Challenges of Chronic Endometritis: Novel Tools

Karen Wolfsdorf, DVM, Dipl. ACT | Hagyard Equine Medical Institute | Published: Issue 1 2024

Editor's Pick

One of the most commonly identified problems associated with infertility in the mare is uterine inflammation or endometritis. This can be caused by acute or chronic infections or persistent mating-induced endometritis (PMIE).

The most common etiological agents found to cause uterine infections are Streptococcus zooepidemicus and Escherichia coli as the result of fecal and genital flora contamination. Normal mares can clear their uterus of bacteria by the uterine defense mechanisms of inflammatory response, uterine contraction, and lymphatic drainage. In susceptible mares, when bacteria infiltrate and contaminate the uterus, persistent or chronic endometritis persists.

Repeated foaling and breeding can cause anatomical defects such as poor perineal conformation, incompetent vestibule-vaginal sphincter, vaginal stretching, incompetent cervix, and a pendulous uterus all contributing to chronic endometritis. Thus, chronic endometritis is a major cause of equine infertility in older and multiparous mares. In addition, certain bacteria are able to utilize methods to survive degradation by the host immune system and antibiotic therapy.

This image is of poor perineal conformation identified by the placement of the tube perpendicular to the vulvar lips, the rectum sunken in relative to the vulva lips, and extreme declination of the vulvar lips.
Images courtesy of Dr. Karen Wolfsdorf

One survival tool used by B-hemolytic Streptococci and identified with Flourescent In Situ Hybridization (FISH) is entering a non-dividing or dormant state. Another may form a biofilm, produced by the adhesion of bacteria (most commonly E.coli, P.aeruginosa, and K. pneumonia) to the endometrial surface with subsequent production of an exopolysaccharide matrix.

Biofilms allow bacteria to become unrecognizable by the host immune system, provide a diffusion barrier that decreases antibiotic penetration, and slow down metabolism and replication, making bacteria more resistant and persistent.

Chronic endometritis can present to the equine practitioner in three different scenarios. The first and most common is routine endometritis when the mare has a vulvar discharge, intraluminal fluid on ultrasonography, and inflammation and bacteria on culture and cytology.

The bacteria are in a planktonic state in which they can move around the uterus, stimulating an inflammatory response and being identified by culture and cytology.

The second is where there is no vulvar discharge or intraluminal fluid, but there are signs of inflammation, mucous, and debris on cytology with no bacteria identified on culture. In this case, the bacteria could be trapped in the inspissated mucous produced with inflammation or within a biofilm. Identification requires the breakdown of the mucous layer or biofilm.

Lastly, a mare may present where there is no vulvar discharge, intraluminal fluid, inflammation, or infection, but shows acute or chronic inflammation on endometrial biopsy. The last scenario is often accompanied by a history of not becoming pregnant when bred to a fertile stallion over numerous estrous cycles or pregnancy loss. Further diagnostics are required to potentially identify the presence of underlying or dormant bacteria within the endometrium.

NEW DIAGNOSTICS: Mucous and Biofilm Disruptors

These are different technique options for performing endometrial culture and cytology.

N-Acetylcysteine (NAC) is a mucolytic agent that decreases mucous viscosity by reducing the disulfide bonds between mucin polymers. It also has antioxidant and possibly antimicrobial properties. NAC can be used for both the identification and treatment of chronic endometritis.

Studies using a 3.3% solution of NAC infusion (20ml of a 20% solution diluted to 120ml with saline or LRS) followed by a low-volume lavage culture/cytology the following day, identified an increase in the number of mares with a positive inflammatory cytology and/or culture.

Pre-infusion, 81% of mares were negative on cytology, and 63% had no growth, while post-infusion, 27% and 31% were negative, respectively. This procedure can therefore potentially aid in identifying persister bacteria sequestered in the inspissated mucous or biofilm, allowing appropriate treatment to be initiated.

In earlier studies, sub-infertile mares infused with a 0.6% NAC solution either on the estrus previous to the estrus used for breeding, or just 48 hours before breeding, improved pregnancy rates. The use of NAC as a treatment has proven to enhance the efficacy of β-lactams but reduce the antibacterial activity of aminoglycosides, fluoroquinolones, and erythromycin. NAC has been suggested to reduce biofilms of E. coli isolates in vitro, but this was not observed for other bacteria.

