CERVICITIS AS A COMPONENT OF POSTPARTUM REPRODUCTIVE DISEASE IN DAIRY COWS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0231319
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Veterinarian Clinical Sciences

Non Technical Summary
Our laboratory identified subclinical endometritis (uterine inflammation diagnosed by endometrial cytology after 4 weeks postpartum) as a common condition that contributed to impaired reproductive performance. During the same period, clinical endometritis, characterized by purulent vaginal exudate, was identified as another condition contributing to subfertility. It was assumed that the clinical form of the disease (with visible purulent exudate in the vagina) was simply a more severe form of cytologically-diagnosed endometritis. Recently, this understanding has been shown to be false. As many as 40% of cows with visible vaginal exudate in fact have no evidence of endometrial inflammation at all. (Similarly, about 40% of cows with cytological evidence of inflammation have no visible vaginal discharge.) If the visible purulent exudate in the vagina is not derived from the uterus, where is it coming from Our hypothesis is that inflammation of the cervix is a cause of purulent vaginal exudate. We propose to confirm the prevalence of cervicitis by adapting our cytological methods used successfully to characterize endometritis to the study of cervicitis. We will distinguish the condition from endometritis and investigate its association with the presence of detectable vaginal exudate. The effects of each condition on subsequent reproductive performance will be determined. We will compare the bacterial population of the uterus and cervix of normal and affected cows, using modern molecular techniques, and identify the risk factors for cervicitis in postpartum dairy cows.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3410	1020	10%
301	3410	1100	20%
301	3410	1170	20%
311	3410	1020	10%
311	3410	1100	20%
311	3410	1170	20%

Knowledge Area
311 - Animal Diseases; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1100 - Bacteriology; 1020 - Physiology; 1170 - Epidemiology;

Keywords

bovine

dairy

reproduction

uterus

cervix

vagina

inflammation

infection

epidemiology

metagenomics

Goals / Objectives
The major objectives of this project are to: 1. Establish the prevalence of cervicitis in postpartum dairy cows, 2. Determine the relationship between cervicitis, endometritis and purulent vaginal exudate, 3. Examine the effect of cervicitis on subsequent reproductive performance, 4. Investigate the bacterial species associated with cervicitis, and 5. Identify risk factors for development of postpartum cervicitis. These will be achieved in a three-year period. The first year will be devoted almost entirely to enrollment of participating herds and collection of the required samples. During the second year we will analyze data and publish reports on prevalence of cervicitis, its impact on reproduction, its relationship with other conditions of the reproductive tract and identified risk factors for cervicitis. The third year will be devoted to the metagenomic investigation of the microbiome of the healthy and diseased cervix, endometrium and vagina, and publication of these results.

Project Methods
With informed owner consent 1000 cows in 5 different commercial dairy herds will be examined at 35-49 days postpartum by transrectal palpation and ultrasonography. The presence of vaginal exudate will be determined by use of the Metricheck device. (A sterile instrument is inserted into the vagina and used to retrieve a sample of vaginal mucus.) Then a sample will be obtained from the cervix using a cytobrush. A sterile disposable soft brush is protected in a stainless steel sheath and advanced under rectal control into the cervix where it is unsheathed and rolled in the cervix to obtain a sample of cervical cells, which will be transferred to a microscope slide to fixing, staining and microscopic examination. Finally, an endometrial sample will be obtained by low-volume flush as established in our laboratory. Cervical and endometrial cytology will be examined. DNA will be isolated from vaginal, cervical and endometrial samples for metagenomic analysis of bacterial species present. This sensitive technique permits ready identification of bacterial species that may be important in pathogenesis, but which are difficult to culture using traditional methods (especially anaerobic bacteria, known to be important in pathogenesis of uterine disease). Sampling will be completed within the first 1.5 years. Cows will be followed and cow files downloaded for analysis 300 days after examination to extract reproductive performance. Risk factors for cervicitis and endometritis and their effects on reproduction will be analyzed.

