ADSA Annual Meeting Abstracts

Shifts in uterine microbiome associated with pregnancy outcomes at first insemination and clinical cure in dairy cows with metritis

C. C. Figueiredo, H. F. Monteiro, F. Cunha, F. S. Lima, K. N. Galvão,  and R. S. Bisinotto  

The objectives were to assess differences in the uterine microbiome associated with clinical cure failure and subsequent pregnancy outcomes in dairy cows treated for metritis. Lactating Holstein cows from 2 herds in FL were diagnosed with metritis (presence of fetid, watery, reddish-brownish vaginal discharge; VD) and paired with cows without metritis (Metritis, n = 43; No Metritis, n = 42). All cows with metritis were treated with antimicrobials. Uterine contents were collected through transcervical lavage with saline solution at the time of diagnosis (day 0), five days after diagnosis (day 5), and at 40 DIM. Cure failure was characterized by persistence of fetid, watery, reddish-brownish VD on day 5 (Not cured, n = 25; Cured, n = 18). Cows were artificially inseminated at 79 ± 1 DIM, and pregnancy outcomes (Pregnant, n = 41; Not pregnant, n = 44), metritis, and cure were used for statistical analysis. After sequencing the 16S rRNA gene and quality control procedures, amplicon sequence variants were created. Richness and diversity were analyzed using ANOVA, and beta-diversity was analyzed using PERMANOVA and linear discriminant analysis. Although richness and diversity on day 0 and day 5 were similar between cows with and without metritis (P ≥ 0.36), Porphyromonas, Bacteroides, and Veillonella were more prevalent in the uterus of cows with metritis, whereas, Streptococcus, Sphingomonas, and Ureaplasma were more prevalent in the uterus of cows without metritis (P < 0.001). Cure was not associated with differences in uterine microbiome (P ≥ 0.15) on day 0 or day 5. At 40 DIM, greater (P < 0.04) diversity of the uterine microbiome was associated with pregnancy success and reduced (P = 0.02) richness of the uterine microbiome was associated with metritis. No differences (P > 0.10) in beta-diversity were associated with pregnancy outcomes or metritis 40 DIM. No relationship between the uterine microbiome and pregnancy outcomes or clinical cure were observed, indicating that there are other mechanisms associated with recovery of fertility, and clinical cure aside from intrauterine changes in bacterial community. 

The association between bovine leukemia virus infection and metritis in dairy cows

M. Hernandez, S. Casaro, F. Cunha, M.B. Ugarte Marin, R. S. Bisinotto, F. Maunsell, C. C. Figueiredo, and K. N. Galvão  

Enzootic bovine leukosis is a cattle disease induced by the bovine leukemia virus (BLV). BLV infection is associated with reduced longevity and reduced milk yield in dairy cows, which leads to economic losses of approximately $500 million annually. The innate and adaptive immune cells of BLV-infected cattle have abnormal phenotypes and infected cattle have been shown to have an increased risk of mastitis, digestive problems, and pneumonia. Metritis is a highly prevalent disease in dairy cows and is associated with neutrophil and monocyte dysfunction. Therefore, we hypothesized that dairy cows infected with BLV have an increased risk of developing metritis. The objectives were to evaluate the association between BLV infection and the risk of metritis, and the association between BLV infection and the risk of leaving the herd across lactations. Holstein heifers with BLV tests from 2012 to 2019 (n = 6,121) were followed until the end of lactation 3. A single agar gel immunodiffusion test was used to diagnose BLV at 382 ± 16 d of age. Individual electronic farm records were reviewed for the main events of metritis diagnosis and leaving the herd. Metritis was diagnosed by examining the uterine discharge expressed after palpation per rectum at 4, 6, and 10 d after parturition, and defined as reddish-brownish, fetid, watery vaginal discharge. Logistic regression was used to analyze the odds of metritis in lactations 1, 2, and 3. The Cox proportional hazards model was used to evaluate the risk of leaving the herd by the end of lactation 3. The odds of metritis in lactation 1 were greater for BLV-positive than for BLV-negative cows (19.3 vs. 15.4%; OR = 1.31; 95% CI = 0.99–1.74; P = 0.05). No differences in the odds of metritis were observed in lactations 2 (P = 0.76) and 3 (P = 0.66). The hazard of leaving the herd by the end of lactation 3 was greater for BLV-positive than for BLV-negative cows (HR = 1.33; 95% CI = 1.20–1.49; P < 0.01). In conclusion, BLV infection was associated with increased odds of metritis in lactation 1 and an increased hazard of leaving the herd by the end of lactation 3.

