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Abstract Background Liver fibrosis is a common pathological process of chronic liver disease, characterized by excessive deposition of extracellular matrix (ECM). Mesenchymal stem cells (MSCs) have been found to have potential therapy effect on liver fibrosis, but the mechanism involved was still unclear. The objective of this study is to investigate the therapeutic efficacy of adipose-derived mesenchymal stem cells (ADMSCs) on the treatment of liver fibrosis, with particular emphasis on elucidating the underlying mechanism of action through which ADMSCs inhibit the activation of hepatic stellate cells (HSCs). Methods ADMSCs were isolated from adipose tissue and injected intravenously into hepatic fibrosis model of rats. The histopathological changes, liver function, collagen deposition, the expression of fibroin and Hippo pathway were evaluated. In vitro, ADMSCs were co-cultured with HSCs activated by transforming growth factor beta 1 (TGF-β1), and the inhibitor of Hippo pathway was used to evaluate the therapeutic mechanism of ADMSCs transplantation. Results The results showed that after the transplantation of ADMSCs, the liver function of rats was improved, the degree of liver fibrosis and collagen deposition were reduced, and the Hippo signaling pathway was activated. In vitro, ADMSCs can effectively inhibit the proliferation and activation of HSCs induced by TGF-β1 treatment. However, the inhibitory effect of ADMSCs was weakened after blocking the Hippo signaling pathway. Conclusions ADMSCs inhibit HSCs activation by regulating YAP/TAZ, thereby promoting functional recovery after liver fibrosis. These findings lay a foundation for further investigation into the precise mechanism by which ADMSCs alleviate liver fibrosis.

Chikungunya virus (CHIKV) is an arbovirus that can lead to chronic arthritis and significantly diminish the quality of life of patients. Given the expanding global prevalence of CHIKV and the absence of specific antiviral therapies, there is an urgent need to explore effective treatment options. This study aimed to evaluate the antiviral effects of shikimic acid (SA) against CHIKV through a combination of network pharmacology, molecular docking, and in vitro assays. Network pharmacology analysis identified 26 potential targets through which SA could inhibit CHIKV, including key pathogenic targets such as TNF, IL-6, and MAPK3. This hypothesis was further supported by molecular docking. The molecular docking analysis revealed that SA could interact with multiple CHIKV-related targets, including EGF, with vina scores generally lower than −6, indicating a high propensity for stable complex formation. The results also suggested that SA could potentially disrupt the IL-17 signaling pathway by engaging with various targets to form complexes. In vitro experiments confirmed that SA significantly enhanced the viability of 293T and BHK-21 cells infected with CHIKV by ~25% and reduced viral load by over 20% at concentrations ranging from 1,000 to 31.25 μM. Additionally, SA was found to markedly downregulate the expression of CHIKV-related attachment factors ACTG1, TSPAN9, and TIM-1 in 293T cells infected with CHIKV. Furthermore, RT-qPCR analysis demonstrated that SA effectively decreased the expression of NFKB1, PTGS2, RELA, and EGF related to the IL-17 signaling pathway. In conclusion, these findings indicate that SA is a promising candidate for developing treatment strategies targeting CHIKV with good clinical application value.

BackgroundProbiotics, which are beneficial to the host, have been shown to benefit the health of cats. Lactic acid bacteria (LAB) are commonly used probiotics, but most strains used for cats are not derived from cats, leading to reduced efficacy and poor adaptation to cats. The objective was to identify LAB with promising probiotic potential specific to cats.MethodLABs were isolated from fecal samples of 20 healthy cats. Gram staining and the survival rate in the simulated gastrointestinal tract were used for preliminary screening. Candidate strains were identified by 16S rDNA sequencing, and further evaluated for adhesion ability, growth characteristics, antibacterial activity, antioxidant capacity, and safety.Results24 Gram-positive isolates were identified, with 10 (F1-F10) showing robust viability in the simulated gastroenteric fluid. These 10 strains exhibited excellent adhesion to Caco-2 cells and strong auto-agglutination properties. They also possessed the capacity to antagonize and aggregate pathogens (Staphylococcus aureus ATCC 25923, Salmonella Braenderup H9812, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa PAO1), Moreover, all strains demonstrated tolerance to H2O2 concentrations ranging from 0.5–2 mmol/L and the ability to scavenge 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, indicating a certain level of antioxidant activity. Safety tests showed no hemolytic activity, and all but F6 were highly sensitive to antibiotics, with over 62.5% sensitivity to 16 antibiotics. Remarkably, F4 (Lactobacillus reuteri) and F10 (Lactobacillus brevis) exhibited exceptional viability in the simulated gastrointestinal tract, coupled with robust growth potential, enhanced adhesion efficiency, significant antibacterial and antioxidant properties.ConclusionOur findings revealed that F4 (Lactobacillus reuteri) and F10 (Lactobacillus brevis) hold promising potential as probiotics. This research lays a solid scientific foundation for the selection and application of probiotics tailored specifically for cats.

Abstract In recent years, dental implants have become a trend in the treatment of human patients with missing teeth, which may also be an acceptable method for companion animal dentistry. However, there is a gap challenge in determining appropriate implant sizes for different dog breeds and human. In this study, we utilized skull computed tomography data to create three-dimensional models of the mandibles of dogs in different sizes. Subsequently, implants of various sizes were designed and subjected to biomechanical finite element analysis to determine the optimal implant size. Regression models were developed, exploring the relationship between the average weight of dogs and the size of premolar implants. Our results illustrated that the regression equations for mean body weight (x, kg) and second premolar (PM2), third premolar (PM3), and fourth premolar (PM4) implant length (y, mm) in dogs were: y = 0.2785x + 7.8209, y = 0.2544x + 8.9285, and y = 0.2668x + 10.652, respectively; the premolar implant diameter (mm) y = 0.0454x + 3.3506, which may provide a reference for determine suitable clinical implant sizes for dogs.

IntroductionLactic acid bacteria (LAB) are Gram-positive bacteria that produce lactic acid during fermentation, with some strains enhancing host health by modulating the gut microbiota, boosting immune responses, and reducing inflammation.MethodsIn this study, 6 LAB strains were isolated from two dog milk samples, and their probiotic properties were comprehensively evaluated. The evaluation included growth properties, stress resistance, antipathogen activity, adhesion activity, safety assessment, antioxidant capacity, and prebiotic metabolites assessment.ResultsIn comparison to the control strain Lactobacillus rhamnosus LGG, all 6 LAB isolates exhibited favorable probiotic properties. Additionally, the results of the antioxidant tests indicated that these strains demonstrated high tolerance to 0.5 mmol/L H2O2 and exhibited significant scavenging abilities for the free radicals 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and hydroxyl (OH−). Furthermore, the 6 LAB isolates were found to produce elevated concentrations of prebiotic metabolites, including exopolysaccharides (EPS), γ-aminobutyric acid (GABA), and bile salt hydrolase (BSH).DiscussionThis study presents a comprehensive analysis of LAB isolates derived from canine milk. These isolates exhibited multifunctional properties, with strain L221 performing the best overall, making it a promising candidate for probiotic use in dogs.

IntroductionGuided endodontics represents an effective method for achieving safe and reliable endodontic surgery in human medicine. However, it is rarely employed in small animal dentistry. This study employed finite element analysis and three-dimensional (3D) printing techniques to explore the feasibility of guided endodontics in Beagle mandibular teeth.MethodsThe methodology included the processing of Computed Tomography (CT) data, the creation of mathematical and 3D printing templates of the root canal pathways, and the evaluation of dog 3D printing guided endodontics compared with classic root canal method using radiograph.ResultsIn this experiment, the coordinates of the central point of pulp crown and apex point for each tooth were recorded. Based on the extension line of the central point of dental root canal orifice and the apex point, guided endodontic templates were designed on each root canal of 20 teeth in the Beagle mandible. Among them, the average relative deviation of guided endodontics and classic root canal method was 4.28% ± 2.75%, and the mean angular deviation was 1.90 ± 0.25°.DiscussionOur research indicated that dog 3D printing guided endodontics has accurate position, direction, and length, which may assist veterinary dentistry in root canal treatment in small animals.

