Sirtuin 5 alleviates apoptosis and autophagy stimulated by ammonium chloride in bovine mammary epithelial cells.

Ammonia (NH3) is an irritating and harmful gas that affects cell apoptosis and autophagy. Sirtuin 5 (SIRT5) has multiple enzymatic activities and regulates NH3-induced autophagy in tumor cells. In order to determine whether SIRT5 regulates NH3-induced bovine mammary epithelial cell apoptosis and autophagy, cells with SIRT5 overexpression or knockdown were generated and in addition, bovine mammary epithelial cells were treated with SIRT5 inhibitors. The results showed that SIRT5 overexpression reduced the content of NH3 and glutamate in cells by inhibiting glutaminase activity in glutamine metabolism, and reduced the ratio of ADP/ATP. The results in the SIRT5 knockdown and inhibitor groups were comparable, including increased content of NH3 and glutamate in cells by activating glutaminase activity, and an elevated ratio of ADP/ATP. It was further confirmed that SIRT5 inhibited the apoptosis and autophagy of bovine mammary epithelial cells through reverse transcription-quantitative PCR, western blot, flow cytometry with Annexin V FITC/PI staining and transmission electron microscopy. In addition, it was also found that the addition of LY294002 or Rapamycin inhibited the PI3K/Akt or mTOR kinase signal, decreasing the apoptosis and autophagy activities of bovine mammary epithelial cells induced by SIRT5-inhibited NH3. In summary, the PI3K/Akt/mTOR signal involved in NH3-induced cell autophagy and apoptosis relies on the regulation of SIRT5. This study provides a new theory for the use of NH3 to regulate bovine mammary epithelial cell apoptosis and autophagy, and provides guidance for improving the health and production performance of dairy cows.

Prevalence and Molecular Characteristics of Enterococci Isolated from Clinical Bovine Mastitis Cases in Ningxia.

Purpose: This study aimed to investigate the prevalence and genetic characterization of enterococcal isolates (Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae) isolated from clinical bovine mastitis cases in Ningxia, China. Patients and Methods: The enterococci were identified by 16S rRNA amplification and sequencing. Antimicrobial resistance was determined by disc diffusion method. Virulence and antimicrobial resistance genes were detected by PCR assays. Results: Overall, 198 enterococcal isolates were identified from 2897 mastitis samples, including 137 (4.7%) E. faecalis, 50 (1.7%) E. faecium and 11 (0.4%) E. hirae. E. faecalis, E. faecium and E. hirae isolates showed high resistance to tetracycline (92.7%, 68.0%, 90.9%), followed by erythromycin (86.9%, 76.0%, 72.7%). The multidrug-resistant strains of E. faecalis and E. faecium were 29 (21.2%) and 13 (26.0%), respectively. The resistance of E. faecalis, E. faecium and E. hirae isolates to tetracycline is mainly attributed to the presence of tetL (alone or combined with tetM and/or tetK), the erythromycin resistance to ermB (alone or combined with ermC and/or ermA). Moreover, cpd (94.2%), gelE (77.4%), efaAfs (93.4%), and esp (79.6%) were the most common virulence genes in E. faecalis. In E. faecium, except for the gene efaAfs (82.0%), other virulence genes are rarely found. Only two strains of E. hirae carrying asa1 gene were detected. Conclusion: The results of this study can provide a reference for the prevention and treatment of bovine mastitis caused by enterococci.

Evaluation of bovine embryo quality based on gene expression profiling using whole-transcriptome amplification.

This study aimed to develop a method to evaluate the quality of bovine in vitro fertilized (IVF) embryos based on gene expression profiling via whole-transcriptome amplification. The expression of 11 developmentally important genes in individual bovine in vivo-derived (IVD) and IVF embryos were examined. Gene expression profiling was conducted by classifying the expression level of each gene in individual embryos as low, medium, or high. The IVF group had a higher (P < 0.01) proportion of embryos with low expression of SOX2, NANOG, and FGF4. In addition, a correlation analysis between the expression levels of each gene in individual embryos demonstrated that the relationship between gene expression differed with respect to IVD and IVF embryos. Our results suggest that the expression profiling of developmentally important genes using IVD embryos as normal controls could be a useful indicator for evaluating the quality of bovine IVF embryos.

Impact of an Injectable Trace Mineral Supplement on the Immune Response and Outcome of Mannheimia haemolytica Infection in Feedlot Cattle.

