Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4?kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.     

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 characteristics in specific pathogen-free chickens 21 and 35 days after intra-tracheal inoculation with vaccine or field strains of infectious laryngotracheitis virus

ABSTRACT

Latency is an important feature of infectious laryngotracheitis virus (ILTV) yet is poorly understood. This study aimed to compare latency characteristics of vaccine (SA2) and field (CL9) strains of ILTV, establish an in vitro reactivation system and examine ILTV infection in peripheral blood mononuclear cells (PBMC) in specific pathogen-free chickens. Birds were inoculated with SA2 or CL9 ILTV and then bled and culled at 21 or 35 days post-inoculation (dpi). Swabs (conjunctiva, palatine cleft, trachea) and trigeminal ganglia (TG) were examined for ILTV DNA using PCR. Half of the TG, trachea and PBMC were co-cultivated with cell monolayers to assess in vitro reactivation of ILTV infection. ILTV DNA was detected in the trachea of approximately 50% of ILTV‐inoculated birds at both timepoints. At 21?dpi, ILTV was detected in the TG only in 29% and 17% of CL9- and SA2-infected birds, respectively. At 35?dpi, ILTV was detected in the TG only in 30% and 10% of CL9- and SA2-infected birds, respectively. Tracheal organ co-cultures from 30% and 70% of CL9- and SA2-infected birds, respectively, were negative for ILTV DNA at cull but yielded quantifiable DNA within 6 days post-explant (dpe). TG co-cultivation from 30% and 40% of CL9-and SA2-infected birds, respectively, had detectable ILTV DNA within 6 dpe. Latency characteristics did not substantially vary based on the strain of virus inoculated or between sampling timepoints. These results advance our understanding of ILTV latency and reactivation.

RESEARCH HIGHLIGHTS

• Following inoculation, latent ILTV infection was detected in a large proportion of chickens, irrespective of whether a field or vaccine strain was inoculated.

• In vitro reactivation of latent ILTV was readily detected in tracheal and trigeminal ganglia co-cultures using PCR.

• ILTV latency observed in SPF chickens at 21 days post-infection was not substantially different to 35 days post-infection.

     

中药复方S-抗毒合剂对传染性喉气管炎的防治作用

目的 探讨中药复方S-抗毒合剂对传染性喉气管炎的防治作用。方法 将患有传染性喉气管炎的孔雀30只随机分为三组，每组10只，S-抗毒合剂组饲喂含中药的基础日粮；西药组饲喂含西药(阿莫西林 利巴韦林)的基础日粮；对照组为基础日粮，连续饲养二周；观察治疗期患有传染性喉气管炎孔雀的发病与死亡情况。结果 中药复方S-抗毒合剂对传染性喉气管炎防治的有效率达98％以上，西药及对照组有效率分别仅为20％、50％。结论 中药复方S-抗毒合剂对防治传染性喉气管炎效果良好。     

复方S-抗毒合剂治疗传染性喉气管炎的血液学影响

目的 探讨中药复方S-抗毒合剂治疗传染性喉气管炎(ILT)后血液细胞和血液化学的变化。方法以孔雀为实验对象，将中药按每日每只2g拌饲料投喂ILT孔雀进行治疗，并于治疗前后取翅静脉血做血细胞和化学检测。结果该药能提高ILT孔雀的RBC、HGB和PCT数，降低肝功酶谱活性．改善损伤的肾功指标，且在用中药2周内使血清IgM升高；4周时IgA显著升高。结论中药复方S-抗毒合剂对孔雀具有止血和抗贫血，恢复肝肾功能，提高机体免疫功能和防治ILT病的作用。     

Sero-epizootiological investigation of infectious laryngotracheitis infection in commercial poultry of Plateau State,north central Nigeria

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens with outbreaks resulting in high economic losses due to increased mortality and drop in egg production. This study reports a survey of ILT virus antibody conducted in nine local government areas (LGAs) of Plateau State involving 67 randomly selected commercial poultry flocks. In all, 938 sera were tested using the Agar Gel Immuno-diffusion (AGID) technique. Overall prevalence of 1.2% (N = 11) was recorded. ILT virus antibody was found in 2.5% (n = 9) and 7.1% (n = 2) of the tested sera from Jos South and Langtang North LGAs, respectively. No detectable ILT virus antibody was found from the other seven LGAs. This is the first report of ILT infection in poultry from the North central part of Nigeria. It is therefore recommended that the economic implication of ILT infection in Nigerian poultry population be conducted in order to know if vaccination should be adopted for control.     

Detection of DNA from infectious laryngotracheitis virus by colourimetric analyses of polymerase chain reactions

A combination of the polymerase chain reaction and a novel ELISA-type DNA colourimetric assay (developed from studies with a retrovirus from man) was used in a preliminary study to detect DNA from avian infectious laryngotracheitis virus. The method is sensitive, specific and easy to perform. Since it can be readily adapted for the detection of DNA from other sources it could be useful for the identification of a variety of pathogens from other species of veterinary importance.     