Background: Biofilm, a common drug-resistant phenotype of Staphylococcus aureus (S. aureus), demonstrates significant drug resistance and recurrence due to its extracellular polymeric substance (EPS) barrier and subsequent bacterial migration. Hence, there is an urgent need for effective solutions to mitigate the hazards posed by biofilms. Result: This study developed a stable, low-toxicity multifunctional nanomicelle, GLA@SOL/EYL, by encapsulating glabridin (GLA) using sophorolipid (SOL) and egg yolk lecithin (EYL). Optimizations were performed for the hydration medium, the ratio of carrier materials to GLA, and EYL additions. GLA@SOL/EYL exhibited a particle size of 122.1 ± 0.8 nm and a surface potential of −66.4 ± 1.7 mV, endowing it with the ability to permeate biofilms EPS effectively. GLA@SOL/EYL encapsulated 98.3 ± 1.2 % of GLA and demonstrated a slow-release effect, significantly enhancing the bioavailability of GLA. The addition of EYL reduced the hemolytic toxicity of GLA@SOL/EYL and improved its encapsulation rate and stability. GLA@SOL/EYL reduced the minimum inhibitory concentration of GLA to 8 μg/mL and extended its inhibitory effect at low concentrations by rapidly disrupting the structural integrity of S. aureus. GLA@SOL/EYL may penetrate biofilms to disperse EPS and remove twice as much biofilm as GLA alone, thereby eliminating 99.99 % of S. aureus within biofilms, compared to 99 % bactericidal efficacy of GLA. Additionally, GLA@SOL/EYL inhibited 63.8 ± 1.8 % of biofilm formation by affecting the expression of the srtA gene, thereby reducing the expression of cell wall-anchoring protein genes. In contrast, the biofilm inhibition rates of GLA and blank micelles were less than 10 %. Conclusion: GLA@SOL/EYL utilizes the nanoparticle effect to penetrate biofilms and deliver antimicrobial GLA. The SOL disperses the biofilm matrix while GLA is released to kill S. aureus, preventing bacterial dissemination and colonization. Thus, GLA@SOL/EYL presents an innovative strategy for effectively eradicating S. aureus biofilms and preventing new hazards in a one-step approach.

BackgroundHeat stress significantly impacts dairy cows, primarily through oxidative stress, which undermines their health. The problem is exacerbated by the ongoing global warming trend. Lactic acid bacteria (LAB) are safe, economical, and readily accessible options for enhancing the host’s antioxidant defenses and preventing oxidative damage. They have been proven effective in alleviating heat stress-related damage, making them an excellent choice for protecting dairy cows from the adverse effects of heat stress.MethodIn this study, five strains of LAB from Holstein cow milk (Lactobacillus plantarum L5, L14, L17, L19, L20) were evaluated for their heat resistance and antioxidant capacity by evaluating the growth characteristics and tolerance of the strains under high-temperature conditions, as well as their H2O2 tolerance, free radical scavenging ability (DPPH, OH−, ABTS), reducing ability, and EPS production ability. Furthermore, we employed Caco-2 cells to assess the adhesion rate of the strain, thereby confirming its ability to successfully colonize the host’s intestinal tract and ensuring the effective execution of its probiotic functions. The strain with excellent heat resistance and antioxidant capacity was then subjected to genomic analysis to gain insight into the molecular mechanisms behind their heat resistance, antioxidant capacity, and safety.ResultsAmong the two strains, Lactobacillus plantarum L19 emerges as a highly promising candidate. The strain exhibits robust growth even at high temperatures at 40°C and maintains a survival rate of 16.42% at the extreme temperature of 65°C. Furthermore, it demonstrates superior tolerance to hydrogen peroxide (27.3%), and possesses a notably higher free radical scavenging capacity with a high adhesion rate to Caco-2 cell (22.19%) compared to the other four strains tested. Genomic analysis revealed its’ genome has 17 genes related to antioxidants and three genes related to heat resistance. Importantly, L19 lacks any resistance genes, ensuring its safety as a probiotic.ConclusionThe results imply that Lactobacillus plantarum L19 has the potential to serve as an effective food additive in mitigating damages associated with heat stress. This research offers a valuable reference for the prevention and management of heat stress in dairy cows, while also expanding the scope of applications for LAB derived from cow milk.

BackgroundIntestinal fungal composition plays a crucial role in modulating host health, and thus is of great significance in the conservation of endangered bird species. However, research on gut fungal composition in birds is limited. Therefore, in this study, we aimed to examine gut fungal community and potential fecal pathogen composition in wild Arborophila rufipectus.MethodsFecal samples were collected from the habitats of wild A. rufipectus and Lophura nycthemera (a widely distributed species belonging to the same family as A. rufipectus) in summer and autumn. Thereafter, RNA was collected and the internal transcribed spacer rRNA gene was sequenced via high-throughput sequencing to investigate seasonal variations in intestinal core fungi, microbial fungi, and potential pathogenic fungi.ResultsThe gut microbiota of A. rufipectus and L. nycthemera were highly similar and mainly consisted of three phyla, Ascomycota (58.46%), Basidiomycota (28.80%), and Zygomycota (3.56%), which accounted for 90.82% of the fungal community in all the samples. Further, the predominant genera were Ascomycota_unclassified (12.24%), Fungi_unclassified (8.37%), Davidiella (5.18%), Helotiales_unclassified (2.76%), Wickerhamomyces (1.84%), and Pleosporales_unclassified (1.14%), and the potential fecal pathogens identified included Candida, Cryptococcus, Trichosporon, and Malassezia.ConclusionOur results provide evidence that the diversity of intestinal fungi in the endangered species, A. rufipectus, is similar to that in the common species, L. nycthemera, and may serve as a basis for monitoring the status of A. rufipectus and for developing conservation measures.

IntroductionThe most common factor leading to renal failure or death is renal IR (ischemia-reperfusion). Studies have shown that mesenchymal stem cells (MSCs) and their exosomes have potential therapeutic effects for IR injury by inhibiting M1 macrophage polarization and inflammation. In this study, the protective effect and anti-inflammatory mechanism of adipose-derived mesenchymal stem cell-derived exosomes (ADMSC-Exos) after renal IR were investigated.MethodInitially, ADMSC-Exos were intravenously injected into IR experimental beagles, and the subsequent assessment focused on inflammatory damage and macrophage phenotype. Furthermore, an in vitro inflammatory model was established by inducing DH82 cells with LPS. The impact on inflammation and macrophage phenotype was then evaluated using ADMSC and regulatory miR-146a.ResultsFollowing the administration of ADMSC-Exos in IR canines, a shift from M1 to M2 macrophage polarization was observed. Similarly, in vitro experiments demonstrated that ADMSC-Exos enhanced the transformation of LPS-induced macrophages from M1 to M2 type. Notably, the promotion of macrophage polarization by ADMSC-Exos was found to be attenuated upon the inhibition of miR-146a in ADMSC-Exos.ConclusionThese findings suggest that miR-146a plays a significant role in facilitating the transition of LPS-induced macrophages from M1 to M2 phenotype. As a result, the modulation of macrophage polarization by ADMSC-Exos is achieved via the encapsulation and conveyance of miR-146a, leading to diminished infiltration of inflammatory cells in renal tissue and mitigation of the inflammatory reaction following canine renal IR.

BackgroundProteus mirabilis is a Gram-negative, rod-shaped bacterium widely found in natural environments. It is known for causing a range of severe illnesses in mammals, particularly urinary tract infections (UTIs). This study evaluates the therapeutic efficacy of phage P2-71 against Proteus mirabilis in vivo and in vitro environments.MethodsThe in vitro therapeutic potential of bacteriophage P2-71 was assessed through the ability of phage to kill Proteus mirabilis by using a plate counting assay, and biofilm inhibition and biofilm lysis assays using a microtitre plate method. Additionally, an in vivo UTI model in C57BL/6Jmice was developed via urethral inoculation of the bacterium. Phage therapy was administered through urethral injection over a period of 5 days. Therapeutic outcomes were measured by analyzing bacterial load, phage titer, inflammatory markers, and histopathological changes in the urine, urogenital tissues, and spleen.ResultsIn vitro, bacteriophage P2-71 achieved significant reductions in P. mirabilis concentrations, with log reductions of 1.537 and 0.7009 CFU/mL in laboratory and urine environments, respectively (p 0.05). Concurrently, phage titers decreased over time. Importantly, phage treatment notably reduced bacterial load in the bladder, kidneys, and spleen (p

Altrenogest is a key regulatory hormone for intensive and batch management of reserve sows in breeding farms. As a synthetic hormone, altrenogest could make ovaries stay at the initial stage of follicles and inhibit estrus and ovulation in animals. However, the currently used oral altrenogest solution needs to be administered continuously every day for more than two weeks in clinical practice. In this study we developed a phospholipid-based injectable gel carrying altrenogest to decrease the number of administrations, sustain release of the drug, and enhance therapeutic efficacy for clinical use. The altrenogest gel had a viscosity of 100 cP before phase transition and over 1,000,000 cP after phase transition. In vitro, altrenogest can be continuously released from gel for over two weeks. The pharmacokinetic results showed that the AUC (0–∞) of the altrenogest gel was almost double that of the altrenogest solution. The MRT (0–∞) was 40.92 ± 7.21 h and the t1/2 of the altrenogest gel was 80.03 ± 20.79 h. The altrenogest gel demonstrated excellent fluidity, ease of injectability, high drug-loading capacity, and appropriate sustained-release characteristics both in vitro and in vivo, making it a potential drug delivery system for swine production.