The study aimed to assess the impact of injectable trace mineral ("ITM"; Multimin90; Fort Collins, CO) supplementation on bacterial infection in cattle. Angus-crossbred steers (n = 32) were organized into two blocks by initial body weight. Steers were maintained on a ryelage and dry-rolled corn-based growing diet without supplementation of Zn, Cu, Mn, and Se for the duration of the study. The steers were transported 6 h, then randomized into three treatment groups: control received sterile saline ("CON"), ITM administered 1 day after transport (6 days before infection, "ITMPRE"), and ITM administered 2 days post infection (dpi) concurrent with antibiotic treatment ("ITMPOST"). Steers were infected with Mannheimia haemolytica on day 0, and all were treated with tulathromycin at 2 dpi. Plasma levels of Zn, Cu, and Se did not differ among treatments (P ≥ 0.74). Liver Se was higher in ITMPRE at 2 dpi (P < 0.05), and both ITM groups had higher liver Se at 5 dpi (P < 0.05) compared to CON. A time × treatment interaction was detected for liver Cu (P = 0.02). Clinical scores were lower (P < 0.05) in ITMPRE on 1 and 8 dpi and ITMPOST on 8 dpi compared to CON. Thoracic ultrasonography scores were lower in ITMPRE at 2 dpi compared to CON (P < 0.05) and ITMPOST (P < 0.1). No treatment effects (P > 0.10) were observed for bacterial detection from bronchoalveolar lavage (BAL) or nasopharyngeal swabs. At 5 dpi, both ITMPRE and ITMPOST showed higher frequencies of γδ T cells and NK cells in BAL compared to CON (P < 0.05). Before infection, leukocytes from ITMPRE steers produced more IL-6 (P < 0.01) in response to stimulation with the TLR agonist, Pam3CSK4. Use of ITM may be an effective strategy for improving disease resistance in feedlot cattle facing health challenges.

Associations of community knowledge, perceptions, and practices related to zoonotic disease with sociodemographic factors in and around Chiro Town, Eastern Ethiopia: a cross-sectional study.

BACKGROUND: Zoonoses are infectious diseases that are transmitted from animals to humans. Studying the knowledge, perceptions and practices of communities related to zoonoses and the associated risk factors is crucial for effective control and prevention. This study aimed to assess the knowledge, perceptions, and practices of respondents on zoonoses and the associated risk factors in and around Chiro town, Ethiopia. Zoonotic diseases, such as rabies, anthrax, bovine tuberculosis, and brucellosis, pose a direct threat to health and livelihoods in the communities where they occur. These diseases emerge due to a combination of human-animal interactions, migration, and contact with wildlife and their respective parasites and vectors. Hence, recognizing residents' perceptions, knowledge, and practices is crucial for effectively minimizing risks. METHODS: A cross-sectional study was conducted from January 2020 to July 2021 in and around Chiro town using a pretested close-ended questionnaire. A total of 350 respondents were selected using simple random sampling methods. The questionnaire included information on the sociodemographic status of the respondents and questions concerning the knowledge, perceptions, and practices of the participants regarding the selected zoonotic diseases. The associations of knowledge, perceptions, and practices related to zoonoses with zoonotic risk factors were analysed using chi-square tests. RESULTS: The study revealed that 82.9% of the respondents had knowledge of bovine tuberculosis, followed by knowledge of rabies (80%), knowledge of anthrax (45.1%), and knowledge of brucellosis (24.3%). Males had greater knowledge of bovine tuberculosis (84.8%), followed by rabies (79.8%) and anthrax (48.6%), while females had greater knowledge of brucellosis (23.6%). The most cited source of information was radio (68%). Most respondents mentioned the outbreaks of rabies (62.5%), bovine tuberculosis (53.2%), anthrax (35.6%), and brucellosis (15.7%). Respondents with higher educational levels and urban residents had more knowledge of zoonoses. More than 75% of respondents had a good perception of the transmission of zoonotic disease from animals, and the practice of consuming raw milk or raw/undercooked meat and sharing the same house with animals was high. CONCLUSION: The majority of respondents reported that they had knowledge of bovine tuberculosis and rabies, but lower knowledge and perceptions were reported for anthrax and brucellosis. These findings illustrate the need for collaboration among animal, human and environmental health offices in one health approach to prevent and control zoonotic disease.

Salmonellosis in adult cattle in Central Argentina: case series.

