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.     

Virulence of infectious laryngotracheitis viruses: comparison of modified-live vaccine viruses and North Carolina field isolates

Virulence of six modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses was compared with that of 11 field isolates (indistinguishable from vaccine viruses by DNA restriction endonuclease analyses) by intratracheal exposure of 4-week-old, specific-pathogen-free chickens. Virulence of ILT viruses was based on an intratracheal pathogenicity index, mortality, and tracheal lesions. Intratracheal pathogenicity indices for ML vaccine viruses ranged from 0.0 to 0.14, while those for field isolates were 0.20 to 0.82. Mortality was a consistent clinical feature of field isolates; all produced mortality, with seven of the 11 isolates causing two or more deaths per inoculation group. In contrast, only one of six ML vaccine viruses produced mortality (one death per inoculation group). In general, tracheal lesions were more severe in chickens inoculated with field isolates and were produced more consistently than in chickens inoculated with vaccine viruses. These studies indicate that virulence of ILT field isolates was greater than that of ML vaccine viruses. Together with previous restriction endonuclease analyses, these findings suggest the possibility that field isolates originated from ML vaccine viruses through reversion to parental-type virulence.     

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 na?ve 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.     

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.     

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.     

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.     

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.     

Vaccines protect chickens against H5 highly pathogenic avian influenza in the face of genetic changes in field viruses over multiple years

Inactivated whole avian influenza (AI) virus vaccines, baculovirus-derived AI haemagglutinin vaccine and recombinant fowlpoxvirus-AI haemagglutinin vaccine were tested for the ability to protect chickens against multiple highly pathogenic (HP) H5 AI viruses. The vaccine and challenge viruses, or their haemagglutinin protein components, were obtained from field AI viruses of diverse backgrounds and included strains obtained from four continents, six host species, and isolated over a 38-year-period. The vaccines protected against clinical signs and death, and reduced the number of chickens shedding virus and the titre of the virus shed following a HP H5 AI virus challenge. Immunization with these vaccines should decrease AI virus shedding from the respiratory and digestive tracts of AI virus exposed chickens and reduce bird-to-bird transmission. Although most consistent reduction in respiratory shedding was afforded when vaccine was more similar to the challenge virus, the genetic drift of avian influenza virus did not interfere with general protection as has been reported for human influenza viruses.     

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.     

Safety and efficacy of the cell-associated vaccine prepared by an attenuated infectious laryngotracheitis virus.

The safety and efficacy of the cell-associated (C-A) vaccine prepared by chicken embryo fibroblast (CEF) cells infected with the tissue-culture-modified strain of infectious laryngotracheitis (ILT) virus were studied in chickens. Over seventy percent of chickens inoculated with the C-A vaccine by the subcutaneous (S.C.) or intramuscular (I.M.) route at 1 day of age was protected against challenge with a virulent strain of ILT virus without any clinical signs. Chickens vaccinated with the C-A vaccine at 1 day of age acquired immunity within 6 days after vaccination, and the protection rate maintained more than 60% until 10 weeks post-vaccination. The C-A vaccine was invariably effective for chickens at various age. There was no evidence that the development of immunity was hindered by further vaccination with Newcastle disease and infectious bronchitis combined live vaccine. In addition, the C-A vaccine was safe when chickens were inoculated with 10 doses. In the field trials of the C-A vaccine, no adverse reaction was observed, and over 65% of vaccinated chickens was protected against the challenge of the virulent ILT virus at 8 weeks after vaccination.     

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.     

Re-evaluation of the pathogenicity of A/chicken/Alabama/75 (H4N8) influenza virus.

Avian influenza (AI) virus A/chicken/Alabama/7395/75 (H4N8), a putatively non-pathogenic virus associated with a self-limiting outbreak of severe disease in commercial layers, was selectively passed in chickens or in cell cultures and then in chickens to determine whether virus with increased pathogenicity would emerge. When 20 derivatives of the parental virus were each inoculated intranasally and intratracheally in leghorn hens, mortality rates ranged from zero (0/24) to 25% (6/24); mortality was 4% (1/24) for hens inoculated with the parental virus. Many virus reisolates (51/144) from hens that died exhibited high pathogenicity, killing at least six of eight intravenously inoculated 4-week-old chickens. Most derivatives examined produced plaques in trypsin-free cell cultures more efficiently than the parental virus, but the highest plaquing efficiencies observed (10%) were lower than would be expected (100%) for highly pathogenic subtype H5 or H7 AI viruses. These results confirm that the Alabama H4N8 virus can acquire increased pathogenicity upon passage in chickens and suggest that it may have acted alone in producing the severe disease observed in laying chickens in Alabama.     

