Dynamics of postpartum endometrial cytology and bacteriology and their relationship to fertility in dairy cows

Accepted Manuscript Dynamics of postpartum endometrial cytology and bacteriology and their relationship to fertility in dairy cows Robert O. Gilbert, Natalia R. Santos PII:

S0093-691X(16)00063-7

DOI:

10.1016/j.theriogenology.2015.10.045

Reference:

THE 13503

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Theriogenology

Received Date: 8 March 2015 Revised Date:

15 October 2015

Accepted Date: 28 October 2015

Please cite this article as: Gilbert RO, Santos NR, Dynamics of postpartum endometrial cytology and bacteriology and their relationship to fertility in dairy cows, Theriogenology (2016), doi: 10.1016/ j.theriogenology.2015.10.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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REVISED

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Dynamics of postpartum endometrial cytology and bacteriology and their relationship to

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fertility in dairy cows

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4 Robert O. Gilbert and Natalia R. Santos1

Department of Clinical Sciences

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College of Veterinary Medicine

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Cornell University

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Ithaca, NY 14853-6401

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USA

12 Address correspondence to:

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R.O. Gilbert

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[email protected]

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Ecole nationale Vétérinaire d'Alfort

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Laboratoire de biologie de la reproduction

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7, avenue du Général de Gaulle

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94700 Maisons-Alfort

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FRANCE

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Present address:

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ABSTRACT

25 Endometrial samples were obtained from 56 consecutively calving dairy cows examined for

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endometrial cytology and for aerobic and anaerobic bacterial growth. Changes over time,

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correlations between different cell types and between cell and bacterial populations and with

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fertility measures were calculated. The proportion of neutrophils in cytologic preparations

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decreased with time postpartum. Other cell types did not change significantly with time. The

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proportion of neutrophils early (day 0 and 7) postpartum was negatively correlated with

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neutrophil proportion at 5 or 7 weeks postpartum and positively correlated with fertility. Cows

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with high proportion of neutrophils at 7 days postpartum (> 40 %) were significantly more likely

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to become pregnant than those with lower proportions of neutrophils.

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Escherichia coli were the bacteria most frequently isolated at 0 or 7 days postpartum but were

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uncommon after that. Trueperella pyogenes were most prevalent at 3 weeks postpartum, and

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were more likely to infect cows that had previously been infected with E. coli. Presence of T.

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pyogenes at 3 weeks postpartum increased the risk of concomitant or later infection with gram

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negative anaerobes. The presence of T. pyogenes at 3 weeks postpartum significantly reduced

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the risk of pregnancy at 150 days in milk (DIM). The presence of alpha-hemolytic Streptoccus

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spp. at 7 days postpartum was associated with improved reproductive performance. The

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proportion of neutrophils at 5 and 7 weeks postpartum was related to concomitant bacterial

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infection.

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These findings suggest that rapid mobilization of neutrophils to the postpartum uterus is a

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beneficial response for uterine health in dairy cows.

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Key words: Cytology; endometritis; infection; inflammation; uterus

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1. INTRODUCTION:

53 Prevalence of endometritis, as diagnosed by endometrial cytology, is high in dairy cows,

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persisting up to and beyond the end of the traditional postpartum voluntary waiting period.

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Several studies have confirmed a high prevalence of endometritis after 40 days postpartum and

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that the condition has a negative impact on subsequent reproductive performance [1-7]. In

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contrast, however, most publications maintain that the bovine uterus, although consistently

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contaminated or infected in the early postpartum period, is essentially sterile by about 28 days

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postpartum [8-11]. The distinction between proportion of infected cows and cows with

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subclinical endometritis is well documented by Sheldon, et al. [9]. Therefore, one aim of this

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study was to examine the relationship between bacterial infection and cytological evidence of

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endometritis and to determine if endometritis persisted after elimination of bacterial infection.

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The relationship between persistent bacterial infection and inflammation is important in devising

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strategies for prevention and treatment of endometritis. For example, antibacterial treatments are

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unlikely to be effective if the inflammation occurs in the absence of bacterial infection, or

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persists long after the infection is resolved. This study was undertaken to further understanding

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of the relationship between uterine infection and evidence of endometrial inflammation

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(measured by endometrial cytology), and the relationships between uterine infection,

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endometritis, postpartum endometrial cytology and fertility in lactating dairy cows. We

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postulated that uterine bacterial populations would account for observed inflammation and that

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inflammation (and infection) would steadily decrease with time postpartum.

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The relationship between bacterial infection and endometritis, which is often subclinical, could

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also cast light on the pathogenesis of endometritis. For example, is endometritis largely a

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reflection of a generalized inflammatory environment attributable to a form of metabolic

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syndrome (extensive fat mobilization and insulin resistance) that is often recognized in

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postpartum cows [12], or is it more simply a consequence of local bacterial infection (which

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itself may follow more generalized immune impairment [13])?

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Finally, characterization of the relationship between uterine infection and endometritis could cast

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light on the mechanism of infertility in endometritis. Do bacteria play a direct role in altering the

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uterine environment, rendering it hostile to spermatozoa or the developing zygote, or is infertility

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mediated by more subtle changes brought about by inflammatory mediators at a uterine [14,15]

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or ovarian [15-17] level?

