2. Universidad Cardenal Herrera-CEU

Indirect assessment of metabolic status using milk samples provides a non-invasive and objective tool for cow-level health monitoring. Milk fat-to-protein ratio (FPR) has been commonly evaluated as an indirect measure for negative energy balance (EB) in confined dairy cows. However, milk component ratios have not been explored for their association with pasture-based cows' metabolic status. The objectives of this observational study were to 1) describe milk component ratios from 0 to 45 d postpartum, 2) evaluate the associations between milk component ratios [FPR, fat-to-lactose (FLR), protein-to-lactose (PLR)] and indicators of EB (serum β-hydroxybutyrate (BHB) concentration at 5–45 d postpartum and body condition score (BCS) change during the transition period), and 3) evaluate the associations between milk component ratios and serum Ca concentration 0–4 d postpartum in spring-calving dairy cows from pasture-based commercial farms. Milk component ratios were determined on samples collected before AM or PM milkings from 548 cows at 0–45 d postpartum (n = 970). Serum BHB and Ca determinations were performed in blood samples collected at the time of milk sample collection at 5–45 d postpartum (n = 918) and 0–4 d postpartum (n = 50), respectively; and BCS change was calculated using BCS assigned between 29 d prepartum and 45 d postpartum (n = 851). Cows' calving date, parity (1st, 2nd–3rd or ≥ 4th) and breed (Holstein-Friesian or dairy crossbred) information was obtained from the farm records. Data was analyzed by multiple linear regression. Average milk FPR, FLR and PLR were 0.70, 0.53 and 0.72, respectively. Milk FPR linearly increased while milk FLR linearly decreased postpartum both at a rate of 0.004 units per day; milk PLR decreased 0.05 units per day for the first 30 d postpartum and moderately increased afterward. Milk FPR and FLR were 0.71 and 0.52 units lower before AM than PM milking, respectively; while milk PLR was similar before AM and PM milking. Milk FPR and FLR were 0.07 to 0.10 units higher for 2nd–3rd compared with 1st and ≥ 4th parity cows. Milk PLR was 0.03 units greater for ≥ 4th compared with 2nd–3rd and 1st parity cows. Further, crossbred cows had 0.07, 0.08 and 0.03 higher milk FPR, FLR and PLR than Holstein-Friesian cows, respectively. Moderate to high P-values along with moderate to small estimated slopes and wide 95% confidence intervals were observed for the associations between milk component ratios and indicators of EB. A positive linear association was observed between milk FPR and serum Ca concentration within 4 d postpartum; milk FPR increased 0.31 units per each mmol/L increase in serum Ca concentration. Cows with low serum Ca concentration within 4 d postpartum had 0.27 units lower milk FPR compared with cows at or above the threshold (2.12 mmol/L), and tended to have 0.15 units lower milk FPR compared with cows at or above the threshold (2.00 mmol/L). In conclusion, further research is needed to reach conclusions on the association between milk component ratios determined before milking and EB indicators. The potential of milk FPR for monitoring blood Ca status warrants further investigation in early-lactation pasture-based dairy cows.

The aim of this study was to investigate the possible genotypic differences between commensal "Pasteurella multocida" isolates from apparently healthy animals (AHA) at the time of entry to feedlots and those from BRD-affected animals (BRD-AA). A total of 20 batches of beef calves in seven feedlots were followed-up during the fattening period. P. multocida was isolated from 28.1% of AHA and 22.9% of BRD-AA. All isolates belonged to the A: L3 genotype. Most isolates from clinical cases (81.0%) grouped into a PFGE cluster were significantly associated with BRD cases (OR, 24.9; 95% CI, 6.4–96.2). The whole genomes of 14 isolates representative of the pulsotypes most frequently detected in BRD-AA and AHA were sequenced and compared with 53 bovine genomes belonging to the identified ST13, ST79, and ST80 genotypes for a global comparison. No differences were found in the virulence-associated gene content between sequence types (STs) globally or between BRD-AA and AHA isolates in this study. Significantly, ST79 isolates harbored ARGs, conferring resistance to different antimicrobials, including macrolides and tetracyclines, which are commonly used for the treatment of BRD. Two Spanish ST79 isolates carried an ICE highly similar to ICE Tn7407, which was recently detected in Germany, suggesting that ST79 P. multocida isolates in Europe and North America may be associated with different ICEs.

