Lining the small intestine with mycobacteriophages protects from Mycobacterium avium subsp. paratuberculosis and eliminates fecal shedding.

Johne's disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry.

Adherence and metal-ion acquisition gene expression increases during infection with Treponema phagedenis strains from bovine digital dermatitis.

Digital dermatitis (DD) is an ulcerative foot lesion on the heel bulbs of dairy cattle. DD is a polymicrobial disease with no precise etiology, although Treponema spirochetes are found disproportionally abundant in diseased tissue. Within Treponema, several different species are found in DD; however, the species Treponema phagedenis is uniformly found in copious quantities and deep within the skin layers of the active, ulcerative stages of disease. The pathogenic mechanisms these bacteria use to persist in the skin and the precise role they play in the pathology of DD are widely unknown. To explore the pathogenesis and virulence of Treponema phagedenis, newly isolated strains of this species were investigated in a subcutaneous murine abscess model. In the first trial, a dosage study was conducted to compare the pathogenicity of different strains across three different treponemes per inoculum (TPI) doses based on abscess volumes. In the second trial, the expression levels of 11 putative virulence genes were obtained to gain insight into their involvement in pathogenesis. During the RT-qPCR analysis, it was determined that genes encoding for two metal-ion import lipoproteins and two adherence genes were found highly upregulated during infection. Conversely, two genes involved in motility and chemotaxis were found to not be significantly upregulated or utilized during infection. These results were supported by gene expression data from natural M2 lesions of dairy cattle. This gene expression analysis could highlight the preference in strategy for T. phagedenis to persist and adhere in the host rather than engage in motility and disseminate.

Proinflammatory CD14highCD16low monocytes/macrophages prevail in Treponema phagedenis-associated bovine digital dermatitis.

Digital dermatitis (DD) is a skin disease in cattle characterized by painful inflammatory ulcerative lesions in the feet, mostly associated with local colonization by Treponema spp., including Treponema phagedenis. The reason why most DD lesions remain actively inflamed and progress to chronic conditions despite antibiotic treatment remains unknown. Herein, we show an abundant infiltration of proinflammatory (CD14highCD16low) monocytes/macrophages in active DD lesions, a skin response that was not mitigated by topical treatment with oxytetracycline. The associated bacterium, T. phagedenis, isolated from DD lesions in cattle, when injected subcutaneously into mice, induced abscesses with a local recruitment of Ly6G+ neutrophils and proinflammatory (Ly6ChighCCR2+) monocytes/macrophages, which appeared at infection onset (4 days post challenge) and persisted for at least 7 days post challenge. When exploring the ability of macrophages to regulate inflammation, we showed that bovine blood-derived macrophages challenged with live T. phagedenis or its structural components secreted IL-1ß via a mechanism dependent on the NLRP3 inflammasome. This study shows that proinflammatory characteristics of monocytes/macrophages and neutrophils dominate active non-healing ulcerative lesions in active DD, thus likely impeding wound healing after antibiotic treatment.

Invited Review: Improved control of Johne's disease in dairy cattle through advancements in diagnostics, testing and management of young stock.

