The bovine foot skin microbiota is associated with host genotype and the development of infectious digital dermatitis lesions.

BACKGROUND: Bovine Digital Dermatitis (BDD) is a prevalent infectious disease, causing painful foot skin lesions and lameness in cattle. We describe herein the bovine foot skin microbiota and its associations with BDD using 16S rRNA gene amplicon and shotgun metagenomic sequencing on samples from 259 dairy cows from three UK dairy farms. RESULTS: We show evidence of dysbiosis, and differences in taxonomy and functional profiles in the bovine foot skin microbiome of clinically healthy animals that subsequently develop BDD lesions, compared to those that do not. Our results suggest that taxonomical and functional differences together with alterations in ecological interactions between bacteria in the normal foot skin microbiome may predispose an animal to develop BDD lesions. Using genome-wide association and regional heritability mapping approaches, we provide first evidence for interactions between host genotype and certain members of the foot skin microbiota. We show the existence of significant genetic variation in the relative abundance of Treponema spp. and Peptoclostridium spp. and identify regions in the bovine genome that explain a significant proportion of this variation. CONCLUSIONS: Collectively this work shows early changes in taxonomic and functional profiles of the bovine foot-skin microbiota in clinically healthy animals which are associated with subsequent development of BDD and could be relevant to prevention of disease. The description of host genetic control of members of the foot skin microbiota, combined with the association of the latter with BDD development offer new insights into a complex relationship that can be exploited in selective breeding programmes. Video Abstract.

The microbiome of Escherichia coli and culture-negative nonsevere clinical mastitis: Characterization and associations with linear score and milk production.

Culture-negative and Escherichia coli cases are uncommonly treated in pathogen-based protocols for nonsevere mastitis. High-throughput 16S rRNA sequencing might reveal the presence of other pathogens and can provide information on microbial diversity. The objective was to explore the milk microbiome at the time of the mastitis event (enrollment) and its association with survival in the herd, milk production, and postevent linear score (LS) for cows with clinical mastitis characterized as negative or E. coli by culture. Fifty E. coli-positive and 35 culture-negative samples from cases were enrolled. No cases were treated with antimicrobials. All E. coli-positive quarters were characterized as transient; microbiological culture of samples taken 15 d postmastitis were negative for this organism. However, a difference in α-diversity (Shannon index) was present between enrollment and follow-up samples (3.8 vs. 5.1). When α-diversity was explored for enrollment E. coli samples, no relationship was observed between the Shannon indices of these samples and postmastitis LS. Alpha-diversity of the enrollment samples was lower for E. coli-positive cows that subsequently had greater losses in milk production. This difference was explained by a greater relative abundance of the family Enterobacteriaceae (67.8 vs. 38.4%) for cows that dropped in production. Analysis of composition of the microbiome identified one phylum, Proteobacteria, that differed between E. coli-positive cows that dropped in production and those that did not. Evaluation of ß -diversity found no statistical relationship between postmastitis LS and the microbiome. When evaluating α- and ß-diversities and composition of the microbiomes for culture-negative quarters, no associations were found for milk production changes and postmastitis LS. Three cows did not remain in the herd, limiting the ability to analyze survival. The findings suggest that a contributing factor to negative outcomes in E. coli-positive cows is relative abundance of this pathogen, and that no single or collective group of bacterial families is associated with milk production changes or postmastitis LS in culture-negative quarters. Although additional studies should be performed, the absence of associations between outcomes explored and microbial profiles in this study suggests that we are not missing opportunities by not treating nonsevere E. coli or culture-negative mastitis cases.

Effects of pegbovigrastim administration on periparturient diseases, milk production, and reproductive performance of Holstein cows.

