Five resistant (mean age 6 years) and 7 susceptible (mean age 19 years) mares were selected through a meticulous screening procedure of research mares of mixed breeds and used in this study. All mares were maintained at the Department of Veterinary Science's Maine Chance Farm, University of Kentucky, Lexington, KY, USA. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.
Selection of mares
A flow chart depicting the steps and tests used for selecting mares for the study, and the timeline for PBS and E. coli inoculation is shown in Figure . In total, 90 non-pregnant mares were screened for resistance and susceptibility to persistent endometritis. Out of these, 12 mares were assigned to either of two groups, resistant or susceptible, based on endometrial histology, bacteriology, cytology and response to insemination with freeze-killed stallion spermatozoa.
Flow chart. Flow chart depicting the steps and tests used for selecting resistant and susceptible mares for the study, and the timeline for PBS and E. coli inoculation.
Uterine swab samples and endometrial biopsies were collected using an alligator jaw biopsy punch introduced into the uterus through a sterile speculum (Equivet ®, Kruuse A/S, Langeskov, Denmark) 3, 12, 24 and 72 h after inoculation. Biopsies were collected from the ventral part of the uterine body. The samples were immediately transported to the laboratory for preparation and analysis.
An endometrial biopsy was obtained in diestrous (day 5 post ovulation) from all mares, fixed in 10% formalin, sectioned at 5 μm and stained with hematoxylin and eosin. Each biopsy was examined for periglandular fibrosis, inflammatory cells, glandular distribution and lymphatic lacunae, then graded according to Kenney and Doig [21
]. The semen was prepared from a fresh ejaculate, washed in phosphate buffered saline (PBS) (pH 7.4), resuspended in milk based semen extender (EquiPro®
; Minitube of America, Verona, WI, USA) to yield 1 × 109
spermatozoa in 35 mL of extender per insemination dose, then stored at -20°C. Before use, the semen was thawed at room temperature. This method has previously been shown to reliably induce a uterine inflammation [22
]. Once in estrus (determined by the presence of uterine edema and a follicle of at least 35 mm in size), mares were inseminated with the killed semen, and then evaluated daily. A uterine swab sample and low volume uterine lavage were obtained 48 or 96 h later depending on uterine biopsy score [21
]. 250 ml of Lactated Ringer were infused into the uterus, and a minimum of 50 ml were recovered. To evaluate the mares' ability to clear uterine inflammation, uterine samples were collected at different time points based on mares' endometrial quality. In mares with a uterine biopsy I and IIa uterine samples were obtained at 48 h to confirm resistance to persistent endometritis, and at 96 h in mares with a uterine biopsy IIb or III to confirm susceptibility.
Gonadotropin releasing hormone (GnRH; Biorelease 1.5 mg, Deslorelin, BET labs, Lexington, KY, USA) was administered to induce ovulation when a 35 mm follicle and uterine edema was present.
Group I consisted of five mares found to be resistant to persistent endometritis. They had endometrial tissue histologically rated as I or IIa [21
], and negative cytology (< 2 polymorphonuclear neutrophils (PMNs) per 5 fields at 400 × magnification) from a low volume lavage and negative bacterial growth obtained from a uterine swab (Minitube) and no intrauterine fluid 48 h after insemination with killed semen. Group II comprised seven mares susceptible to persistent endometritis with a grade IIb or III endometrium [21
] and an impaired ability to clear uterine inflammation (> 2 PMNs per 5 fields at 400 × magnification) and intrauterine fluid 96 h after insemination with killed semen. More than 2 cm of intrauterine fluid was recorded as fluid retention.
PBS/E. coli inoculation and collection of uterine samples
All mares received an intrauterine infusion of PBS in an estrous cycle prior to the E. coli infusion and all experimental procedures and sampling planned for the subsequent E. coli infusion were carried out in this estrous cycle to document the effects of repeated sampling in each individual mare. Ten ml of PBS (pH 7.4) was infused into the uterus via a sterile insemination catheter (Butler Schein Animal Health, Dublin, OH, USA) of each mare.
Mares were examined daily for follicular development, intrauterine fluid, development of uterine edema and cervical and uterine tone. In the absence of a distinct corpus luteum, the presence of a dominant follicle (> 25 mm), uterine edema and decreased uterine and cervical tone, a uterine swab sample was collected for bacterial culture, and E. coli
was infused in presence of a > 30 mm follicle (24-48 h later). Mares were prepared as for artificial insemination immediately prior to inoculation, and a uterine swab sample was collected (0 h) for bacterial culture and cytology to verify a sterile and non-inflamed uterine environment at the time of inoculation. A total of 105
CFU of E. coli
in 10 ml of PBS (pH 7.4) were inoculated via a sterile insemination catheter (Butler Schein Animal Health). The E. coli
strain (241) was originally isolated from a mare with infectious endometritis, and previously used in an experimental infectious endometritis model [9
Transrectal ultrasonography of the reproductive organs was performed -24, 0, 3, 12, 24, 48 and 72 h after inoculation with PBS/E. coli for detection of intrauterine fluid and ovary status. Uterine swab samples and endometrial biopsies were collected 3, 12, 24 and 72 h after inoculation. Biopsies were collected from the ventral part of the uterine body. The samples were immediately transported to the laboratory for preparation and analysis. The mares received 2500 IU of human chorionic gonadotropin (Chorulon; Intervet, Millsboro, DE, USA) as an ovulation-inducing agent when a follicle > 35 mm and pronounced uterine edema were present.