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While cross sectional studies of endometrial cytology abound, few have examined cows in a

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longitudinal fashion and those that have, have concentrated on cows later in lactation, for

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example at 35 and 56 days postpartum [2,18], or at 3, 5 and 7 weeks postpartum [4]. We

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therefore examined endometrial cytology beginning immediately postpartum and continuing

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until 49 days postpartum. Knowledge of the progression of endometrial cytology is important

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for identifying periods in which clinically significant inflammation can be diagnosed and

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distinguished from the physiological inflammation which occurs postpartum and is necessary for

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the extensive uterine tissue remodeling required during uterine involution for return to fertility.

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Our goals therefore were to characterize the progression of endometrial cytology in postpartum

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dairy cows, and to evaluate the relationship between uterine bacterial infection and endometrial

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cytology. The relationship between endometrial cytology or uterine bacterial infection at different postpartum periods and subsequent fertility was also investigated.

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2. MATERIALS and METHODS:

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103 2.1 Animals and animal procedures

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All animal procedures were approved by the Cornell University Institutional Animal Care and

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Use Committee (protocol 2004-0078).

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Consecutively calving cows (n = 56), calving over a 12-month period in 2004-05, in the College

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of Veterinary Medicine Dairy Herd were enrolled in this experiment. Endometrial samples were

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obtained by low volume uterine lavage on the day of calving and 7, 21, 35 and 49 days

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postpartum as previously described [6]. Briefly, a sterile plastic infusion pipette contained in a

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chemise was passed through the cervix under transrectal control and 20 ml sterile saline injected

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into the uterine lumen, gently agitated, and aspirated. Recovered fluid was used for aerobic and

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anaerobic bacterial culture by thoroughly wetting a Tran-Swab culturette (Fisher Scientific;

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Pittsburgh, PA) and a Port-a-cul® (Becton Dickinson, Franklin Lakes, NJ) swab and submitting

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them to the Animal Health Diagnostic Laboratory at Cornell University for aerobic and

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anaerobic bacterial culture, respectively. Remaining fluid was used for cytological evaluation of

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the uterus. An aliquot of fluid was transferred to a glass microscope slide using a cytocentrifuge,

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air dried and stained using a Romanowski staining technique (Diff Quik, VWR, Arlington

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Heights, IL) [6]. Note that on the day of calving it was usually not necessary to inject fluid into

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the uterus before aspirating a sample. A pipette could simply be introduced and uterine content

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aspirated for further processing.

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123 2.2 Bacterial culture

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Bacterial samples were cultured at the Animal Health Daignostic Laboratory as follows. For

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aerobic culture, the swabs were inoculated on to the following agar plates - Blood Agar (BP),

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Chocolate Agar (CHOC), Eosin Methylene Blue Agar (EMB) and Columbia Colistin Nalidixic

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Acid Agar (CNA) and struck for isolation of bacteria. The plates were incubated at 35°C with

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6% carbon dioxide overnight before being examined. Individual bacterial colonies were

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identified using biochemical methods or Trek automated ID or MALDI-TOF biotyper.

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The anaerobic cultures were performed in an anaerobic chamber on Brucella agar, Phenylethyl

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alcohol agar (PEA), Bacteroides bile esculin agar (BBE), and Brucella laked blood agar with

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kanamycin and vancomycin (LKV) at 35C. Individual bacterial colonies were identified using

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biochemical methods and/or MALDI-TOF biotyper.

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2.3 Endometrial cytology

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Endometrial cytology was evaluated by a single examiner blinded to the source of the sample.

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Nucleated cells (n = 200) were identified as endometrial epithelial cells, polymorphonuclear cells

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(presumptive neutrophils), large mononuclear cells (presumptive macrophages) and small

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mononuclear cells (presumptive lymphocytes). Examination was performed independently by

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both authors using bright field microscopy at 400 x magnification and the mean result used for

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analysis [4,6,7,19-25]. Progression of cell population with days postpartum was evaluated by

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linear regression. The relationship of proportion of each cell type to subsequent reproductive

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performance (pregnancy to first artificial insemination, pregnancy by 150 days postpartum) was

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evaluated by calculating spearman correlation coefficients, because pregnancy is a dichotomous

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outcome. Where significant correlations were found, predictive values of selected parameters

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were chosen by Receiver Operating Characteristic curves. The predictor was then dichotomized

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at the predictive value with the highest sum of sensitivity and specificity, and used in Cox

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proportional hazards regression.

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2.4 Statistical analysis

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Demographic information and reproductive performance of cows were recovered from electronic

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herd records (DairyComp305, ValleyAg Software, Tulare, CA).

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Results are presented as proportions or means (and standard errors). Associations between

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bacterial isolates at each time point and bacterial isolates at later time points, and

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contemporaneous and later cytology results, and the relationship between bacterial isolates and

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endometrial cytology results with pregnancy outcome were examined initially by calculating

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correlations, and then by multiple logistic regression (dichotomous outcome) including factors

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indicated by simple correlation coefficients and P-values. The effect of bacterial isolates on

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simultaneous endometrial cytology findings were first examined by univariate analysis.

