Prevalence and spatial distribution of infectious diseases of dairy cattle in Ontario, Canada.

Here we investigated the prevalence and spatial distribution of selected pathogens associated with infectious diseases of dairy cattle in Ontario, Canada. The cross-sectional study surveyed bulk tank milk for antibodies against bovine leukemia virus (BLV), Mycobacterium avium ssp. paratuberculosis (MAP), and Salmonella Dublin, and for the presence of mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis). Between October 2021 and June 2022, bulk tank milk (BTM) samples were obtained from every commercial dairy farm in Ontario (n = 3,286). Samples underwent ELISA testing for presence of BLV, MAP and S. Dublin antibodies, and quantitative PCR testing for the detection of specific antigens of pathogens associated with mastitis. Bayesian models were used to estimate prevalence, and spatial analysis was carried out to identify regional clusters of high pathogen prevalence. Prevalence varied for different pathogens. BLV was widespread across dairy farms in Ontario, with an estimated prevalence of 88.3%. Prevalence of MAP, Staph. aureus and S. Dublin in Ontario dairy herds were 39.8%, 31.5% and 5.1%, respectively. The vast majority of dairy herds in Ontario were free of intramammary infections caused by Strep. agalactiae and M. bovis. Clusters of increased test positivity rates were detected for S. Dublin, MAP, and Staph. aureus, indicating potential geographic risk factors for pathogen transmission. For S. Dublin, an area of increased test positivity rates was detected in southwestern Ontario, close to the Canada-US border where most of the dairy herds in Ontario are located. Conversely, a localized cluster of positive test outcomes involving 14 subdivisions located in the southeastern region of Ontario was detected for Staph. aureus. Findings from our survey highlight the importance of the testing of aggregated samples and spatial analysis as part of disease surveillance programs and for implementing risk-based trading approaches among dairy producers.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China.

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

A scoping review of the testing of bulk tank milk to detect nonbacterial pathogens or herd exposure to nonbacterial pathogens in dairy cattle.

In this scoping review, we characterized the literature reporting on the testing of bulk milk samples to detect microorganisms other than bacteria that can cause diseases in dairy cattle, including viruses, helminths, algae, and protozoa. A search strategy was completed by screening databases, conference proceedings, animal health agency websites, disease surveillance program websites, and handbooks of cattle-related diagnostic tests for potentially relevant articles. Two reviewers independently screened articles in English, Portuguese, or Spanish; original studies reporting on the testing of farm-level, unprocessed bulk milk samples for presence of pathogens or specific antibodies against agents other than bacteria that can cause diseases in cows were retained. From all studies, we used spreadsheets to extract relevant information, including pathogen screened, test used, and country of origin of bulk milk samples. Additionally, for studies reporting sufficient data to estimate test characteristics, we extracted detailed information about herd eligibility, testing protocol, and herd-level infection definition. A total of 8,829 records were identified, from which 1,592 were retained and assessed for eligibility, and 306 were included. Bovine viral diarrhea virus, Fasciola hepatica, Ostertagia ostertagi, and bovine herpesvirus 1 were the most frequently screened agents, reported from 107, 45, 45, and 33 studies, respectively. Sensitivity of bulk milk ELISA to detect herds with animals infected by bovine herpesvirus 1 ranged from 2 to 100%, and was affected mostly by antigen selection, cut-off adopted, herd vaccination status, and seroprevalence of lactating cows. Bulk milk ELISA had very high specificity to detect herds free of bovine leukemia virus, and varying sensitivity to detect herds with infected animals, which depended on the within-herd seroprevalence of lactating cattle. As for bovine viral diarrhea virus, in general, the sensitivity of bulk milk ELISA was moderate to high (>80%) when infection status was defined based on presence of persistently infected cattle or a high proportion of seropositive lactating cattle. Nevertheless, bulk milk ELISA was not able to distinguish infected and noninfected herds based on presence of seropositive unvaccinated weanlings. The PCR or quantitative PCR protocols employed had very low sensitivities (<40%) and very high specificities (>95%) to classify bovine viral diarrhea virus infection status of dairy herds. Sensitivity and specificity of bulk milk ELISA to classify herds with regards to presence of F. hepatica- or O. ostertagi-parasitized cattle were generally high and driven mostly by the definition of herd infection status. Conversely, bulk milk ELISA demonstrated varying characteristics to detect herds with or without Dictyocaulus viviparus-parasitized cattle, depending primarily on the antigen selected and presence of cattle with clinical signs of lungworm infection.