A genomic map of infectious laryngotracheitis virus and the sequence and organization of genes present in the unique short and flanking regions

We present a genomic map of infectious laryngotracheitis virus (ILT) and an 18,912 bp sequence containing the entire unique short region and a portion of the flanking short repeats. In determining the genomic map, an 856 bp region repeated as many as 13 times was identified within the short repeats. The unique short sequence contains nine potential open reading frames (ORFs). Six of these ORFs show homology to other known herpesvirus unique short genes. Using the herpes simplex virus nomenclature, these genes are the US2, protein kinase, and glycoproteins G, D, I, and E (ORF 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-1 UL47 (ORF 3) is found in the unique short. One very large open reading frame (ORF 5) is present and contains a threonine-rich, degenerate repeat sequence. This gene appears to be unique to ILT among sequenced herpesviruses. Two ORFs were identified within the short repeat (SR) region. SRORF 1 is homologous to a gene (SORF3) found in the unique short region in both MDV and HVT, and appears to be specific to avian herpesviruses. SRORF 2 has homology to HSV US10.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data-base and have been assigned the accession number U28832.     

Ocular exposure to infectious laryngotracheitis virus alters leukocyte subsets in the head-associated lymphoid tissues and trachea of 6-week-old White Leghorn chickens

ABSTRACT

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease primarily infecting the upper respiratory tract and conjunctiva. Administration of live attenuated ILTV vaccines via eye drop, drinking water, or by coarse spray elicits protective mucosal immunity in the head-associated lymphoid tissues (HALT), of which conjunctiva-associated lymphoid tissue (CALT) and the Harderian gland (HG) are important tissue components. The trachea, a non-lymphoid tissue, also receives significant influx of inflammatory cells that dictate the outcome of ILTV infection. The objective of this study was to evaluate leukocyte cellular and phenotypic changes in the CALT, HG and trachea following ocular infection with a virulent ILTV strain. At 1, 3, 5, 7 and 9 days post-infection, CALT, HG, and trachea of 6-week-old specific pathogen free (SPF) chickens ocularly-exposed to vehicle or virulent ILTV strain 63140 were dissociated, the cells enumerated and then phenotyped using flow cytometry. The CALT had the highest viral genomic load, which peaked on day 3. In ILTV-infected birds, the CALT had a decreased percentage of leukocytes. This was reflected by decreased numbers of MHCI⁺MHCII^–, MHCI⁺MHCII^low+, and CD4⁺ cells, while IgM⁺ and MHCI⁺MHCII^High+ expressing cell populations increased. In the HG, the most notable change in cells from ILTV-infected birds was a decrease in IgM expressing cells and histologically, an increase in Mott cells. In summary, an acute, ocular exposure to ILTV strain 63140 in young birds shifts subsets of lymphocyte populations in the CALT and HG with minimal impact on the trachea.     

Variability in biological behaviour,pathogenicity, protectotype and induction of virus neutralizing antibodies by different vaccination programmes to infectious bronchitis virus genotype Q1 strains from Chile

In the period from July 2008 to 2010, a disease episode resulting in serious economic losses in the major production area of the Chilean poultry industry was reported. These losses were associated with respiratory problems, increase of condemnations, drops in egg production and nephritis in breeders, laying hens and broilers due to infections with infectious bronchitis virus (IBV). Twenty-five IBV isolates were genotyped and four strains were selected for further testing by pathotyping and protectotyping. Twenty-four IBV isolates were of the Q1 genotype. The experiments also included comparing the ability of six vaccination programmes to induce virus neutralizing antibodies (VNA) in layers against four selected Chilean strains. Despite the high genetic homology in the S1 gene between the four strains, the heterogeneity in biological behaviour of these different Q1 strains was substantial. These differences were seen in embryonated eggs, in cell culture, in pathogenicity and in level of cross-protection by IBV Massachusetts (Mass) vaccination. This variability underlines the importance of testing more than one strain per serotype or genotype to determine the characteristics of a certain serotype of genotype. The combination of Mass and 793B vaccine provided a high level of protection to the respiratory tract and the kidney for each strain tested in the young birds. The combination of broad live priming using Mass and 793B vaccines and boosting with multiple inactivated IBV antigens induced the highest level of VNA against Q1 strains, which might be indicative for higher levels of protection against Q1 challenge in laying birds.     