Progress 10/01/12 to 09/30/15

Outputs
Target Audience:Major target audiences include: (1) Researchers in animal science and veterinary medicine, (2) dairy producers, (3) veterinary students and (4) other dairy professionals. The results of this research are disseminated by peer-reviewedpublications in scientific and professional journals, continuing education lectures to veterinarians, veterinary professional students, graduate students and animal science undergraduate students. During the course of this project, lectureson the subject material have been delivered to veterinarians and producers from the USA (2013), Russia (2014), Brazil (2014), St. Kitts and Nevus (2015). Material has also been incorporated into lectures given to veterinary students of Cornell University and those participating in the Summer Dairy Conference held annually at Cornell University in every year of the project. Lectures on the project material were given to undergraduate animals science students in 2014 and 2015. Changes/Problems:Changes to this project included more in depth investigation of nutritional and immunological components of pathogenesis of reproductive tract disease than had been planned. These results have been reported or are in progress. We did attempt to characterize the effect of uterine disease on ovarian follicular function, but the quality and quantity of isolated mRNA were inadequate for next generation sequencing. What opportunities for training and professional development has the project provided?This project provided training opportunities for Dr. Marcela Bicalho (a veterinarian currently pursuing PhD studies) and Takashi Yasui, a veterinarian who completed hi PhD in 2014. It also allowed a non-degree training opportunity for veterinarian Dr. Lindsay Goodale. How have the results been disseminated to communities of interest?Publications in peer reviewed journals. Lectures to animals science and veterinary students. Lectures to graduate veterinarians. During the course of this project, lectureson the subject material have been delivered to veterinarians and producers from the USA (2013), Russia (2014), Brazil (2014), St. Kitts and Nevus (2015). Material has also been incorporated into lectures given to veterinary students of Cornell University and those participating in the Summer Dairy Conference held annually at Cornell University in every year of the project. Lectures on the project material were given to undergraduate animals science students in 2014 and 2015. An invited lecture will be presented at the 2016 Annual Meeting of the American College of Theriogenologists and Society for Theriogenology. An invited chapter will be published in Veterinary Clinics of North America during 2016, which includes some results of this project. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the first year of this project we established that cervicitis is a prevalent (approximately 15 % of all postpartum cows) postpartum disorder that is distinguishable from endometritis. Approximately one third of cows with purulent vaginal discharge after about 4 weeks postpartum have no evidence of endometritis; these cows generally have cervicitis. In the second year we showed that cervicitis has a distinct and serious detrimental impact on fertility that is separate from and additive to that of endometritis. We compared different diagnostic methods and found that simple visual examination of a low volume uterine lavage was almost as accurate a diagnostic method as more complicated techniques. During this year we commenced metagenomic investigation of bacterial pathogens associated with the disease. Bacterial species associated with both endometritis and cervicitis appear to be the same, namely Trueperella pyogenes, E. coli and gram negative anaerobes, raising the possibility that a single vaccine might protect aqainst both forms of postpartum uterine disorder, an avenue we are interested in pursuing. We also generated some data to suggest that one detrimentaleffect of inflammation on embryo development was interference with normal protein folding due to oxidative stress. This observation was followed up in thelast year of the project, confirming our earlier observation that preimplantation factor (PIF) was able to reverse this damage, at least partially (manuscript in preparation). Metagenomic studies had tended to suggest a smaller role for T. pyogenes in mediating uterine disease than we had expected. A separate study based on conventional bacteriological techniques proved that T. pyogenes indeed had a significant role in pathogenesis of purulent vaginal discharge and its presence, alone, was associated with significantly impaired reproductive performance. The recognition that cervicitis was more closely related to physical damage of the reproductive tract at parturition and endometritis more likely to follow metabolic disturbance and specifically severe negative energy balance reflected in impaired immune functionresulted in a series of studiesintended to improve overall energy metabolism and immune function in the periparturient period. Monensin supplementation tended to improve immune function but in our study did not reduce incidence of endometritis specifically. Finally, we were able to show that rapid recruitment of neutrophils to the reproductive tract early in thepostpartum period was associated with improved postpartum uterine health and reproductive performance. This study also indicated that early infection with specific strains of Streptococcus spp. tended to reduce later incidence of endometritis and improve reproductive performance, suggesting a possible role for a uterine probiotic (manuscript in review).

