Abstract:In recent years, the widespread prevalence of emergence infectious disease has brought unprecedented challenges to human life, social stability and economic development. The prevention and control of zoonoses have become a global focus. The important role of Veterinary Microbiology in public health and human health is highly valued. In order to showcase the latest research and application achievements in the field of zoonotic pathogenic bacteria (diseases), Microbiology China has organized the publication of the “Issue for Zoonotic Bacteria”, which includes reviews and research reports. The review summarizes the latest research advances and achievements of major zoonotic pathogens’ epidemic situation, the regulation of effector molecules, the discovery of diagnostic markers, and the exploration of new vaccine antigens; The research reports cover the development of pathogen diagnosis technology, cell culture and identification, probiotic isolation and identification, bacteriophage isolation and identification, molecular functional exploration, infection and immunity, etc. This thematic issue provides a communication platform for the development and progress of Veterinary Microbiology in China, and better promotes the exploration of new technologies and theories for the prevention and control of zoonotic diseases.

Abstract:Campylobacter jejuni is one of the most common foodborne pathogenic bacteria worldwide. Humans and animals infected by C. jejuni exhibit different clinical symptoms and pathological changes. Domesticated birds infected by this pathogen did not present clinical symptoms, becoming the main source of human infection. Elucidating the mechanisms of infection and colonization plays a key role in the prevention and control of C. jejuni. C. jejuni secretes effectors through the flagellar type III secretion system (T3SS). The effectors are injected into host cells and bind to intracellular proteins, thus regulating the signaling pathways and altering the defense mechanisms of host cells. Therefore, the effectors play an important role in the infection and colonization of C. jejuni. This article reviews the flagellar T3SS of C. jejuni, the types and mechanisms of T3SS effectors, and the roles of T3SS effectors in infection and colonization of C. jejuni. This review is of great significance for a deeper understanding of the disease-inducing mechanism of C. jejuni.

Abstract:African swine fever (ASF) is an acute infectious disease caused by African swine fever virus (ASFV). At present, there is no effective commercial vaccines at home and abroad, resulting in the difficulty in the prevention and control of ASF. Therefore, early diagnosis and regular epidemiological monitoring are essential for the prevention and control of ASF. The research on the diagnostic targets of ASF can provide a reference for the development of diagnostic technology. This article reviews the currently used diagnostic targets of ASF, aiming to provide a reference for the prevention and control of ASF and the development of efficient and feasible diagnostic technologies.

Abstract:Trained immunity, a functional state of the innate immune response, is characterized by epigenetic reprogramming and metabolic reprogramming of innate immune cells. Stimuli such as vaccines and microorganisms or their products can stimulate the trained immunity of innate immune cells, such as monocytes, neutrophils, and bone marrow stem cells. The re-stimulation can enhance the immune responses of innate immune cells to microbial pathogens. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and β-glucan (the main cell wall component of Candida albicans) can activate the nuclear factor-κB (NF-κB) pathway of innate immune cells or dendritic cell-associated C-type lectin 1/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor 1-alpha (Dectin-1/Akt/mTOR/HIF-1α) signaling pathway to regulate the metabolic reprogramming and epigenetic reprogramming, thereby inducing trained immunity. The discovery of trained immunity in innate immune cells provides a new direction for the research and development of new vaccines, the treatment strategies of immune deficiency, and the regulation of autoinflammatory diseases. Therefore, understanding the cytological mechanisms of trained immunity is of great significance for the prevention of unknown diseases.

Abstract:Brucella is a genus of globally widespread zoonotic pathogens capable of infecting wild animals, domestic animals, and humans. To establish and sustain chronic infections within the host, Brucella have evolved multiple strategies to evade host immune responses and extensively replicate in host cells, which primarily rely on the type Ⅳ secretion system (T4SS) and its secreted effectors. The T4SS functions by directly injecting effectors into host cells, modulating various host cell functions to help the pathogen evade immune surveillance, manipulate the intracellular environment, and promote its survival and replication. This article reviews the structure and function of the Brucella T4SS and the latest research progress in the role of Brucella T4SS effectors in regulating host cell functions, exploring how bacteria of Brucella manipulate host cell signaling pathways to control intracellular vacuole dynamics and establish a replicative niche conducive to bacterial survival. These advancements provide new insights into the pathogenesis of Brucella infections and aid in the development of more effective strategies for preventing and treating Brucella-related diseases.

Abstract:Zoonoses pose a serious threat to human health and affect animal husbandry and public health security. Vaccination is an important means of preventing the spread of these diseases. As a novel type of candidate vaccines, bacterial ghost-based vaccines demonstrate broad application prospects because they can induce strong humoral, cellular, and mucosal immune responses. Moreover, they are praised for the simple fermentation production and purification processes and high safety. This paper briefs the preparation technology and immune response-triggering mechanism of bacterial ghosts and reviews the research progress of bacterial ghosts in brucellosis, a major bacterial zoonosis in animals, aiming to provide valuable reference for the development of novel vaccines.

Abstract:Exosomes are the mediators of intercellular communication and play a key role in the spread and immune regulation of Brucella. This article elaborates on the regulatory role of exosomes in Brucella infection and discusses the application value of exosomes in diagnosis, vaccines, and drugs, aiming to provide reference for the application of exosomes in the prevention and control of intracellular bacteria such as Brucella.

Abstract:Brucellosis caused by Brucella infection is a major bacterial zoonosis, presenting a global prevalence situation. Brucellosis poses severe threats to the health of human and livestock, imposing substantial socio-economic burdens in certain regions. The invasion and establishment of chronic or even lifelong infection by Brucella in the hosts largely reply on its sophisticated immune evasion strategies and complex intracellular replication mechanisms. The manipulation of programmed cell death, such as apoptosis, pyroptosis, and autophagy, is a key strategy for the immune evasion of Brucella. Therefore, in-depth research on the ways Brucella regulates programmed cell death in host cells and their significance can provide valuable references for understanding the infection mechanism of Brucella and identifying potential drug targets. This paper summarizes the recent progress in the precise regulatory mechanisms and the “battle” between Brucella infection and various forms of programmed cell death in hosts.