Salmonellosis is an infectious disease caused by bacteria belonging to the Salmonella genus. Bovine salmonellosis is more frequent in young cattle under intensive overcrowd husbandry conditions, and therefore uncommon in adults. We report four outbreaks of clinical salmonellosis due to Salmonella Typhimurium, Salmonella Newport and Salmonella Dublin provoking outbreaks of diarrheic/septicemic disease in adult cattle of Central Argentina. Anamnesis information, clinical, pathological, and bacteriological findings were retrospective analyzed. This report emphasizes the importance to include salmonellosis among the differential diagnosis of clinical enteric/septicemic disease in adult cattle under different husbandry conditions in Argentina. The source of Salmonella could not be established in these outbreaks.

The BLV-miRNAs pathway of BLV inhibits the expression of Lactoferrin, Lactoperoxidase, Alpha-lactalbumin and Beta-lactoglobulin proteins.

Bovine leukemia virus (BLV) is a widespread virus that decreases milk production and quality in dairy cows. As crucial components of BLV, BLV-encoded microRNAs (BLV-miRNAs) affect BLV replication and may impact the synthesis of Lactoferrin (LTF), Lactoperoxidase (LPO), Alpha-lactalbumin (alpha-LA), and Beta-lactoglobulin (beta-LG). In this study, we investigated the targeting relationship between BLV-miRNAs and LTF, LPO, alpha-LA, and beta-LG in cow's milk. Additionally, we investigated the possible mechanisms by which BLV reduces milk quality. The results showed that cow's milk had significantly lower levels of LTF, LPO, and alpha-LA proteins in BLV-positive cows than in BLV-negative cows. BLV-△miRNAs (miRNA-deleted BLV) enhanced the reduction of LPO, alpha-LA, and beta-LG protein levels caused by BLV infection. Multiple BLV-miRNAs have binding sites with LTF and LPO mRNA; however, only BLV-miR-B1-5 P has a targeting relationship with LPO mRNA. The results revealed that BLV-miR-B1-5 P inhibits LPO protein expression by targeting LPO mRNA. However, BLV does not directly regulate the expression of LTF, alpha-LA, or beta-LG proteins through BLV-miRNAs.

IFRD2, a target of miR-2400, regulates myogenic differentiation of bovine skeletal muscle satellite cells via decreased phosphorylation of ERK1/2 proteins.

The proliferation and differentiation of skeletal muscle satellite cells is a complex physiological process involving various transcription factors and small RNA molecules. This study aimed to understand the regulatory mechanisms underlying these processes, focusing on interferon-related development factor 2 (IFRD2) as a target gene of miRNA-2400 in bovine skeletal MuSCs (MuSCs). IFRD2 was identified as a target gene of miRNA-2400 involved in regulating the proliferation and differentiation of bovine skeletal MuSCs. Our results indicate that miR-2400 can target binding the 3'UTR of IFRD2 and inhibit its translation. mRNA and protein expression levels of IFRD2 increased significantly with increasing days of differentiation. Moreover, overexpression of the IFRD2 gene inhibited proliferation and promoted differentiation of bovine MuSCs. Conversely, the knockdown of the gene had the opposite effect. Overexpression of IFRD2 resulted in the inhibition of ERK1/2 phosphorylation levels in bovine MuSCs, which in turn promoted differentiation. In summary, IFRD2, as a target gene of miR-2400, crucially affects bovine skeletal muscle proliferation and differentiation by precisely regulating ERK1/2 phosphorylation.

Human recombinant interleukin-6 improves the morphological quality of cryopreserved in vitro produced bovine blastocysts.

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis.

Isolation, characterization and whole-genome analysis of G9 group a rotaviruses in China: Evidence for possible Porcine-Human interspecies transmission.

Group A rotaviruses (RVAs) are major causes of severe gastroenteritis in infants and young animals. To enhance our understanding of the relationship between human and animals RVAs, complete genome data are necessary. We screened 92 intestinal and stool samples from diarrheic piglets by RT‒PCR targeting the VP6 gene, revealing a prevalence of 10.9%. RVA was confirmed in two out of 5 calf samples. We successfully isolated two porcine samples using MA104 cell line. The full-length genetic constellation of the two isolates were determined to be G9-P[23]-I5-R1-C1-M1-A8-N1-T7-E1-H1, with close similarity to human Wa-like and porcine strains. Sequence analysis revealed the majority of genes were closely related to porcine and human RVAs. Phylogenetic analysis revealed that these isolates might have their ancestral origin from pigs, although some of their gene segments were related to human strains. This study reveals evidence of reassortment and possible interspecies transmission between pigs and humans in China.

Brillouin spectroscopy of medically relevant samples of bovine jugular vein and pericardium.

