Impact of carbon dioxide concentrations on laboratory sensitivity of Mycoplasma species isolated from dairy cows.

Conventional Mycoplasma spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of Mycoplasma bovis. Despite a wide range of carbon dioxide (CO2) supplementation reported in literature, specific impacts of CO2 on Mycoplasma spp. growth remain unexplored. Our objective was to assess the effect of CO2 concentration on growth detection rates of 24 Mycoplasma spp. isolates from dairy cows. These isolates, mainly M. bovis, were incubated at 37°C in triplicate and three dilution ranges under three CO2 conditions: ambient air or 5% CO2 or 10% CO2. Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log10 cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO2, with less variation observed in ambient air compared with 5% or 10% CO2. However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO2, or 10% CO2. Consequently, Mycoplasma spp. isolated from dairy cattle demonstrated growth after the recommended 7-10 days of culture, even in the absence of supplemental CO2. Given the expected concentration of M. bovis in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being M. bovis, we recommend expanding CO2 concentration ranges in M. bovis culture from 10% CO2 to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO2. IMPORTANCE: Current Mycoplasma spp. culture protocols lack empirical derivation concerning carbon dioxide (CO2) supplementation and are primarily based on the initial cultivation of Mycoplasma bovis. This study indicates that the suitable range for CO2 supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7-10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO2, or 10% CO2 supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing Mycoplasma spp. under ambient air conditions in a laboratory setting.

Dairy farmers' considerations for antimicrobial treatment of clinical mastitis in British Columbia and Alberta, Canada.

Introduction: Clinical mastitis (CM) treatment decision-making is a multifaceted process that remains relatively understudied, despite CM being one of the most prevalent diseases on dairy farms worldwide, contributing greatly to the use of antimicrobials in the dairy industry. This study aimed to gain insights into decision-making mechanisms employed by dairy farmers in British Columbia and Alberta, Canada, when dealing with CM. Methods: Interviews were held with 15 dairy farmers in the Canadian provinces of British Columbia and Alberta and analyzed using reflexive thematic analysis to develop both the decision-pathway and overarching themes influencing the CM decisions by farmers in this region. Results and discussion: The analysis generated a decision-making process that begins with identification and classification of CM, guided by visual characteristics of milk and the udder, available milk production and quality data, presence of systemic signs, and additional diagnostics. Subsequently, CM cases are assessed based on the likelihood of cure, value of the cow, and herd goals to decide whether antimicrobial treatment is desired. Next, a treatment choice is made by evaluating severity and urgency of the case, availability of drugs and timing of the case. Finally, definition of treatment success and progression over time following the treatment decision guides the termination of treatment. Three overarching themes were generated that shape the decision-making process: 'Personal attributes', including personal approach and experiential knowledge; 'Inter-actor dynamics', such as shared decision-making and dynamics among producers, veterinarians, and milkers; and 'Moving beyond protocols', which highlights the dynamic nature of mastitis decision-making. These insights have the potential to inform the development of effective interventions to improve CM antimicrobial use that align with the reality of farming operations within Western Canada, and potentially beyond.

Integrated Analysis of Transcriptome Profiles and lncRNA-miRNA-mRNA Competing Endogenous RNA Regulatory Network to Identify Biological Functional Effects of Genes and Pathways Associated with Johne's Disease in Dairy Cattle.