IntroductionProbiotics, especially Lactic Acid Bacteria (LAB), can promote the health of host animals in a variety of ways, such as regulating intestinal flora and stimulating the host’s immune system.MethodsIn this study, 206 LAB strains were isolated from 48 canine fecal samples. Eleven LAB strains were selected based on growth performance, acid and bile salt resistance. The 11 candidates underwent comprehensive evaluation for probiotic properties, including antipathogenic activity, adhesion, safety, antioxidant capacity, and metabolites.ResultsThe results of the antipathogenic activity tests showed that 11 LAB strains exhibited strong inhibitory effect and co-aggregation ability against four target pathogens (E. coli, Staphylococcus aureus, Salmonella braenderup, and Pseudomonas aeruginosa). The results of the adhesion test showed that the 11 LAB strains had high cell surface hydrophobicity, self-aggregation ability, biofilm-forming ability and adhesion ability to the Caco-2 cells. Among them, Lactobacillus acidophilus (L177) showed strong activity in various adhesion experiments. Safety tests showed that 11 LAB strains are sensitive to most antibiotics, with L102, L171, and L177 having the highest sensitivity rate at 85.71%, and no hemolysis occurred in all strains. Antioxidant test results showed that all strains showed good H2O2 tolerance, high scavenging capacity for 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) and hydroxyl (OH−). In addition, 11 LAB strains can produce high levels of metabolites including exopolysaccharide (EPS), γ-aminobutyric acid (GABA), and bile salt hydrolase (BSH).DiscussionThis study provides a thorough characterization of canine-derived LAB strains, highlighting their multifunctional potential as probiotics. The diverse capabilities of the strains make them promising candidates for canine dietary supplements, offering a holistic approach to canine health. Further research should validate their efficacy in vivo to ensure their practical application.

Canine circovirus (CanineCV) is a recently identified member of the Circoviridae family. Since its discovery in 2011, CanineCV has been detected in different countries worldwide, infecting both domestic and wild canids. The virus is potentially associated with gastrointestinal and respiratory illnesses. In 2016, CanineCV was reported in the southwestern region of Guangxi, China. However, its prevalence in other provinces in the Southwest region remained unknown. This study collected a total of 208 serum samples from domestic dogs in Sichuan, China in 2022 to investigate the prevalence of CanineCV. Among these samples, 26 tested positive for CanineCV, resulting in a positivity rate of 12.5%. Additionally, 12 strains were sequenced, 9 of which had a sequence length of 2,063 nucleotides (nt), 2 of the other threes had a length of 2,062 nts and another was 2,064 nt. Notably, a frameshift mutation was identified, resulting in a truncated ORF1 and the occurrence of a novel sequence comprised of 13 amino acids at the end of the replicate protein (Rep). This mutation could affect the replication cycle of the virus. Phylogenetic and evolutionary analyses revealed that the isolates belonged to the CanineCV-3 genotype and were prevalent in the Southeast and the Southwest regions of China, as well as in the neighboring countries alongside other strains of the same genotype. Collectively, this epidemiological investigation widens our understanding of the genetic diversity of CanineCV in Southwest China and provides insights into viral evolution.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) is regarded as one of the most important priority pathogens within the One Health interface. However, few studies have investigated the occurrence of ESBL-EC in giant pandas, along with their antibiotic-resistant characteristics and horizontal gene transfer abilities. In this study, we successfully identified 12 ESBL-EC strains (8.33%, 12/144) out of 144 E. coli strains which isolated from giant pandas. We further detected antibiotic resistance genes (ARGs), virulence-associated genes (VAGs) and mobile genetic elements (MGEs) among the 12 ESBL-EC strains, and the results showed that 13 ARGs and 11 VAGs were detected, of which blaCTX-M (100.00%, 12/12, with 5 variants observed) and papA (83.33%, 10/12) were the most prevalent, respectively. And ISEcp1 (66.67%, 8/12) and IS26 (66.67%, 8/12) were the predominant MGEs. Furthermore, horizontal gene transfer ability analysis of the 12 ESBL-EC showed that all blaCTX-M genes could be transferred by conjugative plasmids, indicating high horizontal gene transfer ability. In addition, ARGs of rmtB and sul2, VAGs of papA, fimC and ompT, MGEs of ISEcp1 and IS26 were all found to be co-transferred with blaCTX-M. Phylogenetic analysis clustered these ESBL-EC strains into group B2 (75.00%, 9/12), D (16.67%, 2/12), and B1 (8.33%, 1/12), and 10 sequence types (STs) were identified among 12 ESBL-EC (including ST48, ST127, ST206, ST354, ST648, ST1706, and four new STs). Our present study showed that ESBL-EC strains from captive giant pandas are reservoirs of ARGs, VAGs and MGEs that can co-transfer with blaCTX-M via plasmids. Transmissible ESBL-EC strains with high diversity of resistance and virulence elements are a potential threat to humans, animals and surrounding environment.

Abstract The objective of this study was to analyze the antimicrobial resistance (AMR) characteristics produced by antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and gene cassettes in Escherichia coli isolated from the feces of captive black bears. Antimicrobial susceptibility testing was performed by using the disk diffusion method, and both MGEs and integron gene cassettes were detected by polymerase chain reaction. Our results showed that 43.7% (62/142) of the isolates were multidrug resistant strains and 97.9% (139/142) of the isolates were resistant to at least one antibiotic. The highest AMR phenotype was observed for tetracycline (79.6%, 113/142), followed by ampicillin (50.0%, 71/142), trimethoprim-sulfamethoxazole (43.7%, 62/142) and cefotaxime (35.9%, 51/142). However, all isolates were susceptible to tobramycin. tetA had the highest occurrence in 6 ARGs in 142 E. coli isolates (76.8%, 109/142). Ten mobile genetic elements were observed and IS26 was dominant (88.0%, 125/142). ISECP1 was positively associated with five β-lactam antibiotics. ISCR3/14, IS1133 and intI3 were not detected. Seventy-five E. coli isolates (65 intI1-positive isolates, 2 intI2-positive isolates and 8 intI1 + intI2-positive isolates) carried integrons. Five gene cassettes (dfrA1, aadA2, dfrA17-aadA5, aadA2-dfrA12 and dfrA1-aadA1) were identified in the intI1-positive isolates and 2 gene cassettes (dfrA1-catB2-sat2-aadA1 and dfrA1-catB2-sat1-aadA1) were observed in the intI2-positive isolates. Monitoring of ARGs, MGEs and gene cassettes is important to understand the prevalence of AMR, which may help to introduce measures to prevent and control of AMR in E. coli for captive black bears.

Porcine circovirus (PCV) typically causes severe immune suppression in pigs, leading to mixed clinical infections with various pathogens that can cause significant harm to the pig industry. PCV has four subgenotypes, with PCV4 being an emerging virus that requires investigation due to its potential for epidemic outbreaks. Therefore, there is a need to develop a method that can detect all four PCV strains simultaneously. In this study, four pairs of specific primers and TaqMan probes were designed based on the conserved sequence of the PCV1–4 ORF2 gene to establish a PCV1–4 TaqMan multiplex real-time quantitative PCR method. The novel method was compared to six commercial testing kits for its efficacy. Then, a total of 595 mixed samples of spleen and lymph node collected from 12 districts in Chengdu from July to December 2021 were tested using the novel method. The results showed that the novel PCV1–4 TaqMan multiplex real-time quantitative PCR detection method has satisfied specificity, sensitivity, and repeatability. The positive rates of PCV1, PCV2, and PCV3 in Chengdu were 2.18%, 31.60%, and 15.29%, respectively, while no positive PCV4 was detected. The mixed infection rate of PCV2 and PCV3 was 5.21%. Our novel method may be as a potential method for PCV1–4 detection. Currently, PCV2 is the main epidemic PCV subtype in Chengdu, while the potential threat of PCV4 should also be considered.

Proteus mirabilis, a prevalent urinary tract pathogen and formidable biofilm producer, especially in Catheter-Associated Urinary Tract Infection, has seen a worrying rise in multidrug-resistant (MDR) strains. This upsurge calls for innovative approaches in infection control, beyond traditional antibiotics. Our research introduces bacteriophage (phage) therapy as a novel non-antibiotic strategy to combat these drug-resistant infections. We isolated P2-71, a lytic phage derived from canine feces, demonstrating potent activity against MDR P. mirabilis strains. P2-71 showcases a notably brief 10-minute latent period and a significant burst size of 228 particles per infected bacterium, ensuring rapid bacterial clearance. The phage maintains stability over a broad temperature range of 30-50°C and within a pH spectrum of 4-11, highlighting its resilience in various environmental conditions. Our host range assessment solidifies its potential against diverse MDR P. mirabilis strains. Through killing curve analysis, P2-71’s effectiveness was validated at various MOI levels against P. mirabilis 37, highlighting its versatility. We extended our research to examine P2-71’s stability and bactericidal kinetics in artificial urine, affirming its potential for clinical application. A detailed genomic analysis reveals P2-71’s complex genetic makeup, including genes essential for morphogenesis, lysis, and DNA modification, which are crucial for its therapeutic action. This study not only furthers the understanding of phage therapy as a promising non-antibiotic antimicrobial but also underscores its critical role in combating emerging MDR infections in both veterinary and public health contexts.