In the estrous following E. coli infusion, a uterine swab sample was collected from the mares to determine the presence of bacterial growth and inflammation (positive exfoliative cytology). If a sterile and non-inflamed uterine environment was detected, a control biopsy was collected as described above; if the mare had positive cytology and/or growth of pathogens from the uterine swab, they were treated with intrauterine lavage and antimicrobials according to sensitivity testing before collecting the control biopsy. All mares were confirmed free from infection and inflammation at the time for collecting the control biopsy.
Preparation of E. coli inocula
E. coli 241 kept at -80°C was streaked on a blood agar plate (5% horse blood) and incubated for 24 h at 37°C. One colony was transferred to 2 ml of sterile Brain Heart Infusion broth (Fischer Scientific, Pittsburgh, PA, USA) and incubated overnight at 37°C. The overnight broth was serial diluted using sterile PBS to a concentration of 106 colony forming units (CFU) per ml and then diluted in 9 ml of sterile PBS to a final concentration of 106 CFU per 10 ml inoculum. The inocula were kept on ice until use (maximum 2 hours).
Bacterial examination and exfoliative cytology of endometrial biopsies and swabs
Immediately after sampling, endometrial biopsies were divided in two pieces with a sterile scalpel. One part of the biopsy was dissected into small pieces (1-2 mm) and stored in RNAlater (Ambion, Austin, TX, USA) at 5°C for 24 h, followed by storage at -20°C until further processing. The other part of the biopsy and the uterine swab were streaked on blood agars (5% horse blood) and incubated aerobically for 24 h at 37°C. Bacterial growth was identified according to colony morphology, Hancock stain-morphology, haemolysis and catalase and potassium hydroxide (3% KOH) tests. Colonies were counted and scored: no growth/sterile: < 5 CFU, mild growth: 5-10 CFU, moderate growth: 11-50 CFU and heavy growth: > 50 CFU. Culture results were recorded as E. coli
, beta-haemolytic streptococci
, other uterine pathogens or no growth. When more than 3 different isolates were present, the culture was recorded as contamination. The biopsies and swabs were smeared on glass slides, which were dried at room temperature and stained with Diff-Quick ®
(Fisher Scientific), and evaluated by light microscopy (×400 magnification). Cytological classification of the uterine biopsies and swabs were based on numbers of PMNs present per 200 endometrial cells examined [23
]. PMNs were counted and scored: no inflammation: 0-1 PMN, mild endometrial neutrophilia: 2 PMNs, moderate endometrial neutrophilia: 3-4 PMNs, and severe endometrial neutrophilia: > 5 PMNs.
Blood samples were obtained at 0, 3, 6, 12, 24, 48, 72, 96, 120, 168 h after inoculation and in the estrous following E. coli inoculation. Blood was drawn from the jugular vein using a Vacutainer® system into tubes containing sodium citrate for the determination of plasma fibrinogen, tubes containing EDTA for analysis of white blood cell count (BD Vacutainer; BD-Vacutainer Systems, Plymouth, USA), tubes containing no additive (Butler Schein Animal Health) for SAA analysis, and PAXgene tubes (Qiagen, Valencia, CA, USA) for subsequent RNA isolation.
Analysis of the total white blood cell count (WBC) was performed using VetAutoread™ Hematology Analyzer (IDEXX, Westbrook, ME, USA) immediately after collection. Serum and plasma were prepared by centrifugation at 3500 × g at 4°C and stored at -20°C until analysis. Fibrinogen was determined by the Clauss method in an automated coagulometric analyzer (ACL 9000; Instrumentation Laboratory, Barcelona, Spain), and the concentration of SAA was determined by an automated analyzer (ADVIA 1650 Chemistry System; Bayer A/S, Lyngby, Denmark) using a commercially available immunoturbidometric assay (LZ test SAA; Eiken Chemical Co., Ltd., Tokyo, Japan) as described by Jacobsen et al. [24
Total RNA was isolated from 60 mg of endometrial tissue stored in RNAlater using 650 μl TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. SV Total RNA Isolation System (Promega, Mannheim, Germany) including DNAse treatment was used for clean-up of the extracted RNA. Total cellular RNA from leukocytes was isolated from approximately 2.5 ml whole blood collected into PAXgene tubes. The tubes were incubated at room temperature for 24 h and then stored at -20°C until assayed. Once thawed, total RNA was extracted and DNAse treated with the PAXgene blood RNA extraction kit (Qiagen) using manufacturer's protocol. RNA was quantified via spectrophotometry using a NanoDrop ND-1000 (Agilent Technologies, Palo Alto, CA, USA). All samples had 260/280 ratio of 1.95 or higher and 260/230 ratio of 2.0 or higher, and were used for further analysis. RNA samples (1000 ng/reaction for endometrial samples and 250 ng/reaction for blood samples) were reverse transcribed using a RT-PCR kit (Promega), Oligo dT (Promega) and random primers (R&D systems, Minneapolis, MN, USA). The total volume of each reaction was 25 μl. Reactions were incubated at 25°C for 10 min, heated at 42°C for 60 min, heated at 95°C for 5 min, then cooled to 4°C and stored at -20°C until qRT-PCR analysis.
