Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV).

The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6month-old lambs. Twelve groups of two 6month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 10(7.6) TCID(50)/lamb down to 10(-3.4) TCID(50)/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 10(0.6) TCID(50) seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 10(0.6) TCID(50) seroconverted during the 12months. The results of this study indicated that 10(0.6) or 4 TCID(50)/lamb given i.v. was capable of establishing infection.

Detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis.

Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer andprobe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virusand PVM , respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, andthe Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.

Pathogenicity, transmissibility, and tissue distribution of avian pneumovirus in turkey poults.

The pathogenicity, transmissibility, tissue distribution, and persistence of avian pneumovirus (APV) in turkey poults were investigated in three experiments. In the first experiment, we inoculated 2-wk-old commercial turkey poults oculonasally with APV alone or in combination with Bordetella avium. In the dually infected group, clinical signs were more severe, the virus persisted longer, the bacteria invaded more respiratory tissues, and the birds had higher antibody titer than the group exposed to APV or B. avium alone. In the second experiment, we studied the distribution of APV in different tissues in experimentally inoculated 2-wk-old commercial turkey poults. Only samples from sinuses, tracheas, and lungs were positive for APV by both reverse transcriptase-polymerase chain reaction and virus isolation. In the third experiment, we studied the ability of APV to spread among birds in 1-wk-old commercial turkey poults inoculated oculonasally. The virus was isolated and the viral RNA was detected in the inoculated and direct contact birds. The virus was not isolated, viral RNA was not detected, and no antibodies were detected in the indirect contact birds. These birds were placed in different cages in the same room where the airflow was directed from the infected toward the uninfected indirect contact group.

Protective efficacy of high-passage avian pneumovirus (APV/MN/turkey/1-a/97) in turkeys.

A U.S. isolate of avian pneumovirus (APV), APV/MN/turkey/1-a/97, was attenuated by serial cell culture passages in chicken embryo fibroblasts (seven passages) and Vero cells (34 passages). This virus was designated as APV passage 41 (P41) and was evaluated for use as a live vaccine in commercial turkey flocks. The vaccine was inoculated by nasal and ocular routes in 2-to-4-wk-old turkeys in 10 turkey flocks, each with 20,000-50,000 birds. Only 2 birds per 1000 birds were inoculated in each flock with the expectation that bird-to-bird passage would help spread the infection from P41-exposed birds to their respective flock mates. The virus did spread from vaccinated birds to the entire flock within 10 days as detected by reverse transcription-polymerase chain reaction. Mild respiratory illness was observed in a few birds 12 days postvaccination in 2 of 10 flocks. Within 3 wk postvaccination, all flocks became seropositive for APV antibodies as measured by enzyme-linked immunosorbent assay. In an additional flock, the virus was administered to all turkeys simultaneously in drinking water and seroconversion occurred within 2 wk. All 11 flocks remained seropositive until 10 wk postvaccination. When compared with unvaccinated flocks on the same farm from the previous year, the medication cost, total condemnation, and mortality rates attributed to APV were lower in P41-vaccinated flocks. When birds from vaccinated flocks were challenged with virulent APV under experimental conditions, no clinical signs were observed at 2, 6, and 10 wk postvaccination, whereas in the control unvaccinated birds, respiratory illness and virus shedding occurred after challenge. These results indicate that P41 administered by the nasal and ocular routes, and by drinking water, causes seroconversion and induces protection from virulent APV challenge for at least 10 wk.

PCR-based detection of an emerging avian pneumovirus in US turkey flocks.

Avian pneumovirus (APV) or turkey rhinotracheitis virus (TRTV) is an important respiratory pathogen of domesticated poultry in many countries in Europe, Africa, and Asia. Until recently, the United States was considered free of APV. In late 1996, an atypical upper respiratory tract infection appeared in turkey flocks in Colorado and shortly thereafter in turkey flocks in Minnesota. An avian pneumovirus (APV-US) that was serologically distinct from the previously described TRTV was isolated as the primary cause of the new syndrome. The nucleotide sequence of a fragment of the APV-US fusion gene was determined and used to develop a polymerase chain reaction-based assay that specifically detects APV-US viral nucleic acid sequences in RNA extracts of tracheal swabs and turbinate homogenates. The assay is highly sensitive in that it can detect <0.01 TCID50 of APV. The availability of this assay enables the rapid and accurate determination of APV-US in infected poultry flocks.

Avian pneumovirus infections of turkeys and chickens.

Avian pneumoviruses (APVs) cause major disease and welfare problems in many areas of the world. In turkeys the respiratory disease and the effect on egg laying performance are clearly defined. However, in chickens, the role of APV as a primary pathogen is less clear, although it is widely believed to be one of the factors involved in Swollen Head Syndrome. The mechanisms of virus transmission over large distances are not understood, but wild birds have been implicated. APV has recently been reported in the USA for the first time and the virus isolated was a different type or possibly a different serotype from the APVs found elsewhere. Good biosecurity is crucial for controlling infection and highly effective vaccines are available for prophylaxis. Although different subtypes and possibly different serotypes exist, there is good cross protection between them. Diagnosis is usually based on serology using ELISAs, but the available kits give variable results, interpretation is difficult and improved diagnostic tests are required.