Exploring associations between the teat apex metagenome and Staphylococcus aureus intramammary infections in primiparous cows under organic directives.

The primary objective of this study was to identify associations between the prepartum teat apex microbiome and the presence of Staphylococcus aureus intramammary infections (IMI) in primiparous cows during the first 5 weeks after calving. We performed a case-control study using shotgun metagenomics of the teat apex and culture-based milk data collected longitudinally from 710 primiparous cows on five organic dairy farms. Cases had higher odds of having S. aureus metagenomic DNA on the teat apex prior to parturition compared to controls (OR = 38.9, 95% CI: 14.84-102.21). Differential abundance analysis confirmed this association, with cases having a 23.8 higher log fold change (LFC) in the abundance of S. aureus in their samples compared to controls. Of the most prevalent microorganisms in controls, those associated with a lower risk of post-calving S. aureus IMI included Microbacterium phage Min 1 (OR = 0.37, 95% CI: 0.25-0.53), Corynebacterium efficiens (OR = 0.53, 95% CI: 0.30-0.94), Kocuria polaris (OR = 0.54, 95% CI: 0.35-0.82), Micrococcus terreus (OR = 0.64, 95% CI: 0.44-0.93), and Dietzia alimentaria (OR = 0.45, 95% CI: 0.26-0.75). Genes encoding for Microcin B17 AMPs were the most prevalent on the teat apex of cases and controls (99.7% in both groups). The predicted abundance of genes encoding for Microcin B17 was also higher in cases compared to controls (LFC 0.26). IMPORTANCE: Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem for the dairy industry. The microbiome on the external skin of the teat apex may play a role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 16S approach, which precludes the detection of genomic features such as genes that encode for AMPs. Therefore, further research using a shotgun metagenomic approach is needed to understand what role prepartum teat apex microbiome dynamics play in IMI risk.

Cow-related factors associated with intramammary infections during the 1st week postpartum in primiparous dairy cows in certified organic herds.

The objectives if this exploratory study were to identify variables associated with intramammary infections (IMI) during the 1st week of lactation in primiparous organic dairy cows, and to evaluate the association of those variables with somatic cell count (SCC) linear scores and milk yield in early lactation. Nulliparous cows (n = 240) were evaluated for: udder edema, teat edema, milk leakage (ML) and udder hygiene at weeks 6, 4, 2, and 1 prepartum and 1st week postpartum; body condition score (BCS) at 6 weeks prepartum and 1st week postpartum; age at calving (days), gestation length, dystocia, stillbirth, calf sex were included in the analysis. Milk samples from the 1st week postpartum were cultured using standard laboratory techniques and bacterial growth was considered IMI. Intramammary infection was observed in 58.7% of cows, with Staphylococcus chromogenes and S. aureus being the most prevalent pathogens. Cows with ML at 1st week postpartum were at 3.42 greater odds of IMI (P < 0.01). Cows with prepartum BCS ≥ 3.75 were at 3.12 greater odds of IMI (P < 0.01). Cows with ML in the 1st week of lactation had increased SCC (P < 0.01) and lower milk production in the second month of lactation (P = 0.05). Intervention studies are needed to evaluate if monitoring prepartum BCS and ML at early postpartum can aid in the control of IMIs in heifers in organic dairies.

Investigating the cow skin and teat canal microbiomes of the bovine udder using different sampling and sequencing approaches.