Abstract:Zoonotic diseases, caused by the same pathogen and naturally transmitted between humans and vertebrate animals, have become a major global issue that urgently needs to be addressed. Currently, zoonotic diseases account for 60% of globally emerging and existing infectious diseases. The zoonotic diseases caused by Brucella, Mycobacterium bovis, Bacillus anthracis, Streptococcus suis serotype 2, Salmonella, and Klebsiella pneumoniae pose serious threats to the healthy development of animal husbandry, public health, and national biosafety. The situation for prevention and management of these diseases is getting increasingly severe. This article reviews the current prevalence of zoonotic bacterial diseases caused by the six pathogens mentioned above, as well as their antibiotic resistance mechanisms and novel antibacterial strategies, serving as a reference for the prevention and control of these diseases.

Abstract:Zoonoses are infectious diseases that naturally transmit between humans and vertebrates, accounting for 60% of all known human infectious diseases. These diseases pose threats to public health, food security, and biodiversity on a global scale. rapid and sensitive methods for early-stage detection are urgently needed and of great significance to controlling infection sources and preventing the spread of zoonoses. Electrochemical biosensors represent an innovative analytical technology that integrates electrochemistry with biotechnology, enabling precise qualitative and quantitative detection of target analytes. Moreover, they are praised for the simple operation, low costs, high specificity, and broad detection ranges. As a notable branch of electrochemical biosensors, electrochemical immunosensors have been extensively applied in clinical diagnosis, food safety test, and environmental monitoring. This article introduces the principles and classification of electrochemical immunosensors and reviews their advancements in detecting zoonotic bacteria, viruses, and parasites. In addition, this article summarizes the advantages and challenges of applying electrochemical immunosensors in the detection of zoonotic pathogens in livestock in China.

Abstract:[Background] Brucella canis, one of the six representative species of Brucella, is a zoonotic pathogen that poses a serious threat to the health of dogs. [Objective] To prepare the B. canis positive serum, providing the reference material for the development and application of diagnostic reagents for canine brucellosis. [Methods] Beagle dogs were immunized multiple times with the inactivated bacterial suspension (approximately 1.5×10¹⁰ CFU/mL) to obtain the positive serum of B. canis. By conducting the serum agglutination test, we calibrated the serum titer with the international standard serum provided by the European Union. We then used the standard positive serum prepared in this study as the reference material for quality control of the detection sensitivity of the B. canis antigen for rose bengal test and the B. canis indirect ELISA antibody detection kit developed by our laboratory. Furthermore, we used the above two methods to detect the B. canis antibody in the serum samples of pet dogs in Beijing and analyzed the coincidence rates of results between the two methods and the complement fixation test. [Results] The results of the serum agglutination test showed that the titer of B. canis positive serum was 2 400 IU/mL. The serum was diluted with the negative dog serum to reach a titer of 1 000 IU/mL, subpackaged, and freeze-dried to give the standard positive serum of B. canis. With the standard positive serum for quality control, the detection sensitivities of the B. canis antigen for rose bengal test and the B. canis indirect ELISA antibody detection kit developed by our laboratory were both 20 IU/mL. The positive rates obtained by both the B. canis antigen for rose bengal test and the complement fixation test was 6.25% and that obtained by the B. canis indirect ELISA antibody detection kit was 5.97%. The coincidence rates of results between the two detection methods and the complement fixation test were 94.31% and 94.60%, respectively. [Conclusion] The standard positive serum of B. canis that we prepared in this study provides a quality control sample for the development of diagnostic reagents for B. canis.

Abstract:[Background] Brucella, infectious bovine rhinotracheitis virus (IBRV), and Neospora caninum are major pathogens that cause abortions in dairy cows. Current serological methods for detecting the above pathogens are characterized by low specificity and complex operation, while PCR and RT-PCR methods are only capable of detecting single pathogens. Multiplex PCR enabling the detection of multiple pathogens at the same time greatly increases the detection efficiency. [Objective] To establish a multiplex PCR assay for rapidly detecting Brucella, IBRV, and N. caninum in clinical samples. [Methods] Specific primers were designed for Brucella omp25, IBRV gB, and N. caninum SRS2, and the annealing temperature, primer concentration, extension time, and number of cycles of the multiplex PCR assay were optimized. The specificity of the multiplex PCR assay was examined with the standard nucleic acids of Salmonella, Escherichia coli, Cryptosporidium, Toxoplasma gondii, and circovirus. The sensitivity of the multiplex PCR assay was tested with the recombinant plasmids carrying the target genes. Finally, the established assay was employed to test 55 vaginal swabs from cows suffering from abortions. [Results] The optimal conditions of the multiplex PCR assay were annealing temperature of 59 ℃, the extension time of 40 s, 30 cycles, and the primer concentration of 1 μmol/L. The lower limit of detection was 4×10¹ copies for omp25 and SRS2 and 4×10² copies for gB. The established assay showed no cross-reactivity with other pathogens. Nine out of 55 samples were detected positive for Brucella, 3 positive for IBRV, and 7 positive for N. caninum. Particularly, one sample was subjected to mixed infection by Brucella and N. caninum. [Conclusion] A multiplex PCR assay for three pathogens was successfully constructed, enabling rapid and accurate detection of clinical samples.

Abstract:[Background] Bovine tuberculosis (BTB) is a chronic infectious disease caused by Mycobacterium bovis, a part of the Mycobacterium tuberculosis complex, posing threats to cattle health, livestock economies, and public health. [Objective] In order to screen new diagnostic molecular markers of Mycobacterium bovis and establish new diagnostic method for bovine tuberculosis. [Methods] Using isotopic labeling-based relative and absolute quantification techniques and article screening, we identified 11 proteins (Mpb70, GroEL2, HspX, DnaK, Mpb83, EsxW, BfrB, Hrp1, EsxL, AtpD, and EsxN) for further evaluation. These recombinant proteins were then assessed for sensitization effects in a guinea pig model. [Results] At a dose of 100 μg, Mpb70, HspX, Mpb83, and EsxN induced marked delayed-type hypersensitivity (DTH) and rupture. At a dose of 50 μg, Mpb70, HspX, Mpb83, EsxW, EsxL, and EsxN provoked significant DTH. At a dose of 12.5 μg, the proteins did not induce DTH. Among the protein combinations, EsxW/EsxL/EsxN showed the highest activity, triggering robust DTH in guinea pigs. Other combinations such as EsxW/EsxL, EsxW/EsxN, and EsxL/EsxN also induced DTH, though to a lesser extent than EsxW/EsxL/EsxN. [Conclusion] Recombinant proteins Mpb70, HspX, Mpb83, EsxW, EsxL, and EsxN elicited notable DTH, demonstrating the potential as tuberculin substitutes.