The aim of this study was to evaluate the effects of treating Holstein cows with pegbovigrastim on periparturient diseases, milk production, and reproductive performance while exploring the mode of action of an immunomodulatory protein. Cows were randomly allocated to 1 of 2 treatments, untreated control (CTR, n = 423) and pegbovigrastim (PEG, n = 417). At 7 d from the anticipated calving date (d -7), cows allocated to PEG received a subcutaneous injection of 15 mg of pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim injection, Imrestor, Elanco Animal Health, Greenfield, IN). A second injection was administered within 24 h after calving (d 0). Blood samples were obtained from a subset of cows (CTR, n = 103; PEG, n = 102) at -7 and 0, 3, 7, and 14 d relative to parturition. Samples were used for hemogram and quantification of haptoglobin, nonesterified fatty acids, ß-hydroxybutyrate, and trace and macro minerals. Vaginal cytobrush was performed on the same subset cows at d 0, 7, and 14 to assess the relative neutrophil count. Additionally, colostrum samples were collected to measure IgG, IgM, IgA, and lactoferrin concentrations. Postpartum disease occurrence was recorded from calving until 30 d in milk (DIM). Weekly milk yield was recorded for the first 12 wk after calving. Cows treated with PEG had a 3- to 4-fold increase in circulating polymorphonuclear leukocyte, lymphocyte, and monocyte numbers, with a peak at 3 d after treatment followed by a gradual decline, but the counts remained significantly greater compared with CTR at 14 DIM. The administration of PEG did not affect the incidence of clinical and subclinical mastitis, retained fetal membranes, metritis, puerperal metritis, and endometritis. Primiparous cows treated with PEG tended to have lower odds of developing hyperketonemia than CTR [odds ratio (OR) = 0.57, 95% confidence interval (CI) = 0.23 to 1.42]. Cows treated with PEG had higher odds of being diagnosed with lameness within 30 DIM compared with CTR (OR = 1.79, 95% CI = 1.16 to 2.76); however, we found no significant differences by 60 DIM. Treatment with PEG increased the odds of displaced abomasum (OR = 8.27, 95% CI = 1.02 to 66.6). Cows treated with PEG had higher odds of being diagnosed with 1 or more clinical diseases compared with CTR cows (OR = 1.39, 95% CI = 1.02 to 1.90). We observed no differences in linear scores or milk composition between treatments. Furthermore, primiparous cows treated with PEG produced more milk than CTR primiparous cows during the first 12 wk postpartum (PEG = 37.51 ± 0.66; CTR = 35.91 ± 0.65 kg), but no differences were observed on energy-corrected milk. Treatment did not alter reproductive performance; additionally, cows diagnosed with metritis or puerperal metritis and treated with PEG tended to have higher proportion of neutrophils in the vaginal mucosa when compared with CTR metritic cows. Although PEG treatment increased circulating polymorphonuclear leukocyte, monocyte, and lymphocyte numbers, as expected, it was detrimental to cow health because it increased morbidity.

Effects of injectable trace mineral supplementation in lactating dairy cows with elevated somatic cell counts.

Objectives of this clinical trial were to evaluate the effects of injectable trace mineral supplementation (ITMS) on somatic cell count (SCC), linear score (LS), milk yield, milk fat and protein contents, subclinical mastitis cure, and incidence of clinical mastitis in cows with elevated SCC. Holstein cows from a commercial dairy farm in New York were evaluated for subclinical mastitis, defined as SCC ≥200×10(3) cells/mL on the test day preceding enrollment. Cows with a history of treatment for clinical mastitis in the current lactation and those pregnant for more than 150d were not eligible for enrollment. Cows fitting inclusion criteria were randomly allocated to 1 of 2 treatment groups. Cows assigned to ITMS (n=306) received 1 subcutaneous injection containing zinc (300mg), manganese (50mg), selenium (25mg), and copper (75mg) at enrollment (d 0). Control cows (CTRL; n=314) received 1 subcutaneous injection of sterile saline solution. Following treatment, visual assessment of milk was performed daily, and cows with abnormal milk (i.e., presence of flakes, clots, or serous milk) were diagnosed with clinical mastitis (CM). Chronic clinical mastitis was defined as cows with 3 or more cases of CM. Milk yield, milk fat and protein contents, SCC, and LS were evaluated once monthly. Additionally, randomly selected animals were sampled to test serum concentrations of selected minerals on d0 and 30 (n=30 cows/treatment). Treatment did not affect serum concentrations of calcium, magnesium, phosphorus, potassium, copper, iron, manganese, selenium, and zinc on d30. Injectable supplementation with trace minerals did not improve overall cure of subclinical mastitis (CTRL=42.8 vs. ITMS=46.5%), although a tendency was observed in cows with 3 or more lactations (CTRL=27.1 vs. ITMS=40.0%). Supplementation did not reduce treatment incidence of CM (CTRL=48.2 vs. ITMS=41.7%); however, it tended to reduce the proportion of cows diagnosed with chronic CM (CTRL=16.9 vs. ITMS=12.0%), particularly among first-lactation cows (CTRL=18.4 vs. ITMS=7.6%). Cure of subclinical mastitis was associated with higher serum concentrations of phosphorus and selenium on d30. Supplementing trace minerals to cows with elevated SCC had no effect on milk yield, milk fat and protein contents, SCC, and LS.