Differences in lactational performance associated with antimicrobial therapy and clinical cure of metritis in dairy cows

A. Martelo Pereira, P. R. Menta, E. B. de Oliveira, J. G. Prim, V. S. Machado, F. S. Lima, K. N. Galvão, and  C. C. Figueiredo

The objective was to assess differences in lactational performance associated with antimicrobial therapy and clinical cure of metritis in dairy cows. Holstein cows from 5 farms in CA, FL, and TX were diagnosed with metritis (fetid, watery, reddish-brownish vaginal discharge; VD) at 4, 7, and 10 DIM (d0), and randomly assigned (regardless of fever) to receive ceftiofur (CEF) or to remain untreated. At d13 ± 1 clinical cure of metritis was assessed and cows with cure failure had persistent fetid, watery, reddish-brownish VD (Cure, C; NoCure, N). Four groups composed this study (CEF-C=381; CEF-N=76; NT-C=329; NT-N=114), and used for statistical analysis. Risk of pregnancy and culling at 300 DIM were analyzed using logistic regression, time to pregnancy by 300 DIM were analyzed using Cox’s proportional hazard, and milk production within 90 DIM was analyzed using ANOVA with repeated measures. Group was included as fixed effect and farm as random effect in all models. Orthogonal contrasts were set to evaluate the effects of cure (CEF-C+NT-C vs. CEF-N+NT-N), treatment (CEF-C+CEF-N vs. NT-C+ NT-N), and their interaction (CEF-C+NT-N vs. CEF-N+NT-C). Differences in risk of pregnancy and culling at 300 DIM (P ≤ 0.06) were associated with cure (CEF-C=74.2 and 13.5; CEF-N=63.7 and 21.9; NT-C=74.1 and 12.7; NT-N=64.5 and 15.1%, respectively), but not with treatment or interaction (P ≥ 0.38). Reduced median time to pregnancy (P =0.03) was associated with cure (CEF-C=131; CEF-N=150; NT-C=145; NT-N=162 days); but not with treatment or interaction (P ≥ 0.53). Milk production was affected (P < 0.001) by the interaction between group, parity, and month. Whereas differences in milk production associated with cure were observed in multiparous cows (CEF-C=43.4; CEF-N=40.9; NT-C=44.3; NT-N=40.5 kg/day), minimal variations among primiparous cows were observed. No differences were associated with treatment or interaction (P ≥ 0.27). The cure of metritis was positively associated with milk production and reproduction, regardless of antimicrobial therapy, warranting further investigation regarding selective therapy of metritis.

Clinical cure failure of metritis is not associated with differences in the uterine metabolome of dairy cows

F. N. S. Pereira, D. Z. Bisinotto, K. N. Galvão, R. S. Bisinotto, and C. C. Figueiredo

The objective was to characterize differences in uterine metabolome associated with clinical cure failure of metritis in lactating Holstein cows. This prospective cohort study was conducted from February to November 2018 in two dairy herds in Florida. Vaginal discharge was evaluated within 15 DIM using the Metricheck device and metritis was characterized by the presence of fetid, watery, reddish-brownish vaginal discharge (study d 0). Cows with metritis (n = 24) were paired with cows without metritis of similar DIM and parity (NoMET; n = 24). On d 0, the uterine lumen of all cows was flushed with 15 mL of saline solution using a plastic infusion pipette connected to a syringe and cows with metritis were treated with ceftiofur. On d 5, clinical cure failure of metritis, characterized by the persistence of fetid, watery, reddish-brownish vaginal discharge (Cure; n = 8 and NoCure; n = 16), was assessed prior to the performance of a second uterine flush in all cows. The uterine metabolome was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Autoscaled and normalized data were analyzed in Metaboanalyst using ANOVA and Principal Component Analysis. Enriched pathway analysis was performed whenever differences in uterine metabolome composition between Cure and NoCure cows was observed. False discovery rate (FDR) was used to adjust P-values for multiple comparisons. A total of 188 annotated primary metabolites, characterized by different peak intensities and charge, composed the dataset in the study. On d0, the abundance of 107 metabolites differed (FDR ≤ 0.05) between the uterus of cows with and without metritis. Most compounds were associated with the metabolism of amino acids, carbohydrates, and fatty acids. No differences in the uterine metabolome were observed between Cure and NoCure cows on d 0. No differences in the uterine metabolome were observed among any of the groups on d 5. This demonstrates that although metritis is associated with differences in the uterine metabolome on the day of diagnosis, clinical cure failure of metritis is not associated with differences in the uterine metabolome.

