Detection and isolation of infectious laryngotracheitis virus on a broiler farm after a disease outbreak.

A broiler farm in North Alabama suffered a mild infectious laryngotracheitis (ILT) outbreak, as determined by clinical disease and PCR. The poultry integrator sought help to control further outbreaks in subsequent flocks. Samples were collected from various areas of the poultry houses on the farm over an 8-wk period. The first sampling was conducted 8 days after the infected farm was depopulated; the second was conducted 2 days prior to subsequent flock placement; and the third was conducted when the new flock was 5 wk of age. Samples were examined for ILT virus (ILTV) DNA by real-time PCR and virus isolation in embryos. The infected houses were cleaned, disinfected, heated, litter composted, and curtains replaced after the first sampling and prior to placement of the next flock. Samples from all periods were positive for ILTV DNA. However, the number of positive samples and crossing point values indicated a decrease in the amount of viral DNA, while virus isolation in embryos was successful only on the first sampling. The subsequent flock was vaccinated against ILTV by in ovo route using a commercial recombinant vaccine. Cleaning and sanitation after the disease outbreak reduced the amount of ILTV on the farm and together with in ovo vaccination of the new flock may have prevented a recurrence of another ILT outbreak.

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex).

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻5(= 280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻4(=2800 ELD50). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1.

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/µl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/µl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

Infectious laryngotracheitis virus in chickens.

Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control.

Development of TaqMan real-time RT-PCR for detection of avian reoviruses.

Avian reoviruses (ARVs) are an important cause of economic losses in commercial poultry. A TaqMan real-time RT-PCR assay for detecting of ARVs was developed. The primer-probe set was from the conserved region of ARV S4 genome segment. Real-time RT-PCR detected ARV strains including CO8 and ss412 strains, which belonged to different serological subgroups, and the test had no cross-reaction with other avian viruses. The detection limit of this assay was 5 ARV genome copies per 5 µl and was 150 times more sensitive than traditional RT-PCR. Statistical analyses indicated excellent reproducibility. For ARV strain 2408, a titer of 50% embryo infection dose and 50% tissue culture infectious dose equivalent to 3.9 ± 0.8, and 2.9 ± 0.3 ARV genome copies, respectively. This test was rapid, specific, and sensitive for the detection of ARVs and will be useful in veterinary diagnostic laboratories and for the quantitation of vaccine viruses for pharmaceutical companies.

Toward the development of a plant-based vaccine against reovirus.

Transgenic lines of Arabidopsis thaliana producing recombinant TC protein were developed. The S1 gene encoding sigmaC protein of an avian reovirus (ARV) was amplified by reverse-transcription PCR (RT-PCR). The amplified product was cloned into a plant-expression vector, pE1857, with a strong promoter for expression. The resulting construct with the BAR gene cassette for bialaphos selection was designated rpE-sigmaC and was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-sigmaC constructs that transform A. thaliana and transgenic plants were selected using bialaphos selection. The presence of S1 transcript in plants and their relative level of expression were determined by real time RT-PCR. Western blot analysis further confirmed the presence of sigmaC protein in the plants. Transgenic lines with high levels of sigmaC protein were selected for immunization experiments using specific-pathogen free chickens. Recombinant sigmaC protein produced in plants induced a variety of immunoglobulin G (IgG) antibody responses in chickens. Recombinant protein administered either subcutaneously or orally in birds showed significant protection against challenge. Results suggested that the recombinant sigmaC protein produced in plants has the potential for large-scale vaccination against ARV in commercial poultry.

Integrity of a recombinant hemagglutinin protein of an avian influenza virus.

