V.S. Machado

Lameness in dairy cattle: A debilitating disease or a disease of debilitated cattle? A cross-sectional study of lameness prevalence and thickness of the digital cushion

Lameness is the most significant challenge for the dairy industry to overcome, given its obvious disruption of animal welfare and severe economic losses. Sole ulcers and white line abscesses are ubiquitous chronic diseases with the highest associated economic losses among all foot lesions. Their underlying causes are still not fully understood. An observational cross-sectional study was carried out to investigate the association between claw horn lesions and the thickness of the digital cushion.The thickness of the digital cushion was evaluated by ultrasonographic examination of the sole at the typical ulcer site. A total of 501 lactating Holstein dairy cows were enrolled in the study. The prevalence of sole ulcers was 4.2 and 27.8% for parity 1 and parity >1, respectively.The prevalence of white line disease was 1.0 and 6.5% for parity 1 and >1, respectively. The prevalence of lameness (visual locomotion score > or = 3) was 19.8 and 48.2% for parity 1 and >1, respectively. The prevalence of sole ulcers and white line diseases was significantly associated with thickness of the digital cushion; cows in the upper quartile of digital cushion thickness had an adjusted prevalence of lameness 15 percentage points lower than the lower quartile. Body condition scores were positively associated with digital cushion thickness.The mean gray value of the sonographic image of the digital cushion had a negative linear association with digital cushion thickness (R2 = 0.14), indicating that the composition of the digital cushion may have changed with its thickness. Furthermore, digital cushion thickness decreased steadily from the first month of lactation and reached a nadir 120 d after parturition.These results support the concept that sole ulcers and white line abscesses are related to contusions within the claw horn capsule and such contusions are a consequence of the lesser capacity of the digital cushion to dampen the pressure exerted by the third phalanx on the soft tissue beneath.

Molecular and epidemiological characterization of bovine intrauterine Escherichia coli

Escherichia coli are believed to be associated with postpartum metritis and endometritis but their role in the pathogenesis of both diseases is still undefined. In this study, uterine swabs for E. coli isolation were collected from 374 lactating Holstein cows housed on 4 commercial farms near Ithaca, New York. A total, 125 of 374 cows (33.4%) were positive for E. coli culture. Standard multiplex PCR protocols were used to screen the isolates for the presence of 32 virulence factor genes. Cows that had twin parturition were 4.4 times more likely to have intrauterine E. coli contamination than those that gave birth to single live female calves. Stillborn parturition and birth of single live male calves also increased the odds of intrauterine contamination by E. coli (3.7- and 1.6-fold, respectively) compared with birth of live female calves. Six virulence factors, common to extraintestinal and enteroaggregative E. coli, were found to be associated with metritis and endometritis: fimH, hlyA, cdt, kpsMII, ibeA, and astA. The virulence factor gene fimH was the most prevalent and the most significant: intrauterine E. coli carrying fimH and at least 1 of the other 5 identified virulence factors were pathogenic, and phylogenetic analysis based on the nucleotide sequence of DNA gyrase from 41 such IUEC revealed 2 clades.

Antimicrobial resistance and presence of virulence factor genes in Arcanobacterium pyogenes isolated from the uterus of postpartum dairy cows.

Arcanobacterium pyogenes is considered the most significant bacterium involved in the pathogenesis of metritis in cattle. Infections caused by antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to present an overview of antimicrobial resistance in A. pyogenes isolated from the uteruses of postpartum Holstein dairy cows and to identify virulence factors. Seventy-two A. pyogenes isolates were phenotypically characterized for antimicrobial resistance to amoxicillin, ampicillin, ceftiofur, chloramphenicol, florfenicol, oxytetracycline, penicillin, spectinomycin, streptomycin and tetracycline by the broth microdilution method. Presence of virulence factor genes of A. pyogenes was investigated. Isolates exhibited resistance to all antimicrobial agents tested; high levels of resistance were found to amoxicillin (56.9%); ampicillin (86.1%), chloramphenicol (100%), florfenicol (59.7%), oxytetracycline (54.2%), penicillin (86.1%) and tetracycline (50%). Of all isolates, 69 (95.8%) were resistant to at least 2 of the antimicrobial agents tested and multidrug resistance (resistant to at least 3 antimicrobials) was observed in 64 (88.9%) of the A. pyogenes isolates. The major multidrug resistance profile was found for chloramphenicol-ampicillin-penicillin-florfenicol-amoxicillin-tetracycline, which was observed in 21 (29.2%) multidrug resistant isolates. No isolate was resistant to all nine antimicrobial agents tested but four isolates (5.6%) were resistant to eight antimicrobials. The information highlights the need for prudent use of specific antimicrobial agents. All four virulence factor genes occurred in isolates from normal puerperium and clinical metritis; however, the fimA gene was present in significantly higher frequency in isolates from metritis cows.

