Daniel P. Shaw

Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs

A recent epizootic of swine infertility and respiratory syndrome (SIRS) in a Minnesota swine herd was investigated. Examination of a sow, neonatal piglets, and stillborn fetuses obtained during the epizootic from the affected herd revealed interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in the piglets and focal vasculitis in the brain of the sow. Fetuses did not have microscopic lesions. No cause for the infertility and respiratory syndrome was determined. Therefore, attempts were made to experimentally reproduce the disease. Eleven 3-day-old gnotobiotic piglets exposed intranasally to tissue homogenates of piglets from the epizootic became inappetent and febrile by 2–4 days postexposure and had interstitial pneumonitis and encephalitis similar to that seen in the field outbreak. After 2 blind passages in gnotobiotic piglets, tissue homogenates were cultured on continuous cell line CL2621, and a cytopathic virus (ATCC VR-2332), provisionally named SIRS virus, was isolated. Gnotobiotic piglets exposed intranasally to the SIRS virus developed clinical signs and microscopic lesions that were the same as those in piglets exposed to the tissue homogenates, and the virus was reisolated from their lungs. This is the first isolate of SIRS virus in the United States that fulfills Kochs postulates in producing the respiratory form of the disease in gnotobiotic piglets and the first report of isolation and propagation of the virus on a continuous cell line (CL2621). The virus is designated as American Type Culture Collection VR-2332.

Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys.

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.

Virulence factors of Escherichia coli associated with colisepticemia in chickens and turkeys

Four hundred twenty turkey and 80 chicken Escherichia coli isolates from colisepticemic birds were examined for the following properties: heat-labile toxin (LT), heat-stable enterotoxin, verotoxin, colicinogenicity, hemolysin, and hydroxamate/aerobactin production. Twenty-four (5.7%) of the 420 turkey isolates and six (7.5%) of the 80 chicken isolates produced an LT that was cytotoxic for both Vero and Y-1 cells. In contrast, 48 (11.4%) of the turkey isolates and 18 (22.5%) of the chicken isolates produced a distinct LT that was cytotoxic only for Vero cells. In addition, 64 (80.0%) of the chicken isolates and 309 (74.0%) of the turkey isolates produced aerobactin. Colicinogenicity occurred in 51 (64.0%) of the chicken isolates, with 41 (51.0%) producing colicin V. By contrast, 254 (61.0%) of the turkey isolates produced a colicin, of which 176 (42.0%) produced colicin V. None of the chicken and turkey isolates produced hemolysin or heat-stable enterotoxin.

Ornithobacterium rhinotracheale Infection in Turkeys: Experimental Reproduction of the Disease

This report details the first experimental production of clinical disease, mortality, and pathology resembling that of field infections by using Ornithobacterium rhinotracheale alone. Twenty-two-week-old male turkeys were exposed to O. rhinotracheale or lung homogenate from O. rhinotracheale-infected turkeys. Within 24 hr after inoculation, turkeys given O. rhinotracheale or lung homogenate intratracheally were depressed and coughing and had decreased feed intake. By 48 hr, several birds were coughing blood and ultimately died. Grossly, the lungs were reddened, wet, and heavy, failed to collapse, and were covered by tenacious tan-to-white exudate. Microscopically, the parabronchi and air capillaries were filled with fibrin, heterophils, macrophages, and small numbers of gram-negative bacteria. The pleura was often covered by a thick layer of fibrin, heterophils, and macrophages. Turkeys that survived to day 7 postinoculation had severe, subacute pneumonia. Ornithobacterium rhinotracheale was recovered from the lungs of most birds with pneumonia and was also cultured from the air sacs, sinuses, tracheas, spleens, and livers. All turkeys inoculated with O. rhinotracheale developed antibodies to O. rhinotracheale detectable by the serum plate agglutination test.

Isolation and identification of Ornithobacterium rhinotracheale from commercial turkey flocks in the upper midwest

Increased death loss was seen in a flock of 22-wk-old tom turkeys. The predominant postmortem lesion was fibrinopurulent pneumonia and pleuritis. Within 5 wk, turkey flocks on 17 other farms developed similar problems. All affected flocks during the 5-wk period were between 14 and 22 wk of age, and the severity of clinical signs and the degree of mortality increased with age. Ornithobacterium rhinotracheale was isolated in pure culture from affected lungs. Further investigation by tracheal swab culture of 261 flocks between 5 and 7 wk of age resulted in detection of O. rhinotracheale in 43% of the flocks.

