Mycoplasma bovis mastitis in dairy cattle.

Mycoplasma bovis has recently been identified increasingly in dairy cows causing huge economic losses to the dairy industry. M. bovis is a causative agent for mastitis, pneumonia, endometritis, endocarditis, arthritis, otitis media, and many other clinical symptoms in cattle. However, some infected cows are asymptomatic or may not shed the pathogen for weeks to years. This characteristic of M. bovis, along with the lack of adequate testing and identification methods in many parts of the world until recently, has allowed the M. bovis to be largely undetected despite its increased prevalence in dairy farms. Due to growing levels of antimicrobial resistance among wild-type M. bovis isolates and lack of cell walls in mycoplasmas that enable them to be intrinsically resistant to beta-lactam antibiotics that are widely used in dairy farms, there is no effective treatment for M. bovis mastitis. Similarly, there is no commercially available effective vaccine for M. bovis mastitis. The major constraint to developing effective intervention tools is limited knowledge of the virulence factors and mechanisms of the pathogenesis of M. bovis mastitis. There is lack of quick and reliable diagnostic methods with high specificity and sensitivity for M. bovis. This review is a summary of the current state of knowledge of the virulence factors, pathogenesis, clinical manifestations, diagnosis, and control of M. bovis mastitis in dairy cows.

Efficacy of novel staphylococcal surface associated protein vaccines against Staphylococcus aureus and non-aureus staphylococcal mastitis in dairy cows.

Mastitis is an inflammation of the mammary gland commonly caused by bacteria or fungi. Staphylococcus aureus is a major bacterium that causes mastitis in dairy cows. Non-aureus staphylococci are also increasingly reported, with Staphylococcus chromogenes being the most common species. Current staphylococcal mastitis control programs are not fully effective, and treatment with antibiotics is not sustainable. Non-antibiotic sustainable control tools, such as effective vaccines, are critically needed. We previously developed S. aureus surface-associated proteins (SASP) and S. chromogenes surface-associated proteins (SCSP) vaccines that conferred partial protective effects. We hypothesized that vaccination with SASP or SCSP would reduce the incidence of S. aureus mastitis throughout the lactation period. The objective of this study was to evaluate the efficacy of SASP and SCSP vaccines against S. aureus and non-aureus staphylococcal mastitis under natural exposure over 300 days of lactation. Pregnant Holstein dairy cows (n = 45) were enrolled and assigned to receive SASP (n = 15) or SCSP (n = 16) vaccines or unvaccinated control (n = 14). Cows were vaccinated with 1.2 mg of SASP or SCSP with Emulsigen-D adjuvant. Control cows were injected with phosphate-buffered saline with Emulsigen-D adjuvant. Three vaccine injections were given subcutaneously at 60, 40, and 20 days before the expected calving. Booster vaccinations were given at 120 and 240 days in milk. Cows were monitored for mastitis at quarter and cow levels, staphylococcal mastitis incidence, changes in serum and milk anti-SASP and anti-SCSP antibody titers, bacterial counts in milk, adverse reactions, milk yield and milk somatic cells count over 300 days of lactation. The SCSP vaccine conferred a significant reduction in the incidence of staphylococcal mastitis. Milk and serum anti-SASP and anti-SCSP antibody titers were increased in the vaccinated cows compared to unvaccinated control cows. Anti-SASP and anti-SCSP antibody titers decreased at about 120 days in milk, indicating the duration of immunity of about four months. In conclusion, the SASP and SCSP vaccines conferred partial protection from natural infection.

Molecular epidemiology and pathogenomics of extended-spectrum beta-lactamase producing- Escherichia coli and - Klebsiella pneumoniae isolates from bulk tank milk in Tennessee, USA.

Introduction: The rise in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in dairy cattle farms poses a risk to human health as they can spread to humans through the food chain, including raw milk. This study was designed to determine the status, antimicrobial resistance, and pathogenic potential of ESBL-producing -E. coli and -Klebsiella spp. isolates from bulk tank milk (BTM). Methods: Thirty-three BTM samples were collected from 17 dairy farms and screened for ESBL-E. coli and -Klebsiella spp. on CHROMagar ESBL plates. All isolates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). Results: Ten presumptive ESBL-producing bacteria, eight E. coli, and two K. pneumoniae were isolated. The prevalence of ESBL-E. coli and -K. pneumoniae in BTM was 21.2% and 6.1%, respectively. ESBL-E. coli were detected in 41.2% of the study farms. Seven of the ESBL-E. coli isolates were multidrug resistant (MDR). The two ESBL-producing K. pneumoniae isolates were resistant to ceftriaxone. Seven ESBL-E. coli strains carry the blaCTX-M gene, and five of them co-harbored blaTEM-1. ESBL-E. coli co-harbored blaCTX-M with other resistance genes, including qnrB19, tet(A), aadA1, aph(3'')-Ib, aph(6)-Id), floR, sul2, and chromosomal mutations (gyrA, gyrB, parC, parE, and pmrB). Most E. coli resistance genes were associated with mobile genetic elements, mainly plasmids. Six sequence types (STs) of E. coli were detected. All ESBL-E. coli were predicted to be pathogenic to humans. Four STs (three ST10 and ST69) were high-risk clones of E. coli. Up to 40 virulence markers were detected in all E. coli isolates. One of the K. pneumoniae was ST867; the other was novel strain. K. pneumoniae isolates carried three types of beta-lactamase genes (blaCTX-M, blaTEM-1 and blaSHV). The novel K. pneumoniae ST also carried a novel IncFII(K) plasmid ST. Conclusion: Detection of high-risk clones of MDR ESBL-E. coli and ESBL-K. pneumoniae in BTM indicates that raw milk could be a reservoir of potentially zoonotic ESBL-E. coli and -K. pneumoniae.