An open reading frame representing cDNA from a hemagglutinin (HA) encoding gene of a low pathogenic avian influenza virus (AIV) subtype H10N7 was cloned in the pNMT1-TOPO vector under the control of thiamine response promoter. This construct was designated as pNMT1-HA. The pNMT1-HA construct was transformed into Schizosaccharomyces pombe for expression of HA antigen. The correct expression of recombinant HA protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The level of expression of recombinant HA protein was approximately 0.2% of total soluble protein. Purified yeast-derived recombinant HA protein showed hemagglutination activity. The 2-D and 3-D scanning images of recombinant HA protein were observed with an atomic force microscope (AFM). The structural integrity of the HA protein under AFM and hemagglutination activity provided support that the recombinant HA protein may be suitable for development of AIV subunit vaccine for mass administration to poultry.

Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana.

Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines. The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens. The VP2 coding sequence was isolated and integrated into A. thaliana genome by Agrobacterium tumefaciens-mediated transformation. Soluble VP2 expressed in transgenic plants was used to immunize chickens. Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk. Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection. Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection. Results supported the efficacy of plant-produced VP2 as a vaccine against IBD.

Evaluation of the immune response and detection of infectious bursal disease viruses by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay after in ovo vaccination of commercial broilers.

Maternal immunity can interfere with infectious bursal disease virus (IBDV) vaccine efficacy. The effect of maternal antibody (MacAb) on the immune response and detection of two vaccine viruses, an IBDV immune complex (cx) and IBDV-2512, was investigated. Vaccines were administered in ovo at 100 mean embryo infectious dose to commerical broiler embryos derived from young (29 wk) or old (63 wk) flocks. Chickens with low MatAb were challenged with an antigenic standard IBDV strain at 21 and 35 days post in ovo vaccination (PIOV). At 28 and 42 days PIOV, birds were euthanatized and bursa weight: body weight ratios were determined. MatAb of progeny from the 29-wk-old flock was higher (P < 0.05) than that from progeny of the 63-wk-old flock. Progeny titers declined by day 21 PIOV. Birds with MatAb vaccinated with either the IBDV-2512 or IBDV-Icx vaccine did not mount an antibody response by 21 days PIOV. The IBDV-Icx vaccination protected chickens against bursal atrophy when they were challenged at 21 and 35 days PIOV (83% and 77%, respectively). Resistance to challenge in the absence of antibody implies that cellular immunity plays a role in IBDV protection. The IBDV-2512 vaccine caused atrophy of the bursae, and, therefore, we could not examine its protection by these criteria. Neither vaccine could be detected by antigen-capture chemiluminescent enzyme-linked immunosorbent assay. Therefore, total RNA was extracted and reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR were conducted to amplify the VP2 (hypervariable) region on viral RNA collected at 3, 6, 9, 15, and 21 days PIOV. Nested PCR detected the VP2 region of both IBDV vaccine viruses at day 3 PIOV. Only IBDV-2512 RNA was detected on day 6 PIOV. However, the IBDV-Icx RNA was detected on days 9 and 15 PIOV but not on day 21 PIOV. The IBDV-Icx RNA was evident in tissues and could induce protection against challenge. This work gives further insight into the mechanism of the IBDV-Icx vaccine.

Immunosuppression in specific-pathogen-free broilers administered infectious bursal disease virus vaccines by in ovo route.

The effect of two infectious bursal disease virus (IBDV) vaccines (IBDV-immune complex [Icx] and IBDV-2512), administered in ovo, on the cell-mediated immunity of specific-pathogen-free (SPF) broilers was examined. A decrease (P < 0.05) in the T-cell mitogenic response occurred in birds vaccinated with both vaccines on days 9 and 21 post in ovo vaccination (PIOV), but an increase (P < 0.05) occurred on day 15 PIOV. The T cells from birds given the IBDV-2512 were less responsive. There were no significant differences in proportions of lymphocytes expressing CT4+CT8 and CT8+CT4- except on day 21 PIOV, where an increase (P < 0.05) in IBDV-2512-vaccinated birds and a decrease (P < 0.05) in percentage of CT4+CT8- in IBDV-Icx-vaccinated birds was observed. There was an increase (P < 0.05) in percentage of CT8+CT4- T cells on day 21 PIOV in both vaccinated groups. A decrease (P < 0.05) in B-cell percentage was observed on day 21 PIOV in birds given both vaccines. Results indicated that although humoral immunosuppression is associated with destruction of B cells (bursal atrophy), cell-mediated immunosuppression induced by these two IBDV vaccines in SPF birds was not associated with altered helper (CT4+CT8-) or cytotoxic (CT8+CT4-) subpopulations of T lymphocytes.

