R.C. Bicalho

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.

Metagenomic analysis of the uterine bacterial microbiota in healthy and metritic postpartum dairy cows

At present, many bacterial species are validly known as etiological agents of dairy cattle metritis, yet the vast uncultured fraction has received no attention so far. The purpose of this study was to use culture-independent methods to describe and compare the uterine bacterial composition in healthy and metritic postpartum Holstein dairy cows. Both group-specific 16S ribosomal DNA PCR-denaturing gradient gel electrophoresis (DGGE) and clone library sequencing of broad-range 16S ribosomal DNA PCR revealed differences in the bacterial communities comparing healthy and metritic cows. Bacterial diversity in healthy and metritic uteri was greater and more complex than described previously by traditional culture methods. Sequences were assigned to 5 major groups (Gammaproteobacteria, Firmicutes, Fusobacteria, Bacteroidetes, and Tenericutes) and to uncultured bacteria. Additionally, DGGE suggested the presence of Actinobacteria. Most clone sequences in the metritic status libraries were affiliated with the phylum Fusobacteria. Many components, especially from other phyla, have not previously been isolated from cases of metritis. In the clone libraries from the healthy status dairy cows, Gammaproteobacteria was the most prominent group and most sequences showed high identity with Mannheimia varigena, Pasteurella hemolytica, and members of the phylum Tenericutes. Our data showed that the uterine bacterial community in postpartum dairy cows differed considerably between healthy and metritic cows and described the occurrence of a previously unrecognized extent of this diversity in the bovine intrauterine microbiota.

Strategies to Analyze Milk Losses Caused by Diseases with Potential Incidence Throughout the Lactation : A Lameness Example

The objective of this study was to test the hypothesis that lameness incidence in lactating dairy cows decreases milk production compared with controls and to determine the best method of the 5 combinations of 2 study designs (retrospective cohort and retrospective matched cohort) and 3 repeated measures statistical methods: ANOVA (with binary classification of lameness); ANOVA with a lameness index (LAMIX) as the variable of interest; and analysis of covariance (ANCOVA, controlling for mean milk production for the first 3 wk of lactation and with a binary classification of lameness). Data were from 1 dairy farm located near Ithaca, NY, and from cows that calved between January 1, 2004 and January 1, 2007. The LAMIX was defined as -1, -2, -3, -4, and -5 = 1, 2, 3, 4, and > or =5 mo before lameness was detected, respectively; 1, 2, 3, 4, and 5 = 1, 2, 3, 4, and 5 mo after diagnosis, respectively; and 0 = cows that were never lame. With the binary lameness classification analyzed by repeated measures ANOVA, there was no effect of lameness on milk yield. The model was biased because lame cows had higher milk yields before lameness compared with nonlame cows. When the LAMIX was used, milk production before lameness was greater than after lameness (3.1 +/- 0.28 kg/d). Yet, point estimates generated for LAMIX were inaccurate because of the multicollinearity detected between LAMIX and week of lactation and because of the inability of adjusting the least squares means for the interaction of LAMIX and week of lactation. Therefore, the most appropriate models were the ANCOVA models (both for the matched and nonmatched retrospective-cohort designs). The estimated losses associated with lameness were 314 and 424 kg/cow per 305-d lactation, respectively, for the matched and nonmatched designs. Furthermore, high milk yield in the beginning of the lactation was a risk factor for lameness.

Diversity and Succession of Bacterial Communities in the Uterine Fluid of Postpartum Metritic, Endometritic and Healthy Dairy Cows

The diversity of the uterine bacterial composition in dairy cows is still poorly understood, although the emerging picture has shown to be increasingly complex. Understanding the complexity and ecology of microorganisms in the uterus of postpartum dairy cows is critical for developing strategies to block their action in reproductive disorders, such as metritis/endometritis. Here, we used PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and DNA pyrosequencing to provide a comprehensive description of the uterine bacterial diversity and compare its succession in healthy, metritic and endometritic Holstein dairy cows at three intervals following calving. Samples were collected from 16 dairy cows housed in a dairy farm located in upstate New York. PCR-DGGE revealed a complex profile with extensive differences in the community structure. With few exceptions, clustering analysis grouped samples from cows presenting the same health status. Analysis of >65,000 high-quality 16S rRNA gene sequences showed that the uterine bacterial consortia, regardless of the health status, is mainly composed of members of the phyla Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Tenericutes. In addition to these co-dominant phyla, sequences from Spirochaetes, Synergistetes, and Actinobacteria appear less frequently. It is possible that some sequences detected in the uterine fluid resulted from the presence of fecal or vaginal contaminants. Overall, the bacterial core community was different in uterine fluid of healthy cows, when compared to cows suffering from postpartum diseases, and the phylogenetic diversity in all the combined samples changed gradually over time. Particularly at the 34–36 days postpartum (DPP), the core community seemed to be specific for each health status. Our finding reveals that the uterine microbiota in dairy cows varies according with health status and DPP. Also, it adds further support to the hypothesis that there is uterine contamination with diverse bacterial groups following calving and emphasizes the role of unidentified microorganisms in this context.

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.

Prepartum and Postpartum Rumen Fluid Microbiomes: Characterization and Correlation with Production Traits in Dairy Cows

ABSTRACT Microbes present in the rumen of dairy cows are essential for degradation of cellulosic and nonstructural carbohydrates of plant origin. The prepartum and postpartum diets of high-producing dairy cows are substantially different, but in what ways the rumen microbiome changes in response and how those changes may influence production traits are not well elucidated. Here, we sequenced the 16S and 18S rRNA genes using the MiSeq platform to characterize the prepartum and postpartum rumen fluid microbiomes in 115 high-producing dairy cows, including both primiparous and multiparous animals. Discriminant analysis identified differences between the microbiomes of prepartum and postpartum samples and between primiparous and multiparous cows. 18S rRNA sequencing revealed an overwhelming dominance of the protozoan class Litostomatea, with over 90% of the eukaryotic microbial population belonging to that group. Additionally, fungi were relatively more prevalent and Litostomatea relatively less prevalent in prepartum samples than in postpartum ones. The core rumen microbiome (common to all samples) consisted of 64 bacterial taxa, of which members of the genus Prevotella were the most prevalent. The Chao1 richness index was greater for prepartum multiparous cows than for postpartum multiparous cows. Multivariable models identified bacterial taxa associated with increased or reduced milk production, and general linear models revealed that a metagenomically based prediction of productivity is highly associated with production of actual milk and milk components. In conclusion, the structure of the rumen fluid microbiome shifts between the prepartum and first-week postpartum periods, and its profile within the context of this study could be used to accurately predict production traits.

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.