Abstract Background Neonatal calf diarrhea (NCD) is typically treated with antibiotics, while long-term application of antibiotics induces drug resistance and antibiotic residues, ultimately decreasing feed efficiency. Pueraria polysaccharide (PPL) is a versatile antimicrobial, immunomodulatory, and antioxidative compound. This study aimed to compare the therapeutic efficacy of different doses of PPL (0.2, 0.4, 0.8 g/kg body weight (BW)) and explore the effect of plasma metabolites in diarrheal calves by the best dose of PPL. Results PPL could effectively improve the daily weight gain, fecal score, and dehydration score, and the dosage of 0.4 g/kg BW could reach curative efficacy against calf diarrhea (with effective rates 100.00%). Metabolomic analysis suggested that diarrhea mainly affect the levels of taurocholate, DL-lactate, LysoPCs, and intestinal flora-related metabolites, trimethylamine N-oxide; however, PPL improved liver function and intestinal barrier integrity by modulating the levels of DL-lactate, LysoPC (18:0/0:0) and bilirubin, which eventually attenuated neonatal calf diarrhea. It also suggested that the therapeutic effect of PPL is related to those differential metabolites in diarrheal calves. Conclusions The results showed that 0.4 g/kg BW PPL could restore the clinical score of diarrhea calves by improving the blood indexes, biochemical indexes, and blood metabolites. And it is a potential medicine for the treatment of calf diarrhea.

Chitooligosaccharides (COS) have attracted significant attention due to their unique biological activities, water solubility, and absorbable properties. The objective of the present study was to investigate the impact of COS-supplemented diets on the immune response, antioxidative capacity, hematology, serum biochemistry, and modulation of intestinal microbiota in growing beagles. Twelve weaning male beagles (6 weeks old; weighing 3.6 ± 0.6 kg) were fed either a control diet (food without COS, n = 6) or a COS-supplemented diet (n = 6) twice daily for 7 weeks. Blood samples collected at weeks 4 and 7 indicated that hematology and serum biochemistry remained unaffected by COS supplementation. Compared with the control group, the test group showed higher levels of serum antibodies against the canine distemper virus and parvovirus, higher levels of immunoglobulin A, G, and M, and increased activities of superoxide dismutase, glutathione peroxidase, and catalase. In addition, COS was observed to modulate the intestinal flora by enhancing the presence of probiotics, such as Muribaculaceae, Prevotellaceae_Ga6A1_group, Lactobacillus, Collinsella, Blautia, and Lachnospiraceae_NK4A136_group. In summary, a COS-supplemented diet could effectively improve dog health by regulating immune function and antioxidant responses and modulating intestinal microbiota. This study highlights the potentiality of using COS as a valuable nutraceutical for growing dogs.

Abstract Background Adipose‐derived mesenchymal stem cells (ADMSCs) and their extracellular vesicles (EVs) are a promising source of therapies for ischaemia‐reperfusion (IR) because of their potent anti‐inflammatory and immunomodulatory properties. Objectives The aims of this study were to explore the therapeutic efficacy and potential mechanism of ADMSC‐EVs in canine renal IR injury. Methods Mesenchymal stem cells (MSCs) and EVs were isolated and characterised for surface markers. A canine IR model administered with ADMSC‐EVs was used to evaluate therapeutic effects on inflammation, oxidative stress, mitochondrial damage and apoptosis. Results CD105, CD90 and beta integrin ITGB were positively expressed in MSCs, while CD63, CD9 and intramembrane marker TSG101 were positively expressed in EVs. Compared with the IR model group, there was less mitochondrial damage and reduction in quantity of mitochondria in the EV treatment group. Renal IR injury led to severe histopathological lesions and significant increases in biomarkers of renal function, inflammation and apoptosis, which were attenuated by the administration of ADMSC‐EVs. Conclusions Secretion of EVs by ADMSCs exhibited therapeutic potential in renal IR injury and may lead to a cell‐free therapy for canine renal IR injury. These findings revealed that canine ADMSC‐EVs potently attenuate renal IR injury‐induced renal dysfunction, inflammation and apoptosis, possibly by reducing mitochondrial damage.

Enterococcus spp., as an opportunistic pathogen, are widely distributed in the environment and the gastrointestinal tracts of both humans and animals. Captive Asian elephants, popular animals at tourist attractions, have frequent contact with humans. However, there is limited information on whether captive Asian elephants can serve as a reservoir of antimicrobial resistance (AMR). The aim of this study was to characterize AMR, antibiotic resistance genes (ARGs), virulence-associated genes (VAGs), gelatinase activity, hemolysis activity, and biofilm formation of Enterococcus spp. isolated from captive Asian elephants, and to analyze the potential correlations among these factors. A total of 62 Enterococcus spp. strains were isolated from fecal samples of captive Asian elephants, comprising 17 Enterococcus hirae (27.4%), 12 Enterococcus faecalis (19.4%), 8 Enterococcus faecium (12.9%), 7 Enterococcus avium (11.3%), 7 Enterococcus mundtii (11.3%), and 11 other Enterococcus spp. (17.7%). Isolates exhibited high resistance to rifampin (51.6%) and streptomycin (37.1%). 50% of Enterococcus spp. isolates exhibited multidrug resistance (MDR), with all E. faecium strains demonstrating MDR. Additionally, nine ARGs were identified, with tet(M) (51.6%), erm(B) (24.2%), and cfr (21.0%) showing relatively higher detection rates. Biofilm formation, gelatinase activity, and α-hemolysin activity were observed in 79.0, 24.2, and 14.5% of the isolates, respectively. A total of 18 VAGs were detected, with gelE being the most prevalent (69.4%). Correlation analysis revealed 229 significant positive correlations and 12 significant negative correlations. The strongest intra-group correlations were observed among VAGs. Notably, we found that vancomycin resistance showed a significant positive correlation with ciprofloxacin resistance, cfr, and gelatinase activity, respectively. In conclusion, captive Asian elephants could serve as significant reservoirs for the dissemination of AMR to humans.

Corynebacterium pseudotuberculosis is a zoonotic pathogen that causes lymphadenitis in humans, livestock, and wildlife. In this study, C. pseudotuberculosis biovar equi strains were isolated from three alpacas. Antibiotic susceptibility tests and pathogenicity tests were also conducted. Moreover, one strain was sequenced using DNBSEQ and Oxford Nanopore technology. The three strains exhibited resistance to aztreonam, fosfomycin, and nitrofurantoin. The median lethal doses (LD50) of strains G1, S2 and BA3 in experimentally infected mice was 1.66 × 105 CFU, 3.78 × 105 CFU and 3.78 × 105 CFU, respectively. The sequencing of strain G1 resulted in the assembly of a chromosomal scaffold comprising 2,379,166 bp with a G + C content of 52.06%. Genome analysis of strain G1 revealed the presence of 48 virulence genes and 5 antibiotic resistance genes (ARGs). Comparative genomic analysis demonstrates a high degree of genetic similarity among C. pseudotuberculosis strains, in contrast to other Corynebacterium species, with a clear delineation between strains belonging to the two biovars (ovis and equi). The data of the present study contribute to a better understanding of the properties of C. pseudotuberculosis biovar equi strains and the potential risk they pose to alpacas and other livestock, as well as the necessity of ongoing surveillance and monitoring of infectious diseases in animals.

