Molecular and phenotypic characterization of infectious bursal disease virus isolates.

Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.

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 Arabidopsis thaliana 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 tumefaciens by 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.

In situ hybridization, immunohistochemistry, and in situ reverse transcription-polymerase chain reaction for detection of infectious bursal disease virus.

Development of molecular techniques for the detection of infectious bursal disease virus (IBCV) is an important area of research. An in situ hybridization (ISH) test was developed with a 491-bp cDNA fragment derived from the VP2 gene of IBDV. The fragment was amplified and simultaneously labeled with incorporation of digoxigenin-11-dUTP in a nested polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 491-bp nested PCR product was used as probe for ISH to detect and localize IBDV RNA in formalin-fixed, paraffin-embedded bursae of Fabricius from chickens both experimentally infected as well as commercially reared. Bursae from six clinically ill commercial broilers suspected to be IBDV infected were examined by ISH and immunohistochemistry. In two samples, IBDV infection was detected by both ISH and immunohistochemistry, whereas in the other two histologically normal bursae, IBDV was detected only by ISH. Two commercial chickens with atrophied bursae were negative by both ISH and immunohistochemistry. No positive IBDV stained cells were in RNase treated sections from infected birds, uninfected chickens, or reovirus-infected chickens. The ISH test developed herein resulted in important modifications, which makes it superior to other previously published procedures. We also described a direct in situ reverse transcription-polymerase chain reaction method for the amplification and detection of IBDV genome in formalin fixed, paraffin-embedded bursae of Fabricius with a single primer pair with direct incorporation of digoxigenin-11-deoxyuraciltriphosphate (dUTP) into the amplicon. Both molecular tests with their important modifications represent improved detection of IBDV.

Sequence analysis of the VP2 hypervariable region of nine infectious bursal disease virus isolates from mainland China.

The VP2 hypervariable region of infectious bursal disease virus (IBDV) from nine Mainland Chinese strains was amplified by reverse transcriptase/nested polymerase chain reaction and cloned into pGEM-T vector. The nine isolates, which were from the center (HN3), the north (Bj-1, B2/28, HD96), the east (JS-18 and AH-2), the northeast (D11-2, C4-2), and the west (Ts) of China, were sequenced and compared with each other and with six reference IBDV sequences. Clustering analysis separated the nine isolate into two groups. The six virulent isolates, propagated in bursae, formed the first group. They revealed only one to three amino acid changes from the very virulent (vv) European and Japanese isolates, suggesting that they might have the same origin as European and Japanese vvIBDV strains. On the basis of their distinct geographic origins, extensive dissemination of vvIBDV in China was indicated. (The other three chicken embryo fibroblast cell cultured isolates with mild pathogenicity were placed in the second group.) Their sequences correlated closely with those of the culture-adapted strains (Cu-1 (4) and Cj-801). None of the nine isolates showed very close sequence relationship with the antigenic variant strains from the USA. Although antigenic variants have been reported in China, the reverse transcriptase/polymerase chain reaction-restriction endonuclease analyses of the nine viruses tested herein were not similar to any U.S.A. variant strains on the basis of computer software analysis. Our results and conclusions agree with a previous molecular study of IBDV isolates from the south of China.

Comparison of several enzymes between normal physeal and tibial dyschondroplastic cartilage of broiler chickens.

Normal physeal and dyschondroplastic cartilage of broiler chickens was examined for six enzymes by isoelectric focusing in thin-layer polyacrylamide slab gels. Acid phosphatase (ACP), esterase (EST), malate dehydrogenase (MDH), and peroxidase (PRX) were present in the normal physeal cartilage but not in the dyschondroplastic cartilage. Staining intensity of glucose-6-phosphate isomerase (GPI) and triose-phosphate isomerase (TPI) was reduced in the dyschondroplastic cartilage compared with that of the physeal cartilage. Differences in the presence of these enzymes possibly demonstrated their roles in processes of bone formation, cartilage resorption, and calcification. ACP could be involved in calcification. Lack of EST and PRX may be related to the failure of vascular invasion in dyschondroplastic cartilage of afflicted birds. A deficiency of MDH and reduced GPI and TPI in dyschondroplastic cartilage may reflect a reduction in the activity of energetic metabolism, causing the dissipation of energy and necrotic cells.

Promoter and transcription of type X collagen gene in broiler chickens with tibial dyschondroplasia.