Paratuberculosis or Johne's disease (JD), a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes huge economic losses and reduces animal welfare in dairy cattle herds worldwide. At present, molecular mechanisms and biological functions involved in immune responses to MAP infection of dairy cattle are not clearly understood. Our purpose was to integrate transcriptomic profiles and competing endogenous RNA (ceRNA) network analyses to identify key messenger RNAs (mRNAs) and regulatory RNAs involved in molecular regulation of peripheral blood mononuclear cells (PBMCs) for MAP infection in dairy cattle. In total, 28 lncRNAs, 42 miRNAs, and 370 mRNAs were identified by integrating gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In this regard, we identified 21 hub genes (CCL20, CCL5, CD40, CSF2, CXCL8, EIF2AK2, FOS, IL10, IL17A, IL1A, IL1B, IRF1, MX2, NFKB1, NFKBIA, PTGS2, SOCS3, TLR4, TNF, TNFAIP3, and VCAM1) involved in MAP infection. Furthermore, eight candidate subnets with eight lncRNAs, 29 miRNAs, and 237 mRNAs were detected through clustering analyses, whereas GO enrichment analysis of identified RNAs revealed 510, 22, and 11 significantly enriched GO terms related to MAP infection in biological process, molecular function, and cellular component categories, respectively. The main metabolic-signaling pathways related to MAP infection that were enriched included the immune system process, defense response, response to cytokine, leukocyte migration, regulation of T cell activation, defense response to bacterium, NOD-like receptor, B cell receptor, TNF, NF-kappa B, IL-17, and T cell receptor signaling pathways. Contributions of transcriptome profiles from MAP-positive and MAP-negative sample groups plus a ceRNA regulatory network underlying phenotypic differences in the intensity of pathogenicity of JD provided novel insights into molecular mechanisms associated with immune system responses to MAP infection in dairy cattle.

Rapid and reliable detection of Leishmania antibodies in canine serum with double-antigen sandwich homogeneous chemical luminescence.

BACKGROUND: Leishmaniasis, caused by Leishmania spp. parasites, is an important zoonotic disease globally, posing severe threats to humans and animals. In the absence of effective vaccines, reliable serological diagnostic methods are critical for disease control. However, the enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay have limitations due to complexity, time required and/or sensitivity. Therefore, our objective was to develop an accurate, rapid and user-friendly detection method of canine leishmania antibody based on double-antigen sandwich homogeneous chemical luminescence. METHODS: Homogeneous chemiluminescent technology was employed, and expressed recombinant fusion proteins containing full-length K9, K39 and K26 repeat sequences were used as diagnostic antigens. To establish a dual-antigen sandwich serological assay capable of detecting various antibody types, a factorial design was used to optimize concentrations of diagnostic antigen-receptor microspheres and of biotinylated diagnostic antigens, as well as of reaction solution composition and reaction duration. To evaluate and validate this newly developed method, we collected 41 Leishmania-positive serum samples, 30 Leishmania-negative control serum samples and 78 clinical serum samples for which no diagnostic information was available. Comparative analyses were performed using parasitological testing and an indirect ELISA as reference methods, focusing on diagnostic sensitivity and specificity. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the purification of the diagnostic antigens, which exhibited clear bands without impurities. Based on results from the 41 Leishmania-positive samples and 30 Leishmania-negative samples, there was sufficient sensitivity to detect samples diluted up to 256-fold, with analytical specificity of 100%. Overall diagnostic sensitivity was 100% and diagnostic specificity was 93.3%. Diagnostic performance was highly consistent between the newly developed method and the indirect ELISA (Kappa = 0.82, P < 0.01). Testing could be completed within 35 min with the new method CONCLUSIONS: We have developed a novel double-antigen sandwich homogeneous chemical luminescence method to detect canine Leishmania antibodies, with high sensitively and specificity, a short incubation interval and a simple protocol. This streamlined approach not only offers a sensitive and efficient method for clinical diagnosis but also has great potential for use in automated testing.

Escherichia coli infection induces ferroptosis in bovine mammary epithelial cells by activating the Wnt/ß-catenin pathway-mediated mitophagy.

Iron overload causes mitochondrial damage, and then activates mitophagy, which may directly trigger and amplify ferroptosis. Our objective was to investigate whether Escherichia coli (E. coli) isolated from clinical bovine mastitis induces ferroptosis in bovine mammary epithelial cells (bMECs) and if so, the underlying regulatory mechanism. E. coli infection caused mitochondrial damage, mitophagy, and ferroptosis. Rapamycin and chloroquine increased and suppressed ferroptosis, respectively, in E. coli-treated bMECs. Moreover, E. coli infection activated the Wnt/ß-catenin pathway, but foscenvivint alleviated it. In conclusion, E. coli infection induced ferroptosis through activation of the Wnt/ß-catenin pathway-promoted mitophagy, and it also suppressed GPX4 expression.