Abstract:[Background] Streptococcus suis type 2 (SS2) is a major zoonotic pathogen leading to global economic and public health problems, causing meningitis, arthritis, and sepsis. Phosphopantothenoylcysteine decarboxylase (PPCDC) was screened out in the previous study about the interaction between the porcine blood-brain barrier model in vitro and S. suis genome-wide phage display random library, while the mechanism of PPCDC in affecting the blood-brain barrier remains unclear. [Objective] To explore the role of PPCDC in the SS2-induced meningitis. [Methods] In this study, the recombinant protein rPPCDC was expressed in Escherichia coli. The monolayer barrier model of human brain microvascular endothelial cells in vitro and the mouse infection model in vivo were established to study the effect of rPPCDC on the permeability of blood-brain barrier. Furthermore, the rPPCDC vaccine was prepared for the immunoprotection test in mice. [Results] The soluble recombinant protein rPPCDC was successfully purified. This protein reduced the transmembrane resistance of cells in the monolayer barrier model, increased the number of SS2 breaking through the monolayer barrier in vitro. In addition, the protein enhanced the pathogenicity of SS2 in mice. The mice vaccinated with rPPCDC had higher serum levels of rPPCDC antibodies and increased survival. [Conclusion] PPCDC can enhance the permeability of blood-brain barrier and promote the destruction of blood-brain barrier and brain infection of SS2 in mice, thus exerting the immunoprotective effect. The findings provide new ideas for the future research on the pathogenic mechanism of SS2 and the development of vaccines.

Abstract:[Background] Streptococcus suis serotype 2 (SS2) is a significant zoonotic pathogen responsible for severe infections in pigs and humans, resulting in pneumonia, septic shock, and even death. [Objective] Despite the diverse virulence factors identified, the underlying pathogenic mechanism of SS2 remains unclear. Therefore, it is urgent to search for new virulence factors or virulence-related genes for elucidating the pathogenic mechanism. [Methods] An insertion mutant with significantly attenuated pathogenicity in Galleria mellonella larvae was selected from the Tn917 transposon insertion mutant library of SS2, and its mutated gene was identified as B9H01_RS10315, which encoded a glycosyl hydrolase (ghA). An isogenic deletion mutant of ghA (∆ghA) and its complementary strain (C∆ghA) were constructed, and the growth and pathogenicity of the mutants were determined to reveal the pathogenic role of ghA. [Results] Compared with the wild type, ∆ghA displayed a growth defect, a 25% decrease in mortality of Galleria mellonella larvae within 24 h, a reduction of about 50% in adhesion to epithelial cells, a decrease of about 35% in survival in whole blood, and increases of about 100% and 200% in phagocytosis by phagocytes at 10:1 and 50:1 infection ratios, respectively. [Conclusion] The glycosyl hydrolase GhA promotes the growth and participates in the pathogenic process of SS2 by promoting the adhesion, improving the survival in blood, and resisting phagocytosis.

Abstract:[Background] Campylobacter is a genus of major foodborne pathogens that colonize the chicken intestine at high levels. The chicken innate immune response to Campylobacter colonization remains to be fully elucidated. [Objective] To investigate the activity of extracellular fatty acid binding protein (Ex-FABP) related to chicken innate immunity in inhibiting the growth of Campylobacter by specifically binding to enterobactin, providing a theoretical basis for revealing the function of the host innate immunity against Campylobacter infection. [Methods] We knocked out the entB gene from Escherichia coli BL21(DE3) to construct a strain that did not secrete enterobactin. Chicken Ex-FABP and human lipocalin-2 (Lcn2) were expressed in the engineered strain and purified. Western blotting was employed to examine the binding of the expressed proteins to enterobactin. The inhibitory activities of Ex-FABP and Lcn2 against E. coli, Salmonella, and their mutant strains with entB knockout were verified under iron-rich and iron-limited culture conditions, respectively. Then, the inhibitory activities of Ex-FABP and Lcn2 against Campylobacter derived from different hosts were examined by addition of enterobactin. [Results] Ex-FABP and Lcn2 were successfully expressed in the constructed BL21(DE3) mutant with entB knockout. The in vitro binding assays confirmed that the expressed proteins could specifically bind to enterobactin. Under the iron-limited culture condition, Ex-FABP and Lcn2 significantly inhibited the growth of E. coli AN102 capable of secreting enterobactin but had no inhibitory effects on E. coli ATCC25922, Salmonella CVCC1806/CVCC1800, and corresponding mutant strains with entB knockout. Finally, it was confirmed that Ex-FABP and Lcn2 inhibited the growth of five Campylobacter strains derived from different host species by specifically binding to enterobactin. [Conclusion] Chicken Ex-FABP has a similar function to Lcn2. Under iron-limited conditions, it can specifically bind to enterobactin and inhibit bacterial growth by blocking the enterobactin-mediated iron uptake, thus triggering the host innate immune function. The findings provide a theoretical basis for developing new approaches to control Campylobacter.

