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During the design of hypothesis-driven, comparative laboratory animal experiments, investigators must control for cage effects, ensure full blinding and full randomization while adhering to established experimental designs, notably variations of the Completely Randomized Design and the Randomized Block Designs. Failure to meet these criteria introduces partial or complete confounding by multiple known and unknown variables, resulting in biased outcome measures and rendering valid statistical analysis impossible. Our analysis of a stratified, random sample of comparative laboratory animal experiments conducted in North America and Europe and published in 2022, shows that as few as 0–2.5% utilized valid, unbiased experimental designs. The failure of investigators to adopt valid, unbiased study designs undermines scientific rigour, squanders resources and animal lives, and impedes the reliable translation of preclinical research findings to human and veterinary medicine. We propose practical, achievable solutions focused on enhancing the rigour and validity of study designs. This includes developing a specialized group of scientists with expertise in the design of laboratory animal experiments and data analysis, to ensure future studies are conducted with the highest scientific standards.

Mycoplasma bovis is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent Mycoplasma spp. such as M. bovigenitalium and M. bovirhinis have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine Mycoplasma spp. using Madin–Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis. Following infection, a gentamicin protection assay was performed and the cells lysed at 6 and 54 h post-infection. The MDBK cell lysates were cultured for Mycoplasma spp. and qPCR was used to estimate the average number of Mycoplasma bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using M. bovis mNeonGreen strain were also performed. All 14 Mycoplasma strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the M. bovis mNeonGreen strain. Unexpectedly, the M. bovis strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than M. bovirhinis and M. bovigenitalium strains. These findings suggest that the ability for mycoplasmas to invade and replicate within host cells does not account for the differences in virulence between species.

In North America, beef production relies on the administration of antimicrobials to manage disease. Bovine respiratory disease (BRD) is the most significant disease of beef cattle, and antimicrobial resistance (AMR) to conventional therapies presents an existential risk to animal welfare and food production. While AMR surveillance programs are poised to help facilitate antimicrobial stewardship and decision making at feedlots, monitoring strategies for large numbers of animals at an individual or group level are time consuming and costly. Accordingly, we completed a pilot investigation of feedlot water bowls, which is an understudied interface between cattle and bacteria. By performing cultivation-dependent and cultivation-independent studies, we demonstrate that water bowl-dwelling bacteria can act as sentinel organisms for clinically relevant antimicrobial resistance genes (ARGs) and that cattle have an impact on the microbial communities in the bowls. Moreover, by sampling water at a feedlot site before animal arrival, we detected resistance to two antibiotics: florfenicol and tulathromycin. After just 4 weeks of operation, multidrug-resistant bacteria were routinely found in most water bowls. A comparison of ARGs encoded by five water bowl bacterial isolates along with previously reported source and wastewater metagenomes to those found in BRD pathogens confirmed the utility of using water samples for AMR surveillance. A better understanding of how environmental reservoirs of ARGs in the feedlot relate to those found in animal pathogens will help inform and improve disease management, treatment strategies, and outcomes. Monitoring individual cattle or small groups is invasive, logistically challenging, expensive, and unlikely to gain adoption by the beef cattle industry. Wastewater surveillance has become standard in public health studies and has inspired similar work to better our understanding of AMR in feedlots. We derived our insights from sampling water bowls in a newly established feedlot: a unique opportunity to observe AMR prior to animal arrival and to monitor its development over 2 months. Importantly, the bacterial community of a single water bowl can be influenced by direct contact with hundreds of animals. Our results suggest that water bowl microbiomes are economical and pragmatic sentinels for monitoring relevant AMR mechanisms.

Numerous antimicrobial resistance (AMR) surveillance studies have been conducted in North American feedlot cattle to investigate the major bacterial pathogens of the bovine respiratory disease (BRD) complex, specifically: Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. While most bacterial isolates recovered from healthy cattle are susceptible to a repertoire of antimicrobials, multidrug resistance is common in isolates recovered from cattle suffering from BRD. Integrative and conjugative elements (ICE) have gained increasing notoriety in BRD-Pasteurellaceae as they appear to play a key role in the concentration and dissemination of antimicrobial resistant genes. Likewise, low macrolide susceptibility has been described in feedlot isolates of M. bovis. Horizontal gene transfer has also been implicated in the spread of AMR within mycoplasmas, and in-vitro experiments have shown that exposure to antimicrobials can generate high levels of resistance in mycoplasmas via a single conjugative event. Consequently, antimicrobial use (AMU) could be accelerating AMR horizontal transfer within all members of the bacterial BRD complex. While metagenomics has been applied to the study of AMR in the microbiota of the respiratory tract, the potential role of the respiratory tract microbiome as an AMR reservoir remains uncertain. Current and prospective molecular tools to survey and characterize AMR need to be adapted as point-of-care technologies to enhance prudent AMU in the beef industry.