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Absalon-Medina VA, Butler WR, Gilbert RO. Preimplantation embryo metabolism and culture systems: experience from domestic animals and clinical implications. Journal of Assisted Reproduction and Genetics, 2014; 31: 393-409. DOI 10.1007/s10815-014-0179-2. PMID: 24682781
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bicalho M L, Lima FS, Ganda EK, Foditsch C, Meira, EB,Jr., Machado VS, Teixeira AG, Oikonomou G, Gilbert RO, Bicalho RC. 2014. Effect of trace mineral supplementation on selected minerals, energy metabolites, oxidative stress, and immune parameters and its association with uterine diseases in dairy cattle. Journal of Dairy Science. 2014; 97: 4281-4295. Doi: 10.3168/jds.2013-7832. PMID: 24835975.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: de Boer M W, LeBlanc SJ, Dubuc J, Meier S, Heuwieser W, Arlt S, Gilbert RO, McDougall S. 2014. Invited review: Systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows. Journal of Dairy Science. 2014; 97: 3983-3999. Doi: 10.3168/jds.2013-7450. PMID: 24835959.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Bicalho ML, Lima FS, Machado VS, Meira EB, Jr., Ganda EK, Foditsch C, Bicalho RC, Gilbert RO. Associations among Trueperella pyogenes, endometritis diagnosis and pregnancy outcomes in dairy cows. Theriogenology. pii: S0093-691X(15)00516-6. doi: 10.1016/j.theriogenology.2015.09.043.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Yasui T, McCarthy MM, Ryan CM, Gilbert RO, Felippe MJ, Mechor GD, Overton TR. Effects of monensin and starch level in early lactation diets on indices of immune function in dairy cows. Journal of Dairy Science. doi: 10.3168/jds.2015-9572. [Epub ahead of print] PMID: 26709159
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Santos NR, Gilbert RO. Dynamics of postpartum endometrial cytology and bacteriology and their relationship to fertility in dairy cows Theriogenology
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yasui T, McArt JA, Ryan CM, Gilbert RO, Nydam DV, Valdez F, Griswold KE, Overton TR. 2014. Effects of chromium propionate supplementation during the periparturient period and early lactation on metabolism, performance, and cytological endometritis in dairy cows. Journal of Dairy Science. 2014; 97: 6400-6410. doi: 10.3168/jds.2013-7796. PMID: 25087037.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yasui T, McCann K, Gilbert RO, Nydam DV, Overton TR. Associations of cytological endometritis with energy metabolism and inflammation during the periparturient period and early lactation in dairy cows. Journal of Dairy Science. 2014; 97: 2763-2770. doi: 10.3168/jds.2013-7322; PMID: 24612816.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yasui T, Ryan CM, Gilbert RO, Perryman KR, Overton TR. Effects of hydroxy trace minerals on oxidative metabolism, cytological endometritis, and performance of transition dairy cows. Journal of Dairy Science. 2014; 97: 3728-3738. doi: 10.3168/jds.2013-7331; PMID: 24731626
• Type: Journal Articles Status: Other Year Published: 2016 Citation: Goodale LF, Barnea ER, Gilbert RO. Preimplantation factor partially reverses the embryotoxic effect of a protein disulfide isomerase inhibitor in bovine embryos.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The major target audiences are (1) researchers in animal science and veterinary medicine, (2) dairy producers, (3) veternary professional students and (4) other dairy professionals. The results of this research are communicated mainly by publication in peer-reviewed scientific and professional journals. Additional means of communicating results are: lectures to graduate veterinarians during continuning professional education seminars and to veterinary students. During the reporting period, lectures were given at the College of Veterinary Medicine to Cornell Veterinary students in elective courses, and to participants from around the country, and indeed the world, in the Summer Dairy Institute. Lectures were also given to veterinarians and producers from Russia incorporating results of this research (Wimm-Bill-Dann-Cornell Dairy Courses (Russian Dairy Group), Thursday April 3, 2014, 4 hours). Finally, lectures were given to graduate students in veterinary reproduciton at the State University of Sao Paulo in Brazil in February 2014 (12 lectures). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Studies funded by the project form the basis of the PhD dissertation of Dr. Marcela Bicalho, expected to be conferred in 2016 or early 2017. A portion of the doctoral dissertation submitted by Dr. Victor Absalon-Medina was supported by this project. Additionally, it has contributed to the scientific training of veterinary residents and research training of undergraduate animal science students. How have the results been disseminated to communities of interest? Formal publications in peer-reviewed journals (3 published during the reporting period, and three more in preparation). Lectures to veterinary students, graduate students, graduate veterinarians. What do you plan to do during the next reporting period to accomplish the goals? Complete experiments on diagnostic modalities, metagenomic characterization of bacterial communities, effect of inflammation on embryo development, and complete publications reporting these findings.

