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The present study aimed to identify, determine the susceptibility, and detect gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis. Arcanobacterium pyogenes isolates were identified first by using the API Coryne Vit system test, and further through PCR. Minimum inhibitory concentrations of 23 antimicrobial agents against A. pyogenes were tested using standard broth microdilution assays according to the protocols of the Clinical and Laboratory Standards Institute. The genes of integrons I and II were amplified by PCR using specific primers. Thirty-two A. pyogenes isolates were isolated from 136 endometritic cows in the Hohhot region. Antibiotic susceptibility tests revealed that all isolates were highly sensitive to fluoroquinolones (100%), macrolides (∼81.2 to 100%) and florfenicol (90.6%), aminoglycosides (∼15.6 to 81.2%), and tetracyclines (∼43.7 to 68.7%). However, 53.1% were resistant to clindamycin, ∼50 to 65.6% were resistant to penicillins, and ∼37.5 to 71.9% were resistant to cephalosporins. One hundred percent were resistant to sulfonamides and bacitracin zinc. The integrons were further confirmed by sequencing. No class II integrons were detected, whereas 50% (n=16) of the A. pyogenes isolates were positive for the presence of the intI I gene, but only 13 contained gene cassettes. Sequence analysis of gene cassettes revealed 6 gene cassettes, 4 of which encode resistant determinants of aminoglycosides (aadA1, aadA5, aadA24, and aadB) and 1 of which encodes the resistance gene of chloramphenicol (cmlA6). The function of the sixth identified cassette, designated ORF1, is unknown. The gene cassette arrays aadA24-ORF1, aadA5, and aadA1-addB-cmlA6 were found in 46.13% (6/13), 38.46% (5/13), and 38.46% (5/13) of the isolates, respectively. These cassettes segregated according to a consistent pattern, with aadA5 always alone, ORF1 always with aadA24, and aadA1-aadB and cmlA6 always together. Most of the positive integrons existed in the multiresistant isolates (n=∼3 to 7), indicating that the integrons played an important role in the dissemination and spread of antimicrobial resistance. This is the first report of A. pyogenes infections in dairy cows in China and of detection of gene cassettes and integrons in A. pyogenes.

Trueperella pyogenes ( T. pyogenes ) is an opportunistic pathogen associated with a variety of diseases in many domestic animals. Therapeutic treatment options for T. pyogenes infections are becoming limited due to antimicrobial resistance, in which efflux pumps play an important role. This study aims to evaluate the inhibitory activity of luteolin, a natural flavonoid, on the MsrA efflux pump and investigate its mechanism. The results of antimicrobial susceptibility testing indicated that the susceptibility of msrA -positive T. pyogenes isolates to six macrolides increased after luteolin treatment, while the susceptibility of msrA -negative isolates showed no change after luteolin treatment. It is suspected that luteolin may increase the susceptibility of T. pyogenes isolates by inhibiting MsrA activity. After 1/2 MIC luteolin treatment for 36 h, the transcription level of the msrA gene and the expression level of the MsrA protein decreased by 55.0–97.7% and 36.5–71.5%, respectively. The results of an affinity test showed that the equilibrium dissociation constant (KD) of luteolin and MsrA was 6.462 × 10 –5  M, and hydrogen bonding was predominant in the interaction of luteolin and MsrA. Luteolin may inhibit the ATPase activity of the MsrA protein, resulting in its lack of an energy source. The current study illustrates the effect of luteolin on MsrA in T. pyogenes isolates and provides insight into the development of luteolin as an innovative agent in combating infections caused by antimicrobial-resistant bacteria.

