Infection and immunity in the bovine genital tract

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

Uterine infection after parturition is one of the most common endemic diseases of dairy cattle, leading to infertility, increased culling, pain and suffering. Dairy cattle are notable amongst mammals because bacterial contamination of the uterus is ubiquitous after calving and up to 40% of animals develop uterine disease. The diagnosis of uterine disease is straightforward, but the incidence of disease is increasing. The treatments have changed little over the last 30 years, and animals are still sub-fertile even after resolution of the clinical signs. However, whilst some animals have severe disease others efficiently eliminate the bacteria from the uterus after calving, don't develop disease, and are highly fertile. The unanswered questions are about how bacteria are detected by the immune system in the uterus and why some cattle are so prone to disease; how the bacteria cause infertility; and, how the impact of uterine disease can by reduced? The aim of the project is to identify the molecular and cellular mechanisms underlying the infertility associated with uterine infection in dairy cattle. The objectives are: 1. Establish the differential regulation of genes associated with uterine infection and immunity in animals with disparate uterine disease outcomes. 2. Measure the immune responses to the two key bacteria (Escherichia coli; Arcanobacterium pyogenes) and their pathogen associated molecules by reproductive tissues and the impact on the function of uterine and ovarian cells, and the embryo. 3. Determine the effect of blocking key immune signaling pathways on the uterine cell inflammatory response. The expected benefits from exploiting the knowledge generated about uterine disease are: 1. Improved animal welfare by reducing the incidence and severity of uterine disease. 2. Increased sustainability of dairy farming, by reducing the impact of disease on fertility and the cost of disease to the farmer. 3. Reducing the impact on the food chain, by reducing disease and the application of treatments in animals producing milk for human consumption. 4. Increased sustainability of the environment by producing milk from fewer more fertile cows, and thus reduce greenhouse gas emissions, including saving 113 tonnes of CO2 emissions per year. To drive commercialisation and development of the knowledge from the project we have developed a partnership with the biggest veterinary pharmaceutical company in the world, Pfizer Animal Health, as well as exploiting collaborations with international experts from the UK, USA and Italy.

Technical Summary

Infection of the uterus by bacteria causes infertility in mammals. This is a particular problem in dairy cattle, where conception rates are at an all time low and bacterial infection of the uterus affects up to 40 percent of animals. Not only is the uterus damaged, but there is perturbation of ovarian follicle and corpus luteum function, and embryo mortality. The front-line defense against bacterial infection rests in the innate immune system and the Toll- like Receptors are a key component for pathogen recognition, which activate MAPK and/or NFkB pathways leading to secretion of cytokines and an inflammatory response. The present project aims to identify the molecular and cellular mechanisms underlying the infertility associated with uterine infection in cattle. The hypothesis that genes associated with the endometrial cell immune responses are differentially regulated in animals with different uterine disease outcome, will be tested by comparing the expression of a panel of genes associated with immunity and inflammation in the endometrium. Endometrium has been collected at 3 time points from animals undergoing in vivo infection that resulted in (1) no pathology, (2) subclinical, or (3) clinical disease, and will be examined using RT-PCR and in situ hybridization. The hypothesis that tissues and cells of the reproductive tract have the immune capability to detect pathogens or their associated molecules and that this leads to modulation of their function, will be explored using pure populations of cells from the uterus and the ovary, and embryos, challenged with specific bacteria or their pathogen associated molecules. Finally we will use siRNA, chemical and pharmaceutical compounds to test the hypothesis that blocking the MAPK or the NFkB pathways in uterine epithelial and stromal cells will suppress the immune and endocrine response to bacteria. The long-term aim is to develop better strategies to limit the impact of disease on the animal and improve animal welfare.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/F005121/1 04/06/2007 03/08/2008 £173,587
BB/F005121/2 Transfer BB/F005121/1 16/12/2008 15/11/2010 £123,009
 
Description Uterine infection after parturition is a common endemic disease of dairy cattle, leading to infertility, increased culling, pain and suffering. Dairy cattle are notable amongst mammals because bacterial contamination of the uterus is ubiquitous after calving and up to 40% of animals develop uterine disease when infected by specific bacteria - such as Escherichia coli and Arcanobacterium pyogenes. The cost of uterine disease to the UK dairy industry is about £193 million per annum. The present project aimed to identify the molecular and cellular mechanisms underlying the infertility associated with uterine infection in dairy cattle.

The main findings during the project were:
1. The endometrium collected in vivo from postpartum cows that subsequently develop uterine disease had a greater expression of genes associated with inflammation such as IL1B, IL6 and IL8, than infected cows that rapidly recovered from infection. In addition, there was greater expression of the gene encoding the TLR4 innate immune receptor that detects the cell wall component of E. coli, known as lipopolysaccharide (LPS, endotoxin). Thus, innate immunity is likely central to combating infection in the female genital tract.
2. Innate immunity was driven by the epithelial and stromal cells of the endometrium, and the granulosa cells in the ovary that surround the egg. These cells expressed TLR4 protein, and produced an inflammatory response to LPS, via key intracellular pathways of the innate immune system (ERK1/2, p38, NFkB, PKC). On the other hand fertilization of eggs and embryo development were resistant to LPS or inflammatory mediators. The implications of these data are that the cells lining the uterus and nurturing the egg have innate immune capabilities.
3. Knocking down the TLR4 pathway using siRNA prevented the inflammatory response. So, in collaboration with industry, inhibitors of the innate immune system were identified that could limit the severity of inflammation in endometrial and ovarian cells treated with LPS.

Impact/Benefit/Knowledge Transfer
a) Industry: The industrial partner now has a proprietary programme to take our work forward to develop products that limit the incidence and/or severity of uterine disease. Reducing the incidence of uterine disease will also increase the profitability of the 16,400 SMEs that keep dairy cattle in the UK.
b) KT: We have generated 9 peer-review publications as well as multiple media open-access approaches (popular journals, website, presentations) for knowledge transfer to veterinarians, who are the key stakeholders for translating our findings for the treatment of uterine disease in cattle.
c) Environment: Reducing the incidence of uterine disease will benefit animal welfare in the UK. In addition, fewer animals need to be kept to replace infertile cows, so reducing the greenhouse gases and water degradation associated with dairy farming.
d) Funding: We developed a research consortium including Glasgow, Hannover, INRA and Pfizer Global Alliances and we have been awarded an EURO 3.2 million ERA-Net to further explore the mechanisms of uterine disease and test potential strategies to limit the severity and incidence of disease.
e) Bioscientist research training: Two postdoctoral researchers were trained during the project, and the work has spawned two BBSRC PhD studentships.

Project Duration
01/06/2007 to 15/11/2010 (3 years with gap for transfer between RVC and Swansea University)
Exploitation Route The work from this project lead into an ERANET award with Zoetis Inc.
Sectors Agriculture, Food and Drink