Larval paralysis factor of sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand

Abstract

This study aimed to identify the origin and molecular nature of larval paralysis factor (LPF), an uncharacterised natural anthelmintic agent(s) known to be secreted by cells in the small intestine of nematode-parasite-immune sheep. The study first confirmed previous findings that gut mucus and small intestinal mucosal cell culture supernatants (CCSs) from parasite-immune sheep contain LPF. An in vitro larval migration inhibition (LMI) assay showed that LPF is active against numerous nematode parasite larvae of ruminants and adults of Trichostrongylus colubriformis. Lamina propria cells (LPCs) were a richer source of LPF than epithelial cells, and release of the factor was specifically triggered by parasite larval antigens. A series of trials was performed to optimise LPF production in vitro. LPF production was highest when LPCs were selectively extracted from the first three metres of small intestine of resistant-line or hyperimmune parasitized sheep three days after oral challenge with T. colubriformis larvae. LPF release in vitro appeared to be related to the percentage of eosinophils present in cell cultures, but not with mucosal mast cells or globule leucocytes. Subsequent in vitro experiments showed that mucus glycoproteins released from goblet cells enhanced and sustained the activity of LPF, which may point to a previously unrecognised effector link between goblet cell hyperplasia and mucosal immune responses to gut nematode parasites. Sequential purification and molecular analysis of LPF showed that the active agent has a molecular mass less than 1kDa, it is polar and heat and enzyme resistant. Further purification using solid phase extraction and HPLC established that LPF did not bind either C18 or ion exchangers, but LPF activity in the flow through from these sorbents was retained on aminopropyl HPLC columns and eluted as a single active peak after flushing with acetonitrile/water mixture (70:30). This peak, analysed by LC-MS, comprised three main compounds of interest: a UV absorbing compound at 254nm with no MS data, and two non UV absorbing compounds at 254nm: one was a component of m/z 104 (ESI+ve) and the other was detected at m/z 113 (ESI-ve).