Impacts
What was accomplished under these goals? We continue to make good progress. Having previously confirmed that cervicitis is prevalent and is separate from endometritis, we have shown that its reproductive consequences are separate from and additive to those of endometritis. A publication is in preparation. We have also continued to characterize involved bacterial species by metagenomic methods, the results of which are also being compiled into formal publications. Investigations comparing different diagnostic modalities and their ability to predict infertility have suggested that cervicitis has a longer-term detrimental effect on reproduction than endometritis alone (publication in preparation). This seems likely to be due to irreversible anatomic damage accompanying cervicitis in comparison with reversible inflammatory changes for endometritis alone. Our research has also indicated that one consequence of an inflamed environment in the reproductive tract is interference in normal protein folding during embryonic development. We have found that pre-implantation factor may be able to reverse this detrimental effect, at least in vitro, opening the door to prospects of novel, non-antibiotic therapies that might enhance reproduction. (Manuscript in preparation)

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Absalon-Medina VA, Butler WR, Gilbert RO. Preimplantation embryo metabolism and culture systems: experience from domestic animals and clinical implications. Journal of Assisted Reproduction and Genetics, 2014; 31: 393-409. DOI 10.1007/s10815-014-0179-2. PMID: 24682781
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bicalho M L, Lima FS, Ganda EK, Foditsch C, Meira, EB,Jr., Machado VS, Teixeira AG, Oikonomou G, Gilbert RO, Bicalho RC. 2014. Effect of trace mineral supplementation on selected minerals, energy metabolites, oxidative stress, and immune parameters and its association with uterine diseases in dairy cattle. Journal of Dairy Science.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: de Boer M W, LeBlanc SJ, Dubuc J, Meier S, Heuwieser W, Arlt S, Gilbert RO, McDougall S. 2014. Invited review: Systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows. Journal of Dairy Science.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The results of this research are communicated to fellow investigators and dairy professionals by publication in peer-reviewed scientific journals. During the reporting period current results of this research were incorporated into lectures given to graduate veterinarians and veterinary students as part of the Cornell University Summer Dairy Institute, and to dairy-oriented veterinary students in a veterinary distribution course (Advanced Dairy Reproduction)of the Cornell University College of Veterinary Medicine. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project will form part of the PhD dissertation of Dr. Marcela Bicalho. It has provided her with opportunities to develop skills in experimental design and management, including both animal work and laboratory work. How have the results been disseminated to communities of interest? Results included in lectures to veterinarians and veterinary students. What do you plan to do during the next reporting period to accomplish the goals? We anticipate continued progress on metagenomic analysis of bacterial communities of the healthy and diseased postpartum uterus and publication of the results.

Impacts
What was accomplished under these goals? We have made considerable progress toward achieving the objectives of this study. It has become clear that cervicitis is a specific condition distinct from endometritis in postpartum dairy cows. Pathogenesis is different in that obstetrical complications predispose to cervicitis, whereas endometritis is more often a reflection of impaired immune response secondary to negative energy balance. Cervicitis has a detrimental effect on reproduction which is separate from and additive to that of endometritis. We have completed much metagenomic analysis of the bacterial flora of postpartum cows with different reproductive conditions. We anticipate completing all objectives within the period of this project.

Publications