Joseph J. Giambrone

Detection of genetic variations in serotype I isolates of infectious bursal disease virus using polymerase chain reaction and restriction endonuclease analysis

Reverse transcription with polymerase chain reaction (PCR) followed by restriction endonuclease analysis detected genetic variations among serotype I isolates of infectious bursal disease virus (IBDV). Using a set of synthetic primers derived from the large genome segment of APHIS-IBDV, the hypervariable region (AccI-SpeI fragment) located in the VP2 gene was amplified. With all strains, a cDNA fragment of approximately 643 bp was amplified, indicating that there were no apparent deletions or insertions in this region among isolates. Fragments amplified from 9 isolates were digested with 14 restriction enzymes. Restriction fragment profiles generated by restriction enzymes NaeI, StuI, TaqI, and SacI, showed genetic variations among isolates. This study provided a simple and sensitive method for detection of genetic variations among isolates that are closely related serologically and could not be differentiated using current serologic methods.

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

Abstract 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.

Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana.

VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsisthaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciensby electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.

Monoclonal antibodies that recognize specific antigens of Mycoplasma gallisepticum and M. synoviae.

The polypeptide profiles of the type strains of Mycoplasma gallisepticum (PG 31) and M. synoviae (WVU 1853) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were compared. Except for a few discrete peptides that were similar, the species varied considerably in peptide profiles. Congruence was observed between the type strains of each species and homologous cloned serotypes. Protein blots of each species were probed with 2 mouse monoclonal antibodies. Monoclonal antibody G 46 was specific for the antigen p 110 (G) in M. gallisepticum, and S 221 was specific for an antigen complex p 45-50 (S) in M. synoviae. The 2 monoclonal antibodies clearly distinguished between all serotypes of M. gallisepticum and M. synoviae that were examined by Western blot transfer. Autoradiographs of 125I-labeled M. gallisepticum and M. synoviae indicated that p 110 (G) and p 45-50 (S) were surface membrane peptides. Indirect immunofluorescence of M. gallisepticum and M. synoviae in Vero cell cultures supported the autoradiographic findings. The p 110 (G) antigen of M. gallisepticum was heat-stable, pronase-sensitive, and resistant to periodate oxidation, suggesting that its chemical composition is protein. In contrast, the p 45-50 antigen complex of M. synoviae appeared as a broad band in protein blots treated with monoclonal antibody S 221, was sensitive to pronase, and responded to Schiffs reagent but was not completely inhibited by periodate oxidation, suggesting that it is a complex of repeating sequences probably composed of glycosylated peptides.

Immunosuppression in Specific-Pathogen-Free Broilers Administered Infectious Bursal Disease Virus Vaccines by In Ovo Route

SUMMARY. 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.

Molecular characterization of avian reoviruses using nested PCR and nucleotide sequence analysis.

A nested polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis identified and differentiated avian reoviruses (ARVs). PCR products amplified from the S1 gene segment of ARV of USA isolates were 738 and 342 bp, respectively. PCR products were conformed by Southern and dot blot hybridizations. The amplified cDNA fragments were cloned into the pUC18 vector and subjected to DNA sequencing. The nucleotide and deduced amino acid sequences of four USA (S1133, 1733, 2408, and CO8) and two Australian isolates (RAM-1 and SOM-4) were compared. Results of paired difference analysis and a predicted dendrogram revealed that USA isolates were closely related, but different from, Australian isolates. The deduced amino acid sequences of the N-terminal region of ARV sigma C showed a heptapeptide repeat of hydrophobic residues in all ARV isolates.

Correlation of Circulating Antibody and Cellular Immunity with Resistance Against Cryptosporidium baileyi in Broiler Chickens

The correlation of circulating antibody and cell-mediated immunity (CMI) with resistance to Cryptosporidium baileyi was studied using hormonal and chemical bursectomy in the one experiment and cyclosporin A in a second experiment. In Expt. 1, there was no correlation between antibody (confirmed by enzyme-linked immunosorbent assay) and resistance to infection as measured by body weight, gross lesions, morbidity, and mortality. Bursectomy altered antibody production, but not CMI, as measured by the delayed-type hypersensitivity skin reaction. In Expt. 2, cyclosporin A reduced CMI, but not antibody production. Chicks treated with cyclosporin A were more susceptible to C. baileyi (more severe respiratory disease) than untreated controls. Results suggested that CMI is more important in resistance to C. baileyi than circulating antibody.