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Bacterial species found to be related to any cytological outcome (P < 0.2) were included in a

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multivariate linear regression (continuous outcome) to explain variation in cell populations using

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manual backward exclusion of variables. Variables were retained in the final model if P <

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0.05. The relationship between bacterial species isolated at one time point with bacteria isolated

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later was examined initially by calculating spearman coefficients because presence or absence of

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a bacterial species was a dichotomous variable. Bacteria with significant relationships (P < 0.2)

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were included in multivariate logistic regression, with manual backward exclusion of

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variables. Variables were retained in the final model if P < 0.05. The relationship between

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bacterial species at different time points and cytological findings at each time point with

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reproductive outcomes was explored in a similar fashion. Pregnancy to first insemination and

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pregnancy at 150 days in milk were used as outcomes. When cytological parameters were found

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to be related to pregnancy outcomes, they were dichotomized using receiver operating

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characteristic (ROC) curve analysis to find the cutoff point with the highest sum of sensitivity

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and specificity. The dichotomized parameters were then used in a Cox regression model to

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calculate hazard of pregnancy. Cows declared “do not breed” were censored on the day that

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decision was made. Statistical significance was assumed at P ≤ 0.05, with tendencies reported at

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0.05 < P < 0.10. All statistical calculations were performed using Stata IC 11.1 (StataCorp,

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College Station TX) or GraphPad Prism 5.04.

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3.1.Demographics

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Cows in this trial ranged in parity from 1 to 6 (median 2, interquartile range: 1-3). All cows

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calved spontaneously and had single, live calves, of which 57 % (32/56) were male. Mean days

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to first insemination were 60.9 (SD = 8.5 days; range 53 – 100 days). Pregnancy to first

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insemination was 31% (15/49; 7 cows were designated as “do not breed” for reasons unrelated to

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this trial) and 49 % of cows bred (24/49) were pregnant by 150 days postpartum.

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3.2.Endometrial cytology Figure 1 depicts the changes in cell populations in endometrial cytology preparations over time

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postpartum. Compared to the proportion of cells on the day of calving, the polymorphonuclear

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neutrophil (PMN) proportion was unchanged at 1 and at 3 weeks postpartum, but decreased by 5

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and 7 weeks postpartum (P < 0.001; Fig. 1A). The proportion of macrophages (P = 0.175; Fig.

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1B) and lymphocytes (P = 0.384; Fig. 1C) did not change significantly with time postpartum but

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the lymphocyte proportion at 7 weeks was greater than that at calving (P = 0.013). The

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proportion of neutrophils and lymphocytes tended to be negatively correlated with each other,

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significantly so at 7 d (r = -0.45, P = 0.003) and 21 d (r = -0.51, P = 0.0002) postpartum.

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The cell populations most closely related to pregnancy outcomes were the proportions of

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PMN. Specifically, the PMN population at 7 d postpartum was positively correlated to

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pregnancy(r = 0.41; P = 0.01) and PMN proportion at 49 d postpartum was negatively correlated

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with pregnancy (r = -0.38; P = 0.01),. The proportion of neutrophils at 7 d postpartum was

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negatively correlated with the proportion at 7 weeks postpartum (r = -0.39, P = 0.02). Since a

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positive effect of higher neutrophil proportion at 7 days postpartum has not previously been

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reported, it was explored further. For PMN at 7 days postpartum 40 % was chosen as a cutoff

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point based on receiver operating characteristic curves. This value provided the greatest sum of

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sensitivity and specificity with pregnancy at 150 days postpartum as the outcome. Using the

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dichotomized values for PMN proportion at 7 days postpartum as a variable in Cox's

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proportional hazards regression (with parity as covariate), cows with high PMN proportion were

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found to have increased hazard of pregnancy (HR = 3.85; P = 0.01). Figure 2 depicts a Kaplan-

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Meier curve showing this result.

213 There was a tendency for a negative correlation between the proportion of macrophages at 7 d

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postpartum and pregnancy to first AI (r = - 0.27, P = 0.08) and the proportion of lymphocytes at

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7 d postpartum was positively correlated to risk of pregnancy by 150 d postpartum (r = 0.32, P =

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0.04) despite the fact that the proportion of lymphocytes at 7 d postpartum was negatively

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correlated to the proportion of PMN at the same time (r= -0.45, P = 0.003). When these cell

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types were combined in Cox’s proportional hazards analysis, the proportions of neutrophils and

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of lymphocytes remained significant, with both exercising a positive effect on hazard of

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pregnancy (Table 1).

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Table 1. Coefficients from Cox’s Proportional Hazards for pregnancy, depicting the influence of

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proportion of neutrophils and of neutrophils at 7 d postpartum.

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Hazard Ratio

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1.01 – 1.08

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1.006 – 1.17

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1.08

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Lymphocytes at 7 d

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No cytological parameter at 21 d postpartum was correlated with any fertility parameter

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(pregnancy to first AI, pregnancy by 150 DIM or pregnancy by 300 DIM).