Invited review: Selective treatment of clinical mastitis in dairy cattle.

Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.

A scoping review of the testing of bulk milk to detect infectious diseases of dairy cattle: Diseases caused by bacteria.

Testing of bulk milk (BM) samples is a convenient, cost-effective strategy that can easily be implemented as part of disease surveillance programs on dairy farms. Here, we performed a scoping review to summarize the literature reporting on the testing of BM samples to detect infectious diseases of dairy cattle caused by bacteria. We also provide a non-exhaustive, albeit significant, list of diagnostic tests that are marketed for BM samples, as well as a list of disease surveillance activities that included testing of BM samples. A literature search was carried out in 5 databases, yielding 8,829 records from which 474 were retained. Overall, 575 eligible bacterial pathogens were screened for using BM samples, ranging from 1 to 6 individual pathogens per study. Staphylococcus aureus, including methicillin-resistant Staph. aureus, were the most studied bacteria (n = 179 studies), followed by Streptococcus agalactiae (86), Mycobacterium avium ssp. paratuberculosis (79), Coxiella burnetii (79), and Mycoplasma spp. (67). Overall, culture-based protocols, ELISA, real-time PCR, and PCR were the most commonly adopted methodologies to screen BM samples. Sensitivity of BM testing for bovine paratuberculosis was generally low and varied greatly according to the ELISA cut-offs adopted and herd-level definition of disease. In general, protocols had low to moderate sensitivities (<50%), which increased for herds with high within-herd seroprevalence. Specificity of BM testing for paratuberculosis was generally high. With respect to mastitis pathogens, BM testing demonstrated high sensitivity and specificity for Strep. agalactiae, in general. However, we observed inconsistency among studies with respect to the sensitivity of BM culture to detect infected herds, which was notably higher if enrolled herds were heavily infected or had history of clinical disease. Among Salmonella spp. pathogens, Salmonella Dublin was the most frequently studied bacterium for which BM testing has been validated. Specificity of BM ELISA was high, ranging from 89.0 to 99.4. In contrast, sensitivity varied greatly among studies, ranging from 50.6% to 100%. Our findings support that one of most important factors affecting sensitivity of BM ELISA for Salmonella Dublin is whether nonlactating cattle are considered in the definition of herd infection status. In general, protocols analyzed in this review suffered from very low sensitivities, which hardly justifies their use as part of disease surveillance as single testing. Nevertheless, test sensitivity can be increased by the adoption of more inclusive definitions of disease-free herds. Further, low-sensitivity and high-specificity methods can be valuable tools for surveillance when used repeatedly over time.

Knowledge Gaps in the Understanding of Antimicrobial Resistance in Canada.

Current limitations in the understanding and control of antimicrobial resistance (AMR) in Canada are described through a comprehensive review focusing on: (1) treatment optimization; (2) surveillance of antimicrobial use and AMR; and (3) prevention of transmission of AMR. Without addressing gaps in identified areas, sustained progress in AMR mitigation is unlikely. Expert opinions and perspectives contributed to prioritizing identified gaps. Using Canada as an example, this review emphasizes the importance and necessity of a One Health approach for understanding and mitigating AMR. Specifically, antimicrobial use in human, animal, crop, and environmental sectors cannot be regarded as independent; therefore, a One Health approach is needed in AMR research and understanding, current surveillance efforts, and policy. Discussions regarding addressing described knowledge gaps are separated into four categories: (1) further research; (2) increased capacity/resources; (3) increased prescriber/end-user knowledge; and (4) policy development/enforcement. This review highlights the research and increased capacity and resources to generate new knowledge and implement recommendations needed to address all identified gaps, including economic, social, and environmental considerations. More prescriber/end-user knowledge and policy development/enforcement are needed, but must be informed by realistic recommendations, with input from all relevant stakeholders. For most knowledge gaps, important next steps are uncertain. In conclusion, identified knowledge gaps underlined the need for AMR policy decisions to be considered in a One Health framework, while highlighting critical needs to achieve realistic and meaningful progress.