IntroductionProteus mirabilis is a multi-host pathogen that causes diseases of varying severity in a wide range of mammals, including humans. Proteus mirabilis is resistant to multiple antibiotics and has acquired the ability to produce expanded spectrum of β-lactamases, leading to serious public health problems. However, the available information on P. mirabilis isolated from feces of dogs, is still poorly understood, as is the correlation between its virulence-associated genes (VAGs) and antibiotic resistance genes (ARGs).MethodIn this study, we isolated 75 strains of P. mirabilis from 241 samples, and investigated the swarming motility, biofilm formation, antimicrobial resistance (AMR), distribution of VAGs and ARGs, as well as the presence of class 1, 2, and 3 integrons in these isolates.ResultsOur findings suggest a high prevalence of intensive swarming motility and strong biofilm formation ability among P. mirabilis isolates. Isolates were primarily resistant to cefazolin (70.67%) and imipenem (70.67%). These isolates were found to carry ureC, FliL, ireA, zapA, ptA, hpmA, hpmB, pmfA, rsbA, mrpA, and ucaA with varying prevalence levels of 100.00, 100.00, 100.00, 98.67, 98.67, 90.67, 90.67, 90.67, 90.67, 89.33, and 70.67%, respectively. Additionally, the isolates were found to carry aac(6′)-Ib, qnrD, floR, blaCTX-M, blaCTX-M-2, blaOXA-1, blaTEM, tetA, tetB and tetM with varying prevalence levels of 38.67, 32.00, 25.33, 17.33, 16.00, 10.67, 5.33, 2.67, 1.33, and 1.33%, respectively. Among 40 MDR strains, 14 (35.00%) were found to carry class 1 integrons, 12 (30.00%) strains carried class 2 integrons, while no class 3 integrons was detected. There was a significant positive correlation between the class 1 integrons and three ARGs: blaTEM, blaCTX-M, and blaCTX-M-2. This study revealed that P. mirabilis strains isolated from domestic dogs exhibited a higher prevalence of MDR, and carried fewer VAGs but more ARGs compared to those isolated from stay dogs. Furthermore, a negative correlation was observed between VAGs and ARGs.DiscussionGiven the increasing antimicrobial resistance of P. mirabilis, veterinarians should adopt a prudent approach towards antibiotics administration in dogs to mitigate the emergence and dissemination of MDR strains that pose a potential threat to public health.

Nitroxynil(NIT) is a commonly used anti-liver fluke drug for cattle and sheep, Its solubility is closely related to its preparation. In this work, the molar solubility of NIT in nine pure solvents (methanol, ethanol, 1,2-propanediolethyl, isopropanol, ethyl acetate, acetonitrile, n-butanol, phemethylol) and two kinds of binary mixtures with different ratio(ethanol + phemethylol; ethanol + acetonitrile) was determined by shake flask method over the temperature from 278.15 ∼ 323.15 K at atmosphere pressure. Results show that the solubility of NIT in all tested solvents was increased with raised temperature. In mono-solvents, the mole fraction solubility of NIT was highest in phemethylol and the solubility order is: phemethylol > acetonitrile > ethyl acetate > methanol > n-butanol > ethanol > 1,2-propanediolethyl > isopropanol > water. In binary solvents, the mole fraction solubility increased with increasing ratio of phemethylol/acetonitrile. In mono-solvents, the modified Apelblat equation, λh equation, Van't Hoff model were applied to correlate the solubility data. In binary solvents, the modified Apelblat equation, λh equation, GSM model and Jouyban-Acree model were to correlate the solubility data. Solubility order of NIT in nine pure solvent and two binary solvent systems were analysed by using the Hansen solubility parameter (HSP). Activity coefficient was to access the solute–solvent molecular interactions. In addition, the dissolution of NIT is an endothermic and entropy-friendly process, since thermodynamic parameters such as enthalpy, entropy, and apparent standard Gibbs free energy are all greater than zero. The results will supply some essential data on recrystallization process, purification and formulation development of NIT in pharmaceutical applications.

IntroductionIntestinal microbiota composition plays a crucial role in modulating the health of the host. This evaluation indicator is very sensitive and profoundly impacts the protection of endangered species. Currently, information on the gut microbiota of wild birds remains scarce. Therefore, this study aimed to describe the gut microbial community structure and potentially, the pathogen composition of wild Arborophila rufipectus.MethodsTo guarantee comprehensive data analysis, we collected fecal samples from wild A. rufipectus and Lophura nycthemera in their habitats for two quarters. The 16S rRNA gene was then sequenced using high-throughput sequencing technology to examine the intestinal core microbiota, microbial diversity, and potential pathogens with the aim of determining if the composition of the intestinal microflora varies seasonally.Results and DiscussionThe gut microbiota of A. rufipectus and L. nycthemera primarily comprised four phyla: Proteobacteria (45.98%), Firmicutes (35.65%), Bacteroidetes (11.77%), and Actinobacteria (3.48%), which accounted for 96.88% of the total microbial composition in all samples. At the genus level, core microorganisms were found, including Shigella (10.38%), Clostridium (6.16%), Pseudomonas (3.03%), and Rickettsiella (1.99%). In these genera, certain microbial species have been shown to be pathogenic. This study provides important indicators for analyzing the health status of A. rufipectus and formulating protective measures.

IntroductionNeonatal calf diarrhea (NCD) is still one of the most critical diseases in calf rearing. Studies have shown that Pueraria lobata polysaccharides (PLP) have intense antioxidant and immunomodulatory activity and modulate gut microbiota. This randomized clinical trial aimed to determine the effect of PLP on the neonatal calf with diarrhea.MethodsIn this study, we recorded the fecal score of experimental calves, and calves with fecal scores ≥ 2 were determined as diarrhea and assessed their serum concentrations of inflammatory cytokines and oxidative damage-related indices. Fecal microbiota and metabolomics of diarrheal calves were further investigated.ResultsResults showed that treatment with PLP decreased the fecal score of diarrheal calves, serum concentrations of IL-1β, TNF-γ, and malondialdehyde, and also elevated the level of superoxide dismutase. In addition, PLP treatment altered the gut microbiota, significantly increased the relative abundances of beneficial bacteria, including the phyla Bacteroidetes and Actinobacteria, the genus Collinsella, Megamonas, and Bifidobacterium; decreased the relative abundances of pathogenetic or diarrhea related bacteria, such as Proteobacteria, Fusobacteria, Clostridium_sensu_stricto_1, and Escherichia_Shigella. Moreover, PLP can increase the fecal concentrations of isobutyric acid, propionic acid, and pantothenate; lower the levels of PC [18:0/18:1(9Z)], arachidonic acid, and docosahexaenoic acid.DiscussionThus, the results suggested that the PLP may perform the therapeutic activity via alleviating intestinal inflammation and regulating gut microbiota, avoiding further dysbiosis to restore the metabolism of gut microbiota, and finally promoting the recovery of diarrhea. The change further mitigated intestinal inflammation and oxidative damage in diarrheal calves. This indicated that PLP might be a promising treatment to attenuate diarrhea in neonatal calves.

The effect of oat β-glucan on intestinal function and growth performance of weaned rabbits were explored by multi-omics integrative analyses in the present study. New Zealand White rabbits fed oat β-glucan [200 mg/kg body weight (BW)] for 4 weeks, and serum markers, colon histological alterations, colonic microbiome, colonic metabolome, and serum metabolome were measured. The results revealed that oat β-glucan increased BW, average daily gain (ADG), average daily food intake (ADFI), and decreased serum tumor necrosis factor-α (TNF-α) interleukin-1β (IL-1β), and lipopolysaccharide (LPS) contents, but did not affect colonic microstructure. Microbiota community analysis showed oat β-glucan modulated gut microbial composition and structure, increased the abundances of beneficial bacteria Lactobacillus, Prevotellaceae_UCG-001, Pediococcus, Bacillus, etc. Oat β-glucan also increased intestinal propionic acid, valeric acid, and butyric acid concentrations, decreased lysine and aromatic amino acid (AAA) derivative contents. Serum metabolite analysis revealed that oat β-glucan altered host carbohydrate, lipid, and amino acid metabolism. These results suggested that oat β-glucan could inhibit systemic inflammation and protect intestinal function by regulating gut microbiota and related metabolites, which further helps to improve growth performance in weaned rabbits.

Trichosporon asahii is a basidiomycete yeast that is pathogenic to humans and animals, and fluconazole-resistant strains have recently increased. Farnesol secreted by fungi is a factor that causes variations in fluconazole resistance; however, few studies have explored the underlying mechanisms. Therefore, this study aims to delineate the fluconazole resistance mechanisms of T. asahii and explore farnesol’s effects on these processes. A comparative metabolome–transcriptome analysis of untreated fluconazole-sensitive (YAN), fluconazole-resistant (PB) T. asahii strains, and 25 μM farnesol-treated strains (YAN-25 and PB-25, respectively) was performed. The membrane lipid-related genes and metabolites were upregulated in the PB vs. YAN and PB-25 vs. PB comparisons. Farnesol demonstrated strain-dependent mechanisms underlying fluconazole tolerance between the YAN and PB strains, and upregulated and downregulated efflux pumps in PB-25 and YAN-25 strains, respectively. Membrane lipid-related metabolites were highly correlated with transporter-coding genes. Fluconazole resistance in T. asahii was induced by membrane lipid bio-synthesis activation. Farnesol inhibited fluconazole resistance in the sensitive strain, but enhanced resistance in the resistant strain by upregulating efflux pump genes and membrane lipids. This study offers valuable insights into the mechanisms underlying fungal drug resistance and provides guidance for future research aimed at developing more potent antifungal drugs for clinical use.