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At 35 and 49 d postpartum the proportion of neutrophils was negatively correlated to pregnancy

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at 150 DIM, consistent with numerous previous studies [6,7,26-30]. Given the well-known

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nature of this relationship and the small size of the current study, the effect of elevated neutrophil

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proportion at 5 or 7 weeks postpartum was not further investigated.

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The most common aerobic bacterial species isolated on the day of calving and at 7 d postpartum

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were alpha-hemolytic Streptococcus spp. and Escherichia coli. These species rapidly became

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less common. By 21 d postpartum E. coli was rare and Trueperella pyogenes was more

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common. Figure 3 depicts a prevalence of the most commonly isolated aerobic bacterial species.

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Figure 4 shows similar results for anaerobic bacteria. Clostridial species (particularly C.

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perfringens) were most common in very early lactation. Other species were present at low

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prevalence throughout the study period.

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The effects of individual bacterial species and major categories (aerobic and anaerobic) at

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different intervals postpartum on subsequent pregnancy were explored. Interestingly, the

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presence of Streptococcus spp. (alpha-hemolytic Streptococci) at 1 week postpartum was

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positively associated with pregnancy (Cox HR = 3.4, P = 0.02). There was a positive correlation

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between the presence of streptocci at 7 d postpartum and proportion of PMN at the same time.

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Cows with Streptoccus spp. infection at 7 d postpartum had 59.5 ± 4 % neutrophils in

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endometrial cytology and those without the presence of this bacterium had 34.8 ± 4 % (P =

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0.04).

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The presence of Trueperella pyogenes at 3 weeks postpartum was associated with reduced risk of

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pregnancy at 150 days postpartum (OR = 0.102, 95% CI = 0.022 – 0.466; P = 0.003). The

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number of anaerobic isolates was small after 3 weeks postpartum. Isolation of Clostridium

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perfringens early postpartum did not influence subsequent reproduction, or subsequent bacterial

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populations. It had no direct effect on endometrial cytology. The presence of anaerobes at 5

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weeks postpartum (predominantly Fusobacterium necrophorum and Prevotella melaninogenica)

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tended to reduce the hazard of pregnancy (HR = 0.157, 95% CI = 0.019 – 1.26; P = 0.081). In

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this study we found no influence of Mycoplasma or Ureaplasma species on reproductive

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performance, although numbers were severely limiting.

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Cows with E. coli infection at 7 d postpartum had increased risk of having T. pyogenes at 21 d

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postpartum (OR = 4.8, 95% CI: 1.3 – 17.4; P = 0.02) and also of being infected with the gram

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negative anaerobes F. necrophorum and P. melaninogenica (OR = 4.5; 95 % CI 1.06 – 19.4; P =

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0.042). In turn, cows with T. pyogenes at 21 d postpartum were more likely to have concomitant

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infection with the gram negative anaerobes (OR = 18.4; 95 % CI 3.0 – 114; P = 0.002) and

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tended to have greater risk of infection with these pathogens at 35 d postpartum (OR = 9.7; 95 %

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CI 0.89 – 105; P = 0.06).

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3.4.Bacterial isolates and cytology

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The relationship between bacterial isolates and endometrial cytology was explored. In particular,

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the bacterial species associated with the PMN proportion at 1 week and 5 or 7 weeks postpartum

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were investigated, since they had positive or negative effects on subsequent reproduction.

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The presence of aerobic bacteria (all species combined) on the day of calving was associated

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with a lower contemporaneous proportion of PMN. Cows with infection had 43 ± 5 % PMN

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compared to 60 ± 5 % PMN for those uninfected (P = 0.04). A similar observation was made for

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anaerobic bacteria. Cows infected with anaerobes had 40 ± 9 % PMN while those free of

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anaerobic infection had 61 ± 4 % PMN (P = 0.03).

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By 1 week postpartum cows with streptocci isolated from the uterus had higher PMN proportions

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in endometrial cytology than those free of streptocci (60 ± 5 % and 35 ± 4 %, respectively; P =

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0.37). This has been alluded to above. Other individual bacterial species had no significant

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effect in very early lactation.

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There was no correlation between cytological parameters at 3 weeks postpartum and any

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reproductive parameter (pregnancy to first AI, pregnancy by 150 days, or pregnancy by 300

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days). However, the presence of bacteria in the uterus at 3 weeks postpartum was associated

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with higher PMN proportion at this time. Cows with no isolated bacteria had 38 ± 5 % PMN and

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those with bacterial isolates of any species had 57 ± 3 % PMN (P = 0.002). Considering only

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aerobic bacteria, the PMN proportions were was 40 ± 5 % and 57 ± 3 %, respectively (P = 0.01),

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and for anaerobes alone 42 ± 4 % and 59 ± 4 %, respectively (P = 0.015). When bacterial

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species were considered individually, the only significant effect observed was due to the

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presence of T. pyogenes. Cows from which T. pyogenes was isolated had 62 ± 3 % PMN while

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those free of the bacterium had 42 ± 4 % PMN (P = 0.003). The presence of T. pyogenes and the

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concurrent presence of gram negative anaerobes (either Prevotella melaninogenica or

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Fusobacterium necrophorum or both) at 3 weeks postpartum was correlated (r = 0.58, P =

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0.0003).