Type X collagen is produced exclusively in hypertrophic chondrocytes of the growth plate of the proximal tibiotarsus and is believed to play an important role during normal development from chondrogenesis to osteogenesis. Chondrocytes of chickens with tibial dyschondroplasia (TD) fail to attain full hypertrophy and the amount of type X collagen, being a marker of hypertrophy, is likely to be reduced. It is not clear whether transcriptional regulation is functional for expression of the type X collagen gene in TD birds. Nucleotide sequence of the type X collagen gene promoter was determined by sequencing PCR-based DNA clones. Nucleotide identity of this fragment between the normal and TD carriers was 97.6%. Both normal and TD birds were similar in a putative transcription start site, the site of TATAA box, and neither had a CCAAT box. However, there were two gaps in TD carriers, four gaps in normals, and five nucleotide substitution sites. By rapid amplification of cDNA ends by PCR (RACE-PCR), transcription of the gene was assessed using total RNA and mRNA from both normal chondrocytes and TD lesions at 3 and 4 wk of age. The RACE-PCR product for type X collagen mRNA was detectable in both normal and TD birds at two stages. No difference was found between them. This result does not support the hypothesis that transcriptional regulation of type X collagen gene is important in TD development of chickens. Variations in the promoter region did not affect transcription of type X collagen gene in TD carrier chickens.

Random amplified polymorphic DNA comparisons among broiler lines selected for incidence of tibial dyschondroplasia.

Lines selected for high (H) and low (L) incidence of tibial dyschondroplasia (TD) for eight generations and a randombred control (C) line of broiler chickens were fingerprinted by random amplification of genomic DNA mixed from 20 individuals of each line with 20 oligonucleotide primers. Among these 20 primers, 15 could distinguish the H from the L line, 14 the H from the C line, and 13 the L from the C line. Band sharing (BS), on the average over 20 primers, was .7 for the H vs L comparison and .8 for both H vs C and L vs C comparisons. The levels of BS calculated from individuals was .6 between the H and L line, .7 between the H and C line, and .7 between the L and C line. The ranking of BS values obtained from individual DNA samples was consistent with that obtained from the mixed DNA samples. Genomic distance between divergently selected lines (H vs L) was larger than that between the divergently selected lines and randombred line (H vs C and L vs C). Individual variation within lines was detected in spite of eight generations of selection. Results showed that eight generations of divergent selection for TD incidence in broiler chickens had resulted in genetic variation among lines. The procedure of random amplified polymorphic DNA assay using mixed DNA samples could be used to evaluate genetic distance among lines of chickens.

Production and characterization of monoclonal antibodies against variant A infectious bursal disease virus.

Monoclonal antibodies (MAbs) were produced against a variant subtype of infectious bursal disease virus (IBDV) from Delaware variant isolate A (Var-A). Splenocytes from immunized mice were fused to myeloma cells, and antibody-producing hybridomas were screened by dot-blot enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IF) against the homologous isolate. Specificity of the MAbs was tested against viral isolates representing all six serologic subtypes of IBDV and three untyped IBDVs--GLS, Ark, and Miss--found in serotype 1 by dot-blot ELISA and IF. The MAb G11 recognized all isolates, whereas H7 did not recognize two viruses in subtype 1, the Lukert strain and APHIS. MAbs G11 and H7 were not neutralizing and identified both the precursor proteins (VP2a) and protein product (VP2b of VP2) in Western immunoblots. Results showed an antigenic determinant in IBDV isolates and antigenic variation between subtype 1 viruses and other subtypes. This confirms and extends previous work that showed that IBDVs evolved from subtype 1 by alteration or substitution of antigenic sites.

Immunohistochemical detection of infectious bursal disease virus in formalin-fixed, paraffin-embedded chicken tissues using monoclonal antibody.

Immunoperoxidase and immunofluorescence techniques detected and localized infectious bursal disease virus (IBDV) in formalin-fixed paraffin-embedded sections of the bursa of Fabricius of experimentally and naturally infected chickens. The primary antibody was a monoclonal antibody that bound all IBDV serologic subtypes, including the GLS isolate. Both techniques were valuable in detecting IBDV. The presence and severity of microscopic lesions in the bursa correlated with the location and number of positive IBDV-infected cells as measured by either test. Mild vaccine strains induced minimal microscopic lesions and resulted in a few cells positive by either test. In contrast, virulent IBDV produced widespread lymphoid necrosis and numerous cells positive by both assays. The immunoperoxidase test was more useful than the immunofluorescence test, since the same section could be stained and examined by immunoperoxidase and then restained and examined for microscopic pathology. The presence of immunoperoxidase-positive stained cells associated with areas of microscopic pathology suggested that microscopic lesions were induced by IBDV.