Abstract:[Background] Caseinolytic protease L (ClpL), a member of the heat shock protein (HSP) 100 family, is involved in various cellular processes, such as the stress tolerance response, long-term survival, virulence, and antibiotic resistance, playing important roles in the infection of pathogenic bacteria. [Objective] The present study aimed to elucidate the contributions of HSP100/ClpL to the stress tolerance and virulence of Streptococcus suis type 2. [Methods] We used the thermo-sensitive suicide vector pSET4s and the Escherichia coli-Streptococcus suis shuttle vector pSET2 to construct an isogenic clpL mutant strain (ΔclpL) and a complemented strain (CΔclpL), respectively. We then compared the growth characteristics of the wild-type strain SS2-1, ΔclpL, and CΔclpL under various stress conditions (including high temperature, acidic environment, and oxidative stress) in vitro and in pig whole blood. Furthermore, we employed a mouse model of infection and challenged mice with SS2-1+ΔclpL or SS2-1+CΔclpL to compare the virulence of SS2-1, ΔclpL, and CΔclpL. [Results] The mutant strain ΔclpL showed reduced survival rates under stress conditions and in pig whole blood. In addition, it showed attenuated virulence in BalB/C mice. Furthermore, ΔclpL had significantly lower colonization ability than SS2-1 in mice. [Conclusion] The data indicate that the SsClpL is involved in the stress response and pathogenicity of SS2.

Abstract:[Background] Campylobacter jejuni, one of the major foodborne pathogens worldwide, can colonize the cecum of poultry, becoming the main source of infection in humans. Currently, the mechanism of C. jejuni colonization in avian intestine has not been elucidated. [Objective] To investigate the roles of different Cia effectors of C. jejuni in the adhesion and invasion of intestinal epithelial cells and immune cells, as well as the activation of inflammatory cytokines, thus elucidating the mechanism of C. jejuni colonization in chicken intestine and providing potential targets for the prevention and control of this pathogen. [Methods] Firstly, homologous recombination was employed to construct gene knockout mutants of different effectors (CiaB, CiaC, CiaD, and CiaI) of the wild-type strain (WT, C. jejuni NCTC 11168). The effects of cia knockout on the biofilm formation and autoagglutination were determined. Subsequently, the adhesion and invasion of cia mutants on intestinal epithelial cell line Caco-2, as well as their invasion in the chicken macrophage line HD11, were determined. Finally, RT-qPCR was employed to determine the expression levels of inflammatory cytokines in HD11 cells infected by the mutants. [Results] The cia gene knockout mutants of C. jejuni were successfully constructed. Compared with WT, ∆ciaD showed reduced biofilm formation, while ∆ciaC, ∆ciaD, and ∆ciaI demonstrated enhanced autoagglutination. The mutant ∆ciaB showcased enhanced adhesion and invasion of Caco-2 cells, while ∆ciaC, ∆ciaD, and ∆ciaI exhibited reduced abilities to adhere and invade Caco-2 cells and invade HD11 cells. The infection of HD11 cells by C. jejuni WT significantly up-regulated the expression levels of different inflammatory cytokines, while ∆ciaB, ∆ciaC, and ∆ciaD showed down-regulated expression levels of different inflammatory cytokines. In contrast, ∆ciaI presented up-regulated expression levels of different inflammatory cytokines. [Conclusion] Different Cia effectors of C. jejuni play different roles in the adhesion and invasion of intestinal epithelial cells and immune cells, as well as the activation of inflammatory cytokine expression. The findings provided a theoretical basis for understanding the interaction mechanism between C. jejuni and host intestinal epithelium, and mining potential targets for the prevention and control of this bacterium.

Abstract:[Background] Brucella spp. are facultative intracellular pathogens causing a worldwide zoonosis, brucellosis. Streptomycin is the recommended antibiotic for treating brucellosis, while streptomycin-resistant isolates have been identified in China (according to the breakpoint recommended by CLSI). [Objective] To screen and identify new genes associated with streptomycin resistance in Brucella melitensis. [Methods] We employed RNA sequencing (RNA-seq) to mine new genes associated with streptomycin resistance in B. melitensis. Homologous recombination and molecular docking were employed to predict and identify the functions of the relevant genes. [Results] B. melitensis M5 restored its proliferation after 12 h of culture in the medium supplemented with 2×MIC of streptomycin. The results of RNA-seq revealed that cell membrane components played a role in the resistance to low-concentration streptomycin. Under streptomycin stress, the expression of genes involved in the ribosome pathway was up-regulated [|log₂FC|≥2.0, P<0.05], while that of genes involved in quorum sensing and type IV secretion system was down-regulated (|log₂FC|≥2.0, P<0.05). The MICs of streptomycin for virB3- and virB5-deleted strains were higher than that for M5, while the MICs of streptomycin for complemented strains were not significantly different from that for the wild-type strain. Molecular docking results demonstrated that VirB3 and VirB5 could bind to streptomycin through hydrogen bond. [Conclusion] Streptomycin mainly affected pathways related to cell membrane components of B. melitensis. B. melitensis develops resistance to streptomycin by down-regulating the expression of virB3 and virB5 of the type IV secretion system. This study provides new insights into the research on the antibiotic-resistant strains of Brucella spp. and offers candidate targets for the development of new agents for treating brucellosis.

Abstract:[Background] Mycobacterium tuberculosis variant bovis is a zoonotic pathogen capable of causing tuberculosis in both humans and animals. [Objective] To construct a mutant library of M. tuberculosis var. bovis with M. tuberculosis var. bovis C68001 as the maternal strain and the MycoMar T7 transposon system, providing a technical platform for further investigation of the gene functions and pathogenic mechanisms of M. tuberculosis var. bovis. [Methods] The whole genome sequence of M. tuberculosis var. bovis C68001 was systematically analyzed. The temperature-sensitive phages were screened and the conditions of phage proliferation and infection were optimized. Finally, a mutant library with high storage capacity for M. tuberculosis var. bovis C68001 was obtained. [Results] Based on Nanopore third-generation and second-generation sequencing technology, the whole genome of C68001 was 4.342 Mb in size and contained 4 093 genes. A temperature-sensitive phage with a titer higher than 1×10¹¹ PFU/mL was successfully screened. After infecting C68001 at MOI=10 for 3 h, 9 216 monoclonal transposon mutants were obtained. Seven clones were randomly selected for PCR, which confirmed that transposons were successfully inserted into C68001. [Conclusion] The mutant library of M. tuberculosis var. bovis C68001 was successfully constructed.