300 The proportion of PMN in endometrial cytology samples at 35 d postpartum was influenced by

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the presence of T. pyogenes at 21 and at 35 d postpartum and by the presence of the gram

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negative anaerobes at 35 d postpartum. Anaerobic pathogens were combined because of small

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numbers in each species. The final regression model for predicting percentage of PMN at 35 d

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postpartum included only T. pyogenes at 3 weeks and anaerobes at 5 weeks postpartum. It is

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summarized in Table 2.

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Table 2. Linear regression model for effect of uterine bacteria on the proportion of neutrophils at

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D 35 postpartum. (Overall r2 = 0.36; P = 0.0005)

Variable T. pyogenes at 21 d

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P

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1.12 – 29.57

0.035

17.31

11.56 – 57.85

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3.88 – 18.25

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Gram negative anaerobes at 35 d

Coefficient

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The proportion of PMN in endometrial cytology samples at 49 d postpartum was affected by the

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contemporaneous presence of T. pyogenes or Prevotella melaninogenica. Bacteria present before

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7 weeks did not significantly affect PMN at 7 weeks. The effect of these two organisms was

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evaluated using multiple linear regression, the results of which are summarized in Table 3.

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Table 3. Linear regression model for effect of uterine bacteria on the proportion of neutrophils at

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D 49 postpartum. (Overall r2 = 0.43; P < 0.0001)

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95 % CI

T. pyogenes at 49 d

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0.046 – 25.30

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P. melaninogenica at 49 d

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5.70 – 34.36

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Constant

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3.42 – 10.95

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Perhaps the most unexpected finding of this study was the observation that high proportions of

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neutrophils in endometrial cytology samples obtained soon after parturition are beneficial to

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uterine health and subsequent pregnancy. The fact that bacterial infection with any species of

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bacterium was less likely in cows with high proportions of uterine neutrophils early postpartum

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is consistent with this finding, as is the observation that PMN proportion at 7 d postpartum is

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negatively associated with PMN at 7 weeks postpartum. High uterine PMN population in the

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later postpartum period is well known to be associated with poor reproductive outcome [4-

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6,22,26,27,31]. It seems that cows that are capable of recruiting large numbers of neutrophils

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rapidly to the uterus in the immediate postpartum period are likely to have the healthiest

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postpartum course. This is also consistent with the well-established finding that retention of the

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fetal membranes in cows is associated with failure to recruit adequate numbers of neutrophils to

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the placental interface [32]. Furthermore, it has been clearly shown that negative energy balance

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in the periparturient period is a major risk factor for postpartum endometritis [13,22,23,28] and

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that cows with improved periparturient energy balance had higher proportions of neutrophils in

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cytological preparations at 7 d postpartum [24]. Additionally, it appears that diminished

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expression of inflammatory cytokines in the periparturient period may increase risk of

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postpartum uterine disease [33]. Therapeutic interventions to increase mobilization of

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neutrophils to the early postpartum uterus may be beneficial to uterine health and reproduction,

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for example by local application of chemokines such as CXCL8 (formerly IL-8), which is

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involved in neutrophil recruitment to the placental interface at parturition [32]. Conversely, this

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finding might explain why therapeutic interventions with the potential to inhibit neutrophil

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recruitment to the uterus, such as non-steroidal inflammatory treatment in the periparturient

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period, have been found to be detrimental to health and fertility of cows in some studies [34-36].

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We found that the proportion of neutrophils in postpartum endometrial cytology remained high

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for the first three weeks postpartum, declining thereafter. This finding suggests that neutrophils

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at this time are a reflection of physiological inflammatory processes associated with uterine

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remodeling and that it is not appropriate to diagnoses pathological inflammation before 21 days

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postpartum.

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Another novel finding of this study is that infection with alpha-hemolytic streptococci, generally

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regarded as non-pathogenic, opportunist contaminants of the bovine uterus [37], had a positive

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association with subsequent pregnancy as well as being associated with the recruitment of large

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numbers of neutrophils to the uterus in the early postpartum period. Bonnett et al. previously

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reported that alpha-hemolytic streptocci isolated at 26 and 40 d postpartum were associated with

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reduced endometrial inflammation in endometrial biopsies obtained at the same time [38], but

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our observation is the first to associate very early presence of alpha hemolytic streptocci with

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beneficial effects on subsequent inflammation and on fertility, occurring much later in lactation.

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This raises the possibility of successful probiotic treatment of early postpartum cows with alpha-

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hemolytic streptococci to mediate recruitment of uterine neutrophils with a potentially beneficial

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effect on subsequent uterine health and reproductive performance. A similar approach has

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yielded success in colonizing the nasopharynx of infants with a beneficial flora [39].

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A major goal of this project was to determine whether endometritis, as indicated by endometrial

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cytology, persisted after resolution of bacterial infection. Our results indicate that PMN

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proportion in cytological preparations was related to contemporaneous or immediately preceding

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bacterial infection from 3 weeks postpartum onwards. Specifically, the presence of T. pyogenes

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in the uterus at 3 weeks postpartum mediated the increase in uterine PMN at that time and was

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also a risk factor for concurrent and subsequent infection with the gram negative anaerobes P.