Non-aureus Staphylococci and Bovine Udder Health: Current Understanding and Knowledge Gaps.

Despite considerable efforts to control bovine mastitis and explain its causes, it remains the most costly and common disease of dairy cattle worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are not entirely understood. These Gram-positive bacteria have become the most frequently isolated group of bacteria in milk samples of dairy cows and are associated with (mild) clinical and subclinical mastitis. Different species and strains of NAS differ in their epidemiology, pathogenicity, virulence, ecology and host adaptation, and antimicrobial resistance profiles. They have distinct relationships with the microbiome composition of the udder and may also have protective effects against other mastitis pathogens. Some appear to persist on the skin and in the teat canal and udder, while others seem to be transient residents of the udder from the environment. Analyzing genotypic and phenotypic differences in individual species may also hold clues to why some appear more successful than others in colonizing the udder. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.

Virulence profiles of Klebsiella pneumoniae isolated from 2 large dairy farms in China.

We recently reported on the diversity of Klebsiella pneumoniae isolated from dairy herds in China. In our previous work, isolates from subclinical mastitis (SCM) had lower indices of diversity when compared with bacteria from other sources, possibly due to a contagious-like spread of udder adapted strains. Here we explored the virulence profile and capsular types of K. pneumoniae isolated from different sources on 2 dairy farms in China. Our overarching goal was to gain insights on the role of virulence genes toward the severity of mastitis caused by K. pneumoniae. A total of 1,484 samples were collected from clinical mastitis (CM; n = 355), SCM (n = 561), bulk tank milk (BTM; n = 130), and environmental and extramammary (EE) sites (n = 438). From those, 431 K. pneumoniae isolates were obtained, including 129, 77, 66, and 159 isolates from CM, SCM, BTM, and EE samples, respectively. Polymerase chain reactions were used to determine the capsular types and to detect potential virulence genes in all isolates. No significant farm effects were observed when comparing the distribution of most virulence genes in K. pneumoniae isolated from each source. K57 was the most prevalent capsular type in K. pneumoniae from all sources, but with increased detection rate in isolates from CM. entB, kfu, fimH1, mrkD, and ß-d-lacZ were frequently detected in K. pneumoniae from all sources. ß-d-lacZ, entB, and ituA were more prevalent in isolates from CM, whereas kfu, allS, and nif were more frequently detected in isolates from SCM. ybtS, aerobactin, and rpmA had increased prevalence in K. pneumoniae from BTM when compared with bacteria from other sources. No association was detected between virulence genes and the severity of CM. K57 and the nif gene had the highest discriminatory power to classify isolates from CM and SCM, respectively. Based on our findings, it is likely that K57 is the dominant capsular type in K. pneumoniae causing CM in large Chinese dairy herds. Likewise, we demonstrated that ß-d-lacZ is disseminated in K. pneumoniae isolated from large Chinese dairy farms, irrespectively of the source of bacteria. Our results also suggest a low contribution of the virulence profile of K. pneumoniae toward CM severity. Finally, the role of nif in increasing the adaptability to the udder and promoting a contagious-like spread of K. pneumoniae warrants further investigation.

Genetic diversity and molecular epidemiology of outbreaks of Klebsiella pneumoniae mastitis on two large Chinese dairy farms.

