bBovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive etiologic agent associated with the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study details the deep sequencing-based metagenomic evaluation of 48 staged DD biopsy specimens collected during a 3-year longitudinal study of disease progression. Over 175 million sequences were evaluated by utilizing both shotgun and 16S metagenomic techniques. Based on the shotgun sequencing results, there was no evidence of a fungal or DNA viral etiology. The bacterial microbiota of biopsy specimens progresses through a systematic series of changes that correlate with the novel morphological lesion scoring system developed as part of this project. This scoring system was validated, as the microbiota of each stage was statistically significantly different from those of other stages (P < 0.001). The microbiota of control biopsy specimens were the most diverse and became less diverse as lesions developed. Although Treponema spp. predominated in the advanced lesions, they were in relatively low abundance in the newly described early lesions that are associated with the initiation of the disease process. The consortium of Treponema spp. identified at the onset of disease changes considerably as the lesions progress through the morphological stages identified. The results of this study support the hypothesis that DD is a polybacterial disease process and provide unique insights into the temporal changes in bacterial populations throughout lesion development.

Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease.

Incidence rates for bovine respiratory disease (BRD) in dairy cattle have remained essentially unchanged over the last 20 years. Dairy calves are more commonly affected than adult animals, with BRD being the principal cause of death in weaned dairy calves. The lack of progress in controlling respiratory disease demonstrates that there continues to be significant room for improvement in controlling this multifactorial syndrome, and that dairy producers need assistance in applying evolving husbandry practices to improve the health of dairy cattle. Therefore, it seems prudent to focus the management strategies on preventing disease through sound management of the transition period, along with sound vaccination and biosecurity programs.

The study objective was to compare the efficacy of 3 commercial dry cow mastitis formulations regarding quarter-level prevalence of intramammary infections (IMI) postcalving, cure of preexisting infections over the dry period, prevention of new infections during the dry period, and risk for a clinical mastitis case between calving and 100d in milk (DIM). A total of 1,091 cows (4,364 quarters) from 6 commercial dairy herds in 4 different states (California, Iowa, Minnesota, and Wisconsin) were enrolled and randomized to 1 of the 3 treatments at dry-off: Quartermaster (QT; 1,000,000 IU of procaine penicillin G and 1 g of dihydrostreptomycin; Pfizer Animal Health, New York, NY), Spectramast DC (SP; 500 mg of ceftiofur hydrochloride; Pfizer Animal Health), or ToMorrow Dry Cow (TM; 300mg of cephapirin benzathine; Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO). Quarter milk samples were collected for routine bacteriological culture before dry cow therapy treatment at dry-off, 0 to 6 DIM, and 7 to 13 DIM and an on-farm record-keeping system was used to retrieve data on clinical mastitis cases. Noninferiority analysis was used to evaluate the effect of treatment on the primary outcome, risk for a bacteriological cure during the dry period. Multivariable logistic regression techniques were used to describe the effect of treatment on risk for presence of IMI postcalving and risk of a new IMI during the dry period. Cox proportional hazards regression was used to describe the effect of treatment on the risk and time for quarters to experience an episode of clinical mastitis between calving and 100 DIM. The overall crude quarter-level prevalence of infection at dry-off was 19.2%. The most common pathogen isolated from milk samples at dry-off was coagulase-negative Staphylococcus, followed by Aerococcus spp. and other Streptococcus spp. Noninferiority analysis showed no effect of treatment on risk for a cure between dry-off and calving [least squares means (LSM): QT=93.3%, SP=92.6%, and TM=94.0%] and secondary analysis showed no effect of treatment on risk for presence of an IMI at 0 to 6 DIM (LSM: QT=16.5%, SP=14.1%, and TM=16.0%), risk for development of a new IMI between dry-off and 0 to 6 DIM (LSM: QT=14.8%, SP=12.3%, and TM=14.2%), or risk of experiencing a clinical mastitis event between calving and 100 DIM (LSM: QT=5.3%, SP=3.8%, and TM=4.1%). In conclusion, no difference was observed in efficacy among the 3 products evaluated when assessing the aforementioned quarter-level outcomes.

