Exploring potential biomarkers of inflammatory disease in sheep presenting with pleurisy/pneumonia and parasitic infections : A thesis submitted in fulfilment of the requirements for the Degree of Master of Applied Science

Inflammatory diseases of sheep, specifically the pneumonia-pleurisy complex and parasitic infections, impact productivity, health and welfare. Infected sheep can have reduced growth rates and an increased predisposition to other infections. The immune system is a key mechanism for defending against invading pathogens, and within this system, inflammation plays a crucial role in defence. However, when inflammation becomes excessive or uncontrolled, it can compromise the ability to mount an effective immune response and cause tissue damage. Ovine pneumonia is a respiratory disease characterised by inflammation of the lung tissue. It can progress to pleurisy, which is the inflammation of the pleura, the membranes that surround the lungs. Inflammation of the pleura can generate fibrous ‘adhesions’ between the lungs and walls of the thoracic cavity. Parasitic infections of sheep, including nematode parasite infections, can cause inflammation of the gastrointestinal tract. This can result in the loss of condition and productivity. The effect on health is similar to that observed for pneumonia-pleurisy. The diagnosis of pneumonia-pleurisy can be difficult, because many cases are subclinical until they develop to the stage where damage to the lungs has occurred. In New Zealand, pleurisy-affected carcasses are often only detected at slaughter, which results in the carcasses being downgraded. This results in financial loss for farmers and for the industry. Similarly, parasitic infections from nematode parasites like Teladorsagia spp. are often undetectable until clinical signs like weight-loss or decreased growth are observed. These too can cause financial loss for farmers. This study explored potential biomarkers of inflammation that might have value in identifying sub- clinical inflammatory diseases like pneumonic-pleurisy and parasitism. The lambs investigated were selected from three separate studies, the ‘Lincoln University cohort’ (n = 33) and a ‘Parasite cohort’ (n = 6 control, n = 6 infected), which were compared to a third ‘Kaikōura cohort’ (n = 34). All the lambs had blood samples collected to determine the concentration of four pro-inflammatory cytokines (IL-1β, TNF-α, IFN-y and IL-17A), and an inflammatory marker CRP (C-reactive protein), using commercially available enzyme-linked immunosorbent assays (ELISAs). It was hoped these kits would illustrate the inflammatory response/immune status of the sheep to distinguish animals with pneumonia-pleurisy or parasitism from non-affected animals, and to determine whether these potential biomarkers were associated with other performance measurements, including carcass weights for all three cohorts and the faecal egg count of lambs in the parasite cohort. ELISAs are widely used to detect and quantify specific proteins, with the kits tested potentially illustrating the inflammatory status of the individual lambs. Nevertheless, ELISAs do have several limitations, including the availability of appropriate kits and whether they are effective and robust for the intended use. The results obtained with the selected ELISA kits varied across the three groups. At slaughter, 11 out of 123 lambs from the Lincoln University cohort were recorded as having pleurisy. The Lincoln University cohort lambs had detectable levels of TNF-α, IL-1β and CRP; the Parasite cohort had detectable levels of TNF-α; and the Kaikōura cohort had detectable levels of TNF-α and IL-17A. The Parasite cohort had changes in tissue derived from their abomasum, but these changes were not reflected in fluctuations in serum biomarker levels. Collectively, the findings of this study highlighted some challenges associated with using commercially available sheep ELISA kitsets, and their ability to produce meaningful results when used on lambs. The tests are costly, both for the reagents required and because of the time associated with performing them. This suggests that in their current form they may be unfavourable as diagnostic tools. Further research into the use of the currently available ELISA kitsets is required before they have improved value as diagnostic tests in sheep.