Peripheral leukocyte transcriptomic changes in preweaned Holstein dairy calves with varying stages of Bovine Respiratory Disease

Richmond, Lindsey C.,Joel A. Velasco, Stephen P. Ficklin, Neils C. Stegelmeier, Holly R. Hinnant, Carolyn D. Fisher, Corinna T. Cauchy, Lindsay M. Parrish, Craig S. McConnel

Bovine Respiratory Disease (BRD) is a key contributor to calf morbidity and mortality. Historically, BRD diagnosis has relied on clinical assessments and Clinical Respiratory Scoring Charts (CRSC), which may underrepresent BRD prevalence and subclinical pathology. Thoracic ultrasonography has helped overcome CRSC limitations. This study aimed to identify 1) peripheral leukocyte transcriptomic changes in preweaned Holstein heifer calves with varying stages of lobar consolidation diagnosed via thoracic ultrasonography, and 2) potential molecular biomarkers of preweaning BRD. Calves born May 2021 and May 2022 on two conventional dairies in Washington were enrolled at birth and followed for the first 12 weeks of life. Weekly physical examinations and thoracic ultrasonography were performed alongside biweekly jugular venipuncture and blood chemistry. Representative calves were retrospectively categorized based on the presence or absence (onset vs healthy), and duration (healthy vs chronic or resolved) of lobar consolidation (≥1 lobe) between 5 to 9 weeks of age:  healthy (2021: n = 8; 2022: n = 15), onset (2021: n = 16; 2022: n = 15), chronic  (2021: n = 8; 2022: n = 9) or resolved (2021: n = 7; 2022: n = 11). Leukocyte isolation of whole blood samples from each representative calf health state was conducted via modified Ficoll-Paque separation followed by RNA extraction (miRNeasy kit, Qiagen) and sequencing (Novogene Corporation). Differentially expressed genes (DEG) and predictive features were identified by EdgeR and random forest classification modeling. The analysis identified 536 DEG for healthy vs onset, 152 DEG for healthy vs chronic, and 99 DEG for healthy vs resolved, of which predictive features related to each health state were identified among known enrichment pathways. Enrichment pathways related to immunological functions such as positive regulation of natural killer cell mediated cytotoxicity, c-type lectin receptor signaling pathways, and immunoglobulin regulation were observed. Identification of potential biomarkers underlying disease susceptibility and progression may help guide calf management.

Differences in reproductive and productive performance, and culling associated with time to clinical cure in dairy cows treated for metritis

F. N. S. Pereira, V. R. Merenda, E. B. de Oliveira, F. S. Lima, R. C. Chebel, K. N. Galvão, J. E. P. Santos, R. S. Bisinotto, and C. C. Figueiredo

The objective was to assess differences in reproductive and productive performance, and culling associated with time to clinical cure in dairy cows treated for metritis. This retrospective cohort study included data from 3 studies conducted in 5 dairies in FL. Metritis was characterized by the presence of fetid, watery, reddish-brownish vaginal discharge (VD) within 12 DIM (study d 0). Cows with metritis were treated with ampicillin (AMP) or ceftiofur (CEF). Clinical cure (absence of fetid, watery, reddish-brownish VD) was assessed on d 5 and 12 post-diagnosis, resulting in 3 study groups (CureD5, n = 705; CureD12,n = 406; NoCure, n = 299). Risk of pregnancy and culling by 300 DIM were analyzed using logistic regression, hazard of pregnancy by 300 DIM was analyzed using the Cox’s proportional hazard regression, and milk production within 90 DIM was analyzed using ANOVA with repeated measures. Orthogonal contrasts were set to assess the effects of cure (CureD5 + CureD12 vs. NoCure) and time to cure (CureD5 vs. CureD12). Risk of pregnancy was higher for cured cows than for NoCure cows. CureD5 had higher risk of pregnancy than CureD12. Risk of culling was lower for cured cows than for NoCure cows. CureD5 had lower risk of culling than CureD12. Cured cows got pregnant faster than NoCure cows, but no differences were observed by time to cure. An interaction between cure status, parity, and month postpartum was associated with milk production (P < 0.001). In primiparous cows, milk production was similar among the three cure groups. In multiparous cows, milk production was lesser in NoCure than in CureD5 and CureD12. No differences in milk production were observed by time to cure. Hastened time to clinical cure of metritis after antimicrobial therapy was associated with improved reproductive performance and reduced culling in dairy cows.

Table 1.