Pathogenesis of Avian Pneumovirus Infection in Turkeys

Avian pneumovirus (APV) is the cause of a respiratory disease of turkeys characterized by coughing, ocular and nasal discharge, and swelling of the infraorbital sinuses. Sixty turkey poults were reared in isolation conditions. At 3 weeks of age, serum samples were collected and determined to be free of antibodies against APV, avian influenza, hemorrhagic enteritis, Newcastle disease, Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, Ornithobacterium rhinotracheale, and Bordetella avium. When the poults were 4 weeks old, they were inoculated with cell culture–propagated APV (APV/Minnesota/turkey/2a/97) via the conjunctival spaces and nostrils. After inoculation, four poults were euthanatized every 2 days for 14 days, and blood, swabs, and tissues were collected. Clinical signs consisting of nasal discharge, swelling of the infraorbital sinuses, and frothy ocular discharge were evident by 2 days postinoculation (PI) and persisted until day 12 PI. Mild inflammation of the mucosa of the nasal turbinates and infraorbital sinuses was present between days 2 and 10 PI. Mild inflammatory changes were seen in tracheas of poults euthanatized between days 4 and 10 PI. Antibody to APV was detected by day 7 PI. The virus was detected in tissue preparations and swabs of nasal turbinates and infraorbital sinuses by reverse transcription polymerase chain reaction, virus isolation, and immunohistochemical staining methods between days 2 and 10 PI. Virus was detected in tracheal tissue and swabs between days 2 and 6 PI using the same methods. In this experiment, turkey poults inoculated with tissue culture-propagated APV developed clinical signs similar to those seen in field cases associated with infection with this virus.

Avian pneumovirus infection in Minnesota turkeys: experimental reproduction of the disease.

Avian pneumovirus (APV) is an emerging viral respiratory disease agent of turkeys in Minnesota. Clinical signs of APV infection include open mouth breathing, ocular and nasal discharge, and swelling of infraorbital sinuses. The virus spreads rapidly among flocks of susceptible turkeys and is associated with increased mortality rates. A flock of 11-wk-old turkeys experienced a respiratory problem characterized by coughing, sneezing, swollen sinuses, and nasal discharge. The reverse transcriptase-polymerase chain reaction (RT-PCR) performed on tissues from the nasal turbinates and tracheal tissues was positive for avian pneumovirus. Turbinate tissue was inoculated into chicken embryo fibroblasts, and cytopathic effect was observed after five blind passages. In an attempt to reproduce the disease, 50 microl of this cell culture-propagated virus was instilled into each conjunctival space and nostril of 23-day-old turkey poults. The poults were sacrificed at 2-day intervals for 12 days, and serum, tissues, and tracheal and cloacal swabs were collected. Between days 2 and 10 after exposure, the poults developed ocular and nasal discharge and swollen sinuses. The virus was detected by RT-PCR and virus isolation from the nasal turbinates of poults sacrificed on days 4 and 6 postinoculation. Antibodies to APV were detected by enzyme-linked immunosorbent assay.

Specific detection of avian pneumovirus (APV) US isolates by RT-PCR.

Summary. This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 × 10−5 TCID50 (0.0323 μg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota.

Isolation of avian pneumovirus from mallard ducks that is genetically similar to viruses isolated from neighboring commercial turkeys.

Our earlier studies demonstrating avian pneumovirus (APV) RNA in wild geese, sparrows, swallows, starlings and mallard ducks suggested that wild birds might be involved in the circulation of APV in the United States. To determine whether turkey virus can be transmitted to the free flying birds, we placed APV-negative mallard ducks next to a turkey farm experiencing a severe APV outbreak and in an area with a large population of waterfowls. The sentinel ducks did not develop clinical APV disease but infectious APV (APV/MN-12) was recovered from choanal swabs after 2 weeks, and anti-APV antibodies detected after 4 weeks. Four APV isolates recovered from the neighboring turkeys that were experiencing an APV outbreak at the same time shared 95-99% nucleotide identity and 97-99% predicted amino acid identity with the duck isolate. In addition experimental infection of turkey poults with APV/MN-12 resulted in detection of viral RNA in nasal turbinates and APV-specific IgG in serum. These results indicate that the APV isolates from turkeys and ducks shared a common source, and the viruses from different avian species can cross-infect.

Biological and molecular characterization of H13N2 influenza type A viruses isolated from turkeys and surface water.

The pathogenicity potential of two H13N2 influenza viruses, one isolated from turkeys and the other isolated from surface water, was evaluated in turkeys, chickens, and mallard ducks (Anas platyrhynchos) after intracranial and oculonasal inoculation. Both isolates replicated in turkey poults, causing depressed weight gain, morbidity and mortality; both also caused histopathological lesions, such as mild to severe pancreatitis, hepatitis, and nephritis in turkeys. These isolates replicated in mallard ducklings but not in chickens. There was depressed weight gain in ducklings given the H13N2 isolate from water. Neither isolate caused morbidity or mortality in ducklings or chicks after inoculation.