抗禽流感或传染性喉气管炎两种重组鸡痘病毒疫苗联合免疫的相互影响

“表达H5N1禽流感病毒HA和NA基因的重组鸡痘病毒（rFPV—AI）疫苗”以及“表达传染性喉气管炎病毒gB基因的重组鸡痘病毒（rFPV—ILT）疫苗”均已在实现商业化生产。由于两种疫苗使用了相同的载体病毒,如何使用才能减少相互干扰而产生最好的免疫效果。本研究设计了三个试验组：1）免疫rFPV—AI后间隔4周接种rFPV—ILT;2）rFPV—AI和rFPV—ILT混合后接种;3）将rFPV—AI和rFPV—ILT分别在两个翅膀同时免疫。结果表明,试验鸡接种rFPV—AI后4周接种rFPV—ILT,对传染性喉气管炎病毒WG株攻击的保护率为60％（6／10）,低于rFPV—ILT单独免疫组的保护率（100％,10／10）;rFPV—AI和rFPV—ILT混合后免疫组对禽流感病毒强毒攻击的保护率为80％（8／10）,对传染性喉气管炎病毒强毒攻击的保护率为70％（7／10）,而rFPV—AI和rFPV—ILT分两点同时接种对两种病毒攻击均能产生完全保护。本试验结果建议将这两种相同载体的重组病毒疫苗分两点同时接种以避免相互之间的干扰。     

Mild infectious laryngotracheitis in broilers in the southeast

During 2001, a mild infectious laryngotracheitis virus (ILTV) infection occurred in broiler flocks in the southeastern United States. Clinical signs included mild tracheitis, swollen sinuses, and conjunctivitis, with no increased mortality and minimal serologic response. Infrequent intranuclear inclusion bodies with or without syncytial cell formation were observed in eyelid, trachea, and larynx and in the chorioallantoic membrane of infected embryos. Immunohistochemistry and a nested infectious laryngotracheitis polymerase chain reaction (ILT PCR) were utilized to confirm the presence of ILTV nucleic acid in fixed tissues. In addition, 2-wk-old specific-pathogen-free (SPF) birds inoculated with field material exhibited the mild signs observed in broilers in the field. Tracheal swabs and tissues taken from these SPF birds were also positive by nested ILT PCR. Restriction fragment length polymorphism analysis of ILT PCR products indicated that ILT virus associated with mild respiratory disease in the southeast is related to the chicken embryo origin vaccine type strains.     

Virulence of pigeon-origin Newcastle disease virus isolates for domestic chickens.

The virulence of six pigeon-origin isolates of Newcastle disease virus (NDV) was evaluated before and after passage in white leghorn chickens. Four isolates were defined as pigeon paramyxovirus-1 (PPMV-1) and two isolates were classified as avian paramyxovirus-1 (APMV-1) with NDV monoclonal antibodies. The four PPMV-1 isolates were passaged four times in chickens, and the APMV-1 isolates were passaged only once. Infected birds were monitored clinically and euthanatized. Tissues were collected for histopathology, in situ hybridization with a NDV matrix gene digoxigenin-labeled riboprobe, and immunohistochemistry with an anti-peptide antibody to the nucleoprotein. Mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. Sequence analysis of the fusion protein cleavage site of all six isolates demonstrated a sequence typical of the virulent pathotype. Although the pathotyping results indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and some nervous signs in only some of the 4-wk-old specific-pathogen-free white leghorns inoculated intraconjunctivally. However, an increased frequency of clinical signs and some mortality occurred in 2 wk olds inoculated intraconjunctivally with passaged virus. Histologically, prominent lesions in heart and brain were observed in birds among all four groups inoculated with the PPMV-1 isolates. The behavior of the two pigeon-origin APMV-1 isolates when inoculated into chickens was characteristic of velogenic viscerotropic NDVs and included necro-hemorrhagic lesions in the gastrointestinal tract.     