There is a need for standardized, efficient, and practical sampling methods to support large population-based studies of the internal and external epithelial microbiomes of the bovine udder. The primary objective of this study was to evaluate different sampling devices for the isolation of microbial DNA originating from the internal and external teat epithelium. Secondary objectives were to survey and compare the microbial diversity of external and teat canal epithelial microbiomes using amplicon and shotgun metagenomic sequencing approaches. To address these objectives, we enrolled a convenience sample of 24 Holstein dairy cows and collected samples from the external epithelium at the base of udder, the external teat barrel epithelium, the external teat apex epithelium, and the teat canal epithelium. Extracted DNA was quantified and subjected to PCR amplification of the V4 hypervariable region of the 16S rRNA gene and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA). A subset of samples was subjected to a shallow shotgun metagenomic assay on the Illumina HiSeq platform. For samples collected from the external teat epithelium, we found that gauze squares consistently yielded more DNA than swabs, and Simpson's reciprocal index of diversity was higher for gauze than for swabs. The teat canal epithelial samples exhibited significantly lower diversity than the external sampling locations, but there were no significant differences in diversity between teat apex, teat barrel, and base of the udder samples. There were, however, differences in the microbial distribution and abundances of specific bacteria across external epithelial surfaces. The proportion of shotgun sequence reads classified as Bos taurus was highly variable between sampling locations, ranging from 0.33% in teat apex samples to 99.91% in teat canal samples. These results indicate that gauze squares should be considered for studying the microbiome of the external epithelium of the bovine udder, particularly if DNA yield must be maximized. Further, the relative proportion of host to non-host DNA present in samples collected from the internal and external teat epithelium should be considered when designing studies that utilize shotgun metagenomic sequencing.

Effect of subclinical mastitis detected in the first month of lactation on somatic cell count linear scores, milk yield, fertility, and culling of dairy cows in certified organic herds.

It is well established that subclinical mastitis (SCM), characterized by somatic cell count (SCC) >200,000 cells/mL, has a negative effect on the productivity, reproductive performance, and survivability of cows from conventional dairy herds. However, in organic herds, where the use of antimicrobial drugs is restricted for the treatment and control of intramammary infections (IMI) in dairy cows, little is known about the effect of SCM on performance and survivability. The objective of this study was to evaluate whether SCM diagnosed during the first month of lactation was associated with SCC linear score dynamics, milk production, fertility, and culling of dairy cows in USDA-certified organic herds. We collected data from 2 organic herds in New Mexico and Texas. A total of 1,511 cows that calved between June 2018 and May 2019 were included in the study and were followed until month 10 of the current lactation. Cows with SCC >200,000 cells/mL in the first month of lactation were considered to have SCM. We used mixed linear regression models accounting for repeated measures to assess the effect of SCM on monthly milk production and SCC linear scores. We used Cox proportional hazards models to evaluate the effect of SCM on the risk of pregnancy and culling. We considered parity, farm, previous gestation length, stillbirth, twinning, dystocia, and 2- and 3-way interactions as potential confounders. Cows diagnosed with SCM during the first month of lactation produced less milk than cows without SCM. Cows with SCM had elevated SCC linear scores during their previous lactation and throughout the subsequent months of lactation compared to cows without SCM. The effect of SCM on SCC linear scores was more pronounced in multiparous than primiparous cows. Subclinical mastitis during the first month of lactation did not affect the likelihood of pregnancy during the first 300 d in milk. Cows with SCM in the first month were more likely to die or be culled during the 300 d of lactation than cows without SCM. We observed that elevated SCC in the first month of lactation had detrimental effects on the milk yield and survivability of dairy cows in USDA organic herds, but it did not affect reproductive performance. We demonstrated that cows with SCM diagnosed in the first month of lactation continued to have elevated SCC linear scores throughout their entire lactation, and that elevated SCC was carried over from the previous lactation.

Modelling considerations in the analysis of associations between antimicrobial use and resistance in beef feedlot cattle.

A number of sophisticated modelling approaches are available to investigate potential associations between antimicrobial use (AMU) and resistance (AMR) in animal health settings. All have their advantages and disadvantages, making it unclear as to which model is most appropriate. We used advanced regression modelling to investigate AMU-AMR associations in faecal non-type-specific Escherichia coli (NTSEC) isolates recovered from 275 pens of feedlot cattle. Ten modelling strategies were employed to investigate AMU associations with resistance to chloramphenicol, ampicillin, sulfisoxazole, tetracycline and streptomycin. Goodness-of-fit statistics did not show a consistent advantage for any one model type. Three AMU-AMR associations were significant in all models. Recent parenteral tetracycline use increased the odds of finding tetracycline-resistant NTSEC [odds ratios (OR) 1·1-3·2]; recent parenteral sulfonamide use increased the odds of finding sulfisoxazole-resistant NTSEC (OR 1·4-2·5); and recent parenteral macrolide use decreased the odds of recovering ampicillin-resistant NTSEC (OR 0·03-0·2). Other results varied markedly depending on the modelling approach, emphasizing the importance of exploring and reporting multiple modelling methods based on a balanced consideration of important factors such as study design, mathematical appropriateness, research question and target audience.