Mastitis is one of the most important infectious diseases and one of the diseases that causes the greatest use of antibiotics in dairy cows. Therefore, updated information on the bacteria that cause mastitis and their antibiotic susceptibility properties is important. Here, for the first time in over 10 years, we updated the bacterial findings in clinical mastitis in Swedish dairy cows together with their antibiotic resistance patterns and risk factors for each bacterial species. During the period 2013–2018, samples from clinical mastitis were collected, together with information on the cows and herds of origin. The samples were cultured, and a total of 664 recovered bacterial isolates were subjected to susceptibility testing. Staphylococcus aureus (S. aureus) was the most common pathogen and accounted for 27.8% of diagnoses, followed by Streptococcus dysgalactiae (S. dysgalactiae) (15.8%), Escherichia coli (E. coli) (15.1%), Streptococcus uberis (S. uberis) (11.4%), Trueperella pyogenes (T. pyogenes) (7.7%), non-aureus staphylococci (NAS) (2.8%), Klebsiella spp. (2.7%), Enterococcus spp. (1.3%), and Streptococcus agalactiae (S. agalactiae) (1.2%). Various other bacteria accounted for 2.6%. Staphylococci were, in general, susceptible to most antibiotics, but 2.6% of S. aureus and 30.4% of NAS were resistant to penicillin. No methicillin-resistant staphylococci were found. All S. agalactiae were susceptible to penicillin. Bimodal and trimodal MIC distributions for penicillin in S. dysgalactiae and S. uberis, respectively, indicate acquired reduced susceptibility in some isolates. The mostly unimodal MIC distributions of T. pyogenes indicate that acquired resistance does usually not occur in this species. Among E. coli, 14.7% were resistant to at least one antibiotic, most often ampicillin (8.7%), streptomycin (7.8%), or sulphamethoxazole (6.9%). Klebsiella spp. had low resistance to tetracycline (9.1%) but is considered intrinsically resistant to ampicillin. Pathogen-specific risk factors were investigated using multivariable models. Staphylococcus aureus, S. dysgalactiae, and T. pyogenes were more common, while E. coli was less common in quarters with more than one pathogen. S. aureus and T. pyogenes were mostly seen in early lactation, while E. coli was more common in peak to mid lactation and S. dysgalactiae in early to peak lactation. Trueperella pyogenes and Klebsiella spp. were associated with a previous case of clinical mastitis in the current lactation. Staphylococcus aureus was associated with tie stalls and T. pyogenes with loose housing. All pathogens except E. coli and S. dysgalactiae had a seasonal distribution. In conclusion, the aetiological agents for clinical bovine mastitis have remained relatively stable over the last 10–15 years, S. aureus, S. dysgalactiae, E. coli and S. uberis being the most important. Resistance to penicillin among Gram-positive agents was low, and in general, antibiotic resistance to other compounds was low among both Gram-positive and Gram-negative agents.

Trueperella pyogenes, an opportunistic pathogen causes various ailments in different animals. Different strains from different animals have distinct characters phenotypically and genotypically. Hence understanding the strains in a particular geographical location helps in framing the preventive measures. Comparative genomics of all the available T. pyogenes genome in the NCBI was conducted to understand the relatedness among strains. Whole genome phylogeny showed host associated clustering of strains recovered from swine lungs. Core genome phylogeny also showed host associated clustering mimicking whole genome phylogeny results. MLST analysis showed that there was higher diversity among cattle strains. Multidimensional scaling revealed five swine clusters, two cattle and buffalo clusters. Pangenome analysis also showed that T. pyogenes had an open genome with 57.09% accessory genome. Host specific genes were identified by pangenome analysis, and (R)-citramalate synthase was specific for swine strains of Asian origin. Host specifc genes identified by pangenome analysis can be exploited for developing a molecular assay to specifically identify the strains. The study shows that MLST having higher discriminatory power can be used as an epidemiological tool for strain discrimination of T. pyogenes.

Trueperella pyogenes (T. pyogenes) is an important opportunistic bacterial pathogen that causes infections in livestock and wildlife. The increasing antimicrobial resistance of this bacterium poses significant challenges to the prevention and control of T. pyogenes-related diseases. Vaccines are widely used to control infectious diseases. Therefore, the development of vaccines against T. pyogenes would be valuable for the prevention of these diseases. In the current study, the genes encoding the predicted T. pyogenes serine metalloprotease (SMP) and anchor M domain-containing protein (AMD) were cloned. Recombinant SMP (rSMP), AMD fragments (rAMD-1 and rAMD-2), and rPLO D123 (a truncated form of pyolysin containing domains 1, 2, and 3, but not domain 4) were expressed in Escherichia coli cells and purified. The purified recombinant proteins were formulated into genetic engineering subunit vaccines with aluminum hydroxide adjuvant and administered subcutaneously to mice. The vaccines induced high levels of anti-rPLO D123, anti-rSMP, and anti-rAMD antibodies. These antibodies could agglutinate T. pyogenes cells and/or inhibit PLO-induced hemolysis. Mice were challenged intraperitoneally or subcutaneously. The results showed that vaccines containing rSMP provided better immunoprotection than the others. Since the mice receiving rSMP-containing vaccines exhibited attenuated tissue damage, faster resolution of infectious inflammation, and higher survival rate compared to those in other groups. In contrast, the incorporation of rAMD-1/rAMD-2 did not significantly improve the immunoprotective effect of the vaccines. Our findings confirm the potential of SMP as a promising protective antigen for the development of vaccines against T. pyogenes. •For the first time cloned genes encoding SMP and AMD protein of T. pyogenes.•Confirm the potential of SMP as a candidate antigen for T. pyogenes vaccines.•Confirm the vaccines containing recombinant SMP protein help to the rapid resolution of infectious inflammation caused by T. pyogenes.