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 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, S. Dublin, and N. caninum using ELISAs. Herd-level apparent prevalence was calculated as positive samples divided by total tested samples 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 S. 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, S. 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 S. 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/day milk delivered ∼ > 250 cows) were 1.1 times more often BLV-positive, whereas small herds (≤3,600 L/day milk delivered ∼ ≤ 125 cows) were 3.2 times more often N. caninum-positive. For S. Dublin, Hutterite-colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large stratum but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) S. 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.

Epidemiology, linkage to care and natural history of women of childbearing age with chronic hepatitis B: A population-based study.

Pregnant women with chronic hepatitis B (CHB) are a priority population for hepatitis B care. Identification of HBV status prior to pregnancy would facilitate timely maternal interventions and perinatal care. In our study, we aimed to study the epidemiology of CHB among women of childbearing age (WoCBA, 18-49 years) in Alberta, Canada. We retrospectively analysed Alberta Analytics databases to study CHB epidemiology, natural history and care linkage among WoCBA in Alberta, between April 2012 and March 2021. A Poisson regression was conducted to estimate incidence of newly identified CHB cases and prevalence trends, whereas predictors of care linkage were determined using logistic regression. Age/sex-adjusted incidence of newly identified CHB among WoCBA between 2015 and 2020 was 36.2/100,000 person/years, highest among individuals aged 30-39 years. Incidence of newly identified CHB decreased from 52.6 to 18.2/100,000 between 2015 and 2020, but prevalence increased from 131.7 to 248.6/100,000 in the same period. Newly identified CHB incident cases (n = 2124) had lower survival rates than age/sex-matched Canadians, with a standardized mortality ratio of 5.7 (95% CI 2.6-11.0). Increasing age (years) at diagnosis (HR, 1.2; 95% CI 1.1-1.3) was independently associated with mortality. Comorbid hepatocellular carcinoma, anti-HBV treatment and year of diagnosis were not significantly associated with mortality. Of the 1927 women with 2436 hepatitis B surface antigen-positive pregnancies from 2012 to 2020, only 27.6% had recommended HBV assessment during pregnancy. Of those women meeting criteria for antiviral therapy to prevent mother-to-child transmission (MTCT), only 66.4% received treatment. Suboptimal management during pregnancy and overall lower survival rates highlight the need to address care linkage barriers in women of childbearing age living with CHB.

Biological and genomic characterization of 4 novel bacteriophages isolated from sewage or the environment using non-aureus Staphylococci strains.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of "phage tail fiber." In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

Global losses due to dairy cattle diseases: A comorbidity-adjusted economic analysis.

An economic simulation was carried out over 183 milk-producing countries to estimate the global economic impacts of 12 dairy cattle diseases and health conditions: mastitis (subclinical and clinical), lameness, paratuberculosis (Johne's disease), displaced abomasum, dystocia, metritis, milk fever, ovarian cysts, retained placenta, and ketosis (subclinical and clinical). Estimates of disease impacts on milk yield, fertility, and culling were collected from the literature, standardized, meta-analyzed using a variety of methods ranging from simple averaging to random-effects models, and adjusted for comorbidities to prevent overestimation. These comorbidity-adjusted disease impacts were then combined with a set of country-level estimates for lactational incidence or prevalence or both, herd characteristics, and price estimates within a series of Monte Carlo simulations that estimated and valued the economic losses due to these diseases. It was estimated that total annual global losses are US$65 billion (B). Subclinical ketosis, clinical mastitis, and subclinical mastitis were the costliest diseases modeled, resulting in mean annual global losses of approximately US$18B, US$13B, and US$9B, respectively. Estimated global annual losses due to clinical ketosis, displaced abomasum, dystocia, lameness, metritis, milk fever, ovarian cysts, paratuberculosis, and retained placenta were estimated to be US$0.2B, US$0.6B, US$0.6B, US$6B, US$5B, US$0.6B, US$4B, US$4B, and US$3B, respectively. Without adjustment for comorbidities, when statistical associations between diseases were disregarded, mean aggregate global losses would have been overestimated by 45%. Although annual losses were greatest in India (US$12B), the United States (US$8B), and China (US$5B), depending on the measure of losses used (losses as a percentage of gross domestic product, losses per capita, losses as a percentage of gross milk revenue), the relative economic burden of these dairy cattle diseases across countries varied markedly.