Johne's disease (JD; paratuberculosis) control programs have been regionally implemented across the globe, but few have successfully eradicated the pathogen (Mycobacterium avium ssp. paratuberculosis (MAP)) causing this disease. The limited success may partly be attributed to excluding young stock (calves and replacement heifers or bulls) from testing strategies aimed at identifying MAP-infected cattle. Young stock can shed MAP in feces and can have detectable MAP-specific antibodies in blood, as confirmed in experimentally and naturally infected cattle. Furthermore, MAP transmission causes new infections in young stock. Calves and heifers are often included in JD management strategies on dairy farms but excluded from conventional diagnostic tests due to a presumed lag between infection and detection of MAP shedding and/or MAP-specific serum antibodies. We summarize evidence of MAP shedding early in the course of infection and discuss promising diagnostics, testing and management strategies to support inclusion of young stock in JD control programs. Improvements in fecal Polymerase Chain Reaction, interferon-gamma release assay (IGRA), and enzyme-linked immunosorbent assay (ELISA) enable earlier detection of MAP and specific early immune responses. Studies on IGRA and ELISA have focused on evaluation of new antigens and optimal age of testing. There are new diagnostics, including phage-based tests to detect viable MAP, and gene expression patterns and metabolomics to detect MAP-infected young stock. In addition, refinements in testing and management of calves and heifers may enable reductions in MAP prevalence. We provide recommendations for dairy farmers, researchers, veterinarians, and other stakeholders that may improve JD control programs with an objective to control and potentially eradicate JD. Additionally, we have identified the most pressing gaps in knowledge that currently hamper inclusion of young stock in JD prevention and control programs. In summary, transmission among young stock may cause new MAP infections, and appropriate use of new diagnostic tests, testing and management strategies for young stock may improve the efficacy of JD control programs.

Herd-level prevalence of bovine leukemia virus, Salmonella Dublin, and Neospora caninum in Alberta, Canada, dairy herds using ELISA on bulk tank milk samples.

Endemic infectious diseases remain a major challenge for dairy producers worldwide. For effective disease control programs, up-to-date prevalence estimates are of utmost importance. The objective of this study was to estimate the herd-level prevalence of bovine leukemia virus (BLV), Salmonella enterica ssp. enterica serovar Dublin (Salmonella Dublin), and Neospora caninum in dairy herds in Alberta, Canada, using a serial cross-sectional study design. Bulk tank milk samples from all Alberta dairy farms were collected 4 times, in December 2021 (n = 489), April 2022 (n = 487), July 2022 (n = 487), and October 2022 (n = 480), and tested for antibodies against BLV, Salmonella Dublin, and N. caninum using ELISA. Herd-level apparent prevalence was calculated as positive herds divided by total tested herds at each time point. A mixed-effect modified Poisson regression model was employed to assess the association of prevalence with region, herd size, herd type, and type of milking system. Apparent prevalence of BLV was 89.4%, 88.7%, 86.9%, and 86.9% in December, April, July, and October, respectively, whereas for Salmonella Dublin apparent prevalence was 11.2%, 6.6%, 8.6%, and 8.5%, and for N. caninum apparent prevalence was 18.2%, 7.4%, 7.8%, and 15.0%. For BLV, Salmonella Dublin, and N. caninum, a total of 91.7%, 15.6%, and 28.1% of herds, respectively, were positive at least once, whereas 82.5%, 3.6%, and 3.0% of herds were ELISA positive at all 4 times. Compared with the north region, central Alberta had a high prevalence (prevalence ratio [PR] = 1.13) of BLV antibody-positive herds, whereas south Alberta had a high prevalence (PR = 2.56) of herds positive for Salmonella Dublin antibodies. Furthermore, central (PR = 0.52) and south regions (PR = 0.46) had low prevalence of N. caninum-positive herds compared with the north. Hutterite colony herds were more frequently BLV positive (PR = 1.13) but less frequently N. caninum-positive (PR = 0.47). Large herds (>7,200 L/d milk delivered ∼>250 cows) were 1.1 times more often BLV positive, whereas small herds (≤3,600 L/d milk delivered ∼≤125 cows) were 3.2 times more often N. caninum positive. For Salmonella Dublin, Hutterite colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large strata but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) Salmonella Dublin-positive than smaller herds only in non-colony stratum but not in colony stratum. Moreover, N. caninum prevalence was 1.6 times higher on farms with conventional milking systems compared with farms with an automated milking system. These results provide up-to-date information of the prevalence of these infections that will inform investigations of within-herd prevalence of these infections and help in devising evidence-based disease control strategies.

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.

Effects of different culture media on growth of Treponema spp. isolated from digital dermatitis.