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melaninogenica and F. necrophorum as was also reported by Bonnett [38]. This triad of bacteria

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accounted for most of the variance in PMN proportion observed at 5 and 7 weeks postpartum.

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High proportion of uterine PMN at this stage postpartum has consistently been related to poor

374

reproductive performance. We therefore conclude that, at least for the period studied here,

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endometrial cytological evidence of endometritis reflects concurrent bacterial infection. This

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also suggests that uterine infection with these pathogens persists longer than has previously been

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recognized. Earlier workers regarded the uterus as essentially sterile after 4 weeks postpartum

378

[40-42].

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This study confirmed the well-established observations that the presence E. coli immediately

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postpartum or during the first postpartum week predisposes cows to T. pyogenes infection at 3

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weeks postpartum [43-46] and that T. pyogenes after 21 days postpartum is associated with

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concomitant or subsequent infection with gram negative anaerobes F. necrophorum and P.

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melaninogenica, with increased risk of endometritis, and with reduced risk of pregnancy

385

[37,38,45,47,48]. Modern studies have employed metagenomic methods to define the

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microbiome of the postpartum uterus. Not surprisingly, these have suggested that the gram-

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negative anaerobes are much more common than indicated by conventional culture methods

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[45,47], but the importance of these organisms and of T. pyogenes in mediating uterine disease

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and infertility in our study is consistent with more comprehensive modern knowledge.

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Rapid recruitment of neutrophils to the uterine lumen, reflected in a high neutrophil proportion at

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calving and 7 d postpartum was associated with improved fertility. Persistent high neutrophil

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proportions at 35 and 49 d postpartum were detrimental to fertility. Trueperella pyogenes at and

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after 21 d postpartum and gram negative anaerobes (F. necrophorum and P. melaninogenica) at

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and after 21 d postpartum mediated increased neutrophil proportion at 35 and 49 d postpartum

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and reduced fertility. Presence of alpha-hemolytic streptocci in the early postpartum period was

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beneficial to subsequent fertility.

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6. ACKNOWLEDGEMENTS

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This research was funded, in part, by grants from the National Association of Animal Breeders

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and the USDA National Institute of Food and Agriculture, Hatch project 231319. Funders had

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no input into experimental design, interpretation of data, preparation of the manuscript or

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decision to publish. Any opinions, findings, conclusions, or recommendations expressed in this

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publication are those of the authors and do not necessarily reflect the view of the National

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Institute of Food and Agriculture (NIFA) or the United States Department of

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Agriculture(USDA).

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408 7. REFERENCES:

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Cheong SH, Nydam DV, Galvão KN, Crosier BM, Gilbert RO. Cow-level and herd-level risk factors for subclinical endometritis in lactating Holstein cows. J Dairy Sci 2011;94: 762-70. Dubuc J, Duffield TF, Leslie KE, Walton JS, LeBlanc SJ. Definitions and diagnosis of postpartum endometritis in dairy cows. J Dairy Sci 2010;93: 5225-33. Dubuc J, Duffield TF, Leslie KE, Walton JS, Leblanc SJ. Effects of postpartum uterine diseases on milk production and culling in dairy cows. J Dairy Sci 2011;94: 1339-46. Galvão KN, Frajblat M, Brittin SB, Butler WR, Guard CL, Gilbert RO. Effect of prostaglandin F-2 alpha on subclinical endometritis and fertility in dairy cows. Journal of Dairy Science 2009;92: 4906-13. Vieira-Neto A, Gilbert RO, Butler WR, Santos JEP, Ribeiro ES, Vercouteren MM, Bruno RG, Bittar JHJ, Galvao KN. Individual and combined effects of anovulation and cytological endometritis on reproductive performance of dairy cows. Journal of Dairy Science 2014;97: 1-11. Gilbert RO, Shin ST, Guard CL, Erb HN, Frajblat M. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology 2005;64: 1879-88. Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology 2006;65: 1516-30. Sheldon IM, Dobson H. Postpartum uterine health in cattle. Animal Reproduction Science 2004;82-83: 295-306. Sheldon IM, Williams EJ, Miller AN, Nash DM, Herath S. Uterine diseases in cattle after parturition. Vet J 2008;176: 115-21. Sheldon IM. The postpartum uterus. The Veterinary clinics of North AmericaFood animal practice 2004;20: 569-91. Sheldon IM, Rycroft AN, Zhou C. Association between postpartum pyrexia and uterine bacterial infection in dairy cattle. The Veterinary record 2004;154: 289-93. Sordillo LM, Contreras GA, Aitken SL. Metabolic factors affecting the inflammatory response of periparturient dairy cows. Anim Health Res Rev 2009;10: 53-63. Galvão KN, Flaminio MJ, Brittin SB, Sper R, Fraga M, Caixeta L, Ricci A, Guard CL, Butler WR, Gilbert RO. Association between uterine disease and indicators of neutrophil and systemic energy status in lactating Holstein cows. J Dairy Sci 2010;93: 2926-37. Hill J, Gilbert R. Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium. Aust Vet J 2008;86: 312-6.