Selective dry-cow therapy (SDCT) could be used to reduce antibiotic use on commercial dairy farms in the United States but is not yet widely adopted, possibly due to concerns about the potential for negative effects on cow health. The objective of this study was to compare culture-and algorithm-guided SDCT programs with blanket dry-cow therapy (BDCT) in a multi-site, randomized, natural exposure, non-inferiority trial for the following quarter-level outcomes: antibiotic use at dry-off, dry period intramammary infection (IMI) cure risk, dry period new IMI risk, and IMI risk at 1 to 13 d in milk (DIM). Two days before planned dry-off, cows in each of 7 herds were randomly allocated to BDCT, culture-guided SDCT (cult-SDCT), or algorithm-guided SDCT (alg-SDCT). At dry-off, BDCT cows received an intramammary antibiotic (500 mg of ceftiofur hydrochloride) in all 4 quarters. Antibiotic treatments were selectively allocated to quarters of cult-SDCT cows by treating only quarters from which aseptically collected milk samples tested positive on the Minnesota Easy 4Cast plate (University of Minnesota, St. Paul, MN) after 30 to 40 h of incubation. For alg-SDCT cows, antibiotic treatments were selectively allocated at the cow level, with all quarters receiving antibiotic treatment if the cow had either a Dairy Herd Improvement Association test somatic cell count >200,000 cells/ mL during the current lactation or 2 or more clinical mastitis cases during the current lactation. All quarters of all cows were treated with an internal teat sealant. Intramammary infection status at enrollment and at 1 to 13 DIM was determined using standard bacteriological methods. The effect of treatment group on dry period IMI cure, dry period new IMI, and IMI risk at 1 to 13 DIM was determined using generalized linear mixed models (logistic), with marginal standardization to derive risk difference (RD) estimates. Quarter-level antibiotic use at dry-off for each group was BDCT (100%), cult-SDCT (45%), and alg-SDCT (45%). The crude dry period IMI cure risk for all quarters was 87.5% (818/935), the crude dry period new IMI risk was 20.1% (764/3,794), and the prevalence of IMI at 1 to 13 DIM was 23% (961/4,173). Non-inferiority analysis indicated that culture-and algorithm-guided SDCT approaches performed at least as well as BDCT for dry period IMI cure risk. In addition, the final models indicated that the risks for each of the 3 IMI measures were similar between all 3 treatment groups (i.e., RD estimates and 95% confidence intervals all close to 0). These findings indicate that under the conditions of this trial, culture-and algorithm-guided SDCT can substantially reduce antibiotic use at dry-off without negatively affecting IMI dynamics.

Bovine Digital Dermatitis (DD) is a leading cause of lameness in dairy cattle. DD is reportedly increasing in prevalence in beef cattle feedlots of the US. The exact etiologic agent(s) responsible for the disease have yet to be determined. Multiple studies have demonstrated the presence of a variety of Treponema spp. within lesions. Attempts to reproduce clinically relevant disease using pure cultures of these organisms has failed to result in lesions that mirror the morphology and severity of naturally occurring lesions. This manuscript details the systematic development of an experimental protocol that reliably induces digital dermatitis lesions on a large enough scale to allow experimental evaluation of treatment and prevention measures. In total, 21 protocols from five experiments were evaluated on their effectiveness in inducing DD lesions in 126 Holstein calves (504 feet). The protocols varied in the type and concentration of inoculum, frequency of inoculation, duration the feet were wrapped, and type of experimental controls need to validate a successful induction. Knowledge gained in the first four experiments resulted in a final protocol capable of inducing DD lesions in 42 of 44 (95%) feet over a 28 day period. All induced lesions were macroscopically and microscopically identified as clinical DD lesions by individuals blinded to protocols. Lesions were also located at the site of inoculation in the palmer aspect of the interdigital space, and induced clinically measurable lameness in a significant portion of the calves. Collectively these results validate the model and provide a rapid and reliable means of inducing DD in large groups of calves.

Study objectives were to evaluate the effects of intentionally reduced intestinal barrier function on productivity, metabolism, and inflammatory indices in otherwise healthy dairy cows. Fourteen lactating Holstein cows (parity 2.6 ± 0.3; 117 ± 18 d in milk) were enrolled in 2 experimental periods. Period 1 (5 d) served as the baseline for period 2 (7 d), during which cows received 1 of 2 i.v. treatments twice per day: sterile saline or a gamma-secretase inhibitor (GSI; 1.5 mg/kg of body weight). Gamma-secretase inhibitors reduce intestinal barrier function by inhibiting crypt cell differentiation into absorptive enterocytes. During period 2, control cows receiving sterile saline were pair-fed (PF) to the GSI-treated cows, and all cows were killed at the end of period 2. Administering GSI increased goblet cell area 218, 70, and 28% in jejunum, ileum, and colon, respectively. In the jejunum, GSI-treated cows had increased crypt depth and reduced villus height, villus height-to-crypt depth ratio, cell proliferation, and mucosal surface area. Plasma lipopolysaccharide binding protein increased with time, and tended to be increased 42% in GSI-treated cows relative to PF controls on d 5 to 7. Circulating haptoglobin and serum amyloid A concentrations increased (585- and 4.4-fold, respectively) similarly in both treatments. Administering GSI progressively reduced dry matter intake (66%) and, by design, the pattern and magnitude of decreased nutrient intake was similar in PF controls. A similar progressive decrease (42%) in milk yield occurred in both treatments, but we observed no treatment effects on milk components. Cows treated with GSI tended to have increased plasma insulin (68%) and decreased circulating nonesterified fatty acids (29%) compared with PF cows. For both treatments, plasma glucose decreased with time while β-hydroxybutyrate progressively increased. Liver triglycerides increased 221% from period 1 to sacrifice in both treatments. No differences were detected in liver weight, liver moisture, or body weight change. Intentionally compromising intestinal barrier function caused inflammation, altered metabolism, and markedly reduced feed intake and milk yield. Further, we demonstrated that progressive feed reduction appeared to cause leaky gut and inflammation.

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