Partial herd hoof trimming results in a higher economic net benefit than whole herd hoof trimming in dairy herds.

Sole ulcers, a common cause of lameness is the costliest non-infectious foot lesion in dairy cows and one of the most prevalent non-infectious foot lesions in freestall housing systems. Costs associated with sole ulcers are treatment costs, plus increased labor and decreased productivity and fertility. Routine hoof trimming is part of a strategy to manage sole ulcers. However, hoof trimming strategies differ among farms. The two most frequently applied strategies are: 1) partial herd hoof trimming with a 2-month interval between trims; and 2) whole herd hoof trimming at 6-month intervals. A Markov model was developed to investigate whether every 2 months partial herd hoof trimming or whole herd hoof trimming every 6 months was the most cost-effective strategy to avoid costs associated with sole ulcers. In this model, the net benefits for a 100-cow herd and the average productive life span of a dairy cow in intensive dairy systems of 3 years were evaluated. Partial herd hoof trimming was the most cost-effective strategy 100% of the time compared to whole herd hoof trimming, with a difference in 3-year net benefits of US$4,337 (95% CI: US$2,713-US$5,830). Based on sensitivity analyses, variables that were the sources of the biggest uncertainty in the model were herd size, the probability of being trimmed in a partial herd trim, and the prevalence of sole ulcers. To further investigate the impacts of herd size and of probability of being trimmed, various scenario analyses were conducted. With increasing herd size, the difference in net benefits in favor of partial herd hoof trimming increased even more. Scenario analyses about the probability of getting trimmed all indicated that targeted intervention increased the difference in net benefits in favor of partial herd hoof trimming. However, if the selection of cows to be trimmed in a partial herd trim was random, the whole herd hoof trimming strategy became cost-effective. Therefore, targeted selection and early intervention are necessary to decrease costs associated with sole ulcers.

Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Patient compliance with the implementation of a decolonization strategy for Staphylococcus aureus in hip and knee replacements.

BACKGROUND: A preoperative, in-community antimicrobial decolonization protocol combining chlorohexidine gluconate (CHG) sponges and mupirocin ointment to reduce surgical site infections amongst hip and knee replacement patients has been adopted in Alberta, Canada. Patient compliance with the protocol is essential for effectiveness. It is, therefore, important to understand patterns, and reasons why, patients do, and do not, comply. METHODS: A descriptive survey of patients having elective total hip or knee replacement at seven clinics in Alberta was conducted to determine patient compliance and reasons for noncompliance. Descriptive statistics and multivariate logistic regression were computed. RESULTS: Patient compliance was assessed in 3,427 patients. There were no differences in compliance based on the baseline protocols and enhanced protocols, but there was a difference based on clinic location. The odds of compliance with three CHG sponges were 4.47 times higher in rural versus urban clinics (P < .001). The most common reason for noncompliance for patients instructed to use 3 CHG sponges was "patient forgot". CONCLUSIONS: Compliance did not change when enhanced protocols were introduced; however, compliance differed by clinic location. Reasons for noncompliance included "sponges not provided", "patient forgot", and "surgery date moved". Results may inform clinics on areas where improvements could be made to increase patient compliance.

Adoption and decision factors regarding selective treatment of clinical mastitis on Canadian dairy farms.