Detection of Duck Hepatitis B Virus DNA on Filter Paper by PCR and SYBR Green Dye-Based Quantitative PCR

ABSTRACT 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-μl 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 ⩾4°C. The optimal condition for storage was −70°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.

Detection and characterization of avian influenza and other avian paramyxoviruses from wild waterfowl in parts of the southeastern United States

Cloacal swabs were taken from migratory hunter-killed, nonmigratory, nesting waterfowl and migratory shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Samples were processed in embryonated eggs followed by hemagglutination (HA), Directigen, and real-time reverse transcription-PCR tests. Sequence analysis of the hemagglutinin (H) gene of the H10N7 Alabama isolate revealed that it was closely related (98%) to recent isolates from Delaware and Canada, but only 90% related to an H10N7 isolated 30 yr ago. Four isolates had 94 to 97% similarity to published H1N1 isolates including one from swine. No H5 or H7 isolates were found. One sample was highly pathogenic in embryos, produced a high HA titer, and was positive for both avian influenza (AIV) and Newcastle disease virus or avian paramyxovirus (APMV)-1. In recent (2008) sampling, more (14%) AIV, APMV, or both were isolated than in 2006 to 2007 (1% isolation rate). The higher isolation rate during 2008 may be attributed to optimized sample collection, storage in dry ice, new egg incubator, healthier eggs, time or habitat for isolation, species sampled, migratory status of birds, and more experience with detection procedures. An additional egg passage resulted in increased viral titer; however, no HA-negative samples became HA positive. The chance of transmission of APMV or low-nonpathogenic AIV from wild waterfowl to commercial poultry is possible. However, the chance of transmission of H5 or H7 AIV isolates from waterfowl to commercial farms in Alabama, Georgia, or Florida is unlikely. Therefore, continual testing of these birds is justified to ensure that H5 or H7 AIV are not transmitted to commercial poultry.

Detection of Infectious Bursal Disease Vaccine Viruses in Lymphoid Tissues After In Ovo Vaccination of Specific-Pathogen-Free Embryos

Control of infectious bursal disease virus (IBDV) by vaccination is important for poultry production worldwide. Two vaccines, an IBDV immune complex (ICX) vaccine and an IBDV-2512 vaccine, were administered at 100 mean embryo infectious dose to specific-pathogen-free 18-day-old broiler embryos in ovo. At 3, 6, 9, 15, and 21 days post in ovo vaccination (PIOV), bursa, spleen, and thymus tissues were collected and analyzed for virus protein by antigen capture chemiluminescent enzyme-linked immunosorbent assay (ELISA). Chicks were bled and antibody titers were determined by the antibody ELISA. At 21 days PIOV, chickens were challenged with a 1:500 dilution of an antigenic standard IBDV strain. At 28 days PIOV, birds were euthanatized and bursa weight:body weight ratios were determined. Embryos vaccinated with either vaccine exhibited 92% hatchability; however, within 1 wk of hatch, birds vaccinated with IBDV-2512 showed 56% mortality, whereas those given IBDV-ICX had only 3.2% mortality. Both IBDV-ICX and IBDV-2512 vaccines were detected in bursa, spleen, and thymus at day 3 PIOV. A 5-day delay in virus replication was observed with IBDV-ICX vaccine. By day 15 PIOV, the IBDV-ICX was no longer detectable in the bursa and spleen but persisted in the thymus. The IBDV-2512 vaccine persisted in the spleen and thymus on day 15 PIOV. By day 21 PIOV, neither vaccine virus was detected in any lymphoid organ. This assay can be useful in the early detection of vaccine virus in the tissues of chickens vaccinated via the in ovo route. Both vaccines caused bursal atrophy at all times PIOV. The IBDV-2512 caused splenomegaly at day 6 PIOV, whereas splenomegaly was not seen in IBDV-ICX-vaccinated birds until day 9 PIOV. Thymus atrophy was observed in IBDV-2512-vaccinated chicks from day 3 PIOV, whereas this occurred on day 15 PIOV in IBDV-ICX-vaccinated birds. Bursa weight: body weight ratios in IBDV-ICX-vaccinated unchallenged and vaccinated challenged birds were not different (P < 0.05).