Mycoplasma pneumonia, caused by Mycoplasma bovis ( Mycoplasmopsis bovis ; M. bovis ), is linked with severe inflammatory reactions in the lungs and can be challenging to treat with antibiotics. Biofilms play a significant role in bacterial persistence and contribute to the development of chronic lesions. A recent study has shown that polymicrobial interactions between species are an important factor in biofilm formation, yet the precise mechanism of biofilm formation in M. bovis remains unknown. By assuming multiple pathogen infections in the bovine respiratory disease complex (BRDC), this study examined the characterisation of the polymicrobial relationship between M. bovis and Trueperella pyogenes ( T. pyogenes ) during biofilm formation. Autopsies were performed on four Holstein calves (two chronic Mycoplasma pneumonia calves and two control calves). Bacterium-like aggregation structures (> 10 μm), which were assumed to be biofilms of M. bovis in vivo, were observed adhering to the cilia in calves with Mycoplasma pneumonia. M. bovis released an extracellular matrix to connect with neighbouring bacteria and form a mature biofilm on the plate. Biofilm formation in the co-culture of M. bovis and T. pyogenes (strain T1: 1 × 10 5 and 1 × 10 6  CFU/well) significantly increased ( p  < 0.05 and p  < 0.01; 64.1% and 64.8% increase) compared to that in a single culture of these bacteria. Furthermore, some large aggregates (> 40 μm), composed of M. bovis and T. pyogenes , were observed. The morphological characteristics of this biofilm were similar to those observed in vivo compared to a single culture. In conclusion, the polymicrobial interaction between M. bovis and T. pyogenes induces biofilm formation, which is associated with increased resistance to antimicrobial agents, and this exacerbates the progression of chronic Mycoplasma pneumonia.

Trueperella pyogenes is an opportunistic pathogen frequently associated with bovine endometritis, yet the mechanisms by which it induces uterine inflammation remain incompletely understood. In this study, we investigated the effects of T. pyogenes and its membrane vesicles (MVs) on bovine endometrial epithelial cells (BEECs) and explored the underlying inflammatory pathways involved. Bovine endometrial epithelial cells (BEECs) were treated with T. pyogenes (MOI = 100) or MVs at various concentrations (1 × 10 8 , 1 × 10 7 , or 1 × 10 6 particles/mL) for 6–24 h. Inflammatory cytokines (IL-1β, IL-6, IL-18, or TNF-α) and the activation of the NLRP3 and MyD88/NF-κB signalling pathways were analysed by ELISA, qRT‒PCR, and western blotting. Cell death mechanisms were assessed by flow cytometry and scanning electron microscopy. T. pyogenes significantly upregulated inflammatory cytokine mRNA expression at 6 and 12 h and protein expression at 12 and 24 h. Compared with bacterial stimulation at 12 h, MVs induced earlier activation of the NLRP3 inflammasome at 6 h. High-concentration MVs induced necrosis-like membrane disruption, whereas moderate concentrations promoted apoptosis and pyroptosis. Both T. pyogenes and its MVs activated the MyD88/NF-κB signalling pathway, with significantly increased phosphorylation of P65 at 12 h. Cytokine secretion exhibited time- and dose-dependent trends, aligning with transcriptional changes. Collectively, these findings demonstrate that T. pyogenes MVs contribute to endometrial inflammation through the NLRP3 and MyD88/NF-κB signalling pathways, with distinct forms of cell death determined by MV concentration. These findings highlight MVs as key virulence factors and potential therapeutic targets for bovine endometritis.

Trueperella pyogenes (T. pyogenes) is a zoonotic pathogen that is cause a variety of pyogenic diseases in animals. The complex pathogenicity and various virulence factors are important challenges to produce an effective vaccine. According to previous trials, inactivated whole-cell bacteria or recombinant vaccines were unsuccessful in preventing disease. Thus, this study aims to introduce a new vaccine candidate based on a live-attenuated platform. For this purpose, first T. pyogenes was subjected to sequential passage (SP) and antibiotic treatment (AT) to lose their pathogenicity. Second, Plo and fimA expressions as virulence genes were evaluated by qPCR and then mice were challenged with bacteria from SP and AT culture by intraperitoneal route. Compared to the control group (T. pyogenes-wild type), plo and fimA gene expressions were downregulated and vaccinated mice have a normal spleen appearance in contrast to the control group. In addition, there was no significant difference between bacterial count from spleen, liver, heart and peritoneal fluid in vaccinated mice and the control group. In conclusion, this study introduces a new T. pyogenes vaccine candidate based on a live-attenuated strategy that mimics natural infection without pathogenicity for further investigation on vaccines against T. pyogenes infections.