There is the rapid growth in application of Brillouin scattering spectroscopy to biomedical objects in order to characterize their mechanoelastic properties in this way. However, the possibilities and limitations of the method when applied to tissues have not yet been clarified. Here, applicability of Brillouin spectroscopy for testing the elastic response of medically relevant tissues of bovine jugular vein and pericardium was considered. Parameters of the Brillouin peak were studied for samples untreated, diepoxide-fixed, and preserved after treatment in alcohol solutions. It was found that diepoxide cross-linking resulted to a slight tendency to increase the Brillouin position for hydrated tissues. The variations in the position and width of the Brillouin peaks, associated with local fluctuations in water concentration, were reduced after diepoxide treatment in the case of the pericardium, but not in the case of the vein wall. To obtain more information about the elastic response of the protein scaffold without the participation of water, dried samples were also studied. Brillouin spectra of the dried pericardium and vein wall revealed a significant increase in the Brillouin peak position (elastic modulus) after conservation in alcohol. In the case of the vein wall, this effect was found for both collagen and elastin-related peaks, which were identified in the Brillouin spectrum. This result corresponds to a denser packing of fibrous proteins after preservation in alcohol solutions. The ability of Brillouin spectroscopy to independently characterize the effect of treatment on the instantaneous elastic modulus of various tissue components is also attractive for its application in the development of new materials for bioimplants. A comparison of the Brillouin longitudinal and Young's elastic moduli determined for the hydrated samples of the vein and pericardium showed that there is no clear correspondence between these material parameters. The usefulness of using both experimental methods to obtain new information about the elastic response of the material is discussed.

Allicin attenuates the oxidative damage induced by Aflatoxin B1 in dairy cow hepatocytes via the Nrf2 signalling pathway.

Aflatoxin B1 (AFB1) is known to inhibit growth, and inflict hepatic damage by interfering with protein synthesis. Allicin, has been acknowledged as an efficacious antioxidant capable of shielding the liver from oxidative harm. This study aimed to examine the damage caused by AFB1 on bovine hepatic cells and the protective role of allicin against AFB1-induced cytotoxicity. In this study, cells were pretreated with allicin before the addition of AFB1 for co-cultivation. Our findings indicate that AFB1 compromises cellular integrity, suppresses the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, allicin attenuates oxidative damage to bovine hepatic cells caused by AFB1 by promoting the expression of the Nrf2 pathway and reducing cell apoptosis. In conclusion, the results of this study will help advance clinical research and applications, providing new options and directions for the prevention and treatment of liver diseases.

Efficacy of Three Antimicrobials Against two SARS-COV-2 Surrogates, Bovine Coronavirus and Human Coronavirus OC43, on Hard or Soft Nonporous Materials.

The efficacy of three antimicrobials was evaluated against two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogates - bovine coronavirus (BCoV) and human coronavirus (HCoV) OC43 - on hard and soft nonporous materials. Three antimicrobials with three different active ingredients (chlorine, hydrogen peroxide, and quaternary ammonium compound + alcohol) were studied. Initially, a neutralization method was optimized for each antimicrobial. Then, we determined their efficacy against BCoV and HCoV OC43 in both suspension and on surfaces made with polyethylene terephthalate (PET) plastic and vinyl upholstery fabric. All tests were conducted under ambient environmental conditions with a soil load of 5% fetal bovine serum. After a 2-min exposure, all three antimicrobials achieved a >3.0 log10 reduction in viral titers in suspension. All three also reduced virus infectivity on both surface materials below the detection limit (0.6 log10 TCID50/carrier). Treatments in which the reduction in virus titer was <3.0 log10 were attributed to a decreased dynamic range on the carrier during drying prior to disinfection. The carrier data revealed that both surrogates were inactivated more rapidly (p <0.05) on vinyl or under conditions of high relative humidity. Three classes of antimicrobials were efficacious against both SARS-CoV-2 surrogate viruses, with BCoV demonstrating slightly less sensitivity compared to HCoV OC43. These findings also illustrate the importance of (1) optimizing the neutralization method and (2) considering relative humidity as a key factor for efficacy testing.

Assessing the feasibility of test-and-cull and test-and-segregation approaches for the control of high-prevalence bovine tuberculosis in Ethiopian intensive dairy farms.