Retained fetal membranes (RFM), dystocia, and twinning are common postpartum events that increase the risk of metritis, impair reproductive performance, and contribute to antimicrobial use on dairies. The overall objective of this study was to describe treatment decisions after RFM, severe dystocia (cesarean section and fetotomy), nonsevere dystocia (nonmechanical and mechanical assistance to extract the calf), and twinning. A total of 44 dairies from California’s San Joaquin Valley (39 Holstein and 6 Jersey or crossbreed herds) with 450 to 9,500 lactating cows were enrolled in this study. Researchers visited each dairy once to observe cow-side fresh cow health evaluations and to interview health evaluators and maternity workers, using a standardized survey tool. The survey included questions about antimicrobial (class, dose, and duration) and nonantimicrobial therapies for calving-related events. Antimicrobial therapy was used in all 44 dairies to treat RFM at 24 (n = 23), 48 (n = 10), or 72 h (n = 5) after calving, or when puerperal metritis signs were observed (n = 6). Antimicrobial therapy was used after all severe dystocia cases, and after nonsevere dystocia (n = 27) and twinning (n = 15). Ceftiofur products were the most common antimicrobial class; they were used to treat RFM cases (n = 29), nonsevere dystocia (n = 13), and twinning (n = 10). Supportive therapy for calving-related events included nonantimicrobial intrauterine treatments, nonsteroidal antiinflammatory drugs, oxytocin, i.v. calcium, or oral drenches. Our study highlights opportunities to reduce extra-label use of antimicrobials in postpartum cows affected with RFM, and the need for education and outreach efforts on judicious use of antimicrobials. Furthermore, antimicrobial treatment choices differed largely across dairies, indicating a need to reach consensus and promote standardized practices within the industry.

Bovine respiratory disease (BRD) is an important viral and/or bacterial disease that mainly affects feedlot calves. The involvement of Mycoplasma bovis in BRD can lead to chronic pneumonia poorly responsive to antimicrobial treatment. Caseonecrotic bronchopneumonia is a pulmonary lesion typically associated with M. bovis. In Spain, M. bovis is widely distributed in the feedlots and circulating isolates are resistant to most antimicrobials in vitro. However, the role of this species in clinical respiratory disease of feedlot calves remains unknown. Furthermore, available data are relative to a fixed panel of antimicrobials commonly used to treat BRD, but not to the specific set of antimicrobials that have been used for treating each animal. This study examined 23 feedlot calves raised in southeast Spain (2016–2019) with clinical signs of respiratory disease unresponsive to treatment. The presence of M. bovis was investigated through bacteriology (culture and subsequent PCR), histopathology and immunohistochemistry. The pathogen was found in 86.9% (20/23) of the calves, mainly in the lungs (78.26%; 18/23). Immunohistochemistry revealed M. bovis antigens in 73.9% (17/23) of the calves in which caseonecrotic bronchopneumonia was the most frequent lesion (16/17). Minimum inhibitory concentration assays confirmed the resistance of a selection of 12 isolates to most of the antimicrobials specifically used for treating the animals in vivo. These results stress the importance of M. bovis in the BRD affecting feedlot calves in Spain.

Studies have shown that ruminants constitute reservoirs of Listeria monocytogenes, but little is known about the epidemiology and genetic diversity of this pathogen within farms. Here we conducted a largescale longitudinal study to monitor Listeria spp. in 19 dairy farms during three consecutive seasons (N = 3251 samples). L. innocua was the most prevalent species, followed by L. monocytogenes. Listeria monocytogenes was detected in 52.6% of farms and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was most prevalent in environmental samples. Sixtyone different L. monocytogenes cgMLST types were found, 28% common to different animals and/or surfaces within the same farm and 21% previously reported elsewhere in the context of food and human surveillance. Listeria monocytogenes prevalence was not affected by farm hygiene but by season: higher prevalence was observed during winter in cattle, and during winter and spring in sheep farms. Cows in their second lactation had a higher probability of L. monocytogenes faecal shedding. This study highlights dairy farms as a reservoir for hypervirulent L. monocytogenes.