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.

Microbiota of Cow’s Milk; Distinguishing Healthy, Sub-Clinically and Clinically Diseased Quarters

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.

Effect of an injectable trace mineral supplement containing selenium, copper, zinc, and manganese on the health and production of lactating Holstein cows

The objective of this study was to evaluate the effect of a subcutaneous injection of a multimineral preparation containing 300 mg of zinc, 50mg of manganese, 25mg of selenium, and 75 mg of copper at 230 and 260 days of gestation and 35 days postpartum, on the health, milk production and reproductive performance of lactating Holstein cows. A randomized field trial was conducted on three large commercial dairy farms located near Ithaca, New York, USA, with 1416 cows enrolled. All cows were housed and offered a total mixed ration consisting of approximately 55% forage and 45% concentrate on a dry matter basis of the diet, which supplied 2-6 times the NRC requirements for the supplemented elements. Dry cows and pregnant heifers were blocked by parity and randomly allocated to one of two treatments: Trace mineral supplemented (TMS) or control. For multiparous cows, subcutaneous TMS significantly decreased linear somatic cell count scores (normalized data) as compared to control cows. The incidence of subclinical mastitis for TMS and control cows was 10.4% and 8.0%, respectively (P=0.005). The main effect of treatment on clinical mastitis was not significant but the interaction of treatment and parity was significant. For primiparous cows, the incidence of clinical mastitis was 11.8% and 15.6% for control and TMS cows, respectively (P=0.33); for multiparous cows, the incidence of clinical mastitis for control and TMS cows was 25.4% and 19.7%, respectively (P=0.03). Additionally, control cows had increased odds of stillbirth and endometritis (odds ratios 1.69 and 1.30, respectively). The incidence of endometritis was 34.2% and 28.6% for control and TMS cows, respectively (P=0.039) but treatment had no effect on reproductive performance, milk production or other health traits. Further research is required to confirm these findings and to establish whether the response seen in this study was related to the supplementation of a particular mineral.

Efficacy of formalin, copper sulfate, and a commercial footbath product in the control of digital dermatitis

The objective was to evaluate the relative efficacy of a novel, commercially available disinfectant agent (T-Hexx Dragonhyde HBC, Hydromer, Branchburg, NJ) compared with formalin and copper sulfate. The hypothesis was 2 sided; therefore, the hypothesis was that the new agent would be better or worse compared with the industry gold standard footbath agents, formalin and copper sulfate. The study was conducted in a large commercial dairy farm located near Ithaca, New York, from June 18, 2009 to October 26, 2009. Two identical studies were conducted, the first comparing Dragonhyde (5% solution, twice weekly) and formalin (5% solution, twice weekly) and the second comparing Dragonhyde (5% solution, twice weekly) and copper sulfate (10% solution, twice weekly). The study design was identical for both studies with 4 pens (physically identical), 2 treatments (Dragonhyde vs. formalin and Dragonhyde vs. copper sulfate), 2 periods (crossing over the treatment within pen), and 3 repeated measures (3 observations per cow: enrollment, wk 2, and wk 4). For study 1, 406 cows were enrolled (n=201 formalin and 205 Dragonhyde). For study 2, 356 cows were enrolled (n=189 copper sulfate and 167 Dragonhyde). The adjusted odds of digital dermatitis lesion (DDL) throughout the study period were analyzed by mixed logistic regression model. In study 1, the odds of DDL were 1.36 times higher for the formalin group compared with the Dragonhyde group. In study 2, the data were analyzed by a similar statistical model and the variable treatment did not significantly affect the overall prevalence of DDL. In conclusion, the performance of 3 hoof care products was evaluated and it was concluded that Dragonhyde performed better than formalin and that there was no difference between copper sulfate and Dragonhyde.