Klebsiella pneumoniae is an opportunistic and environmental mastitis-causing pathogen, with potential for contagious transmission. Repetitive element sequence-based PCR was used to determine genetic diversity and explore potential transmission and reservoirs for mastitis caused by K. pneumoniae on 2 large Chinese dairy farms. A total of 1,354 samples was collected from the 2 dairy farms, including milk samples from cows with subclinical and clinical mastitis, bedding, feces, feed, teat skin, and milking liners. Environmental samples were collected from all barns and milking parlors and extramammary samples from randomly selected dairy cows on both farms. In total, 272 and 93 K. pneumoniae isolates were obtained from Farms A and B, respectively (with ~8K and 2K lactating cows, respectively). Isolation rates from clinical mastitis (CM), subclinical mastitis (SCM), and environmental or extramammary samples were 34, 23 and 37%, respectively for Farm A and 42, 3, and 34% for Farm B. The K. pneumoniae isolated from CM milk and extramammary or environmental sources had high genetic diversity (index of diversity >90%) on the 2 farms and from SCM on Farm A. However, on Farm B, 9 SCM isolates were classified as 2 genotypes, resulting in a relatively low index of diversity (Simpson's index of diversity = 0.39; 95% CI = 0.08-0.70). Genotypes of K. pneumoniae causing mastitis were commonly detected in feces, bedding, and milking liners (Farm A), or from teat skin, sawdust bedding, and feed (Farm B). Based on its high level of genetic diversity, we inferred K. pneumoniae was an opportunistic and environmental pathogen causing outbreaks of CM on these 2 large Chinese dairy farms. Nevertheless, that only a few genotypes caused SCM implied some strains had increased udder adaptability and a contagious nature or a common extramammary source. Finally, control of intramammary infections caused by K. pneumoniae on large Chinese dairy farms must consider farm-level predictors, as the 2 outbreaks had distinct potential environmental sources of infection.

Prevalence of antimicrobial resistance genes and its association with restricted antimicrobial use in food-producing animals: a systematic review and meta-analysis.

BACKGROUND: There is ongoing debate regarding potential associations between restrictions of antimicrobial use and prevalence of antimicrobial resistance (AMR) in bacteria. OBJECTIVES: To summarize the effects of interventions reducing antimicrobial use in food-producing animals on the prevalence of AMR genes (ARGs) in bacteria from animals and humans. METHODS: We published a full systematic review of restrictions of antimicrobials in food-producing animals and their associations with AMR in bacteria. Herein, we focus on studies reporting on the association between restricted antimicrobial use and prevalence of ARGs. We used multilevel mixed-effects models and a semi-quantitative approach based on forest plots to summarize findings from studies. RESULTS: A positive effect of intervention [reduction in prevalence or number of ARGs in group(s) with restricted antimicrobial use] was reported from 29 studies for at least one ARG. We detected significant associations between a ban on avoparcin and diminished presence of the vanA gene in samples from animals and humans, whereas for the mecA gene, studies agreed on a positive effect of intervention in samples only from animals. Comparisons involving mcr-1, blaCTX-M, aadA2, vat(E), sul2, dfrA5, dfrA13, tet(E) and tet(P) indicated a reduced prevalence of genes in intervention groups. Conversely, no effects were detected for ß-lactamases other than blaCTX-M and the remaining tet genes. CONCLUSIONS: The available body of scientific evidence supported that restricted use of antimicrobials in food animals was associated with an either lower or equal presence of ARGs in bacteria, with effects dependent on ARG, host species and restricted drug.

Critically important antimicrobials are generally not needed to treat nonsevere clinical mastitis in lactating dairy cows: Results from a network meta-analysis.

There is ongoing debate regarding whether critically important antimicrobials (CIA) should be used to treat infections in food-producing animals. In this systematic review, we determined whether CIA and non-CIA have comparable efficacy to treat nonsevere bovine clinical mastitis caused by the most commonly reported bacteria that cause mastitis worldwide. We screened CAB Abstracts, Web of Science, MEDLINE, Scopus, and PubMed for original epidemiological studies that assessed pathogen-specific bacteriological cure rates of antimicrobials used to treat nonsevere clinical mastitis in lactating dairy cows. Network models were fit using risk ratios of bacteriological cure as outcome. A total of 30 studies met inclusion criteria. Comparisons of cure rates demonstrated that CIA and non-CIA had comparable efficacy for treatment of nonsevere clinical mastitis in dairy cattle. Additionally, for cows with nonsevere clinical mastitis caused by Escherichia coli and Klebsiella spp., bacteriological cure rates were comparable for treated versus untreated cows; therefore, there was no evidence to justify treatment of these cases with CIA. Our findings supported that CIA in general are not necessary for treating nonsevere clinical mastitis in dairy cattle, the disease that accounts for the majority of antimicrobial usage in dairy herds worldwide. Furthermore, our findings support initiatives to reduce or eliminate use of CIA in dairy herds.