Therefore, NAC may best be used to help identify bacteria but not as a therapeutic agent at the same time as antibiotics.

EDTA-tris or Tricide. chelates calcium and magnesium in the bacterial cell wall, which may potentiate antimicrobials and break down biofilm. EDTA disrupts the lipopolysaccharide membrane of P. aeruginosa, increasing cell permeability and potentially making the organism more susceptible to 200 to 500ml of tris-EDTA to allow for dispersion completely throughout the uterus. Combination with antibiotics (amikacin, ticarcillin with clavulonic acid, and ceftiofur) minimal inhibitory concentration over each alone. Most recently Tricide has been identified to potentiate the effect of antimicrobials against fungal keratitis.

Hydrogen peroxide at a 1% concentration is known to disrupt preformed biofilms and has been proposed to be beneficial in chronic endometritis; however, the sensitivities to disruption vary greatly between strains of bacteria. It is active against bacteria, fungi, yeast, viruses, and spores by the release of hydroxyl radicals. Other properties include potent oxidants that react with macromolecules such as membrane lipids and DNA. Bacteria that produce catalases render H2O2 ineffective. Using an in vitro system with P.aeruginosa, a 1% hydrogen peroxide solution was capable of disrupting the biofilm in 50% of cases.

Dimethyl sulfoxide (DMSO) acts as an excellent solvent in water and organic matter, an anti-inflammatory, and an oxygen scavenger. The antimicrobial properties, bactericidal versus bacteriostatic, are dependent on concentration (10-30%). DMSO was effective at reducing colony-forming units (CFU) in S.equi zooepidemicus and showed significant reduction in overall biofilm biomass in E.coli infection. Therefore, it can be used in combination therapy with antibiotics.

Ceragyn belongs to a class of peptides that cause depolarization of bacterial cell wall membranes and activate apoptotic pathways. A 60mL vial is labeled for use up to 24 hours before or 12-48 hours after breeding. The drug has become popular among equine practitioners, but the author has found inconsistent data to support its effect on most organisms associated with equine endometritis.

NEW DIAGNOSTICS: Activation/Identification of Dormant/Deep Bacteria

The top image shows the cytology of top yeast within a neutrophil (inflammatory cell), and the bottom image is of fungal hyphae and inflammatory cells.

The top image is the top neutrophils (inflammatory cell) identified on cytology and the bottom image is of a gram stain identifying gram-positive bacteria.

Misoprostil or prostaglandin E2(PGE2) has been used via laparotomy or deep horn application to the oviduct or oviductal papillae to increase pregnancy rates by dilating the oviductal canal and enhancing blockage clearance.

Deep horn application, using one Cytotec tablet (200 mcg of Misoprostol, Pfizer, USA) diluted in three mL of sterile saline per horn, performed during diestrus with mares bred the next cycle had 68% of mares produce embryos or became pregnant within two cycles, with 67% of mares producing embryos in three cycles.

However, while utilizing this technique, it became evident that the procedure/PGE2 may “activate” bacteria and cause mild inflammation. In a recent study performed using low-volume lavage culture/cytology the day after misoprostil deposition on the oviductal papillae, 22% of mares had no bacterial growth, 16% had scant growth, 19% had light growth, 31% moderate, and 13% heavy growth, which was significantly different from pre-infusion.

This illustrated that the procedure may have the additional benefit of stimulating bacteria aiding in identifying the underlying cause of endometritis affecting fertility.

Bactivate can be used in mares with a history of infertility and inflammation without a source. These mares potentially have chronic endometritis due to dormant B. Streptococcus Zooepidemicus. Identification of inflammation on cytology or biopsy without a positive culture is an indication for use.

Infusion of Bactivate when the mare is in estrus activates the dormant B.strep, increasing replication and growth, providing identification at 24 hours with a low-volume lavage culture and cytology, allowing initiation of enhanced antibiotic therapy. Treatment usually consists of systemic antibiotics for two to three weeks in addition to intra-uterine infusions during estrus.

