Holly S. Sellers

Data from 11 years of molecular typing infectious bronchitis virus field isolates.

Abstract In 1993, a new molecular typing method for infectious bronchitis virus (IBV) was introduced. This method uses reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis of the spike gene to obtain RFLP patterns that correlate with serotype. Using that test at the Poultry Diagnostic and Research Center (PDRC, University of Georgia, Athens, GA), we have identified a total of 1523 IBV isolates in the past 11 yr. The data were obtained from clinical samples submitted to our laboratory from birds with clinical signs characteristic of IBV infection. The samples are primarily from the southeastern United States but are also from many other states as well as from outside the United States. Most of the isolations occurred during July, followed by May, April, November, October, and January. The fewest number of isolates identified on an annual basis was 20 in 2003. An unusually high number of isolations occurred in 1997 (318 isolations) and 1999 (246 isolations), which coincided with the GAV variant virus and GA98 variant virus outbreaks respectively. By far, the Ark-DPI strain was the most frequently identified type of IBV and ranged from 23% to 65% of total isolations per year. Ark-like isolates, defined as having a similar but unique RFLP pattern from the Ark-DPI vaccine strain were identified every year of the study except in 1996. In addition, new Ark-like isolates continued to emerge each year (except in the year 2000) beginning in 1997, reflecting the ability of that IBV type to undergo genetic drift. Eighty-two different variant viruses were identified although only two (GAV and GA98) became persistent and caused widespread disease. Some viruses tended to be geographically restricted to a given area (CAV in California and MX97-8147 in Mexico), whereas others were widespread (Ark-DPI, Conn, DE072, and Mass). The Florida, Gray, Holte, Iowa, and JMK types were not detected during the 11-yr period, and no foreign virus types were detected in the United States. These data show that IBV variant viruses are consistently circulating in commercial poultry and are capable of causing disease outbreaks. Our observations highlight the importance of constantly monitoring IBV as well as other coronaviruses like severe acute respiratory syndrome-coronavirus that have the ability to change and emerge to cause disease in a susceptible host.

Multiplex Real-Time Reverse Transcription–Polymerase Chain Reaction for the Detection of Three Viruses Associated with Poult Enteritis Complex: Turkey Astrovirus, Turkey Coronavirus, and Turkey Reovirus

Abstract Poult enteritis complex (PEC) is an economically important disease of young turkeys characterized by diarrhea, poor weight gain, and, in some cases, high mortality. Although PEC is considered to be a polymicrobial disease, numerous viruses, including turkey coronavirus (TCV), turkey astrovirus type 2 (TAstV-2), and avian reoviruses (ARVs), have been associated with PEC-like disease. Real-time reverse transcription–polymerase chain reaction (RRT-PCR), a highly sensitive and specific detection method for viral RNA, was developed in a multiplex format for the simultaneous detection of TAstV-2 and TCV and for the detection of two genetic types of ARV. Assay sensitivity was determined using in vitro transcribed RNA and varied by target between 150 gene copies for TAstV-2 alone and 2200 gene copies for TCV when multiplexed. Virus detection was evaluated with samples collected from poults inoculated at 1 day of age with each of the viruses. Cloacal swabs and intestinal samples were obtained at 1, 2, 3, 4, 6, 9, 14, 17, and 21 days after inoculation, processed, and tested for virus detection by RRT-PCR. Cloacal swabs from TAstV-2– and TCV-infected poults were shown to have sensitivity for virus detection similar to that of intestinal samples when compared directly. ARV detection by RRT-PCR was compared with virus isolation and had similar sensitivity.

Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.

Abstract We have constructed a DNA vaccine (pDKArkS1-DPI) expressing the S1 glycoprotein (Arkansas DPI) of infectious bronchitis virus (IBV) to examine protective immunity after in ovo and intramuscular DNA immunization. Birds receiving in ovo DNA followed by live virus vaccination at 2 wk of age were 100% protected from clinical disease. Birds receiving only live virus vaccine or only in ovo DNA vaccination were ≤80% protected. IBV was detected up to 10 days postchallenge in unvaccinated control groups, whereas birds receiving in ovo DNA and live virus vaccination cleared IBV from tracheal samples before day 5 postchallenge. Transcription of the S1 gene was confirmed in lung tissue after in ovo vaccination by an antisense riboprobe, and the S1 protein was detected by immunohistology in the heart and bursa. In a separate experiment, birds were injected intramuscularly with either 50, 100, or 150 µg of the DNA vaccine at 1 day of age and then again with either 100, 200, or 300 µg of the DNA vaccine, respectively, at 14 days of age. At 10 days postchallenge, no clinical signs were observed and no challenge virus was reisolated from the birds vaccinated with 150 µg and 300 µg of DNA. Between DNA-vaccinated birds and nonvaccinated control birds, no statistical differences were observed for IBV-specific serum antibodies as detected by enzyme-linked immunosorbent assay or the virus neutralization test. These data indicate that DNA vaccination with the S1 gene either in ovo or intramuscularly can provide birds with some protection against clinical disease after homologous IBV challenge.