Abstract Ovariectomy (spaying) using the trans-vaginal dropped ovary technique (DOT) is performed to prevent pregnancy in cull female beef cattle that are not retained for breeding stock in areas practicing extensive grazing management. There are no reports describing analgesia for this surgical procedure. The objective of this study was to measure behavioral and physiological responses to determine whether an analgesic protocol of BXK [butorphanol (0.01 mg/kg), xylazine (0.02 mg/kg), and ketamine (0.04 mg/kg)] injected intramuscularly (i.m.) before spaying could mitigate procedural and immediate postsurgical pain, and whether oral meloxicam (1 mg/kg) administered at the time of spaying could mitigate postsurgical inflammatory pain. Forty-four red Angus and Angus crossbred yearling heifers (322 ± 27.0 kg BW) were randomly allocated to 1 of 3 groups: PALP (control; palpated but not spayed; n = 14), SPAY (spayed with no analgesia; n = 15), and BXKM (spayed with analgesia; n = 15). Behavioral measurements included visual analog scale (VAS) score, flight speed (FS), stride length (SL), and gait score (GS), as well as activity (lying, standing) and feeding behavior. Physiological measurements included salivary cortisol (SC), haptoglobin (Hp), serum amyloid A (SAA), substance P (SP), complete blood count (CBC), and rectal temperature (RT). Saliva and blood samples were collected, and RT, FS, SL, and GS were measured on day −1, day 0 (time of palpation/spaying), and hours 1, 2, 4, and days 1, 2, 4, and 7 after palpation/spaying. The BXKM heifers had lower SC concentrations than SPAY heifers at 1 h (P = 0.01) and 2 h (P = 0.004). Heifers treated with BXKM had Hp concentrations lower than SPAY heifers at 2 d (P = 0.01), 4 d (P < 0.001), and 7 d (P = 0.008), and lower Hp concentrations than PALP heifers at 4 d (P < 0.001). Concentrations of SAA were greater (P = 0.04) in BXKM heifers than in PALP heifers at 1 h and lower in PALP heifers than in BXKM heifers (P = 0.02) and SPAY heifers (P = 0.05) at 1 d. Heifers in the BXKM group had higher RT than PALP and SPAY heifers at 1 h (P < 0.001) and 2 h (P = 0.004). Results suggest that DOT ovariectomy is acutely stressful and painful and administration of BXK before spaying and meloxicam at the time of spaying mitigated the procedural and postsurgical stress, pain, and inflammation.

An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are administered multiple antimicrobial treatments for BRD. A number of factors are responsible for BRD antimicrobial treatment failure, with formation of biofilms possibly being one. It is widely accepted that biofilms play a role in chronic infections in humans and it has been hypothesized that they are the default lifestyle of most bacteria. However, research on bacterial biofilms associated with livestock is scarce and significant knowledge gaps exist in our understanding of their role in AMR of the bacterial BRD complex. The four main bacterial species of the BRD complex, Mannheimia haemolytica , Pasteurella multocida , Histophilus somni , and Mycoplasma bovis are able to form biofilms in vitro and there is evidence that at least H. somni retains this ability in vivo . However, there is a need to elucidate whether their biofilm-forming ability contributes to pathogenicity and antimicrobial treatment failure of BRD. Overall, a better understanding of the possible role of BRD bacterial biofilms in clinical disease and AMR could assist in the prevention and management of respiratory infections in feedlot cattle. We review and discuss the current knowledge of BRD bacteria biofilm biology, study methodologies, and their possible relationship to AMR.

