Pilot study to assess the performance of PCR to dect Jaagsiekte Sheep Retrovirus (JSRV) infected sheep as part of a “thin ewe” diagnostic package

Project number:                    61110018

Lead contractor:                   Moredun Research Institute

Partners:                                   Animal and Plant Health Agency (APHA)

Start and end date:              01 October 2015 – 30 September 2016

 

The Problem:

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumour of sheep caused by Jaagsiekte sheep retrovirus (JSRV). There is no treatment or vaccine and once clinical signs appear the disease is invariably fatal.  Affected flocks may lose as many as 20% of stock in the first couple of years that OPA is seen, and the disease may continue to account for loss of a few per cent of sheep every year for many years thereafter.  The disease appears to be increasingly common throughout the UK and a means of ascertaining flock status is a recognised need.  Ideally producers should be able to identify if the infection is present in their flock and thereafter work with their vet to minimise losses.  Perhaps more importantly, the 70-90% of flocks that are estimated to be free of the disease can only maintain this status by being able to buy replacement breeding stock from other OPA-free flocks.  An OPA flock test based on testing thin ewes could facilitate an appropriate assurance scheme.

 

Aims and Objectives:

The objectives are:

  1. to refine the existing PCR test currently used as a research tool for transfer to a commercial laboratory
  2. explore if JRSV can be identified in nasal swabs
  3. to compare results from nasal swabs and blood for the presence of JRSV in a subset of thin ewes to evaluate the best sample type
  4. to investigate if the test used in a subset of thin ewes can differentiate between infected and uninfected flocks and form the basis of an assurance scheme
  5. to communicate the outcomes to sheep vets / producers

 

Approach:

  • Blood samples and nasal swabs will be collected from 20 thin sheep in six flocks with confirmed OPA and from three uninfected flocks (based on previous laboratory investigations / post-mortem results).  Thin animals will be targeted as poor body condition is one of the clinical signs that may represent early OPA.
  • These will be tested using the PCR test to evaluate the best sample type to take forward.
  • Some of the cull ewes will be purchased and post mortem examinations will be conducted to determine OPA status.
  • Test performance will be evaluated.
  • All the results will be communicated back to the producers.

 

Results:

Key message – The nasal swab test proved better than the blood test and was more suitable for testing in a commercial laboratory setting.  Although, like the blood test, it does not identify every OPA-positive individual, it performed well enough as a flock test applied to thin ewes to have potential as the basis of a flock assurance scheme.

Optimal extraction and PCR protocols were established in the commercial laboratory and samples were obtained for testing from 3 OPA-negative and 6 OPA-positive flocks.  Post mortem examination(PME), thoracic ultrasonography or wheelbarrow test data made available from a concurrent study added value by enabling us to assess the efficacy of testing at an individual level as well as for a flock test.

Initial testing on samples of known OPA-positive and negative individual sheep showed that the nasal swab test was better than the blood test and therefore the remaining work focused on the nasal swab testing.

In 5 of the 6 known OPA-affected flocks tested a positive result was obtained from at least one nasal swab sample of the 20 samples tested.  There were no positive results in animals from OPA-negative flocks or in OPA-negative individuals from affected flocks.  That is, there were no false positives with the nasal swab test.

Statistical analysis estimated the sensitivity of the nasal swab test on individuals as approx. 40% and the specificity as 98-100%.  The sensitivity as a flock test is dependent on the number of true positives within the sample group; If only 1 of the 20 ewes has OPA then the probability of the flock test being positive is 0.65 whereas if 5 of the 20 have OPA the probability of the flock test being positive is 0.9.  Pooling the samples as groups of 5 and doing single rather than replicate PCR reactions can increase the lab throughput and reduce reagent usage, and therefore cost to the end user, but this balances against a small reduction in the sensitivity of the flock test.  A preliminary test of transport conditions did not indicate a significant difference when samples were stored at 4oC or ambient temperature for 24 or 48 hours.