Abstract:[Background] Porcine transmissible pleuropneumonia is a respiratory disease caused by Actinobacillus pleuropneumoniae infection, and the mortality rate of the disease can reach 80%–100%. The ISG15 protein inhibits viral replication, but its study in Actinobacillus pleuropneumoniae is unknown. [Objective] To build a prokaryotic expression vector for porcine ISG15, purify the protein, and study its effect on Actinobacillus pleuropneumoniae (APP) infection. [Methods] A prokaryotic expression vector pET-28a-ISG15 was constructed for the expression of ISG15. Flow cytometry and fluorescence quantitative PCR were used to detect the death of alveolar macrophages and the expression of inflammatory cytokines after ISG15 treatment. A mouse model of APP infection was established. After intranasal administration of ISG15, the body weight, clinical symptoms, and survival of the mice were measured to analyze the influence of ISG15 on the pathogenicity of APP. [Results] ISG15 was obtained successfully after prokaryotic expression and purification. The death of alveolar macrophages and the expression levels of inflammatory cytokines interleukin-6 (IL-6) and interferon-gamma (IFN-γ) were increased after ISG15 treatment (P<0.05). Compared with the APP challenge group, the mice treated with medium- and high-dose ISG15 post-infection showed severe clinical symptoms, increased mortality, and up-regulated expression levels of IL-6, IFN-γ, and IL-1β in the lung tissue. [Conclusion] ISG15 aggravates the APP infection by promoting the death of alveolar macrophages and the expression of inflammatory cytokines. The findings provide a theoretical basis for developing methods for the prevention and treatment of APP.

Abstract:[Background] Acinetobacter spp. are common opportunistic pathogens of hospital infection, and the emergence of drug-resistant strains of Acinetobacter poses a serious threat to public health. [Objective] To understand the prevalence and drug resistance of Acinetobacter spp. in the pet hospital environments in Shanghai. [Methods] Environmental samples were collected from pet hospitals in Shanghai, and the isolated strains were identified by a mass spectrometer for bacterial identification. The minimum inhibitory concentration (MIC) was determined by the broth dilution method, and then the genome sequence and genetic characteristics of a carbapenem-resistant isolate were analyzed. [Results] A total of 124 strains of Gram-negative bacteria were isolated from 730 environmental samples, and 14 strains were identified as Acinetobacter spp., of which 9 strains were A. lwoffii. All the 14 Acinetobacter strains were sensitive to minocycline. Two A. pittii strains and two A. lwoffii strains demonstrated multidrug resistance to carbapenems. Five A. lwoffii strains were resistant to sulfamethoxazole trimethoprim, and one A. johnsonii strain was resistant to ciprofloxacin. The results of genome sequencing showed that the A. pittii strain with multidrug resistance to carbapenems carried drug resistance genes such as NDM-1, OXA-500, and OXA-727. The multilocus sequence typing (MLST) results showed that the isolate was of ST1037 and the single nucleotide polymorphism (SNP) analysis revealed that the isolate was close to A. pittii AP43 isolated from clinical urine in Zhejiang. [Conclusion] The drug resistance rate of Acinetobacter spp. in the environments of five pet hospitals in Shanghai was <40%, and there was multi-drug resistance (Five Acinetobacter strains were resistant to ≥3 drugs). A. pittii carried multiple drug resistance genes.

Abstract:[Background] Diarrhea is one of the main causes of death in newborn calves, and Escherichia coli, as an opportunistic pathogen, is the main pathogen of intestinal infection in newborn calves. Although antibiotics are commonly used to treat this pathogen, the multiple serotypes of E. coli and the enhancing resistance to multiple antibiotics have brought great difficulties to treatment. [Objective] To investigate the clinical symptoms of E. coli-induced diarrhea and the virulence genes, serotypes, drug resistance, and phylogenetic characteristics of E. coli in calves in Jilin Province. [Methods] PCR, drug sensitivity test, and other methods were employed to determine the serotypes, phylogeny, virulence factors, and drug resistance of E. coli. [Results] A total of 92 strains of E. coli were isolated from 150 samples, with the detection rate of 61.3%. The main virulence genes of intestinal pathogenic E. coli were K88 (10%), K99 (48%), F17 (85%), F41 (20%), fimH (20%), csgA (87%), eaeA (4.5%), bfpA (5%), hlyA (4.5%), hlyE (61.8%), stx1 (22%), stx2 (34%), fyuA (3%), STa (15%), and STb (92%), while F6 and F18 were not detected. Among the isolates, 86% (79/92) were of O-antigen serotypes, among which O101 (32%) was the dominant serotype, followed by O103 (15%). Most of the isolates had multidrug resistance, with the resistance rates to ampicillin, tetracycline, gentamicin, and streptomycin being 52.7%, 53.3%, 40.7%, and 50%, respectively. The strains carried ampC (45.21%), bla_CTX (42.47%), bla_TEM (56.85%) of β-lactams, tetA (55.48%) of tetracyclines, and addA (55.48%), aph(3')-Ia (43.15%), and aphA3 (30.22%) of aminoglycosides. Among the strains, 36 (24%), 27 (18%), 6 (4%), and 20 (13.3%) strains belonged to groups A1, B1, B2, and D, respectively. [Conclusion] The infection rate of E. coli causing diarrhea in calves in Jilin Province is serious, and most of the pathogen strains belonged to A1 and B1 groups. O101 was the dominant serotype, and the isolates carried a variety of virulence genes and drug resistance genes, demonstrating serious multidrug resistance. Feeding management and vaccine immunization should be strengthened for calves, and the use of antibiotics should be reduced.