Efficacy of simultaneous administration of Marek's disease and viral tenosynovitis vaccines to day-old broiler chickens.

Experiments were conducted to determine the efficacy of simultaneous administration at 1 day of age of the herpes virus of turkeys vaccine (HVT) and viral tenosynovitis (VT) vaccine. Day-old broilers from commercial breeders, which were immunized against both MD and VT, were used. The vaccines were injected at full dosage or diluted 1 to 4. Challenge with either Marek's disease virus (MDV; intraperitoneal) or VT virus (footpad) was at 7 days of age. At 7 wk of age, birds were weighed, killed, and examined for gross lesions. Either vaccine given at full dosage alone, or in combination, rendered birds resistant to homologous viral challenge. However, when either vaccine was diluted and administered alone, the efficacy of the VT but not the HVT vaccine was reduced, resulting in increased lesions and mortality and decreased weight gain after VT challenge. When the HVT vaccine was diluted and combined with undiluted VT vaccine, interference occurred, resulting in reduced efficacy of the HVT vaccine (increased tumors and mortality and reduced body weight). In contrast, combining the HVT at full dosage and the diluted VT vaccine given alone. The data indicate that caution should be maintained when mixing HVT and VT vaccines. For maximum efficacy, both vaccines should be mixed undiluted when administered simultaneously.

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 Schiff's reagent but was not completely inhibited by periodate oxidation, suggesting that it is a complex of repeating sequences probably composed of glycosylated peptides.

Effects of purified aflatoxin on broiler chickens.

Purified aflatoxin B1 (AFB1) or AFB1 plus aflatoxin B2 (AFB2) was given daily for 5 weeks in gelatin capsules to 2-week-old feather-sexed broilers. In Experiment 1, pure AFB1 was given in doses equivalent to the quantity of toxin received, if diets containing either 0, 200, 500 or 1000 ppb of AFB1 were consumed. In Experiment 2, pure AFB1 or AFB1 plus B2 was administered in capsules in doses equivalent to the quantity of toxin received, if diets containing either 0, 100, 200, or 400 ppb of AFB1 were consumed. In Experiment 1, pure AFB1 greater than or equal to 500 ppb was only mildly toxic. These levels produced a significant decrease in the 5-week weight gain and microscopic lesions indicative of alfatoxicosis. No morbidity, mortality, or effects on feed conversion or immune responses, however, were noted in birds given pure AFB1 at these levels. Gross liver lesions indicative of aflatoxin toxicity occurred at the 1000 ppb only. Results of Experiment 2 were similar to the first. Weight gain and feed conversion were not affected for broilers receiving pure AFB1 as low as 200 ppb. No morbidity, mortality, or gross lesions were evident in birds given either pure AFB1 or AFB1 plus AFB2 as high as 400 ppb. However, cell-mediated immunity as measured by a delayed hypersensitive skin test was significantly affected in birds receiving 400 ppb AFB1 plus AFB2. No effects on humoral immunity or the development of acquired immunity to Newcastle disease or fowl cholera vaccination were noted.

Effect of purified aflatoxin on turkeys.

Purified aflatoxin B1 (AFB1) or AFB1 plus aflatoxin B2 (AFB2) was given daily for 5 weeks in gelatin capsules to 2-week-old Nicholas X Nicholas turkeys. In Experiment 1, pure AFB1 was given in dosages equivalent to the quantity of toxin received if diets containing either 0, 200, 500, or 1000 ppb of AFB1 were consumed. In Experiment 2, either pure AFB1 or AFB1 plus AFB2 was given in capsules equal to the quantity of toxin received if a diet containing either 0, 100, 150, or 200 ppb of AFB1 was consumed. In Experiment 1, pure AFB1 administered by capsule equal to 500 and 1000 ppb was highly toxic and by the second week, it resulted in 100% morbidity, mortality, and gross and microscopic lesions. Aflatoxin B1 at 200 ppb caused none of these changes. However, it did cause a significant depression in feed conversion, but not weight gain, during all weeks and also a reduction in cell-mediated immunity (CMI) as measured by a delayed hypersensitive skin test to mycobacterium. Results in Experiment 2 were similar to the first. Aflatoxin B1 and AFB1 plus AFB2 as high as 200 ppb resulted in no morbidity, mortality, or gross or microscopic lesions. Also, no significant reductions in either weight gain or feed conversion were evident in any level of either AFB1 or AFB1 plus AFB2. However, numerical, but not statistically significant, dose-related reductions in all CMI tests were noted in birds receiving either AFB1 or AFB1 plus B2 at as low as 100 ppb.