Genomic Analysis of Bovine Staphylococcus aureus Isolates from Milk To Elucidate Diversity and Determine the Distributions of Antimicrobial and Virulence Genes and Their Association with Mastitis.

Staphylococcus aureus causes persistent clinical and subclinical bovine intramammary infections (IMI) worldwide. However, there is a lack of comprehensive information regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus in bovine milk in Canada. Here, we performed whole-genome sequencing (WGS) of 119 Canadian bovine milk S. aureus isolates and determined they belonged to 8 sequence types (ST151, ST352, ST351, ST2187, ST2270, ST126, ST133, and ST8), 5 clonal complexes (CC151, CC97, CC126, CC133, and CC8), and 18 distinct Spa types. Pan-, core, and accessory genomes were composed of 6,340, 1,279, and 2,431 genes, respectively. Based on phenotypic screening for AMR, resistance was common against beta-lactams (19% of isolates) and sulfonamides (7% of isolates), whereas resistance against pirlimycin, tetracycline, ceftiofur, and erythromycin and to the combination of penicillin and novobiocin was uncommon (3, 3, 3, 2, and 2% of all isolates, respectively). We also determined distributions of 191 virulence factors (VFs) in 119 S. aureus isolates after classifying them into 5 functional categories (adherence [n = 28], exoenzymes [n = 21], immune evasion [n = 20], iron metabolism [n = 29], and toxins [n = 93]). Additionally, we calculated the pathogenic potential of distinct CCs and STs and determined that CC151 (ST151 and ST351) had the highest pathogenic potential (calculated by subtracting core-VFs from total VFs), followed by CC97 (ST352 and ST2187) and CC126 (ST126 and ST2270), potentially linked to their higher prevalence in bovine IMI worldwide. However, there was no statistically significant link between the presence of VF genes and mastitis.IMPORTANCE Staphylococcus aureus is a major cause of bovine intramammary infections, leading to significant economic losses to dairy industry in Canada and worldwide. There is a lack of knowledge regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus isolated from bovine milk in Canada. Based on whole-genome sequencing and genomic analysis, we have determined the phylogeny and diversity of S. aureus in bovine milk and concluded that it had a large accessory genome, limited distribution of AMR genes, variable VF gene profiles and sequence types (ST), and clonal complex (CC)-specific pathogenic potentials. Comprehensive information on the population structure, as well as the virulence and resistance characteristics of S. aureus from bovine milk, will allow for source attribution, risk assessment, and improved therapeutic approaches in cattle.

Comparison of different approaches to antibiotic restriction in food-producing animals: stratified results from a systematic review and meta-analysis.