Traditional Chinese medicine (TCM) formulas can be adjusted on the basis of TCM basic theory to achieve the best curative effect, especially for diseases with complex pathogenesis, such as post-weaning diarrhea (PWD). Shugan Jianwei Sijunzi decoction (SJ-SJZD) can be recognized as modified Sijunzi Decoction (SJZD) supplemented with Astragalus mongholicus Bunge, Bupleurum chinense DC, Citrus × aurantium L., and Crataegus pinnatifida Bunge (fruit) in a fixed dosage ratio. The inactive ingredients were subsequently added to make granule, which was Shugan Jianwei Sijunzi granule (SJ-SJZG). Previous studies have confirmed the antagonism of SJ-SJZG to PWD. However, the mechanism of SJ-SJZG protective effects on small intestine in weaned Rex rabbits remained unclear. Animals were randomly divided into negative control (NC), low dose (LD), medium dose (MD), high dose (HD), and positive control (PC). SJ-SJZG significantly increased the intestinal length and the jejunum villi length. The SIgA level was statistically increased in duodenum and jejunum with the ELISA. Immunohistochemical detection showed that SIgA protein expression was also increased significantly in jejunum. Meanwhile, the relative expression of Zo1 in duodenum and jejunum of SJ-SJZG group increased significantly. SJ-SJZG significantly increased the relative expression of occludin in duodenum and jejunum as well. Moreover, real-time PCR results showed a significant increase in GLUT2 and SGLT1 relative expression in ileum. SJ-SJZG could also obviously enhance the expression of GLUT2 in jejunum and the expression of SGLT1 in duodenum. In conclusion, SJ-SJZG had been proven to be effective in promoting the development of small intestine and improving the immunity of small intestine. Moreover, SJ-SJZG could ensure the integrity of mucosal barrier and increase the ability of intestine to absorb glucose in small intestine.

This study evaluated the protective effect of Bacillus subtilis HH2 on beagles orally challenged with enterotoxigenic Escherichia coli (ETEC). We assessed the physiological parameters and the severity of diarrhea, as well as the changes in three serum immunoglobulins (IgG, IgA, and IgM), plasma diamine oxidase (DAO), D-lactate (D-LA), and the fecal microbiome. Feeding B. subtilis HH2 significantly reduced the severity of diarrhea after the ETEC challenge (p < 0.05) and increased serum levels of IgG, IgA, and IgM (p < 0.01). B. subtilis HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge (p < 0.05), but no significant changes were observed in D-LA (p > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with B. subtilis HH2 (p < 0.05), while B. subtilis HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles (p < 0.01). Moreover, 14 days of B. subtilis HH2 feeding reduced the relative abundance of Deinococcus-Thermus in feces. This study reveals that B. subtilis HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition.

Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidian pathogens with zoonotic potential that pose significant public health concerns. To ascertain the occurrence and genotypes of E. bieneusi and Encephalitozoon spp., we used nested PCR to amplify the internal transcribed spacer (ITS) gene and DNA sequencing to analyze 198 fecal samples from red pandas from 6 zoos in China. The total rate of microsporidial infection was 15.7% (31/198), with 12.1% (24/198), 1.0% (2/198), 2.0% (4/198) and 1.0% (2/198) for infection rate of E. bieneusi, Encephalitozoon cuniculi, Encephalitozoon intestinalis and Encephalitozoon hellem, respectively. One red panda was detected positive for a mixed infection (E. bieneusi and E. intestinalis). Red pandas living in semi-free conditions are more likely to be infected with microsporidia (χ2 = 6.212, df = 1, p < 0.05). Three known (SC02, D, and PL2) and one novel (SCR1) genotypes of E. bieneusi were found. Three genotypes of E. bieneusi (SC02, D, SCR1) were grouped into group 1 with public health importance, while genotype PL2 formed a separate clade associated with group 2. These findings suggest that red pandas may serve as a host reservoir for zoonotic microsporidia, potentially allowing transmission from red pandas to humans and other animals.

Streptococcus agalactiae is a significant pathogen that can affect both human beings and animals. The extensive current use of antibiotics has resulted in antibiotic resistance. In our previous research, we found that zinc oxide quantum dots (ZnO QDs) had inhibitory effects on antibiotic-resistant microorganisms. In this study, a strain of Streptococcus agalactiaeWJYT1 with a broad antibiotic-resistant spectrum was isolated and identified from Lama glama at Sichuan Agricultural University Teaching Animal Hospital. The genome for the resistance and virulence genes was analyzed. Additionally, the antibacterial effects and anti-virulence mechanism of ZnO QDs for S. agalactiaeWJYT1 were investigated. The results showed that the genome of S. agalactiaeWJYT1 is 1,943,955 bp, containing 22 resistance genes and 95 virulence genes. ZnO QDs have a good antibacterial effect against S. agalactiaeWJYT1 by reducing bacterial growth and decreasing the expression of virulence genes, including bibA, hylB, sip, and cip, which provides a novel potential treatment for S. agalactiae.

Abstract Background Blastocystis is a common anaerobic colonic protist in humans with controversial pathogenicity. Clostridium difficile (C. difficile) is the commonest cause of infectious diarrhea in healthcare settings. The prevalence and subtype (ST) characteristics of Blastocystis in patients with C. difficile infection (CDI) are rarely documented. Therefore, the present study was conducted to investigate the prevalence and subtype characteristics of Blastocystis in patients with suspicion of CDI in Singapore. Methods Fecal samples were collected from 248 patients presenting with suspected CDI from a single tertiary hospital in Singapore. C. difficile was diagnosed through positive glutamate dehydrogenase (GDH) with or without toxin A/B using enzyme immunoassay methods. The prevalence and subtype genetic characteristics of Blastocystis were determined by polymerase chain reaction (PCR) amplification and analysis of the barcode region of the SSU rRNA gene. Results The proportion of C. difficile in patients with healthcare-associated diarrhea in this study was 44% (109/248). Among the 109 C. difficile-positive patients, 59 (54.1%, 59/109) tested positive for toxigenic C. difficile, which was considered CDI. Based on the sequence analyses of the barcode region of the SSU rRNA gene, 10.1% (25/248) of the patients were found to be Blastocystis-positive, and three subtypes were identified: ST7 (64%, 16/25), ST1 (20%, 5/25), and ST3 (16%, 4/25). Remarkably, we found five patients with Blastocystis and C. difficile coinfection, and further subtype analysis showed two with ST7, two with ST1, and one with ST3. Conclusions To the best of our knowledge, this is the first study to investigate the subtype distributions of Blastocystis in patients with CDI in Singapore. We found ST7 to be the predominant subtype in diarrheal patients. The pathogenicity of ST7 has been strongly suggested in previous in vitro and mouse model experiments, further confirming its potential pathogenicity to humans.

There are massive bacteria in the raw milk, especially the lactic acid bacteria (LABs), which have been considered probiotics in humans and animals for a long time. Novel probiotics are still urgently needed because of the rapid development of the probiotic industry. To obtain new LABs with high probiotic potential, we obtained 26 LAB isolates, named L1 ~ L26, from local Holstein raw milk collected from a farm whose milk had never been used for LAB isolation. We identified them at the species level by biochemical and 16S rDNA sequencing methods. Their antagonistic activities against four target pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa PAO1, and Salmonella enterica H9812), co-aggregative ability with these target pathogens, survivability in the simulated gastrointestinal tract conditions and phenol, auto-aggregation and hydrophobicity, hemolytic activity, and antibiotic susceptibility, were evaluated in vitro. Five Lactiplantibacillus plantarum isolates (L5, L14, L17, L19, and L20) showed more promising probiotic potential than others. Specifically, these five isolates conglutinated with and inhibited all the target pathogens, and survived in the simulated gastric juice (92.55 ~ 99.69%), intestinal juice (76.18 ~ 83.39%), and 0.4% phenol (76.95 ~ 88.91%); possessed considerable auto-aggregation (83.91 ~ 90.33% at 24 h) and hydrophobicity (79.32 ~ 92.70%); and were non-hemolytic, sensitive to kinds of common antimicrobials. Our findings demonstrated that these five isolates could be preliminarily determined as probiotic candidates because they have better probiotic potential than those previously reported. Again, this study highlighted the potential of raw milk for probiotic isolating and screening and provided the probiotic industry with five new LAB candidates.