Pathogenicity of attenuated peste des petits ruminants virus in sheep and goats

Progressive loss of virulence for goat kids was noticed when peste des petits ruminants (PPR) virus was passaged in Vero cells. While goats inoculated with the 60th passage suffered from the clinical PPR disease and mortality, goats inoculated with the 80th passage did not show any sign of the disease. If the progressive loss of virulence of the virus with passage continues, it will not be long before a homologous PPR vaccine will be obtained at the National Veterinary Institute, Vom.     

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.     

Experimental selection of virus derivatives with variations in virulence from a single low-pathogenicity H6N1 avian influenza virus field isolate

A mixture of viruses with variations in virulence is likely present within a low-pathogenicity avian influenza virus (LPAIV) population. An H6N1 AIV was isolated from a field case showing 3.8% weekly mortality and a 33% egg production drop in Taiwan. The pathologic lesions included proventricular hemorrhage and urate deposition in the kidneys and on visceral organs. From the field isolate, a done (2838N) that caused no lesions or death was obtained using limit dilution in chicken embryos and a done (2838V) that caused renal lesions and death was obtained using contact infection in chicks. Both clones were inoculated intranasally in 4-week-old specific pathogen free (SPF) chickens to test their virulence. Renal urate deposition was found in chickens inoculated with 2838V but not in chickens inoculated with 2838N. In situ hybridization, polymerase chain reaction, and virus isolation were used to confirm the spread of 2838V from the respiratory tract to the renal tissue. We found that contact infection in chickens is a good method to obtain a more virulent done from a heterogeneous virus population.     

Induction of strong protection by vaccination with partially attenuated serotype 1 Marek's disease viruses

Studies were conducted to better understand the relationship among Marcek's disease (MD) vaccine strains between induction of protective immunity and the degree of attenuation (or virulence). To obtain viruses at different stages of attenuation, very virulent plus MD strains 584A and 648A and selected clones of these strains were serially passaged in chicken and duck cells. These viruses were considered fully attenuated after passage for 70-100 times in chicken embryo cell cultures until they no longer induced gross lesions in susceptible, maternal antibody-negative (ab-) chickens. Lower passages of the same strains were considered partially attenuated, provided their virulence was less than that of the parent strain. Four of five partially attenuated preparations derived from MD virus strains 584A and 648A or the previously attenuated Md11 strain induced 28%-62% higher levels of protection in maternal antibody-positive (ab+) chickens against virulent MD challenge than the fully attenuated counterpart viruses. The partially attenuated 584A/d2/3 strain replicated in chickens but was totally nonprotective. Data from two subsequent trials in ab+ chickens confirmed that protection induced by the partly attenuated (passage 80) preparations was 79% and 118% higher, respectively, than that induced by the fully attenuated (passage 100) preparations of strain 648A. However, in one trial with ab- chickens, no difference in protection between partially and fully attenuated virus was observed. Strong protection (up to 85%) against highly virulent challenge also was provided by preparations of 648A at passages 40-60, which were moderately oncogenic when used alone. Partially attenuated strains tended to replicate to higher titers in both ab+ and ab- chickens compared with fully attenuated vaccines. Also, ab+ and ab- chickens vaccinated with partially attenuated strains developed three- to nine fold more extensive microscopic lesions in peripheral nerves at 14 and 22 days after virulent challenge than chickens vaccinated with fully attenuated strains. When measured in ab+ chickens, loss of lesion induction by 648A was achieved 30 passages earlier (at passage 70) than when measured in ab- chickens. Thus, maternal antibodies appeared to abrogate the pathogenicity of some partially attenuated strains. These studies establish for MD the principle that at least some partially attenuated MD viruses may replicate better and induce stronger immunity against virulent challenge than fully attenuated preparations of the same strain, at least when tested in ab+ chickens. Moreover, depending on passage level, partially attenuated vaccine strains may be relatively innocuous for ab+ chickens, causing few or no lesions.