This is concordant with several archaeological indicators showing long-term increases in population density in the Upper Paleolithic and Late Stone Age, including increased small-game exploitation, greater pressure on easily collected prey species like tortoises and shellfish, and more intense hunting of dangerous prey species. We further note that much of the literature pointing to sudden increases in effective population size beginning earlier in the Pleistocene in subSaharan Africa is based on mtDNA data, which tends to show unimodal mismatch distributions and a skew in the frequency distribution towards rare alleles in many African farming and non-African populations. However, this mtDNA signal of demographic expansion is typically absent from samples of African hunter-gatherers. Our autosomal data provide a very different picture of more recent population growth in both sub-Saharan African hunter-gatherers and food producers. Preliminary simulations indicate that a model of population growth similar to that tested here does not result in elevated values of Tajima’s D and Rozas’ R2 for mtDNA as a result of its smaller effective population size relative to the autosomes. On the contrary, the four-fold smaller Ne of mtDNA means that it should reflect population growth more prominently . Consequently, pipp mobile systems mtDNA data may not accurately tell us when and to what extent human populations expanded, either as a result of evolutionary stochasticity , or as a result of natural selection at functional sites .
We specifically avoid these issues by considering multiple, independent, neutral regions from the autosomes. In sum, the ,1000-fold increase in human population size that has taken place over the last 10 kya is unlikely to be detectable with current resequencing data. The finding that autosomal resequencing data from all sub-Saharan African populations so far tested contain a signal of exponential size increase beginning in the Late Stone Age is concordant with archaeological data showing intensification in the number of LSA sites on the African landscape, an increased abundance of blade-based lithic technologies, and enhanced long-distance exchange after 50 Kya. Interestingly, there is mounting evidence that many of the individual elements of complex behavior first appear earlier in the Middle Stone age, 70–100 Kya. This suggests that the demographic effects manifest in these indicators of modern culture were felt only sporadically in the MSA, and that they did not become the general condition until the LSA, coincident with the significant population growth that is detectable in the autosomes of contemporary sub-Saharan Africans.Antimicrobial therapy is critical for the treatment of bacterial infections in veterinary medicine; however, resistance to these treatments has been increasing for decades and, as a result, there are concerns about the efficacy of antimicrobials in food-producing animals. It has been well documented that use of antimicrobial drugs is associated with increases in antimicrobial resistant bacteria , but increases in antimicrobial resistance genes can also develop in the absence of antimicrobial drug use .
There is consensus that applying pressure on a population of bacteria through antimicrobials will enrich ARGs within that population ; however, AMR cannot be attributed to exposure to antimicrobials alone . Although Enterococcus spp. are part of the normal flora in the bovine gastrointestinal tract, there are reports of Enterococcus spp. as a causative agent of mastitis in cattle and diarrhea in neonatal calves . Enterococcus has, however, been primarily used as a sentinel and potential source of AMR genes for other Gram-positive pathogens. Enterococcus spp. are known to be intrinsically resistant to many antibiotics and can develop and confer AMR status to other pathogens . Thus, Enterococcus spp. have the potential to cause disease and serve as sentinels for the status of AMR pathogens in an environment. E. coli is another component of the normal flora of the bovine enteric system with many strains that have varying pathogenicity. Various E. coli strains can cause mastitis or metritis in cows as well as neonatal diarrhea or septicemia in calves . E. coli has also been used as an indicator of AMR prevalence in fecal bacteria and a potential source for transmission of ARGs to other Gram-negative organisms , as it acquires resistance easily and can inhabit many types of animals . California is an important contributor to the U.S. beef industry, with approximately 590,000 beef cows and contributing $3.4 billion in cash receipts from total cattle and calf sales in 2015 . In beef cow-calf operations in the U.S., calves are born and stay at the same location with their dam usually until 6–8months of age, at which point they are weaned, removed from the dam, and are often placed in a group with animals of approximately the same age and/or size.