Abstract:[Background] In our previous study, we passaged the virulent strain NP03 of novel duck reovirus (NDRV) on muscovy duck embryo fibroblast (MDEF) cells and obtained an attenuated strain NDRV S. Compared with that of the virulent strain, the p18 protein of NDRV S misses 35 residues at the C-terminus. [Objective] To prepare anti-p18 polyclonal antibodies and identify the differences in p18 between the virulent and attenuated strains of NDRV. [Methods] RT-PCR was employed to amplify the p18 gene sequences of the virulent and attenuated strains of NDRV. The obtained sequences were then cloned into the prokaryotic expression vector pET-28a for induced expression. The target proteins were purified by nickel columns and identified by SDS-PAGE and Western Blot. The polyclonal antibodies were prepared by immunizing the BALB/c mice with the purified proteins, and the antibodies were detected and identified by indirect ELISA, Western Blot, and indirect immunofluorescence assay (IFA). [Results] The SDS-PAGE and Western Blot results showed that the recombinant p18 proteins of the virulent and attenuated strains of NDRV were obtained. The indirect ELISA result showed that the serum titer of the polyclonal antibodies was 1:51 200. The result of Western Blot demonstrated that the prepared polyclonal antibodies could specifically detect the p18 proteins expressed by cells infected with virulent and attenuated strains. The molecular weights of the p18 proteins of virulent and attenuated strains varied, being 18 kDa and 14 kDa, respectively. The IFA results confirmed that the prepared polyclonal antibodies could recognize p18 proteins expressed in cells infected with virulent and attenuated strains. [Conclusion] We successfully prepared the anti-p18 polyclonal antibodies and identified the significant differences in its composition between virulent and attenuated strains of NDRV. The findings lay a solid foundation for investigating the biological function of p18 as well as the pathogenic mechanism of NDRV.

Abstract:[Background] In recent years, efforts have been made to protect wildlife and improve the eco-environment. Accordingly, the number of wild boars has been increasing year by year, and it has even exceeded the number of protection in some areas, which has caused a certain impact on agricultural production. Wild boars have strong disease resistance, environmental adaptability, and rich gut microbiota, demonstrating a potential value of improving the quality of domestic pigs. However, there are few reports on the research and development of gut microbiota of wild boars in China. [Objective] To explore the diversity of gut microbiota in wild boars and isolate strains with good probiotic performance from the gut based on the omnivorous habit and disease resistance of wild boars. [Methods] Five feces samples were collected from wild boars, foreign three-way cross hybrid pigs, and commercial pigs of new American pig lines, respectively. Bacillus strains with cellulase activity were isolated from the guts of wild boars and tested for their probiotic properties such as acid tolerance and enzyme production. [Results] The alpha diversity of gut microbiota in wild boars and foreign three-way cross hybrid pigs was higher than that in the pigs of new American pig lines, and the richness of gut microbiota was the highest in wild boars. At the phylum level, Firmicutes, Bacteroidota, and Fusobacteriota had the highest abundance in the guts of wild boars, foreign three-way cross hybrid pigs, and pigs of new American pig lines, respectively. At the genus level, unclassified_f_Lachnospiraceae, UCG-005, Prevotelaceae_NK3B31_group, and Ruminococcus had high abundance in the guts of wild boars. The β diversity clustering analysis results showed that the similarity of gut microbiota between the wild boar samples was the lowest among the three groups of samples. Several strains of Bacillus velezensis isolated from the guts of wild boars exhibited high cellulase and amylase activities and acid tolerance. [Conclusion] The richness of gut microbiota in wild boars is higher than that in commercial pigs, and several strains of B. velezensis isolated from the guts of wild boars have the potential to be developed as probiotic preparations.

Abstract:[Background] Lactic acid bacteria as probiotics can be developed as microecological preparations for animals. The lactic acid bacteria in the gut of Bombyx mori, a model insect of Lepidoptera, remain to be studied. [Objective] To isolate the lactic acid bacteria with good biological properties from the gut of B. mori for developing microecological preparations. [Methods] The lactic acid bacteria isolated from the gut of B. mori were screened based on morphological, physiological, and biochemical characteristics and identified by 16S rRNA gene sequencing. The safety of the strain screened out was evaluated by the hemolysis test, and the growth characteristics and acid production of the strain were determined by growth curve and acid production curve. The stress tolerance of the strain was determined by temperature and pH sensitivity tests. The adhesion of the strain was evaluated based on self-aggregation, hydrophobicity, and coagglutination with pathogens. Furthermore, the drug resistance of the strain was examined by the drug sensitivity test, and the drug resistance genes carried by the strain were detected. [Results] Among the 56 strains isolated, one strain A2 was screened out and identified as Enterococcus farcalis. The strain did not present hemolysis, experienced rapid growth and acid production 3–9 h after inoculation. It could survive at a high temperature of 80 ℃. The survival rate of the strain decreased in the case of below pH 4.0 and was over 78% at pH 10.0, which indicated that the strain had strong alkaline tolerance. The strain demonstrated strong adhesion, with the self-aggregation of 18%, the hydrophobicity of 70%, and the coagglutination of 32%, 34%, and 40% with Escherichia coli, Salmonella, and Staphylococcus aureus, respectively. It was resistant to ampicillin, erythromycin, and vancomycin, moderately sensitive to gentamicin, and sensitive to sulfafurazole, chloramphenicol, tetracycline, and ofloxacin. The strain carried five drug resistance genes conferring resistance to four categories of antibiotics: β-lactam (bla_SHV and bla_TEM), chloramphenicol (cat), aminoglycosides (aph(3')-IIa), and tetracycline (tetE). [Conclusion] The lactic acid bacterial strain isolated from the gut of B. mori was safe, thermostable, and alkali-tolerant and it had strong adhesion. Therefore, it served as a candidate strain for the subsequent development of microbial fermented feed and microecological preparations for silkworm. This study provided a scientific basis and high-quality resources for the development and utilization of gut probiotics.

Abstract:[Background] The innate immune system is the first defense line and plays a key role in the host defense against microbial early infection. Enhancing the innate immunity from trained immunity is an effective way to improve the resistance of animals to diseases. [Objective] To isolate the bacteria capable of inducing trained immunity from healthy porcine intestinal flora and thus provide new insights for the prevention and control of microbial diseases by trained immunity. [Methods] Bacteria were isolated from healthy pig feces, and the strains capable of inducing trained immunity were screened with Galleria mellonella, a well-established model. The strain screened out was identified by 16S rRNA gene sequencing and characterized by growth curve. Its capability of inducing trained immunity was then evaluated in a mouse model. [Results] A strain SC-2 capable of inducing trained immunity in G. mellonella was identified as Enterococcus faecium. Administration of SC-2 significantly increased the percentages of neutrophils and macrophages in the peripheral blood and lung, decreased the bacteria loads in main organs, and increased the survival rate of mice infected by Streptococcus suis. [Conclusion] SC-2 had the effect of inducing trained immunity in mice. Our study provides new insights for immune regulation and disease treatment from the perspective of trained immunity.