Altered microbiomes in bovine digital dermatitis lesions, and the gut as a pathogen reservoir.

Bovine digital dermatitis (DD) is the most important infectious disease associated with lameness in cattle worldwide. Since the disease was first described in 1974, a series of Treponema species concurrent with other microbes have been identified in DD lesions, suggesting a polymicrobial etiology. However, the pathogenesis of DD and the source of the causative microbes remain unclear. Here we characterized the microbiomes of healthy skin and skin lesions in dairy cows affected with different stages of DD and investigated the gut microbiome as a potential reservoir for microbes associated with this disease. Discriminant analysis revealed that the microbiomes of healthy skin, active DD lesions (ulcerative and chronic ulcerative) and inactive DD lesions (healing and chronic proliferative) are completely distinct. Treponema denticola, Treponema maltophilum, Treponema medium, Treponema putidum, Treponema phagedenis and Treponema paraluiscuniculi were all found to be present in greater relative abundance in active DD lesions when compared with healthy skin and inactive DD lesions, and these same Treponema species were nearly ubiquitously present in rumen and fecal microbiomes. The relative abundance of Candidatus Amoebophilus asiaticus, a bacterium not previously reported in DD lesions, was increased in both active and inactive lesions when compared with healthy skin. In conclusion, our data support the concept that DD is a polymicrobial disease, with active DD lesions having a markedly distinct microbiome dominated by T. denticola, T. maltophilum, T. medium, T. putidum, T. phagedenis and T. paraluiscuniculi. Furthermore, these Treponema species are nearly ubiquitously found in rumen and fecal microbiomes, suggesting that the gut is an important reservoir of microbes involved in DD pathogenesis. Additionally, the bacterium Candidatus Amoebophilus asiaticus was highly abundant in active and inactive DD lesions.

Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

The effect of claw horn disruption lesions and body condition score at dry-off on survivability, reproductive performance, and milk production in the subsequent lactation

The objective of this study was to evaluate the effects of claw horn disruption lesions (CHDL; sole ulcers and white line disease) and body condition score (BCS) at dry-off on survivability, milk production, and reproductive performance during the subsequent lactation. An observational prospective cohort study was conducted on a large commercial dairy in Cayuga County, New York, from September 2008 until January 2009. A total of 573 cows enrolled at dry-off were scored for body condition and hoof trimmed; digits were visually inspected for the presence of CHDL. The BCS data were recategorized into a 3-level variable BCS group (BCSG), with cows with BCS<3 placed in BCSG 1 (n=113), cows with BCS=3 placed in BCSG 2 (n=254), and cows with BCS>3 placed in BCSG 3 (n=206). Cows in BCSG 2 were 1.35 and 1.02 times more likely to conceive than cows in BCSG 1 and 3, respectively. The cull/death hazard for BCSG 1 cows was 1.55 and 1.47 times higher than for cows in BCSG 2 and BCSG 3, respectively. Milk yield for cows in BCSG 2 (44.6 kg/d, 95% CI 43.4-45.8) was significantly greater than that for cows in BCSG 1 (41.5 kg/d, 95% CI 39.8-43.3). Cows with previous lactation days open<or=91 had 1.6 times higher odds of being classified into BCSG 1 at dry-off; cows with previous lactation mature-equivalent 305-d milk>14,054 kg had a similar 1.6 times higher odds of being classified into BCSG 1. Claw horn disruption lesions were found in 24.4% of the cows (n=140) at dry-off. Cows without CHDL were 1.4 times more likely to conceive than cows with CHDL. Additionally, lesion cows were 1.7 times more likely to die or be culled than nonlesion cows. Absence of CHDL did not have a significant effect on milk yield. These findings highlight the importance of claw health and BCS at the end of lactation on future survival and performance.