Digital dermatitis (DD) is an emerging disease in feedlot cattle. Our objective was to identify animal- and feedlot-level risk factors for DD by analyzing individual animal health records (n = 1,209,883) and feedlot-level records from western Canadian feedlots (n = 28) between 2014 and 2018, inclusive. The risk of a DD diagnosis was higher (incidence rate ratio (IRR) = 2.08, 95% CI 1.52 to 2.86) in cattle sourced from confined background operations (CB) versus cattle sourced from auction markets (AM). Conversely, ranch direct (RD) cattle were (IRR = 0.02, 95% CI 0.04 to 0.30) lower risk than AM cattle of being diagnosed with DD. The risk of being diagnosed with DD was higher in females than in males. The magnitude of the risk in females over males was influenced by annual DD incidence in low morbidity years (2014, 2017, and 2018) (IRR = 2.02, 95% CI 1.27 to 3.19), medium morbidity years (2016) (IRR = 2.95, 95% CI 1.64 to 5.33), and high morbidity years (2015) (IRR = 5.41, 95% CI 3.27 to 8.95). At the feedlot-level, the risk of a diagnosis of DD was lower in small capacity (SCF) versus large capacity feedlots (LCF) (IRR = 0.24, 95% CI 0.05 to 0.76). Future research should focus on identifying factors that may propagate disease transmission between cattle of different sexes and from different acquisition sources.

Among more than twenty species belonging to the class Mollecutes, Mycoplasma bovis is the most common cause of bovine mycoplasmosis in North America and Europe. Bovine mycoplasmosis causes significant economic loss in the cattle industry. The number of M. bovis positive herds recently has increased in North America and Europe. Since antibiotic treatment is ineffective and no efficient vaccine is available, M. bovis induced mycoplasmosis is primarily controlled by herd management measures such as the restriction of moving infected animals out of the herds and culling of infected or shedders of M. bovis. To better understand the population structure and genomic factors that may contribute to its transmission, we sequenced 147 M. bovis strains isolated from four different countries viz. USA (n = 121), Canada (n = 22), Israel (n = 3) and Lithuania (n = 1). All except two of the isolates (KRB1 and KRB8) were isolated from two host types i.e., bovine (n = 75) and bison (n = 70). We performed a large-scale comparative analysis of M. bovis genomes by integrating 103 publicly available genomes and our dataset (250 total genomes). Whole genome single nucleotide polymorphism (SNP) based phylogeny using M.agalactiae as an outgroup revealed that M. bovis population structure is composed of five different clades. USA isolates showed a high degree of genomic divergence in comparison to the Australian isolates. Based on host of origin, all the isolates in clade IV was of bovine origin, whereas majority of the isolates in clades III and V was of bison origin. Our comparative genome analysis also revealed that M. bovis has an open pangenome with a large breadth of unexplored diversity of genes. The function based analysis of autogenous vaccine candidates (n = 10) included in this study revealed that their functional diversity does not span the genomic diversity observed in all five clades identified in this study. Our study also found that M. bovis genome harbors a large number of IS elements and their number increases significantly (p = 7.8 × 10−6) as the genome size increases. Collectively, the genome data and the whole genome-based population analysis in this study may help to develop better understanding of M. bovis induced mycoplasmosis in cattle.

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.

Antimicrobials are crucial for treating bovine respiratory disease (BRD) in beef feedlots. Evidence is needed to support antimicrobial use (AMU) decisions, particularly in the early part of the feeding period when BRD risk is highest. The study objective was to describe changes in prevalence and antimicrobial susceptibility of BRD bacterial pathogens at feedlot processing (1 day on feed (1DOF)), 12 days later (13DOF), and for a subset at 36DOF following metaphylactic antimicrobial treatment. Mixed-origin steer calves (n = 1599) from Western Canada were managed as 16 pens of 100 calves, receiving either tulathromycin (n = 1199) or oxytetracycline (n = 400) at arrival. Deep nasopharyngeal swabs collected at all time points underwent culture and antimicrobial susceptibility testing (AST). Variability in the pen-level prevalence of bacteria and antimicrobial susceptibility profiles were observed over time, between years, and metaphylaxis options. Susceptibility to most antimicrobials was high, but resistance increased from 1DOF to 13DOF, especially for tetracyclines and macrolides. Simulation results suggested that sampling 20 to 30 calves per pen of 200 reflected the relative pen-level prevalence of the culture and AST outcomes of interest. Pen-level assessment of antimicrobial resistance early in the feeding period can inform the evaluation of AMU protocols and surveillance efforts and support antimicrobial stewardship in animal agriculture.