Digital dermatitis (DD) lesions in cattle are characterized by the presence of multiple Treponema species. Current culture media for isolating treponemes generally uses serum supplementation from different animals to target particular Treponema sp.; however, their suitability for DD Treponema isolation has not been fully determined. We studied the effect of culture media (OTEB, NOS and TYGV) and serum supplementation on mixed Treponema spp. dynamics. Bacterial growth was evaluated by direct microscopic count, optical density, wet weight and a species-specific qPCR and the correlations between these independent methods were calculated. Wet weight, optical density and bacterial count correlated best with each other. Different Treponema species performed differently under the tested culture media. T. phagedenis growth was enhanced in OTEB media supplemented with bovine fetal serum (BFS) or horse serum (HS). T. medium had lower generation time when culture media were supplemented with rabbit serum (RS). Lowest generation time for T. pedis and T. denticola were obtained in NOS media supplemented with HS and OTEB media supplemented with BFS, respectively. Detection of cystic forms observed after 5 days of culture did not differ among the culture media. Correlation between different Treponema spp. growth quantification techniques indicated that alternative quantification methods such as qPCR and wet weight could be used depending on the purpose. We conclude that effects of culture media and serum supplementation on mixed Treponema spp. communities should be taken into account when isolating a specific Treponema species.

Oxytetracycline reduces inflammation and treponeme burden whereas vitamin D3 promotes ß-defensin expression in bovine infectious digital dermatitis.

Digital dermatitis (DD), a common ulcerative disease of the bovine foot causing lameness and reducing productivity and animal welfare, is associated with infection by spirochete Treponema bacteria. Topical tetracycline, the most common treatment, has inconsistent cure rates; therefore, new therapeutic options are needed. We compared effects of topical oxytetracycline and vitamin D3 on innate immunity in DD-affected skin. Cows with active DD lesions were treated topically with oxytetracycline or vitamin D3 and skin biopsies were collected from lesions. Tissue samples were examined histologically, transcriptional expression of pro-inflammatory cytokines, Toll-like receptors (TLRs), and host defense peptides assessed, and the presence of specific treponeme species determined. Effects of treatments at a mechanistic level were studied in a human keratinocyte model of treponeme infection. Oxytetracycline promoted hyperplastic scab formation in ulcerated DD lesions and decreased transcriptional expression of Cxcl-8 (neutrophil chemoattractant). Oxytetracycline also reduced numbers of Treponema phagedenis and T. pedis and enhanced Tlr2 mRNA expression. Vitamin D3 did not modify expression of cytokines or Tlrs, or bacterial loads, but enhanced transcription of tracheal antimicrobial peptide (Tap), a key bovine ß-defensin. Combing oxytetracycline and vitamin D3 provides complementary clinical benefits in controlling DD through a combination of antimicrobial, immunomodulatory, and pro-healing activities.

Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1ß cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.

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.

Quantifying transmission of Mycobacterium avium subsp. paratuberculosis among group-housed dairy calves.

Johne's disease (JD) is a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), with control primarily aimed at preventing new infections among calves. The aim of the current study was to quantify calf-to-calf transmission of MAP among penmates in an experimental trial. Newborn Holstein bull calves (n = 32) were allocated into pens of 4, with 2 inoculated (IN) calves and 2 calves that were contact exposed (CE). Calves were group-housed for 3 months, with frequent collection of fecal and blood samples and tissue collection after euthanasia. The basic reproduction ratio (R0) was estimated using a final size (FS) model with a susceptible-infected model, based on INF-γ ELISA and tissue culture followed by qPCR. In addition, the transmission rate parameter (ß) for new shedding events was estimated using a general linearized method (GLM) model with a susceptible-infected-susceptible model based on culture, followed by qPCR, of fecal samples collected during group housing. The R0 was derived for IN and CE calves separately, due to a difference in susceptibility, as well as differences in duration of shedding events. Based on the FS model, interferon-γ results from blood samples resulted in a R 0 IG of 0.90 (0.24, 2.59) and tissue culture resulted in a R 0 T of 1.36 (0.45, 3.94). Based on the GLM model, the R0 for CE calves to begin shedding (R 0 CE ) was 3.24 (1.14, 7.41). We concluded that transmission of MAP infection between penmates occurred and that transmission among calves may be an important cause of persistent MAP infection on dairy farms that is currently uncontrolled for in current JD control programs.