Bovine tuberculosis (bTB) is endemic and has a substantial impact on the livestock sector in Ethiopia and other low and middle-income countries (LMICs). With a national emphasis on dairy farm intensification to boost milk production and spur economic growth, the incidence of bTB is anticipated to rise. However, Ethiopia, like other LMICs, lacks a comprehensive national bTB control strategy due to the economic and social infeasibility of traditional test-and-cull (TC) approaches. To inform the development of such a strategy, we evaluated the effectiveness and feasibility of TC and test-and-segregation (TSg) strategies for bTB control on Ethiopian dairy farms. A TC approach was used at Farm A [N = 62; comparative cervical test (CCT) > 4 mm, starting prevalence 11.3%] while TSg was implemented at Farm B (N = 45; CCT > 4 mm, prevalence 22.2%), with testing intervals of 2-4 months. Both strategies achieved a reduction in bTB prevalence to 0%, requiring seven rounds of TC over 18 months at Farm A, and five rounds of TSg over 12 months at Farm B's negative herd. The results show that adopting more sensitive thresholds [CCT > 0 mm or single cervical test (SCT) > 2 mm] during later rounds was pivotal in identifying and managing previously undetected infections, emphasizing the critical need for optimized diagnostic thresholds. Cost analysis revealed that TC was approximately twice as expensive as TSg, primarily due to testing, labor, and cow losses in TC, versus construction of new facilities and additional labor for TSg. This underscores the economic and logistical challenges of bTB management in resource-limited settings. Taken together, our study highlights an urgent need for the exploration of alternative approaches including TSg and or vaccination to mitigate within herd transmission and enable implementation of bTB control in regions where TC is not feasible.

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.

Evaluation of bovine coronavirus in Korean native calves challenged through different inoculation routes.

Bovine coronavirus (BCoV) is a pneumoenteric virus that can infect the digestive and respiratory tracts of cattle, resulting in economic losses. Despite its significance, information regarding BCoV pathogenesis is limited. Hence, we investigated clinical signs, patterns of viral shedding, changes in antibody abundance, and cytokine/chemokine production in calves inoculated with BCoV via intranasal and oral. Six clinically healthy Korean native calves (< 30 days old), initially negative for BCoV, were divided into intranasal and oral groups and monitored for 15 days post-infection (dpi). BCoV-infected calves exhibited clinical signs such as nasal discharge and diarrhea, starting at 3 dpi and recovering by 12 dpi, with nasal discharge being the most common symptoms. Viral RNA was detected in nasal and fecal samples from all infected calves. Nasal shedding occurred before fecal shedding regardless of the inoculation route; however, fecal shedding persisted longer. Although the number of partitions was very few, viral RNA was identified in the blood of two calves in the oral group at 7 dpi and 9 dpi using digital RT-PCR analysis. The effectiveness of maternal antibodies in preventing viral replication and shedding appeared limited. Our results showed interleukin (IL)-8 as the most common and highly induced chemokine. During BCoV infection, the levels of IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1ß were significantly affected, suggesting that these emerge as potential and reliable biomarkers for predicting BCoV infection. This study underscores the importance of BCoV as a major pathogen causing diarrhea and respiratory disease.

Selection and application of small non-coding RNAs for normalizing RT-qPCR data of bovine preimplantation embryo conditioned medium.

Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly-CCC-1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly-CCC-1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly-CCC-1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.

Identification of genetic markers of resistance to macrolide class antibiotics in Mannheimia haemolytica isolates from a Saskatchewan feedlot.

Mannheimia haemolytica is a major contributor to bovine respiratory disease (BRD), which causes substantial economic losses to the beef industry, and there is an urgent need for rapid and accurate diagnostic tests to provide evidence for treatment decisions and support antimicrobial stewardship. Diagnostic sequencing can provide information about antimicrobial resistance genes in M. haemolytica more rapidly than conventional diagnostics. Realizing the full potential of diagnostic sequencing requires a comprehensive understanding of the genetic markers of antimicrobial resistance. We identified genetic markers of resistance in M. haemolytica to macrolide class antibiotics commonly used for control of BRD. Genome sequences were determined for 99 M. haemolytica isolates with six different susceptibility phenotypes collected over 2 years from a feedlot in Saskatchewan, Canada. Known macrolide resistance genes estT, msr(E), and mph(E) were identified in most resistant isolates within predicted integrative and conjugative elements (ICEs). ICE sequences lacking antibiotic resistance genes were detected in 10 of 47 susceptible isolates. No resistance-associated polymorphisms were detected in ribosomal RNA genes, although previously unreported mutations in the L22 and L23 ribosomal proteins were identified in 12 and 27 resistant isolates, respectively. Pangenome analysis led to the identification of 79 genes associated with resistance to gamithromycin, of which 95% (75 of 79) had no functional annotation. Most of the observed phenotypic resistance was explained by previously identified antibiotic resistance genes, although resistance to the macrolides gamithromycin and tulathromycin was not explained in 39 of 47 isolates, demonstrating the need for continued surveillance for novel determinants of macrolide resistance.IMPORTANCEBovine respiratory disease is the costliest disease of beef cattle in North America and the most common reason for injectable antibiotic use in beef cattle. Metagenomic sequencing offers the potential to make economically significant reductions in turnaround time for diagnostic information for evidence-based selection of antibiotics for use in the feedlot. The success of diagnostic sequencing depends on a comprehensive catalog of antimicrobial resistance genes and other genome features associated with reduced susceptibility. We analyzed the genome sequences of isolates of Mannheimia haemolytica, a major bovine respiratory disease pathogen, and identified both previously known and novel genes associated with reduced susceptibility to macrolide class antimicrobials. These findings reinforce the need for ongoing surveillance for markers of antimicrobial resistance to support improved diagnostics and antimicrobial stewardship.