The mRNA expression of IL-1β
in leukocytes and endometrial tissue and the expression of SAA
in endometrial tissue were measured by qRT-PCR. A specific primer for IL-1ra was designed using Primer3 http://frodo.wi.mit.edu/
Oligonucleotide primer sequences for amplification of various equine cytokines, SAA and reference genes
All primers were commercially synthesized (Invitrogen). qRT-PCR was completed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with the following cycling conditions: 95°C for 10 min; 45 cycles of 95°C for 10 s, 60°C for 10 s, 72°C for 30 s; 55-95°C for dissociation. The qRT-PCR reactions were performed in 96-well plates (for cDNA synthesized from leukocyte extracted RNA) and 384-well plates (for endometrial samples), with a final volume of 20 μl per reaction. Each reaction contained a diluted (10×) cDNA sample (4 μl), 10 μl SYBR green, 2 μl of each primer (forward and reverse, 10 μM) and 2 μl ddH2O. A no-template control (RNase-free water) was included for every qRT-PCR run. Samples were done in duplicates. Efficiency of amplification for each primer was monitored through the analysis of serial dilutions (10-fold). The melting curves of the amplified PCR products were obtained for confirmation of specific amplification. Negative controls containing no template (H2O) and non-reverse transcribed RNA were included to verify amplification of a single product. The product sizes of specific products were verified on a 1% agarose gel. A pool of all endometrial samples (from here on referred to as calibrator) was generated and added as internal control during each qRT-PCR analysis.
All gene amplifications from endometrial samples were normalized to β-actin
, selected as the most stable reference gene across all uterine samples from a panel of three potential reference genes (glyceraldehydes-3-phosphate dehydrogenase (GAPDH
), beta glucoronidase (B-GUS
), beta-actin (β-actin
)) analyzed using GeNorm [28
was used as an endogenous control gene for the leukocyte samples [29
Data analyses and statistical methods
Cycle threshold (Ct) values were obtained through the auto Ct function. Following efficiency correction, the mean threshold cycle (CT
) was calculated and then normalized to the reference gene using delta (Δ) CT
. The calibrator was used to carry out an additional normalization step in order to account for differences in amplification dynamics between PCR reactions between different PCR reaction plates. Changes in relative expression were calculated using the 2-ΔΔCt
]. The specific transcripts are presented as n-fold change relative to pre-inoculation level (leukocytes) and estrous baseline levels (controlbiopsy, endometrium).
Outliers were defined as relative gene expression levels differing more than 3 × standard deviation, and were excluded for further data analyses. A high degree of individual variation in endometrial gene expression was observed, and in total 47 out of 752 (6%) gene expression levels were defined as outliers and excluded from data analyses.
The effect of intrauterine infusion of E. coli on repeated measurements of blood variables, SAA and cytokine mRNA expression in endometrial biopsies and leukocytes and cytological response was statistically analyzed using a repeated measures analysis of variance procedure in SAS (PROC MIXED). A first order autoregressive covariance structure was defined to take into account significant autocorrelation between measurements within mares. Differences in least squares mean estimates from the repeated measurement analyses were used to identify time points where the analyzed marker increased/decreased significantly from the pre-inoculation level. Bonferroni's multiple comparison procedure was used in order to control for Type I errors. To test if intrauterine infusion of PBS/E. coli elicited any upregulated endometrial gene expression of SAA and cytokines compared to estrous baseline levels, data was analyzed using the repeated measures analysis of variance as described above.
A non-parametric t-test (Mann Whitney U test) was used to identify specific time points where the gene expression differed significantly between resistant and susceptible mares and between different treatments (PBS and E. coli).
The effects of intrauterine infusion of PBS or E. coli on the presence on intrauterine fluid, bacterial growth of E. coli, S. zooepidemicus and other pathogens were statistically analyzed using linear logistic regression (PROC GENMOD) in SAS. A logit transformation of data was used to describe the relationship between the outcome and the explanatory variable. A generalized score test (Wald's test) was used in the type 3 analysis, and significant differences between the time points for sample collection were identified by using least square means. Goodness-of-fit tests were performed to control the model of analyses of a dichotomous outcome.
All values are presented as means ± standard error of the means (SEM). Assumptions were checked on residual plots and tested for normality. Initial inspection of the data revealed that SAA and cytokine mRNA expression varied markedly between individuals. Because variances were heterogenous, 2-ΔΔCt values were log transformed, and geometric least square means statistically compared. All statistical calculations were made with the software SAS 9.2 (SAS Institute, Cary, NC, USA). Graphs were made using the software GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). The level of significance was set to p ≤ 0.05.