Mannheimia haemolytica in feedlot cattle: prevalence of recovery and associations with antimicrobial use, resistance, and health outcomes.

BACKGROUND: Mannheimia haemolytica is an important etiological agent in bovine respiratory disease. OBJECTIVES: Explore risk factors for recovery of susceptible and resistant M. haemolytica in feedlot cattle and explore associations with health outcomes. ANIMALS: Cattle (n = 5,498) from 4 feedlots sampled at arrival and later in feeding period. METHODS: Susceptibility of M. haemolytica isolates tested for 21 antimicrobials. Records of antimicrobial use and health events analyzed using multivariable regression. RESULTS: M. haemolytica recovered from 29% of cattle (1,596/5,498), 13.1% at arrival (95% CI, 12.3-14.1%), and 19.8% at second sampling (95% CI, 18.7-20.9%). Nearly half of study cattle received antimicrobial drugs (AMDs) parenterally, mostly as metaphylactic treatment at arrival. Individual parenteral AMD exposures were associated with decreased recovery of M. haemolytica (OR, 0.2; 95% CI, 0.02-1.2), whereas exposure in penmates was associated with increased recovery (OR, 1.5; 95% CI, 1.05-2.2). Most isolates were pan-susceptible (87.8%; 95% CI, 87.0-89.4%). AMD exposures were not associated with resistance to any single drug. Multiply-resistant isolates were rare (5.9%; 95% CI, 5.1-6.9%), but AMD exposures in pen mates were associated with increased odds of recovering multiply-resistant M. haemolytica (OR, 23.9; 95% CI, 8.4-68.3). Cattle positive for M. haemolytica on arrival were more likely to become ill within 10 days (OR, 1.7; 95% CI, 1.1-2.4). CONCLUSIONS AND CLINICAL IMPORTANCE: Resistance generally was rare in M. haemolytica. Antimicrobial drug exposures in penmates increased the risk of isolating susceptible and multiply-resistant M. haemolytica, a finding that could be explained by contagious spread.

Rapid Salmonella detection in experimentally inoculated equine faecal and veterinary hospital environmental samples using commercially available lateral flow immunoassays.

REASONS FOR PERFORMING STUDY: Salmonella enterica is the most commonly reported cause of outbreaks of nosocomial infections in large animal veterinary teaching hospitals and the closure of equine hospitals. Rapid detection may facilitate effective control practices in equine populations. Shipping and laboratory testing typically require ≥48 h to obtain results. Lateral flow immunoassays developed for use in food-safety microbiology provide an alternative that has not been evaluated for use with faeces or environmental samples. OBJECTIVES: We aimed to identify enrichment methods that would allow commercially available rapid Salmonella detection systems (lateral flow immunoassays) to be used in clinical practice with equine faecal and environmental samples, providing test results in 18-24 h. STUDY DESIGN: In vitro experiment. METHODS: Equine faecal and environmental samples were inoculated with known quantities of S. enterica serotype Typhimurium and cultured using 2 different enrichment techniques for faeces and 4 enrichment techniques for environmental samples. Samples were tested blindly using 2 different lateral flow immunoassays and plated on agar media for confirmatory testing. RESULTS: In general, commercial lateral flow immunoassays resulted in fewer false-negative test results with enrichment of 1 g faecal samples in tetrathionate for 18 h, while all environmental sample enrichment techniques resulted in similar detection rates. The limit of detection from spiked samples, ∼4 colony-forming units/g, was similar for all methods evaluated. CONCLUSIONS: The lateral flow immunoassays evaluated could reliably detect S. enterica within 18 h, indicating that they may be useful for rapid point-of-care testing in equine practice applications. Additional evaluation is needed using samples from naturally infected cases and the environment to gain an accurate estimate of test sensitivity and specificity and to substantiate further the true value of these tests in clinical practice.