Prevalence and antimicrobial resistance profiles of extended-spectrum beta-lactamase-producing Escherichia coli in East Tennessee dairy farms.

Introduction: The extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, such as Escherichia coli, are emerging as a serious threat to global health due to their rapid spread and their multidrug-resistant (MDR) phenotypes. However, limited information is available regarding the prevalence and antimicrobial resistance (AMR) profile of ESBL-E. coli in the United States dairy farms. This study aimed to determine the prevalence and AMR pattern of ESBL-E. coli in East Tennessee dairy cattle farms. Methods: Rectal fecal samples from dairy cattle (n = 508) and manure (n = 30), water (n = 19), and feed samples (n = 15) were collected from 14 farms. The presumptive E. coli was isolated on CHROMagar™ ESBL and confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed on the ESBL-E. coli isolates. Results and discussion: From 572 fecal and farm environmental samples, a total of 233 (41%, n = 572) ESBL-E. coli were identified. The prevalence of fecal ESBL-E. coli was 47.5% (95% CI: 46.2-49.2). The within-farm prevalence of ESBL-E. coli ranged from 8 to 100%. Recent treatment history with third-generation cephalosporins (3GC), cow parity ≥3, and calves were the independent risk factors associated (P < 0.05) with fecal carriage of ESBL-E. coli. Overall, 99.6% (n = 231) ESBL-E. coli tested were phenotypically resistant to at least one of the 14 antimicrobial agents tested. The most common AMR phenotypes were against beta-lactam antibiotics, ampicillin (99.1%; n = 231 isolates), and ceftriaxone (98.7%, n = 231). Most ESBL-E. coli isolates (94.4%) were MDR (resistance to ≥3 antimicrobial classes), of which 42.6% showed co-resistance to at least six classes of antimicrobials. ESBL-E. coli isolates with concurrent resistance to ceftriaxone, ampicillin, streptomycin, tetracycline, sulfisoxazole, and chloramphenicol are widespread and detected in all the farms. The detection of MDR ESBL-E. coli suggests that dairy cattle can be a reservoir for these bacteria, highlighting the associated public health risk.

Regulation Mechanisms of Virulence Genes in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common E. coli pathotypes reported to cause several outbreaks of foodborne illnesses. EHEC is a zoonotic pathogen, and ruminants, especially cattle, are considered important reservoirs for the most common EHEC serotype, E. coli O157:H7. Humans are infected indirectly through the consumption of food (milk, meat, leafy vegetables, and fruits) and water contaminated by animal feces or direct contact with carrier animals or humans. E. coli O157:H7 is one of the most frequently reported causes of foodborne illnesses in developed countries. It employs two essential virulence mechanisms to trigger damage to the host. These are the development of attaching and effacing (AE) phenotypes on the intestinal mucosa of the host and the production of Shiga toxin (Stx) that causes hemorrhagic colitis and hemolytic uremic syndrome. The AE phenotype is controlled by the pathogenicity island, the locus of enterocyte effacement (LEE). The induction of both AE and Stx is under strict and highly complex regulatory mechanisms. Thus, a good understanding of these mechanisms, major proteins expressed, and environmental cues involved in the regulation of the expression of the virulence genes is vital to finding a method to control the colonization of reservoir hosts, especially cattle, and disease development in humans. This review is a concise account of the current state of knowledge of virulence gene regulation in the LEE-positive EHEC.

Prevalence of Antimicrobial Resistant and Extended-Spectrum Beta-Lactamase-producing Escherichia coli in Dairy Cattle Farms in East Tennessee.

Antimicrobials have been widely used in dairy farms to prevent and control dairy cattle diseases since 1960s. This led to the emergence of antimicrobial resistant bacteria (ARB) that, along with their antimicrobial resistance genes (ARGs), can spread from dairy farms to humans. Therefore, regular antimicrobial resistance (AMR) monitoring is important to implement proper mitigation measures. The objective of this study was to determine the prevalence of AMR and extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli in dairy cattle. A cross-sectional study was conducted in four dairy cattle farms (A-D) in East Tennessee. A total of 80 samples consisting of 20 samples each of bulk tank milk, feces, dairy cattle manure-amended soil, and prairie soil adjacent to the farms were collected and cultured for the isolation of E. coli. Tetracycline (TETr)-, third-generation cephalosporin (TGCr)- and nalidixic acid (NALr)-resistant E. coli (n = 88) were isolated and identified on agar media supplemented with TET, cefotaxime, and NAL, respectively. TGCr E. coli were tested for ESBLs and other coselected ARGs. TETr (74%, n = 88) was the most common, followed by TGCr (20%) and NALr (8%). Farms had significant (p < 0.001) differences: the highest prevalence of TGCr (55%) and TETr (100%) were observed in farm D, while all NALr isolates were from farm C. Over 83% of TGCr isolates (n = 18) harbored ESBL gene blaCTX-M. Majority (78%) of the E. coli isolates were multidrug-resistant (MDR), being positive for beta-lactams (blaCTX-M), TETs tet(A), tet(B), tet(M)), sulfonamides (sul2), aminoglycosides (strA), and phenicols (floR). This study indicated the widespread occurrence of MDR ESBLs-E. coli in dairy cattle farms. AMR surveillance of more dairy farms and identification of farm-level risk factors are important to mitigate the occurrence and spread of ARB of significant public health importance, such as ESBLs-E. coli.