Immunization of Turkeys with a DNA Vaccine Expressing Either the F or N Gene of Avian Metapneumovirus

Abstract SUMMARY. In this study we compared protection by DNA vaccination with the F (pCMV-F) or N (pCMV-N) gene from avian metapneumovirus (aMPV) in turkeys. One-week-old turkey poults received two intramuscular injections 2 wk apart. Birds were challenged with a turkey-embryo-adapted aMPV at 5 wk of age. Birds vaccinated with pCMV-F had decreased clinical signs of disease as well as significantly reduced virus load in tracheal swabs compared with birds vaccinated with pCMV-N or unvaccinated control birds. Serum neutralizing antibodies were significantly higher in birds receiving pCMV-F compared with all other groups. These results indicate that DNA vaccination with the F, but not N, gene of aMPV can induce significant protection against aMPV infection.

Mild Infectious Laryngotracheitis in Broilers in the Southeast

Abstract 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.

Development of a Multiplex Reverse Transcription–Polymerase Chain Reaction Diagnostic Test Specific for Turkey Astrovirus and Coronavirus

Abstract A multiplex reverse transcription–polymerase chain reaction (RT-PCR) assay was developedfor the simultaneous detection of two enteric viruses of poultry: turkey enteric coronavirus (TCV) and turkey astrovirus (TAstV). PCR primers were designed to conserved regions within the nucleocapsid gene of TCV and to the polymerase gene of TAstV-2. The primer pairs were successfully used in a multiplex RT-PCR to detect nucleic acid of TAstV-2 and TCV. The test was optimized for use with intestines/feces from naturally infected turkeys. The primers were specific and did not amplify other common RNA or DNA avian viruses. The detection limit was determined to be 10 ng of RNA used as starting template. The use of this specific test allows the rapid and early diagnosis of two financially costly viruses affecting the commercial turkey industry.

Investigation into the aetiology of runting and stunting syndrome in chickens

Currently, the aetiology of runting and stunting syndrome (RSS) in chickens is unknown. The impact of RSS on weight gain and microscopic lesions in immunological organs and the duodenum, was investigated in 1-day-old commercial broilers at 12 days following exposure to RSS-contaminated litter. Furthermore, the presence of the viral nucleic acids of three astroviruses and one parvovirus was analysed by in situ hybridization from days 1 through 5 post exposure. A 70% decrease in weight was observed in the RSS-exposed group at the end of the experiments when compared with the unexposed controls. Lesions in the bursa of Fabricius and thymus were present in both groups but were significantly higher at the end of the study in the RSS-exposed group. In contrast, no significant difference in Harderian gland lesions was observed between the groups. Histological lesions in the duodenum were already present 24 h after exposure in the RSS-exposed group only, peaked at day 4 and declined until the end of the study. Results of the in situ hybridization studies clearly indicate replication of three astroviruses (chicken astrovirus, avian nephritis virus [ANV]-1, ANV-2) in the duodenum but not in other organs evaluated. Chicken astrovirus nucleic acids were detected on days 1 and 2 post exposure, while ANV-1 and ANV-2 nucleic acids were observed on several days during the period investigated. Surprisingly, no viral nucleic acid specific for the chicken parvovirus was observed. The results indicate that astroviruses probably play an important role during RSS due to the concurrence of viral RNA detection and lesions in the duodenum.

Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates.

ABSTRACT Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolates pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

Phylogenetic Analysis of the Sigma 2 Protein Gene of Turkey Reoviruses

Abstract The open reading frame of the S3 segment encoding the σ2 protein of four turkey reovirus field isolates was analyzed for sequence heterogeneity. The turkey reoviruses we present here have a 97% amino acid identity to turkey NC 98. The S3 nucleotide and amino acid sequence similarity was ≤61% and 78%–80%, respectively, when compared to the chicken reovirus isolates. Comparison of amino acid sequences from chickens and turkeys with that of a duck isolate revealed a 53% and 55% similarity, respectively. Phylogenetic analyses, based on both nucleotide and amino acid sequence, resulted in three major groups among the avian reoviruses; these groups were clearly separated by species. The results of this study provide further evidence, based on the deduced σ2 sequence, that turkey reoviruses form a distinct, separate group relative to chicken and duck isolates. In addition, as a result of the limited sequence identity with their avian counterparts, turkey reoviruses could potentially be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.

A purified recombinant baculovirus expressed capsid protein of a new astrovirus provides partial protection to runting–stunting syndrome in chickens

Abstract A new viral sequence likely belonging to a virus of the family Astroviridae was determined using the gut content of chickens affected with the runting–stunting syndrome (RSS) in chickens. Since the appropriate virus could not be isolated in cell culture the open reading frame of the viral capsid protein was cloned to generate a recombinant baculovirus. The protein was purified and used as an experimental vaccine in broiler breeders to provide maternal derived antibodies for the protection of the offspring. The presence of specific antibodies was monitored by an ELISA. The offspring of vaccinated breeder hens were partially protected in a RSS challenge model.