Detecting total immunoglobulins in diverse animal species with a novel split enzymatic assay.

BACKGROUND: Total immunolobulin G concentration is a useful, albeit underutilized, diagnostic parameter for health assessments of non-domestic animal species, due to a lack of functional diagnostic tools. Traditional assays, including enzyme-linked immunosorbent assay or radial immunodiffusion, require development of specific reagents (e.g., polyclonal antisera and appropriate protocols) for each animal species, precluding wide and easy adoption in wildlife welfare. As an alternative, bacterial virulence factors able to bind IgGs in antigen-independent manner can be used. To further simplify the diagnostic procedure and increase the number of species recognized by an assay, in this study a recently developed Split Trehalase immunoglobulin assay (STIGA) with bIBPs as a sensing elements was used to detect antibodies in 29 species from 9 orders. Three bacterial immunoglobulin binding proteins (protein G, protein A and protein L) were incorporated into STIGA reagents to increase the number of species recognized. RESULTS: IgG concentrations were detected through glucose production and produced signals were categorized in 4 categories, from not active to strong signal. Activation was detected in almost all tested animal species, apart from birds. Incorporation of Protein G, Protein A and Protein L allowed detection of IgGs in 62, 15.5 and 6.9% of species with a strong signal, respectively. Assays combining 2 bacterial immunoglobulin binding proteins as sensing element generally gave poorer performance than assays with the same bacterial immunoglobulin binding proteins fused to both trehalase fragments. CONCLUSIONS: STIGA assays have potential to be further developed into an easily adoptable diagnostic test for total amount of IgGs in almost any serum sample, independent of species.

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.

Split trehalase as a versatile reporter for a wide range of biological analytes.

In health care, biosensors are envisioned as universal diagnostic devices with AAAA characteristics (i.e., available for anything, anywhere, anytime, to anyone). Despite numerous attempts to develop such a diagnostic device, none have managed to fulfill all four criteria and be commercialized. Glucometers, the most successful class of biosensor currently marketed monitor blood glucose concentrations. Their performance in clinical samples, including sensitivity and specificity, has been optimized and they are small and relatively inexpensive. We aimed to develop a technology that uses this existing biosensor, but adds versatility in detection of a wide range of analytes. Herein, we report the periplasmic trehalase of E. coli as a novel split enzyme reporter capable of converting a wide variety of analytes into glucose. Conditional complementation of trehalase fragments induced by detection of analytes, resulting in trehalose hydrolysis and glucose production, was used to detect antibodies and bacterial cells. We also demonstrated retention of split TreA activity in undiluted clinical samples. In conclusion, a trehalase-based biosensor platform offers a versatile and convenient method for point-of-care applications as it does not require sample preparation or handling and can be integrated with existing glucometers or sensors.

Associations between digital dermatitis lesion grades in dairy cattle and the quantities of four Treponema species.

Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity.

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.

Quantifying fecal shedding of Mycobacterium avium ssp. paratuberculosis from calves after experimental infection and exposure.

