Isolation-reared mallards (Anas platyrhynchos) were placed on ponds in turkey-rearing areas in Minnesota, and their cloacae were periodically swabbed to attempt isolating virus from embryonated chicken eggs. Nearby turkeys were sampled by taking cloacal and tracheal swabs as well as blood samples. Hemagglutinating viruses were identified at the National Veterinary Services Laboratory, U.S. Department of Agriculture, Ames, Iowa. During this two-year study, the weekly influenza virus-isolation rate from ducks varied from 0 to 24.4%. A total of 213 influenza viruses were isolated from the ducks. Twenty-six influenza virus subtypes were detected. Ninety-seven flocks of turkeys were diagnosed as having influenza by virus isolation and/or serology. Eight influenza virus subtypes were involved in the turkey outbreaks, and seven of these were also detected in the ducks and/or other avian species. The weekly infection rate of the sentinel ducks correlated directly with observations of wild ducks at the monitoring sites. Influenza virus was isolated from water samples collected near the sentinel duck sites during the study.

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To detect avian pneumovirus (APV) in central North America, nasal turbinates or choanal deft tissues from domestic turkeys and wild birds were examined for the presence of APV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas serum samples from domestic turkeys were analyzed for APV antibodies by enzyme-linked immunosorbent assay (ELISA). In 2002, the seroprevalence of disease in domestic turkeys in Minnesota remained high (42.3% of the flocks). In addition, there is evidence the disease has spread to turkey flocks in North Dakota (8.2%), South Dakota (7%), Iowa (10%), and Wisconsin (8.6%) as detected by RT-PCR and/or ELISA. House sparrows and ring-billed gulls sampled in Minnesota and snow geese from Saskatchewan, Canada, were found to harbor APV RNA. Sequence analysis of wild bird APV strains showed high amino acid sequence identity among wild bird isolates (<97%) and between wild bird and turkey viral isolates (93.2%-99.3%). This study demonstrated that APV infections were present in domestic turkey flocks and wild birds outside the state of Minnesota; however, the role of wild birds in spreading APV to domestic turkeys remains unclear.

Abstract. Antibodies to avian pneumovirus (APV) were first detected in Minnesota turkeys in 1997. Virus isolation was attempted on 32 samples (28 tracheal swabs, 4 pools of trachea and turbinates) that were positive for APV by reverse transcriptase polymerase chain reaction (RT-PCR). The cell cultures used were chicken embryo fibroblast (CEF), Vero cells, and QT-35 cells. Five virus isolates were obtained from these samples, and the identity of the isolates was confirmed by RT-PCR. Four isolates were obtained by inoculation of CEF cells, and 1 isolate was obtained in QT-35 cells after 3-7 blind passages in cell cultures. Vero cells did not yield any isolate on primary isolation; however, all 5 isolates could be adapted to grow in Vero cells following primary isolation in CEF or QT-35 cells. This is the first report of isolation of APV in Minnesota and also the first report of primary isolation of APV in QT-35 cells.An acute, upper respiratory tract disease of turkeys, turkey rhinotracheitis (TRT), has been reported from many countries since the late 1970s. 10 The clinical signs consist of profuse ocular and nasal discharge, facial edema or swelling, and respiratory distress. In laying birds, a drop in egg production associated with slight respiratory distress is observed. The clinical signs become more severe if complicated by secondary bacterial infections. The morbidity is often 100%, and the mortality ranges from 0.4% to 90%. 11 The disease is caused by TRT virus (TRTV), which is classified on the basis of polypeptide and messenger RNA analyses as an avian pneumovirus (APV) in the subfamily Pneumovirinae of family Paramyxoviridae. 11 Besides being the cause of TRT in turkeys, TRTV is also suggested to cause swollen head syndrome in chickens. 2,3,13,15 The TRTV infection of turkeys has been reported in many countries, e.g., Great Britain, South Africa, France, Israel, Spain, Germany, Italy, and The Netherlands. 11 Nucleotide sequencing and serologic studies using polyclonal and monoclonal antibodies showed that TRTV could be separated into 2 groups. Subgroup A includes the early British and French strains, and subgroup B includes the other continental European strains. 9 In the USA, clinical signs of APV infection were first reported in turkey flocks in Colorado in 1996. Before that, the USA, 12 Canada, 7 and Australia 1 were considered to be free from APV. However, APV was isolated from turkey flocks in Colorado in February 1997. 14 Outbreaks of similar illness were reported in Minnesota in March 1997 with clinical signs of depression, coughing, sinusitis, airsacculitis, and mortality. Because Minnesota is one of the major turkeyproducing states, APV infections in that state can have a significant economic impact.Routine methods for the primary isolation of APV consist of specimen inoculation (tracheal swabs, trachea, turbinate, lungs) in embryonated chicken eggs (ECE) or turkey eggs (ETE) or in tracheal organ cultures (TOC). Subsequently, the virus is adapted to chicken embryo fibroblast (CEF) or Vero cells....

BackgroundThe rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains.Methodology and Principal FindingsWe evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of ∼230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs.ConclusionsWe describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.