ABSTRACT Blastocystis is a common protistan parasite inhabiting the gastrointestinal tract of a wide range of hosts including humans and domestic and wild animals. Many studies have revealed the associations between Blastocystis and gut microbiome in humans. However, only a few studies have focused on the associations between Blastocystis and gut microbiome of animals, especially in forest musk deer (Moschus berezovskii). We investigated the effects of the Blastocystis colonization on the intestinal bacterial community compositions using amplicon sequencing targeting the V4 variable region of the 16S rRNA. Two subtypes of Blastocystis (ST5 and ST10) and Blastocystis-free (control) were included in this study. We found that compared with the forest musk deer without Blastocystis, ST10-colonized forest musk deer had higher bacterial richness and diversity, while ST5-colonized forest musk deer showed a comparable bacterial diversity. Likewise, beta diversity revealed significant differences in bacterial community structure between ST10-colonized and Blastocystis-free forest musk deer. The proportion of Bacteroidetes were significantly enriched in ST10-colonized forest musk deer. Bacterial community structure between ST5-colonized and Blastocystis-free forest musk deer did not differ significantly. The present study explored the associations between Blastocystis and gut microbial community of forest musk deer for the first time, and revealed ST10 colonization, instead of ST5, is associated with higher bacterial diversity and shifted microbial structure. Our data provides valuable insights into the associations between gut microbiomes and parasites. IMPORTANCE Forest musk deer is listed as an endangered species by International Union for Conservation of Nature Red List, and the Chinese government has introduced captivity breeding measures to curb the rapid decline of the musk deer population since the 1950s. It has been suggested that Blastocystis colonization can modulate the composition of the host's intestinal microbiota, thereby affecting the host health. The present study investigated the effects of the Blastocystis colonization on the gut microbiota in the feces of forest musk deer in Sichuan Province, China. Two subtypes (ST5 and ST10) have differential effects on the bacterial diversity and community composition, suggesting that the study of Blastocystis should be distinguished at the subtype level. Because the pathogenicity of Blastocystis is controversial, pathogenic, or commensal, continuous monitoring of the impact of Blastocystis colonization on the intestinal microbiota is of great significance to assess its health effects on forest musk deer.

Abstract Cladosporium cladosporioides causes asthma and superficial and deep infections, mostly in immunodeficient individuals and animals. This study aimed to investigate whether C. cladosporioides spores can enter the lungs through pulmonary circulation and influence pulmonary immune response. We intravenously injected mice with C. cladosporioides spore suspension and conducted several assays on the lungs. Pulmonary hemorrhage symptoms and congestion were most severe on days 1, 2, and 3 post-inoculation (PI). Extensive inflammatory cell infiltration occurred throughout the period of infection. More spores and hyphae colonizing the lungs were detected on days 1, 2, and 3 PI, and fewer spores and hyphae were observed within 21 d of infection. Numerous macrophages, dendritic cells, and neutrophils were observed on day 5 PI, along with upregulation of CD54, an intercellular adhesion molecule. Th1 and Th2 cells increased after infection; specifically, Th2 cells increased considerably on day 5 PI. These results suggest that days 2 and 5 PI represent the inflammatory peak in the lungs and that the Th2 and Th1 signaling pathways are potentially involved in pulmonary immune responses. In conclusion, the further adaptive immune responses played important roles in establishing effective pulmonary immunity against C. cladosporioides systemic infections based on innate immune responses.

Abstract Background Blastocystis sp. is an anaerobic protozoan that parasitizes many animal hosts and the human gastrointestinal tract, and its pathogenicity is controversial. Captive wildlife may be potential reservoirs for human infection with Blastocystis sp. The present study was performed to investigate the prevalence and subtype distribution of Blastocystis sp. in zoo animals in Sichuan Province, southwestern China. Methods A total of 420 fresh fecal samples were collected from 54 captive wildlife species in four zoos in southwestern China between June 2017 and September 2019. The prevalence and subtype (ST) genetic characteristics of Blastocystis sp. were determined by PCR amplification of the barcode region of the SSU rRNA gene and phylogenetic analysis. Results Overall, 15.7% (66/420) of the animal samples and 20.7% (14/54) of the species tested were shown to be infected with Blastocystis sp. The highest prevalence of Blastocystis sp. was found in Panzhihua Zoo (24.3%), which was significantly higher than that in Chengdu Zoo (6.9%), and Xichang Zoo (2.9%) (P

Abstract Background Microsporidia are common opportunistic parasites in humans and animals, including rabbits. However, only limited epidemiology data concern about the prevalence and molecular characterization of Enterocytozoon bieneusi and Encephalitozoon spp. in rabbits. This study is the first detection and genotyping of Microsporidia in pet rabbits in China. Results A total of 584 faecal specimens were collected from rabbits in pet shops from four cities in Sichuan province, China. The overall prevalence of microsporidia infection was 24.8% by nested PCR targeting the internal transcribed spacer (ITS) region of E. bieneusi and Encephalitozoon spp. respectively. E. bieneusi was the most common species (n = 90, 15.4%), followed by Encephalitozoon cuniculi (n = 34, 5.8%) and Encephalitozoon intestinalis (n = 16, 2.7%). Mixed infections (E. bieneusi and E. cuniculi) were detected in five another rabbits (0.9%). Statistically significant differences in the prevalence of microsporidia were observed among different cities (χ2 = 38.376, df = 3, P

Blastocystis sp. is a common eukaryotic parasite, which infects humans as well as various other animals. To date, epidemiological data regarding the detection rate and distribution of Blastocystis sp. subtypes in pet rodents are lacking in China; the present study aims to fill this gap. A total of 503 fecal samples collected from pets in different locations in southwestern China were screened for the presence of Blastocystis sp. using a nested PCR amplification of SSU rRNA method. Forty-two samples (8.35%) tested positive for Blastocystis sp. colonization. Two subtypes of Blastocystis sp. were identified based on nucleotide sequence homology and phylogenetic analysis: Blastocystis ST4 was present in 41 samples, and Blastocystis ST17 was found in 1 sample. Our results revealed robust host preference of Blastocystis ST4 and confirmed that Blastocystis ST17 can also parasitize rodents. Keywords: Blastocystis sp., Pet rodent, Subtype, China

Porcine deltacoronavirus (PDCoV) is an emerging porcine intestinal coronavirus in recent years, which mainly causes different degrees of vomiting and diarrhea in piglets and has caused great harm to the swine husbandry worldwide since its report. Selenium is an essential trace element for organisms and has been demonstrated to have antiviral effects. In this study, pig kidney epithelial (LLC-PK) cells were used to study the antiviral activity of selenomethionine (Se-Met) (2, 4, 8, and 16 μM) against PDCoV by detecting the replication of the virus, the expression of the mitochondrial antiviral signal protein (MAVS) protein, and the phosphorylation of interferon regulatory factor-3 (IRF-3), IFN-α, and IFN-β, and the changes in glutathione content, glutathione peroxidase, superoxide dismutase activity, and hydrogen peroxide content in the cells. The results showed that Se-Met at higher than physiological concentrations (16 μM) could significantly inhibit the replication of PDCoV in LLC-PK cells and enhance the expression of MAVS protein and the phosphorylation of IRF-3. In addition, Se-Met also improved the intracellular production of IFNα/β and antioxidant capacity with increasing doses. These data suggest that the availability of selenium through selenomethionine supports the antiviral response in porcine kidney cells, and the specific mechanism is attributed to the improved cellular antioxidant capacity and activation of the MAVS pathway by Se-Met.

Fecal microbiota transplantation (FMT) is a potential treatment for many intestinal diseases. In dogs, FMT has been shown to have positive regulation effects in treating Clostridioides difficile infection (CDI), inflammatory bowel disease (IBD), canine parvovirus (CPV) enteritis, acute diarrhea (AD), and acute hemorrhagic diarrhea syndrome (AHDS). FMT involves transplanting the functional components of a donor’s feces into the gastrointestinal tract of the recipient. The effective components of FMT not only include commensal bacteria, but also include viruses, fungi, bacterial metabolites, and immunoglobulin A (IgA) from the donor feces. By affecting microbiota and regulating host immunity, these components can help the recipient to restore their microbial community, improve their intestinal barrier, and induce anti-inflammation in their intestines, thereby affecting the development of diseases. In addition to the above components, mucin proteins and intestinal epithelial cells (IECs) may be functional ingredients in FMT as well. In addition to the abovementioned indications, FMT is also thought to be useful in treating some other diseases in dogs. Consequently, when preparing FMT fecal material, it is important to preserve the functional components involved. Meanwhile, appropriate fecal material delivery methods should be chosen according to the mechanisms these components act by in FMT.