As clinical mastitis (CM) treatments are responsible for a large portion of antimicrobial use on dairy farms, many selective CM treatment protocols have been developed and evaluated against a blanket treatment approach of CM cases. Selective treatment protocols use outcomes of diagnostic tests to exclude CM cases from antimicrobial treatment when they are unlikely to benefit. To tailor interventions to increase uptake of selective treatment strategies, a comprehension of current on-farm treatment practices and factors affecting treatment decisions is vital. Two questionnaires were conducted among 142 farms across 5 provinces participating in the Canadian Dairy Network for Antimicrobial Stewardship and Resistance in this cross-sectional study. Self-reported adoption of selective CM treatments by dairy farmers was 64%, with median of 82% of cows treated in those herds using selective treatment. Using logistic regression models, the odds to implement a selective CM treatment protocol increased with a decreasing average cow somatic cell count. No other associations were identified between use of a selective CM treatment protocol and farm characteristics (herd size, CM incidence, province, milking system, and housing system). Three subsets of farmers making cow-level CM treatment decisions were identified using a cluster analysis approach: those who based decisions almost exclusively on severity of clinical signs, those who used various udder health indicators, and farmers who also incorporated more general cow information such as production, age, and genetics. When somatic cell count was considered, the median threshold used for treating was >300,000 cells/mL at the last Dairy Herd Improvement test. Various thresholds were present among those considering CM case history. Veterinary laboratories were most frequently used for bacteriological testing. Test results were used to start, change, and stop treatments. Regardless of protocol, reasons for antimicrobial treatment withheld included cow being on a cull list, having a chronic intramammary infection, or being at end of lactation (i.e., close to dry off). If clinical signs persisted after treatment, farmers indicated that they would ask veterinarians for advice, stop treatment, or continue with the same or different antibiotics. Results of this study can be used to design interventions targeting judicious mastitis-related antimicrobial use, and aid discussions between veterinarians and dairy producers regarding CM-related antimicrobial use.

Within-herd transmission of Mycoplasma bovis infections after initial detection in dairy cows.

Mycoplasma bovis outbreaks in cattle, including pathogen spread between age groups, are not well understood. Our objective was to estimate within-herd transmission across adult dairy cows, youngstock, and calves. Results from 3 tests (PCR, ELISA, and culture) per cow and 2 tests (PCR and ELISA) per youngstock and calf were used in an age-stratified susceptible-infected-removed/recovered (SIR) model to estimate within-herd transmission parameters, pathways, and potential effects of farm management practices. A cohort of adult cows, youngstock, and calves on 20 Dutch dairy farms with a clinical outbreak of M. bovis in adult cows were sampled, with collection of blood, conjunctival fluid, and milk from cows, and blood and conjunctival fluid from calves and youngstock, 5 times over a time span of 12 wk. Any individual with at least one positive laboratory test was considered M. bovis-positive. Transmission dynamics were modeled using an age-stratified SIR model featuring 3 age strata. Associations with farm management practices were explored using Fisher's exact tests and Poisson regression. Estimated transmission parameters were highly variable among herds and cattle age groups. Notably, transmission from cows to cows, youngstock, or to calves was associated with R-values ranging from 1.0 to 80 secondarily infected cows per herd, 1.2 to 38 secondarily infected youngstock per herd, and 0.1 to 91 secondarily infected calves per herd, respectively. In case of transmission from youngstock to youngstock, calves or to cows, R-values were 0.7 to 96 secondarily infected youngstock per herd, 1.1 to 76 secondarily infected calves per herd, and 0.1 to 107 secondarily infected cows per herd. For transmission from calves to calves, youngstock or to cows, R-values were 0.5 to 60 secondarily infected calves per herd, 1.1 to 41 secondarily infected youngstock per herd, and 0.1 to 47 secondarily infected cows per herd. Among on-farm transmission pathways, cow-to-youngstock, cow-to-calf, and cow-to-cow were identified as most significant contributors, with calf-to-calf and calf-to-youngstock also having noteworthy roles. Youngstock-to-youngstock was also implicated, albeit to a lesser extent. Whereas the primary focus was a clinical outbreak of M. bovis among adult dairy cows, it was evident that transmission extended to calves and youngstock, contributing to overall spread. Factors influencing transmission and specific transmission pathways were associated with internal biosecurity (separate caretakers for various age groups, number of people involved), external biosecurity (contractors, external employees), as well as indirect transmission routes (number of feed and water stations).