Johne's disease, a chronic enteritis caused by Mycobacterium avium ssp. paratuberculosis (MAP), causes large economic losses to the dairy industry worldwide. Fecal shedding of MAP contaminates the environment, feed, and water and contributes to new infections on farm, yet there is limited knowledge regarding mechanisms of shedding, extent of intermittent shedding, and numbers of MAP bacteria shed. The objectives were to (1) compare (in an experimental setting) the frequency at which intermittent shedding occurred and the quantity of MAP shed among pen mates that were inoculated or contact-exposed (CE); and (2) determine whether an association existed between inoculation dose and quantity of MAP shed. In the first experiment, 32 newborn Holstein-Friesian bull calves were allocated to pens in groups of 4, whereby 2 calves were inoculated with a moderate dose (MD; 5 × 108 cfu) of MAP and 2 calves acted as CE. Calves were group-housed for 3 mo, fecal samples were collected and cultured, and culture-positive samples were quantified. In the second experiment, 6 calves were inoculated with either a low (LD) or high (HD) dose of MAP (1 × 108 or 1 × 1010 cfu, respectively), and fecal samples were collected for 3 mo and cultured for detection of MAP. The amount of MAP was quantified using direct extraction (DE) of DNA from fecal samples and F57-specific quantitative PCR. In experiment 1, the average amount of MAP in all culture-positive samples did not differ between MD and CE calves. In experiment 2, when comparing inoculation doses, LD calves had the lowest proportion of MAP-positive culture samples and HD had the highest, but no difference was detected in the average quantity of MAP shed. This study provided new information in regards to Johne's disease research and control regarding shedding from various inoculation doses and from CE animals; these data should inform future trials and control programs.

Udder health in Canadian dairy heifers during early lactation.

Mastitis is the most prevalent and costly disease in dairy cattle worldwide, with implications for animal health and welfare as well as production and economics. Nonlactating heifers are an often-neglected group of animals concerning mastitis management, as they are assumed to be free of mastitis. An observational field study was conducted between 2007 and 2008 on 91 dairy herds across Canada, representative of provincial averages of bulk milk somatic cell count (BMSCC) and barn type. The aims of that study were to (1) estimate in early-lactating heifers overall and pathogen-specific incidence rate of clinical mastitis (IRCM), prevalence of intramammary infection (IMI), and prevalence of subclinical mastitis (SCM; defined as SCC ≥200,000 cells/mL); (2) compare these udder health parameters between heifers and multiparous cows; and (3) determine regional patterns and variations in these udder health parameters across BMSCC categories. During the first day of lactation, IRCM was higher in heifers than in multiparous cows (99 vs. 48 cases per 10,000 quarter-days at risk, respectively). Clinical mastitis affected 4% of heifers (0.73 cases per 100 quarters) in the first 30 d after calving, with the most common pathogens isolated being Staphylococcus aureus and Escherichia coli, whereas S. aureus and non-aureus staphylococci were the most commonly isolated pathogens in multiparous cows. The IRCM in heifers was highest in Ontario heifers, but overall IRCM did not vary by BMSCC category and it was only higher in multiparous cows than heifers in high-BMSCC Ontario herds. Intramammary infections were present in 33% of heifer quarters, with non-aureus staphylococci the most commonly isolated group of bacteria in both heifers (26% of quarters) and multiparous cows (18% of quarters). Pathogen-specific prevalence of IMI did not differ between heifers and multiparous cows, but we noted regional differences and differences across BMSCC categories in pathogen-specific prevalence of IMI. Prevalence of SCM in heifers was 13.6% and was lowest in Alberta herds. In all regions, SCM prevalence was higher in multiparous cows than in heifers. In conclusion, udder health of Canadian dairy heifers was similar to that of other countries, demonstrating the importance of the issue. Differences between heifers and multiparous cows early in lactation highlighted the need for management practices to target the precalving period in heifers, when exposure to risk factors differs from that in lactating cows.

Composition of the teat canal and intramammary microbiota of dairy cows subjected to antimicrobial dry cow therapy and internal teat sealant.