Recent studies showed that Escherichia coli (E. coli) strains isolated from captive giant pandas have serious resistance to antibiotics and carry various antibiotic resistance genes (ARGs). ARGs or virulence-associated genes (VAGs) carried by antibiotic-resistant E. coli are considered as a potential health threat to giant pandas, humans, other animals and the environment. In this study, we screened ARGs and VAGs in 84 antibiotic-resistant E. coli strains isolated from clinically healthy captive giant pandas, identified the association between ARGs and VAGs and analyzed the phylogenetic clustering of E. coli isolates. Our results showed that the most prevalent ARG in E. coli strains isolated from giant pandas is blaTEM (100.00%, 84/84), while the most prevalent VAG is fimC (91.67%, 77/84). There was a significant positive association among 30 pairs of ARGs, of which the strongest was observed for sul1/tetC (OR, 133.33). A significant positive association was demonstrated among 14 pairs of VAGs, and the strongest was observed for fyuA/iroN (OR, 294.40). A positive association was also observed among 45 pairs of ARGs and VAGs, of which the strongest was sul1/eaeA (OR, 23.06). The association of ARGs and mobile gene elements (MGEs) was further analyzed, and the strongest was found for flor and intI1 (OR, 79.86). The result of phylogenetic clustering showed that the most prevalent group was group B2 (67.86%, 57/84), followed by group A (16.67%, 14/84), group D (9.52%, 8/84) and group B1 (5.95%, 5/84). This study implied that antibiotic-resistant E. coli isolated from captive giant pandas is a reservoir of ARGs and VAGs, and significant associations exist among ARGs, VAGs and MGEs. Monitoring ARGs, VAGs and MGEs carried by E. coli from giant pandas is beneficial for controlling the development of antimicrobial resistance.

Dermatomycosis is the second major cause of morbidity in giant pandas (Ailuropoda melanoleuca), and seriously endangers its health. Previous observations indicated that the occurrence of dermatomycosis in the giant panda varies in different seasons. The skin microbiota is a complex ecosystem, but knowledge on the community structure and the pathogenic potentials of fungi on the skin of the giant panda remains limited. In this study, samples from the giant panda skin in different seasons were collected, and the mycobiota were profiled by 18S rRNA gene sequencing. In total, 375 genera in 38 phyla were detected, with Ascomycota, Basidiomycota, Streptophyta, and Chlorophyta as the predominant phyla and Trichosporon, Guehomyces, Davidiella, Chlorella, Asterotremella, and Klebsormidium as the predominant genera. The skin mycobiota of the giant panda changed in the seasons, and the diversity and abundance of the skin fungi were significantly higher in spring, autumn, and summer than in the winter. Several dermatomycosis-associated fungi were detected as opportunists in the skin mycobiota of healthy giant pandas. Clinical dermatomycosis in the giant panda is observed more in summer and autumn. In this study, the results indicated that the high diversity and abundance of the skin fungi may have enhanced the occurrence of dermatomycosis in autumn and summer, and that dermatomycosis-associated fungi are the normal components of the skin mycobiota.

Enterocytozoon bieneusi, a common eukaryotic obligate intracellular parasite, can infect a wide range of hosts, including humans and domestic animals. There have been some reports of this organism in captive wildlife animals worldwide, but few studies have reported its detection in the captive black bears in Sichuan province of southwestern China. The present study was performed to determine the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in captive Asiatic black bears from three farms in Sichuan province. Fecal specimens from Asiatic black bears in three farms were collected and analyzed for the prevalence of E. bieneusi. The overall prevalence of E. bieneusi was 18.7% (57/305) as determined by nested PCR amplification of the Internal Transcribed Spacer (ITS) gene on the rRNA of E. bieneusi, with the highest prevalence in the farm being 47.8% (44/92). Altogether, five genotypes of E. bieneusi were identified among the 57 E. bieneusi-positive samples, comprising three known genotypes (SC02, MJ2, and MJ5) and two novel genotypes named SCBB1 and SCBB2. Phylogenetic analysis showed that the genotypes SC02 and MJ2 were clustered into group 1 of zoonotic potential and that the genotypes MJ5, SCBB1, and SCBB2 were clustered into group 10. In conclusion, two known genotypes, SC02 and MJ2, were found to belong to the zoonotic potential group 1 and this evidence points to the fact that the E. bieneusi from these black bears could infect humans.

Abstract Background Trichosporon is the dominant genus of epidermal fungi in giant pandas (Ailuropoda melanoleuca) and causes local and deep infections. To provide the information needed for the diagnosis and treatment of trichosporosis in giant pandas, the sequence of ITS, D1/D2, and IGS1 loci in 29 isolates of Trichosporon spp. which were isolated from the body surface of giant pandas were combination to investigate interspecies identification and genotype. Morphological development was examined via slide culture. Additionally, mice were infected by skin inunction, intraperitoneal injection, and subcutaneous injection for evaluation of pathogenicity. Results The twenty-nine isolates of Trichosporon spp. were identified as 11 species, and Trichosporon jirovecii and T. asteroides were the commonest species. Four strains of T. laibachii and one strain of T. moniliiforme were found to be of novel genotypes, and T. jirovecii was identified to be genotype 1. T. asteroides had the same genotype which involved in disseminated trichosporosis. The morphological development processes of the Trichosporon spp. were clearly different, especially in the processes of single-spore development. Pathogenicity studies showed that 7 species damaged the liver and skin in mice, and their pathogenicity was stronger than other 4 species. T. asteroides had the strongest pathogenicity and might provoke invasive infection. The pathological characteristics of liver and skin infections caused by different Trichosporon spp. were similar. Conclusions Multiple species of Trichosporon were identified on the skin surface of giant panda, which varied in morphological development and pathogenicity. Combination of ITS, D1/D2, and IGS1 loci analysis, and morphological development process can effectively identify the genotype of Trichosporon spp.

Plant-based natural products represent an alternative to chemical compounds for the control of mites in veterinary medicine. Here, the essential oil of Elsholtzia densa (E. densa) Benth was extracted using hydrodistillation at a rate of 1.2%. The chemical composition of the essential oil was determined by gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS analysis indicated that the principal compounds in the volatile oil of the sample were 4-Pyridinol (28.16%) and thymol (26.58%). The acaricidal activity of E. densa oil against Sarcoptes scabiei (S. scabiei) was tested in vitro. Toxicity test data were analysed using a complementary log-log (CLL) model. The E. densa oil was prepared in five concentrations by dilution with liquid paraffin (1, 2, 4, 8 and 16 mg/ml) and exhibited strong toxicity against S. scabiei with LT50 values of 16.637, 5.075, 2.884, 1.184 and 0.760 h, respectively. The LC50 values were 7.678, 4.623, 2.543, 1.502, 1.298 and 0.981 mg/ml for S. scabiei at 1, 2, 4, 8, 16 and 24 h, respectively. Compared to the control, the essential oil showed significant effects against S. scabiei in vitro. At 16 mg/ml, E. densa oil was found to kill all mites within a 16-h period. The results indicate that E. densa oil possesses potential acaricidal activity in vitro and may be exploited as a novel drug for the effective control of S. scabiei.

The giant panda is one of the rarest animals in the world. Skin diseases seriously endanger the health of giant panda and are considered the second major cause of its morbidity. Skin microbiota is a complex ecosystem, and the community structure and the pathogenic potential of bacteria on giant panda skin remain largely unclear. In order to understand the skin bacterial flora of captive giant pandas, the microbiota in giant panda skin samples collected during different seasons was profiled via 16S rRNA gene sequencing. In total, 522 genera from 53 bacterial phyla were detected, with Proteobacteria (40.5%), Actinobacteria (23.1%), Firmicutes (21.1%), Bacteroidetes (9.5%), Cyanobacteria (2.1%), and Thermi (1.2%) as the predominant phyla and Streptococcus (13.9%), Acinetobacter (9.2%), Staphylococcus (2.9%), Pseudomonas (5.9%), Dermacoccus (4.8%), Brachybacterium (2.9%), Escherichia (2.7%), Chryseobacterium (2.1%), Arthrobacter (1.6%), Kocuria (1.5%), Psychrobacter (1.2%), Deinococcus (1.1%), and Flavobacterium (1.1%) as the predominant genera. The results indicated that the diversity was lower in winter than in other seasons and higher in autumn than in other seasons, and the abundance in spring was significantly higher than that in other seasons. Several skin disease-associated bacteria were detected as opportunists in the skin microbiota of healthy giant pandas. In this study, the results indicated that the high diversity and abundance of the skin bacteria may have enhanced the occurrence of skin disease in autumn and spring and that skin disease-associated bacteria are the normal components of the skin microbiota.

Biofilm-related bacteria show high resistance to antimicrobial treatments, posing a remarkable challenge to human health. Given bacterial dormancy and high expression of efflux pumps, persistent infections caused by mature biofilms are not easy to treat, thereby driving researchers toward the discovery of many anti-biofilm molecules that can intervene in early stage biofilms formation to inhibit further development and maturity. Compared with mature biofilms, early stage biofilms have fragile structures, vigorous metabolisms, and early attached bacteria are higher susceptibility to antimicrobials. Thus, removing biofilms at the early stage has evident advantages. Many reviews on anti-biofilm compounds that prevent biofilms formation have already been done, but most of them are based on compound classifications to introduce anti-biofilm effects. This review discusses the inhibitory effects of anti-biofilm compounds on early stage biofilms formation from the perspective of the mechanisms of action, including hindering reversible adhesion, reducing extracellular polymeric substances production, interfering in the quorum sensing, and modifying cyclic di-GMP. This information can be exploited further to help researchers in designing new molecules with anti-biofilm activity.