Abstract:[Background] Actinobacillus pleuropneumoniae (APP) causes severe inflammatory damage to the lungs. Cellular oxidative stress is closely linked to lung inflammatory damage, while the relationship between APP infection and cellular oxidative stress remains unclear. [Objective] To clarify the relationship between APP infection and cellular oxidative stress and explore its mechanism, thereby laying a foundation for elucidating the pathogenic mechanism of APP. [Methods] After infecting porcine alveolar macrophages (PAMs) with APP wild-type strain 5b (5b WT) and Adh-deleted strain (5bΔAdh), respectively, we observed the levels of intracellular reactive oxygen species (ROS) using fluorescence microscopy, and determined the expression of oxidative stress-related molecules by RT-qPCR and Western blotting. Cells were treated with the NLRP3 inflammasome inhibitor MCC950 and then infected with 5b WT and 5bΔAdh, respectively. Biochemical methods were employed to measure the intracellular lactate and pyruvate levels. Flow cytometry was employed to determine the ROS levels and changes in mitochondrial membrane potential. Additionally, TMT-labeled quantitative proteomics sequencing was conducted to analyze changes in cellular protein expression and detect the release of mitochondrial DNA (mtDNA). [Results] Results from immunofluorescence and flow cytometry indicated that the 5b WT group had higher levels of ROS than the 5b ΔAdh group, and this trend was reversed following MCC950 treatment. Biochemical analysis further revealed that the 5b WT group had significantly lower levels of pyruvate and lactate, which elevated again after NLRP3 was inhibited with MCC950. Additionally, the PAMs infected by 5b WT and 5bΔAdh showcased notable declined levels of Nrf2 and GCLM and up-regulated expression of NOX2, with a more pronounced up-regulation observed in the 5b WT group. Proteomics analysis indicated significant changes in the expression of 15 proteins. GO analysis revealed that these differentially expressed proteins were mainly involved in key biological processes such as ATP metabolism, purine metabolism, and inflammation. KEGG analysis predicted that these proteins were involved in inflammation regulation, energy transport, and oxidative phosphorylation. Notably, 5b WT and 5bΔAdh infections increased the proportions of PAMs with mitochondrial damage to 69.6% and 58.5%, respectively, with mtDNA release into the cytoplasm. [Conclusion] APP induced oxidative stress in PAMs via Adh, and the activation of NLRP3 inflammasome participated in the generation of cellular oxidative stress.

Abstract:[Background] Avian pathogenic Escherichia coli (APEC) could cause inflammatory conditions such as avian airsacculitis and pericarditis, which seriously restricts the healthy development of the poultry industry. Meanwhile, APEC and human extraintestinal pathogenic Escherichia coli (ExPEC) have similar virulence factors, posing a serious threat to public health. Biofilm formation can enhance the antibiotic resistance of APEC and help it escape from the host immune system. Therefore, exploring the key genes and regulatory mechanisms of the biofilm formation of APEC are of great theoretical and practical significance for the prevention and control of APEC. [Objective] To elucidate the role of the outer membrane protein CsgG in the biofilm formation of APEC, reveal the molecular mechanism of its biofilm formation, and provide a theoretical basis for the prevention and control of avian diseases associated with APEC. [Methods] The csgG-deleted strain ΔcsgG and its complementary strain CΔcsgG were constructed by Red homologous recombination. The strains were then used to study the effects of csgG on the growth, motility, membrane permeability, extracellular polysaccharide (EPS) production, and biofilm formation of APEC. [Results] The deletion of csgG did not impact the growth or membrane permeability but enhanced the motility of APEC. The deletion of csgG significantly reduced the biofilm formation of APEC. Scanning electron microscopy revealed that the biofilm changed from multilayers to monolayers after the deletion of csgG, with reduced intercellular adhesion. Confocal laser microscope revealed that the deletion of csgG led to a loose structure of the biofilm and a decrease in the number of adherent bacteria. Furthermore, the deletion of csgG significantly reduced the EPS production of APEC. The RT-qPCR results showed that the deletion of csgG led to significant reductions in the transcript levels of genes related to biofilm formation. [Conclusion] CsgG is a pivotal factor in the biofilm formation of APEC, influencing the structural integrity of the biofilm.

Abstract:[Background] Although a variety of human and murine cell lines have been utilized as models for Brucella infection in vitro, there remains a lack of large animal cell models that can accurately simulate the infection process. [Objective] To obtain a stable passable goat alveolar macrophage line with single cloning characteristics and optimize the goat cell infection model of Brucella infection. [Methods] Goat alveolar macrophages were isolated by perfusing goat lungs with PBS under sterile conditions. An immortalized goat alveolar macrophage cell line was established by transfection with SV40 large T antigen (SV40LT) and puromycin resistance screening. The characteristic surface marker of mononuclear macrophages, CD₁₄, was detected by immunofluorescence and flow cytometry. The phagocytic ability of the established cell line was assessed with chicken red blood cells. Subsequently, the cell line was infected with Brucella vaccine strain M5, and the intracellular bacteria were counted at different time points. [Results] The alveolar macrophage cell line of goat was successfully isolated and constructed, and it had typical macrophage morphological characteristics, showing an irregular shape, pseudopodium protruding, large cells, and strong phagocytosis. After 10 d of cultivation, 95.40% of the primary cells specifically expressed CD₁₄, and 92.4% of the cells specifically expressed CD₁₄ after immortalization. Compared with the other three cell lines, the immortalized alveolar macrophage cells were sensitive to Brucella, with the sensitivity similar to that of RAW264.7 cells and higher than that of sheep testicular interstitial cell (STIC). [Conclusion] An immortalized goat alveolar macrophage cell line was successfully established, providing an in vitro experimental object for studying the pathogenicity and immune escape mechanism of Brucella.