BACKGROUND: We have previously reported, in a systematic review of 181 studies, that restriction of antibiotic use in food-producing animals is associated with a reduction in antibiotic-resistant bacterial isolates. While informative, that report did not concretely specify whether different types of restriction are associated with differential effectiveness in reducing resistance. We undertook a sub-analysis of the systematic review to address this question. METHODS: We created a classification scheme of different approaches to antibiotic restriction: (1) complete restriction; (2) single antibiotic-class restriction; (3) single antibiotic restriction; (4) all non-therapeutic use restriction; (5) growth promoter and prophylaxis restriction; (6) growth promoter restriction and (7) other/undetermined. All studies in the original systematic review that were amenable to meta-analysis were included into this substudy and coded by intervention type. Meta-analyses were conducted using random effects models, stratified by intervention type. RESULTS: A total of 127 studies were included. The most frequently studied intervention type was complete restriction (n=51), followed by restriction of non-therapeutic (n=33) and growth promoter (n=19) indications. None examined growth promoter and prophylaxis restrictions together. Three and seven studies examined single antibiotic-class and single antibiotic restrictions, respectively; these two intervention types were not significantly associated with reductions in antibiotic resistance. Though complete restrictions were associated with a 15% reduction in antibiotic resistance, less prohibitive approaches also demonstrated reduction in antibiotic resistance of 9%-30%. CONCLUSION: Broad interventions that restrict global antibiotic use appear to be more effective in reducing antibiotic resistance compared with restrictions that narrowly target one specific antibiotic or antibiotic class. Importantly, interventions that allow for therapeutic antibiotic use appear similarly effective compared with those that restrict all uses of antibiotics, suggesting that complete bans are not necessary. These findings directly inform the creation of specific policies to restrict antibiotic use in food-producing animals.

Prevalence of Potential Virulence Genes in Klebsiella spp. Isolated from Cows with Clinical Mastitis on Large Chinese Dairy Farms.

Klebsiella spp. is a common cause of clinical mastitis (CM) in dairy cows. However, relatively less information is available about distribution of virulence factors of Klebsiella spp. isolated from cows with CM. Objectives of this study were, therefore, to determine the prevalence of hypermucoviscosity (HMV) phenotype, capsule serotypes, and potential virulence genes in Klebsiella spp. from cows in China with CM. A total of 241 Klebsiella spp. isolates were recovered from cows with CM on 123 dairy farms (each had >500 lactating cows) located in 13 provinces of China. Of the isolates, 124 (51%) and 117 (49%) were identified as Klebsiella pneumoniae and Klebsiella oxytoca, respectively. The prevalence of HMV was 16% for K. pneumoniae and 11% for K. oxytoca; entB (78%), fimH1 (55%), kfu (31%), and mrkD (24%) were the prevalent virulence genes among K. pneumoniae, whereas entB (50%), fimH1 (30%), and mrkD (22%) were prevalent in K. oxytoca. Prevalence of the lac gene was higher for K. pneumoniae (78%) than for K. oxytoca (13%), whereas the nif gene was more prevalent in K. oxytoca than in K. pneumoniae (12% and 1%, respectively). Fifty-six K. pneumoniae isolates were confirmed as K57, the most prevalent capsule serotype (45%). Twenty-one (18%), 20 (10%), and 9 (8%) of 117 K. oxytoca isolates were positive for K57, K5, and K54 serotypes, respectively. As the predominant serotype, K. pneumoniae K57 isolates had a higher prevalence of the HMV phenotype and fimH1 than non-K57 K. pneumoniae. In conclusion, virulence factors were commonly detected for both K. oxytoca and K. pneumoniae causing CM in Chinese dairy herds. HMV isolates were commonly identified, irrespective of species. In addition, as the predominant capsule in bovine K. pneumoniae, the K57 serotype may be better adapted to the udder environment; therefore, further studies targeting pathogenicity to mammary tissue should contribute new knowledge for vaccine development using this serotype.

Examination of unintended consequences of antibiotic use restrictions in food-producing animals: Sub-analysis of a systematic review.

Antimicrobial resistance is considered one of the greatest threats to global and public health today. The World Health Organization, the Food and Agriculture Organization, and the World Organisation for Animal Health, known as the Tripartite Collaboration, have called for urgent action. We have previously published a systematic review of 181 studies, demonstrating that interventions that restrict antibiotic use in food-producing animals are associated with a reduction in antibiotic resistant bacterial isolates in both animals and humans. What remains unknown, however, are whether (and what) unintended consequences may arise from such interventions. We therefore undertook a sub-analysis of the original review to address this research question. A total of 47 studies described potential consequences of antibiotic restrictions. There were no consistent trends to suggest clear harm. There may be increased bacterial contamination of food products, the clinical significance of which remains unclear. There is a need for rigorous evaluation of the unintended consequences of antibiotic restrictions in human health, food availability, and economics, given their possible widespread implications.

Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds.

The prevalence of antimicrobial resistance (AMR) is increasing in human and animal pathogens, becoming a concern worldwide. However, prevalence and characteristics of AMR of bovine mastitis pathogens in large Chinese dairy herds are still unclear. Therefore, our objective was to determine the AMR profile of bacteria isolated from clinical mastitis in large (>500 cows) Chinese dairy herds. A total of 541 isolates of the 5 most common species, Staphylococcus aureus (n = 103), non-aureus staphylococci (NAS; n = 107), Streptococcus species (n = 101), Klebsiella species (n = 130), and Escherichia coli (n = 100), isolated from bovine clinical mastitis on 45 dairy farms located in 10 provinces of China were included. Presence of AMR was determined by minimum inhibitory concentrations using the microdilution method. Prevalence of multidrug resistance (resistance to >2 antimicrobials) was 27% (148/541). A very wide distribution of minimum inhibitory concentrations was screened in all isolates, including Staph. aureus isolates, which were resistant to penicillin (66%). In addition, NAS (30%) were more resistant than Staph. aureus to oxacillin (84%), penicillin (62%), tetracycline (34%), and clindamycin (33%). Prevalence of resistance to tetracycline was high (59%) in Streptococcus spp. Additionally, prevalence of resistance of both E. coli and Klebsiella spp. was high to amoxicillin/clavulanate potassium (81 and 38%, respectively), followed by tetracycline (only Klebsiella spp. 32%). A high proportion (27%) of isolates were multidrug resistant; the most frequent combinations were clindamycin-cefalexin-tetracycline or enrofloxacin-cefalexin-penicillin patterns for Staph. aureus; enrofloxacin-oxacillin-penicillin-tetracycline patterns for NAS; clindamycin-enrofloxacin-tetracycline patterns for Streptococcus spp.; amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for Klebsiella spp.; and amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for E. coli. Resistance for 4 kinds of antimicrobials highly critical for human medicine, including daptomycin, vancomycin, imipenem, and polymyxin B, ranged from 0 to 24%. In conclusion, prevalence of AMR in mastitis pathogens was high on large Chinese dairy farms, potentially jeopardizing both antimicrobial efficacy and public health. Results of this study highlighted the need for improvements in antimicrobial stewardship and infection control programs in large Chinese dairy farms to reduce emergence of AMR.

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.

Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.

Antimicrobial resistance in non-aureus staphylococci isolated from milk is associated with systemic but not intramammary administration of antimicrobials in dairy cattle.

Non-aureus staphylococci (NAS) are the bacteria most frequently isolated from bovine milk. Objectives of this study were to determine herd-level associations between antimicrobial use (AMU) and prevalence of antimicrobial resistance (AMR) and antimicrobial resistance genes in NAS according to antimicrobials and routes of administration. The AMR profile was determined using a micro-broth dilution method against a panel of 23 antimicrobials for 1,702 NAS isolates obtained from 89 herds. A subset of these isolates (n = 405) was submitted to whole-genome sequencing, and the presence of AMR genes was determined using data from 4 databases. Antimicrobial use was determined for all herds using an inventory of empty drug containers and quantified for each antimicrobial as the number of antimicrobial daily doses administered. Generalized linear models were used to estimate antimicrobial and route-specific associations between AMR in NAS and AMU. Prevalence of multidrug resistance in NAS was associated with systemic use of antimicrobials. Estimated relative risk associated with a 1-unit increase in antimicrobial daily doses per cow-year administered systemically was 1.28. No association was present with either intramammary or intrauterine use. Three drug classes, all of high or very high importance for human medicine, were associated with drug-specific AMR when administered systemically: penicillins, third-generation cephalosporins, and macrolides. Prevalence of tet, erm, and blaARL genes in NAS was higher in herds that used more tetracyclines, macrolides, and third-generation cephalosporins, respectively. No association between drug-specific AMU and prevalence of blaZ, mphC, and msrA was identified, irrespective of route of administration. The either weak or nonexistent association between AMR and antimicrobials administered intramammarily suggest that a decrease in AMR of NAS following implementation of selective dry cow therapy would be minimal in comparison to reduced use of systemic antimicrobials.

