Final Activity Report Summary - APHORISM (Ascending Placentitis in the Horse - Involvement of Sialidases and Mucinases) Mucus is made up of mucin glyco-proteins which have a core apoprotein and side chains of sugars such as hexosamines, galactose, fucose, and sialic acid, some of which may be decorated with sulphate groups. The mucus of the reproductive tract has a defensive function. It binds micro-organisms and removes them from the tract by the movement of hair-like cilia on the surface of cells. Mucus is constantly produced by the surface cells in the tract and its turnover may, in part, be regulated by mixtures of enzymes known as mucinases. These include glycosidases that break down the sugar side-chains, sulpatases that remove sulphate groups, and proteases that break down the protein core of the mucin molecules. The physical properties of mucus vary throughout the reproductive cycle, and, during pregnancy in the normal mare, a thick mucus plug seals the cervical canal. The mechanism by which it forms is unclear. The normally sterile intrauterine environment is protected by this plug, which separates the microbially- colonised vagina from the normally sterile uterus. Ascending placentitis (AP) is an important cause of abortion in late term pregnant mares where bacteria from the lower reproductive tract migrate through the cervix and infect the fetal membranes. This leads to abortion or weak and/or unviable foals. AP causes 20% of abortions in late-term pregnant mares in the USA and 10% in the UK/Ireland. The reasons for this geographical difference are unknown. Furthermore, the mechanisms by which normal cervical barrier function is maintained in the mare are poorly understood: impeding a greater understanding of AP. Bacterial vaginosis affects women, and may be analogous to AP in the mare. In this condition, the bacterial population of the vagina becomes abnormal, with an overgrowth of bacteria which produce enzymes capable of degrading mucus. Destruction of the cervical mucus plug then allows micro-organisms to enter the uterus, leading to infection of the chorio-amnion and pre-term delivery. Sialidase and glycosulphatase are the mucinases most associated with bacterial vaginosis and diagnosis can be achieved by the detection of these activities using a dip-stick test. Very little is known about the normal composition of mucus in the reproductive tract of the mare and the normal environment of the vagina during the reproductive cycle and pregnancy. Therefore, this study investigated the factors involved in the formation, turnover, and abnormal degradation of cervical mucus during the oestrus cycle, and in pregnancy by: - Determining the normal expression, glycosylation, and distribution of epithelial mucins in the vagina and cervix. - Characterising the normal microbial flora of the vagina and the vaginal pH, and comparing it with that in AP. - Surveying the presence of mucin degrading enzymes in the vagina in normal mares and a small number of mares affected with AP. For this work lower reproductive tract tissue samples from mares at different stages of the reproductive cycle and during pregnancy were obtained from a commercial abbatoir. Live mares were also swabbed for enzymic and microbial analysis. The stage of the reproductive cycle was determined by measuring blood progestone levels, and by gross or ultrasonographic evaluation of the reproductive tract as necessary. Conclusions: The expression distribution and glycosylation of cervical mucins consistently varied throughout the oestrus cycle. Previously un-described mucus-secreting cells were identified in the vagina. The normal vaginal microflora was catalogued and differentiated from that of other species, and a range of sialidase-producing micro-organisms were identified and characterised. Unlike in human BV, the pH of the reproductive tract did not vary from normal, nor did it vary throughout the oestrus cycle. Cycle-related, geographical, and pathologically driven changes in the profile of mucin-degrading enzymes were identified. Further funding has been obtained to extend this work. This will involve the analysis of further samples from other geographical locations, and additional gene expression profiling.