Adoptive transfer of delayed hypersensitivity and protective immunity to Eimeria tenella with chicken-derived transfer factor.

Delayed hypersensitivity (DH) and protective immunity were transferred to nonimmune 4- and 10-week-old broiler chickens with transfer factor (TF) prepared from splenic leukocytes of chickens immunized with CocciVac D. Only chickens injected with the immune TF showed DH by wattle reaction to oocyst antigen and protective immunity to Eimeria tenella challenge infection. Chickens given a single injection of TF 5 days before challenge infection exhibited a significant (P less than .05) DH response at the time of infection. Immune TF preparations were active at concentrations of 100, 200, or 400 mg. Neither DH nor protective immunity was transferred to chickens injected with the TF-diluent control or nonimmune TF. The nonimmune TF was prepared from chickens kept free of coccidial infection. These findings indicated that TF prophylaxis produced beneficial results in nonimmune chickens by conferring some protection against challenge with E. tenella. The effects of TF on T-lymphocyte mediated protective immunity to coccidia in chickens are discussed.

Adoptive transfer of delayed wattle reactivity in chickens with a dialyzable leukocyte extract containing transfer factor.

Delayed wattle reactions (DWR) to tuberculin, diphtheria toxoid (DT), and keyhole limpet hemocyanin (KLH) were transferred to chickens with dialyzable leukocyte extracts (DLE) prepared from splenic leukocytes of chickens sensitized and reactive by DWR to the tuberculin, DT, or KLH. A DLE prepared from chickens unsensitized and unreactive to tuberculin by DWR failed to transfer DWR to tuberculin in recipients. Only chickens injected with DLE from tuberculin sensitized and reactive chickens exhibited significant (P less than .01) DWR to tuberculin. Chickens that received DLE prepared from DT sensitized and reactive chickens exhibited significant (P less than .01) DWR to DT but not to KLH. Further, DWR to both DT and KLH was transferred with DLE prepared from chickens sensitized to both antigens. Adoptive transfer of DWR to KLH in comparison to DT was more successful. Finally, the serial transfer of DWR to KLH (P less than .05) but not to DT (P greater than .05) was accomplished using DLE prepared from chickens that previously were recipients of DLE prepared from chickens sensitized to KLH and DT. Results indicate that DLE prepared from chickens contain transfer factor (TF) responsible for adoptive transfer of DWR to tuberculin, DT, and KLH.

Field trials with an oil emulsion infectious bursal disease vaccine in broiler breeder pullets.

Two independent field trials were undertaken with commercial broiler companies in separate sections of Alabama in order to test the efficacy of an experimental commercially prepared oil emulsion infectious bursal disease (IBD) vaccine in broiler breeder pullets. The IBD vaccine was tested both for safety and the production of neutralizing antibodies in pullets and their progeny as well as its ability to prevent early field exposure to virulent IBD virus (IBDV) in broiler progeny that may lead to immunosuppression resulting in poor performance. The following was demonstrated in both trials: 1) the IBD vaccine had no adverse effect on breeder performance, 2) the IBD antibody titers in breeders peaked at 10 weeks postvaccination (PV) and remained about 1.5 to 4 times higher than nonvaccinated breeders through 25 weeks (PV), 3) 1-day IBDV maternal titers in broilers were related to the parental titers, 4) the IBD vaccination in pullets prevented early infection to field IBDV in progeny as determined by antibody titers and various performance parameters.

Demonstration of Eimeria tenella in bursa of Fabricius of chickens.

Coccidial life-cytle stages were detected in the bursa of Fabricius of broiler chickens inoculated with Eimeria tenella, whether or not the chickens had previously been infected with infectious bursal disease virus (IBDV). Chickens infected only with E. tenella had developing parasites in the lining epithelium, whereas chickens with both infections had gametocytes also in the epithelial cells surrounding numerous degenerating bursal cysts.

Long term studies comparing the efficacy of cell-free versus cell-associated HVT vaccines against Marek's disease.

In long-term field trials lasting approximately 12 months attempts were made to determine whether the cell-free HVT vaccine was as effective in reducing MD losses as the cell-associated HVT vaccine. Four trials were conducted in which flocks were vaccinated with either the cell-free or the cell-associated HVT vaccine. The cell-free HVT vaccine was as safe and effective in preventing MD as the cell-associated HVT vaccine. Although there were some losses due to MD in all flocks, there were also losses due to lymphoid leukosis, multiple hemangioma, liposarcoma or coccidiosis. Egg production was essentially the same in flocks vaccinated with the cell-free HVT vaccine as in those vaccinated with the cell-associated HVT vaccine.