Glycoprotein D peptide-based diagnostic approach for the detection of avian infectious laryngotracheitis antibodies

ABSTRACT

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens, pheasants, and peafowl. It is caused by the alpha herpesvirus, infectious laryngotracheitis virus (ILTV). Glycoprotein D (gD) of ILTV is immunogenic and helps in its binding to the susceptible host cell receptor. In the present study, a recombinant gD protein was expressed in a prokaryotic system to develop a single serum dilution ELISA. In addition, two immunogenic peptides, corresponding to regions 77–89 and 317–328, were identified in gD protein. The peptides were synthesized using solid-phase peptide synthesis, purified using reversed-phase HPLC, and characterized using mass spectrometry. The peptides displayed a good titre and were found to be promising antigens to coat the ELISA plate to detect the ILTV antibodies in the serum sample. The developed ELISA showed 96.9% sensitivity, 87.5% specificity, and 95.3% accuracy as compared to OIE referenced standard indirect ILTV ELISA (whole viral coated). The assay may not differentiate vaccinated from infected birds when the flocks are administered with live attenuated vaccines. However, the assay could be useful to detect the disease condition in birds vaccinated with recombinant vaccine expressing glycoproteins other than gD. The developed ILTV single serum dilution ELISA could be an alternative to the existing diagnostics for the detection of ILTV antibodies.     

Long-term protection against a virulent field isolate of infectious laryngotracheitis virus induced by inactivated,recombinant, and modified live virus vaccines in commercial layers

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens controlled through vaccination with live-modified attenuated vaccines, the chicken embryo origin (CEO) vaccines and the tissue-culture origin (TCO) vaccines. Recently, novel recombinant vaccines have been developed using herpesvirus of turkey (HVT) and fowl pox virus (FPV) as vectors to express ILTV immunogens for protection against ILT. The objective of this study was to assess the protection efficacy against ILT induced by recombinants, live-modified attenuated, and inactivated virus vaccines when administered alone or in combination. Commercial layer pullets were vaccinated with one or more vaccines and challenged at 35 (35 WCH) or 74 weeks of age (74 WCH). Protection was assessed by scoring clinical signs; and by determining the challenge viral load in the trachea at five days post-challenge. The FPV-LT vaccinated birds were not protected when challenged at 35 weeks; the HVT-LT and TCO vaccines in combination provided protection similar to that observed in chickens vaccinated with either HVT-LT or TCO vaccines when challenged at 35 weeks, whereas protection induced by vaccination with HVT-LT followed by TCO was superior in the 74 WCH group compared with the 35 WCH group. Birds given the inactivated ILT vaccine had fewer clinical signs and/or lower viral replication at 74 WCH when combined with TCO or HVT-LT, but not when given alone. Finally, the CEO-vaccinated birds had top protection as indicated by reduction of clinical signs and viral replication when challenged at 35 weeks (74 weeks not done). These results suggest that certain vaccine combinations may be successful to produce long-term protection up to 74 weeks of age against ILT.     

The route of inoculation dictates the replication patterns of the infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine

Infectious laryngotracheitis virus (ILTV) has a high proclivity to replicate in the larynx and trachea of chickens causing severe lesions. There is a lack of knowledge on the ability of ILTV to replicate in other respiratory associated tissues apart from in the trachea. The objective of this study was to investigate how tissues that first encounter the virus dictate further sites of viral replication during the lytic stage of infection. Replication patterns of the pathogenic strain 63140 and the chicken embryo origin (CEO) vaccine in the conjunctiva, the Harderian gland, nasal cavity and trachea were evaluated after ocular, oral, intranasal or intratracheal inoculation of specific pathogen-free chickens. Viral replication was assessed by detection of microscopic cytolytic lesions, detection of viral antigen and viral genome load. The route of viral entry greatly influenced virus replication of both strain 63140 and CEO vaccine in the conjunctiva and trachea, while replication in the nasal cavity was not affected. In the Harderian gland, independently of the route of viral entry, microscopic lesions characteristic of lytic replication were absent, whereas viral antigen and viral genomes for either virus were detected, suggesting that the Harderian gland may be a key site of antigen uptake. Findings from this study suggest that interactions of the virus with the epithelial-lymphoid tissues of the nasal cavity, conjunctiva and the Harderian gland dictate patterns of ILTV lytic replication.     

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.     

Full genome analysis of hepatitis B virus genotype E strains from South-Western Africa and Madagascar reveals low genetic variability

The eight genotypes of hepatitis B virus (HBV), A-H, have a characteristic geographical distribution. The high prevalence of genotype E within a wide expanse of Africa makes it important to characterize this genotype. Although the preS/S region of a large number of genotype E isolates has been sequenced, to date only eight complete genotype E genomes have been published. The aim of this study, therefore, was to sequence and characterize the complete genome of genotype E isolates obtained from geographically distinct regions in Africa, including Namibia, Angola, and Madagascar. The nine newly sequenced genotype E isolates were closely related with an intra-group nucleotide divergence of 1.2%, reflecting their close geographical origin. All genotype E strains have the same characteristics, an in-frame deletion of three nucleotides (one amino acid) in the 5' pre-S1, a signature pattern of amino acids in the pre-S1 region and the serological subtype ayw4. In addition, we have identified another unique attribute of genotype E strains, the introduction of another start codon Met(83) in the pre-S1 region that may result in the translation of an elongated middle hepatitis B surface protein (MHBs).     

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.     

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5′ conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2 × 10⁸ to 2 × 10² copies/μl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5′ conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV.