Detection of ovine respiratory syncytial virus in pneumonic lungs from apparently healthy sheep slaughtered at 5 abattoirs in Australia

Abstract

Respiratory disease is one of the main diseases of sheep in many regions globally. Respiratory syncytial virus (RSV) causes severe disease in humans and in calves, but little is known about the role of RSV in sheep. We studied the prevalence of ovine RSV in sheep processed at 5 abattoirs in southern Australia. Bronchial swab samples were collected from 182 consignments of lambs up to 12 months of age and 71 consignments of adult sheep; these were tested for the presence of the virus using a qPCR based on the F gene sequence. Six of the 253 abattoir consignments (2.4%) tested positive for ovine RSV. Four of the positive consignments were lambs and 2 were adult sheep. To our knowledge, this is the first report of the ovine strain of RSV in sheep with pneumonia from Australia. Further research is needed to clarify the role of RSV in pneumonia in sheep.

Respiratory disease is common in sheep, in part due to their anatomy (1), and is one of the main diseases of sheep in many regions globally (2). Data collected using post-mortem observations as part of the Australian National Sheep Health Monitoring Program indicate that up to 40% of Australian sheep flocks have endemic pneumonia (3). This aligns with our own observations on pneumonia in Australian sheep flocks, with 50% of the lines of lambs examined at an abattoir in the state of South Australia having evidence of pneumonia/pleurisy (4).

In Australia, bacterial pneumonia in sheep due to the combined effect of Mycoplasma ovipneumoniae, Mannheimia haemolytica, and Pasteurella multocida, is often called summer pneumonia (5). Hot dry weather, raised dust, summer storms, the first shearing and grain feeding can be stressful for lambs, contributing to clinical outbreaks of the disease. Four viruses have also been associated with respiratory disease in sheep: adenovirus, reovirus, parainfluenza virus type 3, and respiratory syncytial virus (6,7). Little is known about the prevalence of viral infections in sheep in Australia, and particularly the prevalence of RSV.

Respiratory syncytial virus causes severe respiratory disease in humans and in calves. Although less is known about how RSV affects sheep, antibodies to the virus are common in sheep in some parts of the world, with 35 to 64% of animals being seropositive (7–9). Sheep are susceptible to infection with bovine (10), human (11,12), and ovine (13) strains of RSV, with neonatal lambs proposed as a model for the study of respiratory disease in humans (14). Infection with ovine strains of the virus is not well-studied in sheep.

We studied the prevalence of ovine respiratory virus in sheep processed at 5 abattoirs in southern Australia in a non-blinded, cross-sectional, observational study with sample collection commencing in October 2020 and concluding in December 2021. Twenty-four abattoir visits to collect samples were conducted, with a minimum of 1500 sets of ovine lungs inspected per abattoir visit. During the 24 visits, samples were collected from 253 abattoir consignments of sheep, including 182 consignments of lambs up to 12 mo of age and 71 consignments of adult sheep over 12 mo old. No consideration was given to the sex or breed of the sheep.

Sample collection was conducted during 4 sampling periods: spring, summer, autumn, and winter, (subject to access restrictions from COVID-19) as lambs from different geographic regions of Australia are generally sent for slaughter at various times of the year. We aimed to collect 5 samples per abattoir consignment, although the fast-processing speed in Australian sheep abattoirs (10 to 11 per min) meant that in some smaller consignments less than 5 samples were obtained.

Samples were collected from ovine lungs with gross pathological signs consistent with pneumonia including cranio-ventral consolidation, and/or widespread lung mottling and areas of consolidation with or without a thickened pleura. Samples were collected by licensed veterinarians after identification of diseased lungs in the offal trays at the point of inspection. The dorsal surface of the trachea was opened with a knife and then the entire bronchial tree swabbed using a sterile viscose tipped sample collection swab, starting with the right cranial lobe and moving clockwise around the bronchial tree. The knife was cleaned at an abattoir boiling water station for a minimum of 5 s after each set of lungs. Swabs were preserved in 1 mL Sucrose Phosphate Glutamate Transport Medium (15) immediately after collection, placed on wet ice and frozen at −20°C within 12 to 24 h of sample collection. Courier delays with samples sent from Tasmania and Western Australia due to COVID-19 led to delays of up to 24 to 48 h in freezing these samples.

After defrosting at 4°C, viral RNA was extracted from transport media using the QIAamp MinElute Virus Spin Kit (Qiagen Australia Pty Ltd, Victoria, Australia) following kit instructions. Samples were processed in pools of 5, concentrated to 200 μL using a Amicon Ultra-2 or Amicon Ultra-4 Centrifugal Filter Unit (Merck Millipore Australia, Bayswater, Australia), depending on availability.

The testing for ovine RSV was based on the F gene sequence of the virus (16). Briefly, the virus F gene sequence was amplified using the forward primer OVRSV F 5′-CAGAGTTGGTTCACTATAC-3′, the reverse primer OVRSV R 5′-CTCTCCCTCTAAATGTAATAC-3′ and the probe 5′-/6-FAM/ATTCCACTCGCAATAGCAGACC-BHQ-3′. Each 20 μL mixture contained 10 μL KiCqStart One-Step Probe RT-qPCR ReadyMix (Sigma Aldrich Pty Ltd, North Ryde, Australia), 0.8 μM of each primer, 0.25 μM probe, 4 μL Water for Molecular Biology (Sigma Aldrich Pty Ltd) and 5 μL sample DNA. The PCR cycling conditions consisted of 50°C for 10 min, 95°C for 3 min, followed by 50 cycles of 95°C for 10 s, 56°C for 30 s and 72°C for 45 s. Samples were checked for inhibition using the QuantiNova IC Probe Assay and IC RNA (Qiagen Australia Pty Ltd) following kit instructions. Viral and inhibition control assays were run as single-plex reactions during the same PCR run. Polymerase chain reaction contamination controls were used, including negative controls and separation of preparation and amplification areas.

Six of the 253 abattoir consignments (2.4%) sampled during the 24 abattoir visits tested positive for ovine RSV. Four of the positive consignments were lambs and 2 were adult sheep. To our knowledge, this is the first report on the ovine strain of RSV in sheep from Australia that had pneumonia.

Lambs are considered a good model to study RSV in humans because of the similarities in lung alveolar development and structure, immune responses, and susceptibility to infection (12,17,18). Pulmonary pathology in lambs after infection with RSV is similar to that observed in humans (11,14). Infection with the bovine strain of RSV has been shown experimentally to facilitate pulmonary infection with M. haemolytica in lambs (19). This suggests that our detection of the ovine strain of RSV in sheep with pneumonia in Australia is significant and that the virus may be contributing to the pneumonia problem in these sheep. Further research is needed to understand the role of RSV in pneumonia in sheep.

There are several limitations to this study. Firstly, sheep are susceptible to infection with bovine, human, and ovine strains of RSV, and we tested only for the ovine strain of the virus. However, most sheep in Australia are managed extensively with limited human contact. This reduces the likelihood of human strains of the virus playing a key role in pneumonia in these sheep. In addition, most Australian sheep do not co-graze with cattle. For these reasons, we believe that our choice of screening for the ovine strain of RSV is valid.

In humans, RSV causes disease in infants and the elderly. In our study we sampled slaughter-age lambs, 4 to 12 mo old, and adult sheep of unknown age. It is possible that the prevalence of infection may have been higher if we had targeted our sampling at neonatal lambs and older adult sheep. However, this is impractical in an Australian context due to distance, the extensive nature of Australian sheep farming, and the fact that lambs are usually not handled until they are at least 2 wk old to prevent mismothering. Instead, abattoirs provide an ideal opportunity for disease surveillance of sheep in Australia because they bring together animals from multiple sources and large geographical areas, allowing for cost-effective sample collection.

Our testing method (PCR) detects active viral infections, not previous infections already cleared by the animals, with the latter better detected by serology. Although we considered using blood sample collection and serology as a testing method, we dismissed this because there is not a commercially available test to detect antibodies to ovine RSV in sheep, and we did not have access to infected sheep to establish an in-house assay. Furthermore, collecting blood samples from sheep in lairage antemortem or from draining carcasses immediately after slaughter presents challenges for animal welfare as well as worker health and safety.

In summary, this study detected 2.4% prevalence of ovine RSV in pneumonic lungs of apparently healthy sheep processed at 5 abattoirs in Australia. Further research is needed to clarify the role of RSV in pneumonia in sheep.

Funding Statement

The study was funded by Animal Health Australia and Meat & Livestock Australia.