A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes

Abstract

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.

Keywords: animal model; clinical endometritis; dairy cow; inflammation; uterine infection.

Figure 1.

Requirements for establishment of experimental uterine infection. (A) Timeline of experimental procedures employed during the experimental period. Gonadotropin-releasing hormone and PGF_2α were used to synchronize estrous cycles in 9 virgin Holstein heifers before intrauterine infusion of treatments. On d 0, intrauterine infusion of either vehicle or live bacteria was performed. Vehicle infusion consisted of 30 mL of sterile Luria-Bertani broth. Bacteria infusion consisted of 10 mL of Escherichia coli MS 499 (4.64 × 10⁷ cfu/mL), 10 mL of Trueperella pyogenes MS249 (3.38 × 10⁷ cfu/mL), and 10 mL of sterile Luria-Bertani broth. Progesterone (P4) was administered at 200 mg/d i.m. Days relative to treatment for evaluation of plasma haptoglobin (hpt), P4 (P4), and hematology (hem) are indicated in italics. (B) The tool used to enable scarification of the endometrium.

Figure 2.

Clinical observations of induced uterine disease. (A) Vaginal mucus was visually confirmed in heifers receiving bacterial infusion. (B) Metricheck (Simcro, Hamilton, New Zealand) tool containing vaginal mucus of a bacteria-infused heifer. (C) Examples of vaginal mucus samples collected from bacteria-infused heifers using the Metricheck tool. (D and E) Representative ultrasound images of a transverse cross-section of a uterine horn from a control (D) and bacteria-infused (E) heifer. The arrow head denotes the uterine lumen; the double-headed arrow denotes the uterine wall. The scale bar represents 5 mm.

Figure 3.

Vaginal mucus grade following intrauterine infusion of bacteria. Vaginal mucus was collected using a Metricheck (Metricheck, Simcro, Hamilton, New Zealand) tool and graded according to Sheldon et al. (2009). Vaginal mucus was graded from 0 to 3 and each heifer is represented by a single circle. Control heifers are represented by open circles (A, ○), and bacteria-infused heifers are represented by filled circles (B, ●). The vertical dotted line denotes the day of treatment; solid horizontal lines indicate the median vaginal mucus score for the day of observation. Data were analyzed using the GLIMMIX procedure following a Poisson distribution to determine the effect of treatment (Trt).

Figure 4.

Effect of treatment on vaginal mucus 16S rRNA and circulating concentrations of haptoglobin and progesterone in plasma. (A) Total 16S rRNA quantification from vaginal mucus on d −5, −1, 1, and 5 relative to treatment in either control (open bars) or bacteria-infused (solid bars) heifers. Total 16S rRNA was normalized to the weight of vaginal mucus processed for nucleic acid extraction (ng of rRNA per mg of mucus). Different letters denote differences within the bacteria-infused group; * denotes difference between treatment groups on a given day (P < 0.05). (B) Haptoglobin plasma concentration (ng/mL) was measured in control (○) and bacteria-infused (●) heifers on d 0, 7, and 13 relative to treatment. (C) Progesterone plasma concentration (ng/mL) was measured in control (○) and bacteria-infused (●) heifers on d −2, 1, 4, 11, and 15 relative to treatment. Progesterone (P4) denotes the period of exogenous administration of 200 mg/d of P4. All data are presented as LSM ± SEM.