Prevalence and Genetic Basis of Antimicrobial Resistance in Non-aureus Staphylococci Isolated from Canadian Dairy Herds.

Emergence and spread of antimicrobial resistance is a major concern for the dairy industry worldwide. Objectives were to determine: (1) phenotypic and genotypic prevalence of drug-specific resistance for 25 species of non-aureus staphylococci, and (2) associations between presence of resistance determinants and antimicrobial resistance. Broth micro-dilution was used to determine resistance profiles for 1,702 isolates from 89 dairy herds. Additionally, 405 isolates were sequenced to screen for resistance determinants. Antimicrobial resistance was clearly species-dependent. Resistance to quinupristin/dalfopristin was common in Staphylococcus gallinarum (prevalence of 98%), whereas S. cohnii and S. arlettae were frequently resistant to erythromycin (prevalence of 63 and 100%, respectively). Prevalence of resistance was 10% against ß-lactams and tetracyclines. In contrast, resistance to antimicrobials critically important for human medicine, namely vancomycin, fluoroquinolones, linezolid and daptomycin, was uncommon (< 1%). Genes encoding multidrug-resistance efflux pumps and resistance-associated residues in deducted amino acid sequences of the folP gene were the most frequent mechanisms of resistance, regardless of species. The estimated prevalence of the mecA gene was 17% for S. epidermidis. Several genes, including blaZ, mecA, fexA, erm, mphC, msrA, and tet were associated with drug-specific resistance, whereas other elements were not. There were specific residues in gyrB for all isolates of species intrinsically resistant to novobiocin. This study provided consensus protein sequences of key elements previously associated with resistance for 25 species of non-aureus staphylococci from dairy cattle. These results will be important for evaluating effects of interventions in antimicrobial use in Canadian dairy herds.

Invited review: Effectiveness of precalving treatment on postcalving udder health in nulliparous dairy heifers: A systematic review and meta-analysis.

Clinical mastitis affects 3% of primiparous dairy cattle (heifers) in the first month after calving. Additionally, the prevalence of intramammary infection (IMI) in the months before first calving is high, resulting in a high prevalence of heifers calving with IMI. Precalving therapy is an accepted recommendation for reducing mastitis in multiparous cows, but prophylactic treatment for heifers is uncommon in North America. Objectives of this study were to (1) quantify changes in postcalving udder health in heifers following application of a precalving treatment; (2) compare effectiveness among various types of treatments; and (3) compare effectiveness of various types of treatments against specific pathogens. A systematic review was conducted comparing interventions aimed at improving udder health in heifers. Of 62 included studies, 48 clinical trials were used in a meta-analysis. Data were synthesized using a random effects model for meta-analysis, followed by sub-group analyses comparing treatment types, and specific pathogens with statistical testing using meta-regression. Occurrence of mastitis (defined as elevated somatic cell count, clinical mastitis, and IMI) was reduced in treated heifers compared with untreated controls with a pooled risk ratio of treated to untreated heifers of 0.56 (95% confidence interval: 0.47 to 0.67). Upon stratification by treatment types, teat sealants and combination therapies (vaccines and antimicrobials; antimicrobials and teat sealants; and all 3) were most effective at improving udder health with pooled risk ratios of 0.40 (95% confidence interval: 0.30 to 0.52) and 0.34 (95% confidence interval: 0.25 to 0.45), respectively. Antimicrobials and vaccines also reduced occurrence of IMI and subclinical and clinical mastitis when compared with untreated heifers. Although variation was observed in the pathogen-specific effectiveness of treatments at reducing rates of disease, antimicrobials, teat sealants, and combinations of vaccines or teat sealants with antimicrobials were consistently effective, whereas vaccines were only effective for contagious pathogens. Recommendations for use of antibiotics should consider their relative benefit while also considering potential for increasing antimicrobial resistance.