Associations among Trueperella pyogenes, endometritis diagnosis, and pregnancy outcomes in dairy cows.

The objective of this study was to evaluate three commonly used methods for endometritis diagnosis by comparing each one's association with the presence of intrauterine Trueperella pyogenes (TP) and reproductive performance. Lactating Holstein cows (n = 452) were evaluated a single time at 35 ± 3 days postpartum to diagnose endometritis on the basis of three criteria: presence of purulent vaginal discharge (PVD) detected by a Metricheck device, presence of purulent uterine lavage fluid (PUL), presence of cytologic endometritis (CE) based on relative abundance of polymorphonuclear leukocytes in uterine lavage fluid. A threshold of polymorphonuclear leukocytes greater than 5% was used to diagnose the occurrence of CE. Also, a swab of the uterine lavage was cultured to evaluate the presence of TP and determine its association with endometritis diagnosis criteria and pregnancy outcomes. The results showed that cows positive for TP had increased prevalence of PVD and PUL and tended to have greater prevalence of CE. Median time to pregnancy was 56 days longer, and hazard of pregnancy was 34% lower for TP-positive cows than for TP-negative cows. Presence of PUL led to a 35% lower hazard of pregnancy and 34-day-longer median time to pregnancy than cows without PUL. Likewise, cows diagnosed with PVD had a 47% lower hazard of pregnancy and 57-day-longer median time to pregnancy than cows without PVD. Cows diagnosed with CE had a 27% lower hazard of pregnancy and 42-day-longer median time to pregnancy than cows without CE. When the three different diagnostic methods were used as independent variables in a Cox's proportional hazard model that evaluated hazard of pregnancy, the variable PVD was the only statistically significant variable. Combined PUL and CE or combined PUL and PVD had no additional effects on hazard of pregnancy when compared with only PUL, only PVD, or only CE as the criterion to determine endometritis. However, combined PVD and CE had an additive detrimental effect on reproductive performance. The sensitivity and specificity of each of TP, PUL, PVD, and CE for predicting nonpregnancy at 300 days postpartum were all similar. In conclusion, TP-positive cows had impaired reproductive performance and increased likelihood of PVD and PUL. Additionally, TP-positive cows tended to have an increased prevalence of CE. Cows with PVD had the lowest reproductive performance when compared to cows with PUL or CE, suggesting that PVD at 35 days postpartum is a better criterion for the diagnosis of endometritis and reproductive failure.

The effect of intrauterine infusion of dextrose on clinical endometritis cure rate and reproductive performance of dairy cows.

The main objective of this study was to evaluate the intrauterine administration use of 200 mL of 50% dextrose solution as a treatment against clinical endometritis (CE); CE cure rate and reproductive performance were evaluated. Additionally, the association of several relevant risk factors, such as retained placenta (RP), metritis, CE, anovulation, hyperketonemia, and body condition score with reproductive performance, early embryonic mortality, and CE were evaluated. A total of 1,313 Holstein cows housed on 4 commercial dairy farms were enrolled in the study. At 7±3 DIM cows were examined for metritis and had blood collected to determine serum ß-hydroxybutyrate concentration. To determine if cows had ovulated at least once before 44±3 DIM, the presence of a corpus luteum was evaluated by ovarian ultrasonography at 30±3 DIM and at 44±3 DIM. At 30±3 DIM, CE was diagnosed using the Metricheck device (SimcroTech, Hamilton, New Zealand); cows with purulent or mucopurulent vaginal discharge were diagnosed as having CE. Cows diagnosed with CE (n=175) were randomly allocated into 2 treatment groups: treatment (intrauterine infusion of 200 mL of 50% dextrose) or control (no infusion). Clinical endometritis cows were re-evaluated as described above at 44±3 DIM, and cows that were free of purulent or mucopurulent vaginal discharge were considered cured. Intrauterine infusion of dextrose tended to have a detrimental effect on CE cure rate, and treatment did not have an effect on first-service conception rate and early embryonic mortality. A multivariable Cox's proportional hazard model was performed to evaluate the effect of several variables on reproductive performance; the variables RP, CE, parity, anovulation, and the interaction term between parity and anovulation were associated with hazard of pregnancy. Cows that did not have RP or CE were more likely to conceive than cows that were diagnosed with RP or CE. Cows that had RP were at 3.36 times higher odds of losing their pregnancy than cows that did not have RP. In addition, cows diagnosed with CE were at 2.16 higher odds of losing their pregnancy than cows without CE. In conclusion, intrauterine infusion of 200 mL of 50% dextrose solution as a treatment for CE had a strong statistical tendency to decrease CE cure rate, did not improve first-service conception rate and early embryonic mortality, and did not decrease calving-to-conception interval.