Draft genome sequences of 148 Klebsiella pneumoniae species complex members from bovine and human hosts.

Klebsiella pneumoniae species complex members, particularly K. pnemoniae sensu stricto, are common bovine clinical mastitis pathogens and often the cause of hospital- and community-acquired infections in humans. Here, we present 148 draft genome assemblies and annotations of K. pneumoniae species complex members from bovine and human hosts in Canada.

Virulent-MDR-ESBL E. coli and Klebsiella pneumoniae report from North Sinai calves diarrhea and in vitro antimicrobial by Moringa oleifera.

The health of calves has a significant impact on the production of cows and livestock. Some desert plants have pharmacological importance, as they can be used to reduce antibiotic resistance. Our hypothesis is designed to detect Virulent- Multidrug-Resistant and Extended- spectrum Beta- lactamase Enterobacteriaceae (Virulent-MDR-ESBL Enterobacteriaceae and to determine whether Moringa oleifera has antibacterial activity against the detected isolates. A total of 39 Enterobacteriaceae isolates from 28 diarrheic samples were collected from calves aged between 20 days and 20 months from 3 different flocks in North Sinai, Sahl-Eltina region, Egypt. E.coli 46% (18/39), O157 13% (5/39), Klebsiella pneumoniae 41% (16/39). MDR members accounted for 87%, while ESBL isolates accounted for 43%. The antibacterial activity is represented by microdilution. Minimum inhibition concentration (MIC) for the methanol extract of Moringa oleifera ranged from 2.5,5,10, and 25mg/ ml among E.coli isolates, and O157 was susceptible to (2.5mg/ ml), Klebsiella pneumoniae isolates were susceptible to (5-50mg/ ml). Analysis of the methanol extract revealed that ferulic acid was the dominant phenolic compound with a concentration of 29,832 parts per million (ppm). In silico docking study expected the active site of ferulic acid to act on the tyrosine bacterial enzyme through Pi-alkyl, Pi-anion, Carbon hydrogen bonds, and extra ionic attractive interactions with copper ions which can stabilize ferulic acid inside the targeted pocket Diverse virulent gene profiles were observed in E. coli. The Shiga toxin-producing Escherichia coli (STEC) was reported in 83% of the isolated E. coli, while the DNA gyrase (gyrA) was harbored in 100% of Klebsiella pneumoniae isolates. Various profiles of antibiotic resistance genes for both E. coli and Klebsiella pneumoniae isolates were distinguished. blaTEM genes were detected in 99% of E. coli and 100% of Klebsiella pneumoniae. Sequence analysis for E. coli strain DRC-North Sinai-Eg was placed in accession numbers (OP955786) for the Shiga toxin 2 gene (Stx2A), (OP997748) and (OP997749) for the Adhesion to host cell gene (Eae). For the hemolysine gene (hylA), the accession number was (OP946183). Klebsiella pneumoniae strain DRC-North Sinai-Eg was placed in (OP946180) for (gyrA). This study has proven the broad range of Moringa oliefera's antibacterial effects in vitro against the virulent-MDR- ESBL E. coli and Klebsiella pneumoniae isolated from North Sinai calves diarrhea. These are congruent with the disability effect on bacterial tyrosinase enzyme through docking study therefore, we recommend the usage of this desert plant as a prospective feed additive, we endorse this as an antibacterial new insight natural source and for the medication of considered pathogens with zoonotic impacts.