At this stage of the beef production cycle, there are many sectors that can involve the movement and mixing of animals. Some calves may be moved directly from cow-calf operations to feedlots after weaning until they reach slaughter weight. Alternatively, if there is high forage availability, they may be moved temporarily to a stocker facility before ultimately finishing at a feedlot facility. The time spent in each sector, size of group, and management of the animals are highly variable and depend on many factors including geographic location, producer goals, and access to pasture and/or facilities. Rates for multi-drug resistance have previously been shown to be higher in California cattle from various types of beef production systems when compared to nearby states, Washington and Oregon . Additionally, many AMR studies thus far have focused on feedlot, stocker, and calf ranch operations, where there may be increased selection pressure on bacterial populations through antimicrobial therapy from treatment and/or prevention of disease that develops likely as a result of mixing of animals, transport, and stress . A 2010 study found that the highest proportion of MDR E. coli isolates originated from calf ranches, followed by feedlots, while the least MDR was found in isolates from adult beef cows . Prophylactic or metaphylactic use of antibiotics and occurrence of disease requiring antibiotic treatment is less common in cow-calf operations than other operation types in the beef production chain . There are far fewer studies investigating the levels of AMR that exist in cow-calf operations and not yet one that exclusively investigated the levels of AMR that exist on cow-calf operations in California. Nevertheless, characterization of AMR in cow-calf operations is essential for evaluating and understanding the contribution of the cow-calf sector to AMR in the beef industry, as well as whether specific management and antimicrobial use patterns may be associated with AMR during this production stage before the calves are moved to feedlots where higher selection pressures exist. Previous studies in cow-calf herds have indicated that management and operation dependent factors can influence the presence of AMR in a group. Specifically, season of collection of samples for testing , age of animal , and intensity of operation have all been shown to be associated with increased AMR detection in beef operations. As part of the ongoing effort on surveillance for AMR, the objective of this cross-sectional study conducted on cow-calf operations in northern California was to assess prevalence of AMR in E. coli and Enterococcus spp. in beef cattle of different life stages, pasture types, and antimicrobial drug exposure on a herd and individual level. The hypothesis was that the prevalence of AMR in fecal samples varies according to the age of the animal as well as the drugs commonly used in the treatment of sick animals on those operations. The results of this study may serve as reference for future studies on the prevalence of AMR genes in the population of cow-calf operations in California and lead to a better understanding of risk factors for shedding of fecal pathogens carrying ARGs. Further surveillance, risk assessment, and interpretation of these results will help to derive more informed decisions and directions for combatting AMR in the future.A convenience sample of beef cow-calf operations in northern California was enrolled in this study either through the network of University of California Cooperative Extension livestock advisors or as clients of the Veterinary Medical Teaching Hospital at the University of California, Davis.
Enrollment criteria included farms with a geographic location in northern California and primary production sector as beef cow-calf. No restrictions were placed on the type of operation , herd size, grazing practices, breed of beef cattle, planting racks or previous antimicrobial use. All experimental protocols regarding animal use were approved by the Institutional Animal Care and Use Committee at the University of California, Davis.Fecal samples were collected between June 2019 and August 2020 from cows and/or calves on each farm as a convenience sample, by either the herd veterinarian, extension veterinarian, or cooperative extension livestock advisor. Fecal samples were collected from a combination of calves aged between 1week to 1year and adult cows aged between 2 and 10 years either from the rectum or from freshly voided manure after the animal was observed to defecate. Number and life stage of animal samples was based on number of animals available for sampling and that could be observed defecating within an hour of observation at the time of farm visit with the goal of sampling 5 cows and 5 calves per farm. Individual animal identifier, age, life stage , and breed were recorded when available.Fecal samples were collected during a single visit to each farm. Individual disposable gloves were used for collection of each sample and samples were stored in individual 15 mL polypropylene sample tubes. Rectal samples were collected on 8 farms from the recto-anal junction with individual rectal palpation sleeves. Pasture samples were collected as fresh feces via gloved hand from the top and center of a freshly voided fecal pat, where the individual animal was observed to defecate. Samples were transported on ice to the laboratory at the University of California, Davis where they were kept refrigerated at 4°C if culture could be performed within 48 h or stored at −80°C in tryptic soy broth with 25% glycerol.At time of fecal sample collection, an in-person survey regarding management and production practices was conducted. Information was collected on herd size, breed, certification status , whether any farm personnel were Beef Quality Assurance certified , type of pasture , whether the farm incorporated feeding of byproducts, water trough cleaning practices, existence of a current veterinarian-client-patient relationship, and whether the farm had submitted samples in the previous 12months to a diagnostic laboratory. The survey also included detailed questions regarding antibiotic practices on the farm, specifically use of antibiotics in feed, use of intramammary antibiotics, use of injectable antibiotics, practices related to injectable antibiotics including indication for treatment, method for determining treatment duration and dosage, information recorded regarding treatment, and specific antibiotic used in each method listed above. Treatment with antimicrobials in the past 6months before sample collection were recorded for all sampled animals based on ranch records, markings on treated animals, or rancher’s recollection of treatments.Selective growth media, E. coli Chromoselect Agar B and Rapid Enterococci Chromoselect Agar, following manufacturer guidelines , were used for culture and isolation of the respective bacterial types as previously described . Briefly, each fecal sample was streaked on the respective selective media using sterile cotton tipped applicators and incubated at 44°C or 35°C for 24h. Both E. coli and Enterococcus colonies were identified by characteristic blue green colony types on the Chromoselect plates. Two discrete colonies of each bacterial type were selected and purified on 5% sheep blood agar plates . The pure colonies were stored in tryptic soy broth with 25% glycerol at −80°C until all farm sampling was complete.After initial culture, a total of 698 bacterial isolates were stored. From these, 528 bacterial isolates were selected for antimicrobial susceptibility testing . Exclusion criteria included isolates from a farm where the rectal sleeve was accidentally not changed between samplings, all fecal samples which did not yield at least 2 identifiable isolates for each bacterial type after two culture attempts, and any samples with missing or unknown treatment information. Of the 528 selected, 482 were selected for antimicrobial susceptibility testing. See Figures 1A,B for flow charts of the isolate selection process for E. coli and Enterococcus isolates, respectively.Antimicrobial susceptibility testing was conducted by the broth microdilution method using Sensititre™ system against a panel of 19 antibiotics on a commercially available BOPO7F Vet Antimicrobial Susceptibility Testing Plate .Quality control steps included checking for bacterial growth and colony purity by plating 1μL of the inoculated MuellerHinton broth on TSA with 5% sheep blood.