Effects of bovine leukemia virus seropositivity and proviral load on milk, fat, and protein production of dairy cows.

The objective was to evaluate the effects of bovine leukemia virus (BLV) infection, as determined by BLV seropositivity and proviral load, on 305-d milk, fat, and protein production of dairy cows. A cross-sectional study was conducted among 1,712 cows from 9 dairy herds in Alberta, Canada. The BLV status was assessed using an antibody ELISA, whereas BLV proviral load in BLV-seropositive cattle was determined with quantitative PCR. Dairy Herd Improvement 305-d milk, fat, and protein production data were obtained for all enrolled cattle. Differences in these milk end points were assessed in 2 ways: first, by categorizing cows based on BLV serostatus (i.e., BLV positive or negative), and second, by categorizing based on BLV proviral load (i.e., BLV negative, low proviral load [LPL] BLV positive, and high proviral load [HPL] BLV positive). A mixed-effect multivariable linear regression model was used to assess differences in milk parameters. We found that BLV positivity, adjusted for parity and natural log-transformed somatic cell count (SCC), was not associated with reduction in 305-d milk, fat, or protein production. However, significant reductions in 305-d milk, fat, and protein yield occurred in HPL cows, but not in LPL cows, compared with BLV-negative cows, when adjusted for parity number and natural log-transformed SCC. In summary, BLV proviral load may predict effects of BLV infection on milk, fat, and protein production.

Frequency of isolation and phenotypic antimicrobial resistance of fecal Salmonella enterica recovered from dairy cattle in Canada.

Salmonellosis is one of the leading causes of gastrointestinal infections in humans. In Canada, it is estimated that approximately 87,500 cases of salmonellosis occur every year in humans, resulting in 17 deaths. In the United States, it is estimated that 26,500 hospitalizations and 420 deaths occur every year. In dairy cattle, infections caused by nontyphoidal Salmonella enterica can cause mild to severe disease, including enteritis, pneumonia, and septicemia. Our study objectives were to determine the proportion of fecal samples positive for Salmonella in dairy cattle in Canada and determine the resistance pattern of these isolates. We used data collected through the Canadian Dairy Network for Antimicrobial Stewardship and Resistance (CaDNetASR). Pooled fecal samples from preweaning calves, postweaning heifers, lactating cows, and manure storage were cultured for Salmonella, and the isolates were identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibilities were determined using the minimum inhibitory concentration test, and resistance interpretation was made according to the Clinical and Laboratory Standards Institute. A 2-level, multivariable logistic regression model was built to determine the probability of recovering Salmonella from a sample, accounting for province, year, and sample source. The proportion of farms with at least one positive sample were 12% (17/140), 19% (28/144), and 17% (24/144) for the sampling years 2019, 2020, and 2021, respectively. Out of the 113 Salmonella isolates, 23 different serovars were identified. The occurrence of Salmonella appeared to be clustered by farms and provinces. The most common serovars identified were Infantis (14%) and Typhimurium (14%). Overall, 21% (24/113) of the Salmonella isolates were resistant to at least one antimicrobial. Resistance to tetracycline was commonly observed (17%); however, very limited resistance to category I antimicrobials (categorization according to Health Canada that includes third-generation cephalosporins, fluoroquinolones, polymyxins, and carbapenems) was observed, with one isolate resistant to amoxicillin and clavulanic acid. The proportion of Salmonella isolates resistant to 2 and 3 antimicrobial classes was 3.5% and 8.8%, respectively. Our study provided valuable information on the proportion of fecal samples positive for Salmonella, the serovars identified, and the associated resistance patterns across CaDNetASR herds, at regional and national levels.

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.