Abstract:[Background] Mycobacterium is a genus of slender, slightly curved, and branching bacilli, some of which seriously threaten human and animal health. Bacteriophages have emerged as a substitute of antibiotics because of their high efficiency, high specificity, and easy preparation. [Objective] To isolate and identify bacteriophages of Mycobacterium smegmatis, analyze their biological characteristics, and evaluate their adaptability in different environments and their antibacterial activity in vitro. [Methods] Using M. smegmatis mc²155 as the host, we isolated virulent bacteriophages from dairy farm wastewater, feces, soil, and bedding and studied their biological characteristics. [Results] Four strains of M. smegmatis bacteriophages were isolated, identified, and named MP2201, MP2202, MP2203, and MP2204, with titers of 1×10⁸, 5×10⁸, 3×10⁸, and 1×10⁸ PFU/mL, respectively. Transmission electron microscopy results showed that the four strains were long-tailed bacteriophages. The optimal multiplicity of infection of MP2204 was 1, while that of the other three strains was 0.1. Strain MP2201 had the highest burst size, which reached 500 PFU/cell. All the four strains remained stable at 40−60 ℃, and their titers did not change significantly within the range of pH 3.0–11.0. Preparing a bacteriophage cocktail significantly inhibited the growth of the host strain. Whole genome sequencing of the phages revealed that MP2201, MP2202, MP2203, and MP2204 had the genome lengths of 69 720, 68 282, 47 622, and 70 950 bp, in which the corresponding G+C content was 59.14%, 66.58%, 63.22%, and 59.02%, respectively. Notably, none of these genomes harbored antibiotic resistance genes. [Conclusion] Four strains of M. smegmatis bacteriophages were successfully isolated and identified, which provided biological materials and a theoretical basis for the development of new biocontrol methods for mycobacterial infection.

Abstract:[Background] Since 2015, disease outbreaks have occurred in Cherry Valley ducks and other commercial meat ducks in the eastern coastal areas of China and have gradually spread inland. The diseased ducks present short beaks, enlarged tongues, stunted growth, and easy fractures of leg bones. The pathogenic agent of this disease was identified as novel duck parvovirus (NDPV). [Objective] To reveal the genetic evolution of NDPV by epidemiological investigation, so as to provide a basis for understanding the genetic evolution laws, enriching the epidemiological data, and deciphering the pathogenic mechanism of NDPV. [Methods] The liver of a diseased duckling was grinded and the supernatant was obtained by centrifugation of the homogenate. The SPF-grade duck embryo was used for virus isolation and the virus titer was measured. PCR was employed to identify the isolated virus and detect exogenous viruses. The whole genome of the isolated virus was amplified and analyzed for the sequence homology. Molecular characteristics and genetic evolution of the VP1 protein were analyzed. [Results] A NDPV strain, SDGT0628, was isolated from a duck farm with suspected short beak and dwarfism syndrome (SBDS) in Shandong Province. The strain was cultured with SPF-grade duck embryos, with the median lethal dose in duck embryos (DELD₅₀) being 10^−4.5/0.2 mL. SDGT0628 showed the genome-wide nucleotide sequence homology of 99.8% with the strain TX2302 isolated in 2023 and 99.7% with strains SD0101 and LYG23. SDGT0628 showed the VP1 amino acid sequence homology of 99.9% with the strain SD0101 isolated in 2018. SDGT0628 had higher homology with goose parvovirus (GPV) than with muscovy duck parvovirus (MDPV). The phylogenetic tree built based on genome-wide nucleotide sequences showed that SDGT0628 formed a small independent branch with NDPV, demonstrating the continuous evolution of GPV. Compared with the amino acid sequence of GPV VP1 protein, those of 13 NDPV strains (including SDGT0628) had three common amino acid mutations, Q89L, D142E, and S450N. SDGT0628 produced a mutation W497R, which was not observed in other NDPV strains or GPV. Five strains of NDPV were newly isolated in our laboratory, and they were compared with SDGT0628 to reveal the amino acid mutations of the VP1 protein. The results showed that all the 5 strains of NDPV had mutations at positions 89, 142, and 450. [Conclusion] In this study, a strain of NDPV was isolated from a duck farm in Shandong Province and named SDGT0628 (GenBank accession number PQ316314). NDPV had 3 mutations in the VP1 protein, and SDGT0628 had a new mutation of W497R.

Abstract:[Background] New pathogens that interfere with scientific research have been constantly discovered in laboratory animals. Corynebacterium kutscheri and Pseudomonas aeruginosa as pathogenic microorganisms affecting the health of laboratory animals are required to be monitored in the national standard. In this study, suspected strains of C. kutscheri and P. aeruginosa were isolated from specific pathogen-free (SPF) mice reared at the Laboratory Animal Center of Jilin University. [Objective] To identify the species and reveal the biological characteristics, phylogenetic relationship, and genomic characteristics of these pathogens, providing valuable biological resources and laying a foundation for developing reference materials and establishing an evaluation system for these pathogens. [Methods] The biological characteristics of the pathogens were studied by Gram staining, drug sensitivity test, and physiological and biochemical methods. PCR, phylogenetic analysis, whole genome sequencing, and bioinformatics analysis were performed to clarify the genetic characteristics of the strains. [Results] Through morphological observation, 16S rRNA sequencing, genome analysis, and phylogenetic analysis, the isolates were identified as C. kutscheri CCPM-B-B-019-2402-1 and P. aeruginosa CCPM-B-B-013-2402-3, respectively. C. kutscheri CCPM-B-B-019-2402-1 was resistant to oxacillin, nitrofurantoin, and benzylpenicillin. P. aeruginosa CCPM-B-B-013-2402-3 showed resistance to ticarcillin/clavulanic acid and tigecycline, while it demonstrated sensitivity to 9 antibiotics including ceftazidime, ciprofloxacin, and levofloxacin. The results obtained from whole genome sequencing indicated that C. kutscheri CCPM-B-B-019-2402-1 carried key virulence genes such as fagA, fagB, fagC, and fagD, whereas P. aeruginosa CCPM-B-B-013-2402-3 harbored virulence genes like plcB, tse6, and tse5. [Conclusion] In this study, two strains of pathogenic bacteria isolated from the tracheal tissue and cecum of SPF mice were identified, and their genomic characteristics were analyzed. This study provides important resources and support for the establishment of standard strains of pathogenic bacteria in laboratory animals, the development of reference materials of pathogenic bacteria in laboratory animals, and the quality control of laboratory animals.