Antimicrobial dry cow therapy (DCT) is an important component of mastitis control programs aimed to eliminate existing intramammary infections and prevent the development of new ones during the dry period. However, to what extent the microbiota profiles of different niches of the udder change during the dry period and following administration of DCT remains poorly understood. Therefore, the main objective of the present study was to qualitatively evaluate dynamics of the microbiota of teat canal (TC) and mammary secretions (i.e., milk and colostrum) of healthy udder quarters subjected to DCT using a long-acting antimicrobial product, containing penicillin G and novobiocin, in combination with internal teat sealant. To this end, TC swabs (n = 58) and their corresponding milk (n = 29) and colostrum samples (n = 29) were collected at the time of drying off and immediately after calving from clinically healthy udder quarters of Holstein dairy cows from a commercial dairy farm. All samples were subjected to DNA extraction and high-throughput sequencing of the V1-V2 hypervariable regions of bacterial 16S rRNA genes. Overall, shifts were more pronounced within the microbiota of mammary secretions than the TC. In particular, microbiota of colostrum samples collected immediately after calving were less species-rich compared with the pre-DCT milk samples. Proportions of several bacterial genera belonging to the phylum Proteobacteria, including Pseudomonas, Stenotrophomonas, and unclassified Alcaligenaceae, were enriched within the microbiota of colostrum samples, whereas Firmicutes genera, including Butyrivibrio, unclassified Clostridiaceae, and unclassified Bacillales, were overrepresented in pre-DCT milk microbiota. Apart from shifts in the proportion of main bacterial genera and phyla, qualitative analysis revealed a high degree of commonality between pre-DCT and postpartum microbiota of both niches of the udder. Most importantly, a considerable number of bacterial genera and species commonly regarded as mastitis pathogens or opportunists (or both), including Staphylococcus spp., unclassified Enterobacteriaceae, and Corynebacterium spp., were shared between pre-DCT and postpartum microbiota of mammary secretions. Percentage of shared bacterial genera and species was even higher between pre-DCT and postpartum microbiota of TC samples, suggesting that the DCT approach of the present study had limited success in eliminating a considerable proportion of bacteria during the dry period.

Environmental sample characteristics and herd size associated with decreased herd-level prevalence of Mycobacterium avium ssp. paratuberculosis.

Environmental sampling is an effective method for estimating regional dairy herd-level prevalence of infection with Mycobacterium avium ssp. paratuberculosis (MAP). However, factors affecting prevalence estimates based on environmental samples are not known. The objective was to determine whether odds of environmental samples collected on farm changed culture status over 2 sampling times and if changes were specific for location and type of housing (freestall, tiestall, or loose housing), the sample collected (i.e., manure of lactating, dry, or sick cows; namely, cow group), and effects of herd size. In 2012-2013 [sampling 1 (S1)] and 2015-2017 [sampling 2 (S2)], 6 environmental samples were collected and cultured for MAP from all 167 (99%) and 160 (95%) farms, respectively, in the province of Saskatchewan, Canada. Only the 148 dairy farms sampled at both sampling periods were included in the analysis. A mixed effects logistic regression was used to determine whether differences between sampling periods were associated with herd size and sample characteristics (cow group contributing to environmental sample, type of housing, and location). In S1 and S2, 55 and 34%, respectively, of farms had at least 1 MAP-positive environmental sample. Correcting for sensitivity of environmental sampling, estimated true prevalence in S1 and S2 was 79 and 48%, respectively. Herds with >200 cows were more often MAP-positive than herds with <51 cows in both S1 and S2. The percentage of positive samples was lower in S2 compared with S1 for all sampled areas, cow groups contributing to samples, types of housing where samples were collected, and herd size categories. However, samples collected from dry cow areas had the largest decrease in MAP-positive samples in S2 compared with all other cow group samples. Herds that were MAP-negative in S1 with a herd size 51 to 100 or 101 to 150 were more likely to stay MAP-negative, whereas MAP-positive herds with >200 cows more frequently stayed MAP-positive. No difference was observed in the odds of a sample being MAP-positive among housing types or location of sample collection in both sample periods. Of all farms sampled, 104 (70%) did not change status from S1 to S2. In conclusion, when herd-level MAP prevalence decreased over the 3-yr interval, the change in prevalence differed among herd size categories and was larger in samples from dry cow areas. It was, however, not specific to other characteristics of environmental samples collected.