Re-activation with subsequent endometrial culture/ cytology can be done to ensure complete resolution of chronic endometritis. A recent clinical trial showed that activation of subclinical bacterial endometritis followed by antimicrobial treatment increased pregnancy rates per cycle and lowered fetal loss in problem mares.

Kerosene produces a chemical curettage which causes glandular activation in mares, with improved conception rates in treated verses control mares as biopsy grades improved. Nine out of 11 mated Grade III mares conceived and five carried to term. It is hypothesized that removal of the luminal epithelium, opening the glands and globlet cells, provides a more normalized state of the mucus blanket. There are different protocols described as to when and how much kerosene should be used. This author prefers to infuse 100ml of pure K1 kerosene during diestrus at least six days post ovulation. At the same time prostaglandin is administered to open the cervix over the next couple of days to allow intra-uterine lavage and treatment. Culture and cytology of the first liter of lavage fluid post kerosene will help identify bacteria released from the deeper tissues/glands of the uterus. S.zooepidemicus has been identified most commonly in the authors experience. Removal of inspissated mucous and biofilm on the epithelium allows for exposure of the deeper bacterial population in the glands.

Recommended Reading

  1. Davis HA, Stanton MB, Thungrat K & Boothe DM 2013 Uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003–2008). Journal of the American Veterinary Medical Association 242 977–983. (https://doi.org/10.2460/javma.242.7.977)
  2. Katila T 2016 Evaluation of diagnostic methods in equine endometritis. Reproductive Biology 16 189–196. (https://doi.org/10.1016/j. repbio.2016.06.002)
  3. Ferris RA, Mccue PM, Borlee GI, Loncar KD, Hennet ML & Borlee BR 2016 In vitro efficacy of nonantibiotic treatments on biofilm disruption of Gram-negative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. Journal of Clinical Microbiology 54 631–639. (https://doi.org/10.1128/JCM.02861-15)
  4. Von Dollen, KA, Wolfsdorf KE, Levkullic S, Fedorka CE, Lu KG. 2021 How to Use N-Acetylcysteine to Enhance Diagnosis of Bacterial Endometritis in Barren Mares. Proceedings AAEP 67 261-263.
  5. Petersen MR, M.ller Nielsen J, Lehn-Jensen H & Bojesen AM 2009 Streptococcus equi subspecies zooepidemicus resides deep in the chronically infected endometrium of mares. Clinical Theriogenology 2009 393–409.
  6. Christoffersen M, Soderlind M, Rudefalk SR, Pedersen HG, Allen J & Krekeler N 2015b Risk factors associated with uterine fluid after breeding caused by Streptococcus zooepidemicus. Theriogenology 84 1283–1290. (https://doi.org/10.1016/j.theriogenology.2015.07.007
  7. Petersen MR, Rosenbrock A, Osborne M & Bojesen AM 2018 High prevalence of subclinical endometritis in problem mares – effect of activation and treatment on fertility. Journal of Equine Veterinary Science 66 117. (https://doi.org/10.1016/j.jevs.2018.05.162)
  8. Bracher V, Neuschaefer A & Allen WR 1991 The effect of intrauterine infusion of kerosene on the endometrium of mares. Journal of Reproduction and Fertility: Supplement 44 706–707.
  9. Bradecamp EA, Ahlschwede SA & Cook JL 2014 The effects of intra- uterine kerosene infusion on endometrial epithelial cilia concentration. Journal of Equine Veterinary Science 34 134. (https://doi.org/10.1016/j. jevs.2013.10.092)
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About the Author

Karen Wolfsdorf, DVM, Dipl. ACT
Hagyard Equine Medical Institute

Dr. Wolfsdorf attended veterinary school at the University of Florida. She completed an equine field internship at North Carolina State University, followed by a theriogenology residency at University of Florida, becoming a diplomate of the American College of Theriogenology in 1995. Dr. Wolfsdorf is a shareholder, field veterinarian and specialist at Hagyard Equine medical institute whose main focus is diagnosing and treating the problem mare. She has authored or co-authored many publications and book chapters as well as lectured at local, national and international meetings.

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