Comparison of cell culture systems for duck hepatitis B virus using SyBr green quantitative PCR.

The Hepadnaviridae family contains DNA viruses such as human hepatitis B virus (HBV), woodchuck hepatitis B virus (WHV), and duck hepatitis B virus (DHBV). DHBV is distributed in both wild and domestic ducks. HBV is a worldwide health problem with carriers at risk of developing cirrhosis and liver cancer. All medical staff and scientists working with HBV must be vaccinated, because of its highly contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Several cell culture systems have been developed to study anti-DHBV drugs and disinfectants. However, differences in their capabilities to support DHBV propagation have not been reported. Therefore, a sensitive and reproducible quantitative PCR based on SyBr green dye was developed. This system does not need electrophoresis for analysis of PCR products, thus reducing processing time and potential for cross-contamination. It allowed precise quantification of DHBV over 8-logarithm dynamic range with a good correlation (R(2) = 0.9689) and showed minimal run-to-run deviation. Sensitivity was 820 copies of DHBV genome and specificity was confirmed by melting curve analysis. It demonstrated good repeatability in quantification of DHBV loads from serum of infected ducks. This assay compared DHBV yields from different cultured cells. All cells had similar kinetic curves for DHBV replication and replication peaks appeared 4 days post-infection. Duck embryonic hepatocytes showed the highest (P > 0.05) replication peak for DHBV. Therefore, duck embryonic hepatocytes and quantitative PCR based on SyBr green dye are a good choice for anti-DHBV drug and disinfectant testing.

Development of viral disinfectant assays for duck hepatitis B virus using cell culture/PCR.

Human hepatitis B virus (HBV) is a worldwide public health problem with chronic carriers at risk for developing cirrhosis and hepatocellular carcinoma. Accidental nosocomial infections from inadequately disinfected equipment or exposure to blood and body fluids from patients are major routes. To solve such problems, disinfectants to inactivate HBV must be validated. Duck hepatitis B virus (DHBV) is accepted as a surrogate for HBV, due to their similar sensitivities to disinfectants and its safety. Ducklings are used for disinfectant efficacy assays; however, the same virus titer is obtained using duck embryonic hepatocytes. Viral titration in disinfectant efficacy assay is conducted using Southern hybridization of infected duck serum. However, this test requires radioisotopes. Therefore, disinfectant assessment protocols were developed using duck embryonic hepatocytes with polymerase chain reaction (PCR) or nested PCR. The ease of handling, lowered cost and enhanced sensitivity make PCR desirable. Chicken embryonic hepatocytes were applied to DHBV disinfectant efficacy assay. Results were consistent and could be used under certain conditions. The virucidal activities of two quaternary ammonium chloride disinfectants, n-alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl benzyl ammonium chloride (10C-12C) were compared and effective concentrations were 1200 and 1800 ppm, respectively. Efficacies of these disinfectants were validated using real-time quantitative PCR. Results confirmed that the efficacy of n-alkyl dimethyl benzyl ammonium chloride was higher than alkyl dimethyl benzyl ammonium chloride (10C-12C). This assay was useful for rapid discrimination of killing potentials of disinfectants. In conclusion, these assays can be applied to other viruses that are unable to cause CPE in cell cultures and broadened the utility of DHBV as animal model for HBV.

Detection of duck hepatitis B virus DNA on filter paper by PCR and SYBR green dye-based quantitative PCR.

Duck hepatitis B virus (DHBV) belongs to the Hepadnaviridae family, which includes human Hepatitis B virus (HBV) and Woodchuck hepatitis virus. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-microl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was >or=4 degrees C. The optimal condition for storage was -70 degrees C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.