and no reversion to virulence. HVT-vectored ILTV recombinant vaccines were originally approved for subcutaneous HVT or transcutaneous (pox) delivery. The increased incidence of ILTV outbreaks in broiler production sites encouraged the broiler industry to deliver the FPV-LT and HVT-LT recombinant vaccines in ovo. The objective of this study was to evaluate the protection induced by ILTV viral vector recombinant vaccines after in ovo application in 18-day-old commercial broiler embryos. The protection induced by recombinant ILTV vaccines was assessed by their ability to prevent clinical signs and mortality; to reduce challenge virus replication in the trachea; to prevent an increase in body temperature; and to prevent a decrease in body weight gain after challenge. In this study, both recombinant-vectored ILTV vaccines provided partial protection, thereby mitigating the disease, but did not reduce challenge virus loads in the trachea.     

Genetic analysis of vesicular stomatitis virus-New Jersey from the 1995 outbreak in the western United States

OBJECTIVE: To compare molecular associations between the vesicular stomatitis virus (VSV)-New Jersey isolates of the 1995 outbreak with those from previous outbreaks between 1982 and 1985 in the western United States. SAMPLE POPULATION: 23 virus isolates considered representative of the 1995 outbreak of vesicular stomatitis. PROCEDURE: Viral gene coding for surface-envelope protein G was evaluated by use of nucleotide sequencing and phylogenetic analysis. RESULTS: Changes in up to 0.77% of the nucleotide bases and 1.35% of the amino acids were detected among the 1995 viral isolates, whereas changes in up to 3.2 and 2.9% of the nucleotides and amino acids, respectively, were found, compared with the 1982 to 1985 viruses. Insertions or deletions were not found in the entire gene, which spanned 1,554 nucleotide bases. CONCLUSIONS AND CLINICAL RELEVANCE: Phylogenetic analysis indicated that the 1995 VSV-New Jersey belongs to a lineage distinct from that of the 1982 to 1985 viruses that caused previous outbreaks in the western United States. Furthermore, it also is distinct from strains from Central America and from the Georgian Hazelhurst strain.     

Phylogenetic analysis of Newcastle disease viruses isolated from wild birds in the Poyang Lake region of China

Newcastle disease virus (NDV) causes a highly contagious viral disease in poultry and wild birds, and it can cause significant economic loss worldwide. Eight viral strains were isolated by inoculating embryonated chicken eggs from the Poyang Lake region of China with swab samples. All eight of the NDV isolates were identified as class I genotype 3 strains, but they diverged notablely from class II viruses. Further analysis revealed that all eight NDV isolates were lentogenic strains containing the ¹¹²ERQER↓L¹¹⁷ motif at the F protein cleavage site. The strains were highly identical and were more species specific (chicken and waterfowl) than site specific (Nanchang and Duchang regions). The close phylogenetic proximity of these isolates indicates that viral transmission may happen between poultry and wild birds. Our study demonstrates that lentogenic class I NDVs exist in clinically healthy wild waterfowl and poultry within the Poyang Lake region. Active surveillance of these viruses to determine their evolution and origin is one of the most realistic strategies for preventing and controlling NDV outbreaks.     

Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis

Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was < 10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines.     

Epidemiological findings of outbreaks of disease caused by highly pathogenic H5N1 avian influenza virus in poultry in Egypt during 2006

This paper describes the first threats of H5N1 avian influenza outbreaks in Egypt recorded from February to December 2006 in commercial and domestic poultry from different species and summarizes the major characteristics of the outbreak. There were 1024 cases from different poultry species (rural and commercial chickens of different breeding types, turkeys, ducks, geese, and quail) either in commercial breeding or in backyards from different locations in Egypt. All tested positive for the H5N1 subtype. From these cases only 12 avian influenza A viruses were isolated and characterized from samples collected during outbreaks. All isolates were characterized, and the data confirmed that the isolated viruses belong to highly pathogenic avian influenza of subtype H5N1. Full hemagglutinin (HA) gene (segment 4) sequencing was also done, and the sequences of these isolates were compared with other strains from Russia, Africa, and the Middle East. The data revealed that all Egyptian strains were very closely related and belonging to subclade 2.2 of the H5N1 virus of Eurasian origin, the same one circulating in the Middle East region and introduced into Africa at the beginning of 2006. This study showed evidence of the wide spread of H5N1 virus infection in domestic poultry in Egypt within a short time. The most obvious features of these outbreaks were severe clinical signs and high mortalities as well as very rapid and widespread occurrence within the country in a very short time. The possible causes of its rapid spread and prospects of disease control are discussed.