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Gilbert RO. The effects of endometritis on the establishment of pregnancy in cattle. Reprod Fertil Dev 2011;24: 252-7. Herath S, Williams EJ, Lilly ST, Gilbert RO, Dobson H, Bryant CE, Sheldon IM. Ovarian follicular cells have innate immune capabilities that modulate their endocrine function. Reproduction 2007;134: 683-93. Sheldon IM, Price SB, Cronin J, Gilbert RO, Gadsby JE. Mechanisms of Infertility Associated with Clinical and Subclinical Endometritis in High Producing Dairy Cattle. Reproduction in Domestic Animals 2009;44: 1-9. Dubuc J, Duffield TF, Leslie KE, Walton JS, LeBlanc SJ. Risk factors for postpartum uterine diseases in dairy cows. J Dairy Sci 2010;93: 5764-71. Gilbert RO, Shin ST, Guard CL, Erb HN. Incidence of endometritis and effects on reproductive performance of dairy cows. Theriogenology 1998;49: 251-. Santos NR, Lamb GC, Roman HB, Gilbert RO. Postpartum endometrial cytology in beef cows. Theriogenology 2005;64: 796-. Galvão KN, Brittin SB, Frajblat M, Gilbert RO. Defining cutoff points for subclinical endometritis at different stages of lactation. Journal of Dairy Science 2007;90: 13-. Cheong SH, Nydam DV, Galvão KN, Crossier BM, Gilbert RO. Cow-level and herdlevel risk factors for subclinical endometritis in lactating Holstein cows. Journal of Dairy Science 2011;94: 762-70. 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. J Dairy Sci 2014;97: 2763-70. Yasui T, McArt JA, Ryan CM, Gilbert RO, Nydam DV, Valdez F, Griswold KE, Overton TR. Effects of chromium propionate supplementation during the periparturient period and early lactation on metabolism, performance, and cytological endometritis in dairy cows. J Dairy Sci 2014;97: 6400-10. de Boer MW, LeBlanc SJ, Dubuc J, Meier S, Heuwieser W, Arlt S, Gilbert RO, McDougall S. Invited review: Systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows. J Dairy Sci 2014;97: 3983-99. Kasimanickam R, Duffield TF, Foster RA, Gartley CJ, Leslie KE, Walton JS, Johnson WH. Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows. Theriogenology 2004;62: 9-23. Barlund CS, Carruthers TD, Waldner CL, Palmer CW. A comparison of diagnostic techniques for postpartum endometritis in dairy cattle. Theriogenology 2008;69: 714-23. Hammon DS, Evjen IM, Dhiman TR, Goff JP, Walters JL. Neutrophil function and energy status in Holstein cows with uterine health disorders. Veterinary immunology and immunopathology; Veterinary immunology and immunopathology 2006;113: 21-9. Lincke A, Drillich M, Heuwieser W. Subclinical endometritis in dairy cattle and its effect on reproductive performance--a review on recent publications. Berliner und Munchener tierarztliche Wochenschrift 2007;120: 245-50. de Boer MW, LeBlanc SJ, Dubuc J, Meier S, Heuwieser W, Arlt S, Gilbert RO, McDougall S. Invited review: Systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows. J Dairy Sci 2014. Dubuc J, Duffield TF, Leslie KE, Walton JS, Leblanc SJ. Randomized clinical trial of antibiotic and prostaglandin treatments for uterine health and reproductive performance in dairy cows. J Dairy Sci 2011;94: 1325-38.