Background Abortion is a major source of economic losses in cattle breeding. Abortion occurs due to a wide range of causes, but infections are the most frequently diagnosed. However, establishing an aetiological diagnosis remains challenging due to the large variety of bacteria, protozoa, viruses, and fungi that have been associated with abortion in cattle. Economic restraints limit the range of diagnostic methods available for routine diagnostics, and decomposition of the conceptus or lack of proper fetal and/or maternal samples further restrict the diagnostic success. In this study, we report recent diagnostic findings from bovine abortions in Denmark, a country that has a large dairy sector and is free from most infectious agents causing epizootic abortion in cattle. The aims of the study were: (i) to identify infectious causes of bovine abortion in Denmark, (ii) to categorise the diagnostic findings based on the level of diagnostic certainty, and (iii) to assess the diagnostic rate. Due to economic restraints, only a limited panel of routine diagnostic methods were available. Placentas and/or fetuses from mid- to late-term abortions and stillbirths (n = 162) were submitted to the Danish National Veterinary Institute between January 2015 and June 2017. The aborted materials were examined macroscopically, histologically, and by bacterial culture. Maternal blood samples were tested for bovine viral diarrhoea virus (BVDV) antibodies. Results The likely aetiology of the abortion was diagnosed in 52 cases, resulting in a diagnostic rate of 33%. The most common cause was protozoal infection (19%) followed by infection with Trueperella pyogenes (3%), Staphylococcus aureus (2%), and non-haemolytic Escherichia coli (2%). Lesions in fetuses with a protozoal infection were consistent with neosporosis. In many cases (38%), inflammatory changes were found in the placenta and/or fetal organs but no specific aetiology was identified. Neither infection with Brucella spp. nor maternal BVDV antibodies were detected. The majority of submitting herds (92%) were each represented by fewer than three abortion cases over the study period. Conclusions Protozoal infection, most likely neosporosis, was the most commonly diagnosed cause of abortion and the only one associated with potential epizootic abortion events. Despite using a reduced number of diagnostic methods in comparison to other abortion studies, the diagnostic rate of this study was within the range reported in an earlier Danish study, as well as in recent international studies. The low number of submitted cases per herd and the sparse anamnestic information provided at submission hampered conclusions on the potential epizootic character of the abortion events in question.

Trueperella pyogenes (T. pyogenes) is an opportunistic pathogen that causes infertility, mastitis, and metritis in animals. T. pyogenes is also a zoonotic disease and is considered an economic loss agent in the livestock industry. Therefore, vaccine development is necessary. Using an immunoinformatics approach, this study aimed to construct a multi-epitope vaccine against T. pyogenes. The collagen adhesion protein, fimbriae, and pyolysin (PLO) sequences were initially retrieved. The HTL, CTL, and B cell epitopes were predicted. The vaccine was designed by binding these epitopes with linkers. To increase vaccine immunogenicity, profilin was added to the N-terminal of the vaccine construct. The antigenic features and safety of the vaccine model were investigated. Docking, molecular dynamics simulation of the vaccine with immune receptors, and immunological simulation were used to evaluate the vaccine’s efficacy. The vaccine’s sequence was then optimized for cloning. The vaccine construct was designed based on 18 epitopes of T. pyogenes. The computational tools validated the vaccine as non-allergenic, non-toxic, hydrophilic, and stable at different temperatures with acceptable antigenic features. The vaccine model had good affinity and stability to bovine TLR2, 4, and 5 as well as stimulation of IgM, IgG, IL-2, IFN-γ, and Th1 responses. This vaccine also increased long-lived memory cells, dendritic cells, and macrophage population. In addition, codon optimization was done and cloned in the E. coli K12 expression vector (pET-28a). For the first time, this study introduced a novel multi-epitope vaccine candidate based on collagen adhesion protein, fimbriae, and PLO of T. pyogenes. It is expected this vaccine stimulates an effective immune response to prevent T. pyogenes infection.