Our objective was to evaluate the effects of a non-specific immune stimulant (IS) administered around transportation on health scores (HS), average daily gain (ADG), disease treatment and mortality of Jersey and Jersey-cross calves during the rearing period. Newborn calves (4 d ± 1) were randomly allocated to receive either 1mL of saline (CON; n = 438), 1mL of IS before transport (BTIS; n = 431), or 1mL of IS immediately after transport (ATIS; n=436). Calves were health scored weekly for 3 weeks after transport. The data were analyzed using multivariable linear mixed models and multivariable logistic regression models. Kaplan-Meier survival analysis was performed for time to event analysis. Treatment, birth weight, breed, site of birth, serum total solids, dam parity, season of enrollment, and metaphylaxis were offered to models. Differences in respiratory and fecal HS, and ADG between treatment groups were not statistically significant. A total of 196 (15.0%) calves were treated at least once for any disease and 52 calves were treated multiple times. The proportion of calves treated for respiratory disease and/or diarrhea were 14.4, 14.4, and 16.2% for BTIS, ATIS and CON groups, respectively. Although the differences in the likelihood of treatment for both respiratory disease and/or diarrhea during the first 9 weeks of life was not statistically different between groups, we observed that more calves in the control group received disease treatments around 15 days of age compared with calves that received IS. The likelihood of treatment for respiratory diseases alone during the first 30 days of life was smaller in the calves that received IS before transportation when compared to the control group. Only 18 (1.4%) calves died within the study period. The calf mortality likelihood was not statistically different between study groups; however, fewer calves in the IS groups died when compared to CON. In conclusion, the use of IS around transportation did not influence weekly HS, ADG, and the number of disease treatments during the rearing period, but administering IS before transportation resulted in fewer treatments of respiratory diseases during the first 30 days post-transport and marginally lower mortality rates during the rearing period.

The vaginal microbiota plays an important role in the health of dairy cattle, and it could be manipulated for the prevention and treatment of reproduction-related infections. The present study profiles and compares the vaginal microbiota of healthy dairy heifers during the estrous cycle focusing the results in follicular (estrus) and luteal (diestrus) phases using 16S rRNA sequencing of the V3–V4 hypervariable region. Twenty 13–16-months-old virgin dairy heifers from a single farm were included in this study. Vaginal swabs and blood samples were obtained during estrus (6–8 h before artificial insemination) and diestrus (14 days after insemination). Estrus was evaluated by an activity monitoring system and confirmed with plasma progesterone immunoassay. Results showed that the taxonomic composition of the vaginal microbiota was different during the follicular and luteal phases. At the phylum level, the most abundant bacterial phyla were Tenericutes, Firmicutes, and Bacteroidetes which comprised more than 75%of the vaginal microbiota composition. The next more abundant phyla, in order of decreasing abundance, were Proteobacteria, Actinobacteria, Fusobacteria, Epsilonbacteraeota, and Patescibacteria. Together with Tenericutes, Firmicutes, and Bacteroidetes represented more than 96% of the bacterial composition. Ureaplasma, Histophilus, f_Corynebacteriaceae, Porphyromonas, Mycoplasma, Ruminococcaceae UCG-005, were the most abundant genera or families. The results also showed that the vaginal microbiota of dairy heifers was non-lactobacillus dominant. The genus Lactobacillus was always found at a low relative abundance during the estrous cycle being more abundant in the follicular than in the luteal phase. Despite more research is needed to explore the potential use of native vaginal microbiota members as probiotics in dairy heifers, this study represents an important step forward. Understanding how the microbiota behaves in healthy heifers will help to identify vaginal dysbiosis related to disease.

Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.