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Kimura K, Goff JP, Kehrli ME, Jr., Reinhardt TA. Decreased neutrophil function as a cause of retained placenta in dairy cattle. Journal of Dairy Science 2002;85: 544-50. Galvao KN, Felippe MJ, Brittin SB, Sper R, Fraga M, Galvao JS, Caixeta L, Guard CL, Ricci A, Gilbert RO. Evaluation of cytokine expression by blood monocytes of lactating Holstein cows with or without postpartum uterine disease. Theriogenology 2012;77: 35672. Shwartz G, Hill KL, VanBaale MJ, Baumgard LH. Effects of flunixin meglumine on pyrexia and bioenergetic variables in postparturient dairy cows. J Dairy Sci 2009;92: 1963-70. Stahringer RC, Neuendorff DA, Randel RD. The effect of aspirin administration and parity on plasma salicylate concentrations and postpartum reproductive parameters in Brahman cows. Prostaglandins Other Lipid Mediat 1999;58: 125-38. Duffield TF, Putnam-Dingwell H, Weary DM, Skidmore AK, Neuder LM, Raphael W, Millman S, Newby N, Leslie KE. Effect of flunixin meglumine treatment following parturition on cow health and milk production. Journal of Dairy Science 2009;92: 118. Williams EJ, Fischer DP, Pfeiffer DU, England GC, Noakes DE, Dobson H, Sheldon IM. Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. Theriogenology 2005;63: 102-17. Bonnett BN, Martin SW, Gannon VP, Miller RB, Etherington WG. Endometrial biopsy in Holstein-Friesian dairy cows. III. Bacteriological analysis and correlations with histological findings. Can J Vet Res 1991;55: 168-73. Sprunt K, Leidy G. The use of bacterial interference to prevent infection. Can J Microbiol 1988;34: 332-8. Griffin JF, Hartigan PJ, Nunn WR. Non-specific uterine infection and bovine fertility. I. Infection patterns and endometritis during the first seven weeks post-partum. Theriogenology 1974;1: 91-106. Elliott L, McMahon KJ, Gier HT, Marion GB. Uterus of the cow after parturition: bacterial content. Am J Vet Res 1968;29: 77-81. Bekana M, Jonsson P, Kindahl H. Intrauterine bacterial findings and hormonal profiles in post-partum cows with normal puerperium. Acta Vet Scand 1996;37: 251-63. Bicalho RC, Machado VS, Bicalho ML, Gilbert RO, Teixeira AG, Caixeta LS, Pereira RV. Molecular and epidemiological characterization of bovine intrauterine Escherichia coli. J Dairy Sci 2010;93: 5818-30. Bicalho ML, Machado VS, Oikonomou G, Gilbert RO, Bicalho RC. Association between virulence factors of Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium pyogenes and uterine diseases of dairy cows. Vet Microbiol 2012;157: 125-31. Machado VS, Oikonomou G, Bicalho ML, Knauer WA, Gilbert R, Bicalho RC. Investigation of postpartum dairy cows' uterine microbial diversity using metagenomic pyrosequencing of the 16S rRNA gene. Vet Microbiol 2012;159: 460-9. Sheldon IM, Rycroft AN, Dogan B, Craven M, Bromfield JJ, Chandler A, Roberts MH, Price SB, Gilbert RO, Simpson KW. Specific strains of Escherichia coli are pathogenic for the endometrium of cattle and cause pelvic inflammatory disease in cattle and mice. PLoS One 2010;5: e9192. Santos TMA, Gilbert RO, Bicalho RC. Metagenomic analysis of the uterine bacterial microbiota in healthy and metritic postpartum dairy cows. Journal of Dairy Science 2011;94: 291-302.

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Machado VS, Bicalho ML, Meira Junior EB, Rossi R, Ribeiro BL, Lima S, Santos T, Kussler A, Foditsch C, Ganda EK, Oikonomou G, Cheong SH, Gilbert RO, Bicalho RC. Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows. PLoS One 2014;9: e91734.

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100

a

80

a

a

b

A b

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Percentage of total cells

Postpartum progression of PMN proportion

40 20 0 D7

D 21

D 35

D 49

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Progression of macrophage proportion

B

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10

0 D7

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Percentage of total cells

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Progression of lymphocyte proportion

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Percentage of total cells

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ab

40

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igure 1. Proportion of leukocytes in endometrial cytology preparations by week postpartum.

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Columns not sharing a common lower case superscript differ (P < 0.05) A. Neutrophil

546

proportion remained high until 21 days postpartum and declined thereafter. At 35 and 49 days

547

postpartum proportion of neutrophils was significantly lower than on Days 0, 7 and 21, which

548

did not differ from each other. B. Macrophage proportion did not change significantly as days

549

postpartum increased. C. Lymphocyte proportion did not change significantly as days

550

postpartum increased. However, lymphocyte proportion on day 49 was higher than on Day 0,

551

but not different from other days.

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Kaplan-Meier survival estimates Days to pregnancy by PMN proportion at 7 d postpartum (P < 0.01)

0.00

Proportion of Cows Not Pregnant

553

200

300

Days postpartum PMN > 40 %

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PMN < 40 %

Figure 2. Proportion of PMN at 7 days postpartum was related to subsequent reproductive

556

performance. Cows with PMN proportion > 40 % had higher likelihood of pregnancy (P = 0.01).

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Kaplan-Meier survival estimates Days to pregnancy by PMN proportion at 7 d postpartum (P < 0.01)

0

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Proportion of Cows Not Pregnant

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200

300

Days postpartum PMN > 40 %

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PMN < 40 %

Figure 3. The presence of alpha-hemolytic streptocci during the first 49 d postpartum was

560

beneficial to future reproductive performance.

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563

Figu

Prevalence of Aerobic Bacteria

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Streptocci E. coli T. pyogenes Aerobes

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40 20 0 D0

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Percentage infected

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Days postpartum

Prevalence of Anaerobic Bacteria C. perfringens F. necrophorum P. melaninogenica Anaerobes

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Percentage infected

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Days postpartum 564 565 566

Figure 4. Prevalence of common bacteria isolated from the uterus of postpartum cows, and total proportion of cows from which aerobes or anaerobic organisms were isolated. 28

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Highlights This paper describes changes in endometrial cell populations and uterine bacterial isolates in postpartum dairy cows.

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Neutrophils decrease as a proportion of total cells with time postpartum. Other leukocyte populations remain more constant. High proportion of neutrophils recruited to the uterus in the early postpartum period is associated with improved reproduction, and lower neutrophil proportion at 35 or 49 d postpartum.

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Early isolation of E. coli increases risk of T. pyogenes at 21 d postpartum, which in turn increases the risk of isolating gram negative anaerobic pathogens. Intrauterine presence of alpha-hemolytic streptocci in the early postpartum period was associated with improved reproductive performance.