Association of digital cushion thickness with sole temperature measured with the use of infrared thermography.

The main objective of this study was to investigate the association between digital cushion thickness and sole temperature measured by infrared thermography. Data were collected from 216 lactating Holstein cows at 4 to 10d in milk (DIM). Cows were locomotion scored and sole temperature was measured after claw trimming (a minimum delay of 3 min was allowed for the hoof to cool) using an infrared thermography camera. Temperature was measured at the typical ulcer site of the lateral digit of the left hind foot. Immediately after the thermographic image was obtained, the thickness of the digital cushion was measured by ultrasonography. Rumen fluid samples were collected with a stomach tube and sample pH was measured immediately after collection. Additionally, a blood sample was obtained and used for measurements of serum concentrations of ß-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), and haptoglobin. To evaluate the associations of digital cushion thickness with sole temperature, a linear regression model was built using the GLIMMIX procedure in SAS software (SAS Institute Inc., Cary, NC). Sole temperature was the response variable, and digital cushion thickness quartiles, locomotion score group, rumen fluid pH, rumen fluid sample volume, environmental temperature, age in days, and serum levels of NEFA, BHBA, and haptoglobin were fitted in the model. Only significant variables were retained in the final model. Simple linear regression scatter plots were used to illustrate associations between sole temperature (measured by infrared thermography at the typical ulcer site) and environmental temperature and between NEFA and BHBA serum levels and haptoglobin. One-way ANOVA was used to compare rumen fluid pH for different locomotion score groups and for different digital cushion quartiles. Results from the multivariable linear regression model showed that sole temperature increased as locomotion scores increased and decreased as digital cushion thickness increased. These results were adjusted for environmental temperature, which was significantly associated with sole temperature. Serum levels of NEFA, BHBA, and haptoglobin were not associated with sole temperature. However, significant correlations existed between serum levels of NEFA and haptoglobin and between serum levels of BHBA and haptoglobin. Rumen fluid pH was not associated with either locomotion score or digital cushion thickness. In conclusion, we show here that digital cushion thickness was associated with sole temperature in cows at 4 to 10 DIM.

Effect of trace mineral supplementation on selected minerals, energy metabolites, oxidative stress, and immune parameters and its association with uterine diseases in dairy cattle.

The objective of this study was to evaluate the relationship between selected minerals' serum levels, energy metabolites, oxidative stress indicators, IL-8 and haptoglobin levels, and the potential for uterine diseases. Additionally, we investigated the effect of injectable trace mineral supplementation (ITMS) on metabolism, immune function, and animal health under field conditions involving a dairy herd with high milk production. The study was conducted in 1 dairy farm located near Ithaca, New York, with 270 multiparous cows were enrolled from October 3, 2012 until January 10, 2013. Cows were randomly allocated into 1 of 2 treatments groups: ITMS or control. Cows randomly assigned to the ITMS group received 2 injections of trace minerals at 230 and 260 d of gestation; each injection contained 300 mg of Zn, 50mg of Mn, 25mg of Se, and 75 mg of Cu. Retained placenta (RP) and metritis were diagnosed and treated by trained farm personnel. Clinical endometritis evaluation was performed by the investigators. Blood mineral levels, plasma nonesterified fatty acids and serum ß-hydroxybutyrate concentrations, plasma IL-8 concentrations, serum haptoglobin concentration, and serum superoxidase dismutase and plasma glutathione peroxidase activities were measured at various time points before and after calving. Four groups of mixed general linear models were fitted to the data using MIXED procedure of SAS. Injectable trace mineral-supplemented cows had increased serum concentration of Cu, Se, and Zn. Conversely, ITMS did not affect energy metabolites or immune and oxidative stress parameters. Serum concentration of Ca, Cu, K, Mg, Mo, Ps, Pt, Se, and Zn varied according to days relative to parturition. Cows with RP had reduced serum concentrations of Ca, Mg, Mo, and Zn when compared with cows without RP. Cows affected with metritis had significantly lower serum concentrations of Ca, Mo, soluble P, total P, Se, and Zn than nonaffected cows. Serum concentration of Ca, Cu, Mo, and Zn were reduced in cows diagnosed with endometritis in comparison to nonaffected ones.