Abstract:2017年12月，《微生物学通报》首次与中国微生物学会兽医微生物学专业委员会合作推出“兽医微生物学主题刊”，集中刊登了兽医微生物学研究领域的病原分离鉴定、诊断方法、分子流行病学及兽用生物制品研发等方面的最新进展及成果。主题刊发表后引起了同行及读者的广泛好评。2019年末，突如其来的新型冠状病毒肺炎疫情给人类的生活、工作与学习带来了巨大影响，也让病毒、变异、核酸检测、疫苗等词汇快速进入大众视野。“同一健康”“公共卫生”“生物安全”在社会层面被广泛认知，兽医微生物学对公共卫生和人类健康的重要作用愈加突显。在这种背景下，时隔五年，《微生物学通报》与兽医微生物学专业委员会再次达成共识，联合推出“兽医微生物学主题刊”第二季，旨在系统梳理我国兽医微生物学研究领域的新理论、新技术与新产品，搭建兽医微生物学科研工作者学术思想与科研成果的交流与展示平台，推进学科发展与繁荣，为健康中国、健康世界贡献智慧与力量。

Abstract:[Background] Escherichia coli is the main pathogen causing diarrhea and the emerging of the drug-resistant strains has aroused wide concern. [Objective] To understand the prevalence of drug resistance and drug resistance genes of E.coli causing calf diarrhea in Tongliao city, Inner Mongolia Autonomous Region. [Methods] Forty samples of calves suffering from diarrhea were collected from multiple banners and counties of Tongliao, from which 20 strains of E.coli were isolated and identified via 16S rRNA gene sequencing. Drug sensitivity test and PCR were conducted to study the drug resistance and drug resistance genes of the isolated strains. The whole genome of a multi-drug resistant strain was sequenced. [Results] All the 20 isolates had multi-drug resistance and showed the drug resistance rate above 80% to streptomycin, ciprofloxacin, enrofloxacin, and compound sulfamethoxazole. The detection rates of aphA1, strB, TEM-1, and qnrS were 100%. The strain TL-13 had the genome size of 4 897 185 bp and the GC content of 50.68%, and it carried two plasmids with the sizes of 108 288 bp (pTL13-1) and 64 018 bp (pTL13-2), respectively. The plasmids carried 18 mobile drug resistance genes. [Conclusion] The E.coli strains causing calf diarrhea in Tongliao generally have multi-drug resistance, and the four common resistance genes were prevalent.

Abstract:[Background] Bovine respiratory disease complex (BRDC) is a multi-causal and polysymptomatic disease caused by the joint action of pathogens and environments, which brings serious economic losses to the beef cattle industry. The viral pathogens of BRDC include bovine respiratory syncytial virus (BRSV), infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus 3 (BPIV-3), and bovine viral diarrhea virus (BVDV). The pathogen spectrum of BRDC varies regarding different places, time, and breeds. [Objective] To evaluate the epidemic status and dominant pathogens for the prevention and control of BRDC. [Methods] We obtained and tested 196 samples (including 167 nasal swabs, 13 lung tissue samples, 1 nasal mucus sample, 5 tracheal swabs, 1 saliva sample, and 9 serum samples) of 179 cattle with respiratory symptoms from 21 farms in Hubei, Hunan, Anhui, Henan, Guangdong, Guangxi, and Guizhou from September 2021 to July 2022 for BRSV, IBRV, BPIV-3, and BVDV by using reverse transcription-polymerase chain reaction (RT-PCR). [Results] The positive rates of BRSV, IBRV, BPIV-3, and BVDV in the samples were 7.14% (95% CI: 3.96, 11.69), 0.51% (95% CI: 0.01, 2.81), 4.08% (95% CI: 1.78, 7.88), and 6.63% (95% CI: 3.58, 11.07), respectively. The positive rates of the four viruses in the cattle were 7.82% (14/179) (95% CI: 4.34, 12.77), 0.56% (1/179) (95% CI: 0.01, 3.07), 4.47% (8/179) (95% CI: 1.95, 8.62), and 7.26% (13/179) (95% CI: 3.92, 12.10), respectively. The co-infection with BVDV and BRSV occurred in 0.56% (1/179) (95% CI: 0.01, 3.07) of the cattle. One strain of BVDV and 6 strains of BPIV-3 were then isolated from the positive samples. RT-PCR results showed that the BVDV strain was of type 1d and all the 6 BPIV-3 strains were of type C. [Conclusion] This study revealed that the dominant viruses of BRDC were BRSV, BVDV, and BPIV-3 in some regions in China. BVDV-1d and BPIV-3C were the dominant subtypes of BVDV and BPIV-3, respectively. This study provides a basis for the development of related vaccines and further the prevention and control of BRDC.

Abstract:[Background] Proteus mirabilis is an opportunistic pathogen ubiquitous in the environment and often causes infections in animals and humans. [Objective] To explore the cause of death of sick chickens in a chicken farm in Changchun city, Jilin province, and provide reference for disease prevention and control. [Methods] The pathogenic bacterial strain TSA-1 was isolated from the organs of dead young chickens and identified by Gram staining, biochemical tests, and 16S rRNA gene sequencing. Furthermore, drug sensitivity test, virulence gene detection, cytotoxicity and adhesion tests, and challenge in larvae of Galleria mellonella were performed for this strain. [Results] TSA-1 was a short rod-shaped or spherical Gram-negative bacterium, with the biochemical properties consistent with those of P.mirabilis. The 16S rRNA sequence alignment showed that TSA-1 shared the similarity of 100% with P.mirabilis. The drug susceptibility test results showed that TSA-1 was resistant to 14 antibiotics including ampicillin, tetracycline, kanamycin, and cefazolin and sensitive to 7 antibiotics including ciprofloxacin and enrofloxacin. The strain had strong biofilm formation ability and carried 8 virulence genes: ireA, ucaA, pmfA, atfA, ptA, zapA, hpmA and flhC. It showed cytotoxicity to RAW 264.7 macrophages and had strong adhesion to Caco-2 cells. In addition, it had stronger lethal effect on larvae of G.mellonella than avian pathogenic Escherichia coli O78. [Conclusion] P.mirabilis TSA-1 isolated from the organs of dead chickens has multi-drug resistance and carries a variety of virulence genes. It is the one of the major pathogenic bacteria causing chicken death and deserves more attention.

Abstract:[Background] Blaptica dubia can be used for producing living feed, cosmetics, and healthcare products. Studying the gut bacteria is essential for the breeding of B.dubia and development and utilization of gut bacterial resources. [Objective] To disclose the culturable bacterial species from the gut of B.dubia, and the strains capable of producing digestive enzymes, so as to provide a scientific basis and research materials for research on the influence of gut bacteria on the host and for utilization of the functional bacterial strains. [Methods] The gut bacteria of B.dubia were isolated with the culture method and identified via the methods of morphology and molecular biology. The hydrolysis circle method was employed to screen out the strains capable of producing cellulase, protease, amylase, and lipase, respectively. [Results] A total of 7 species of bacteria belonging to 4 genera were isolated from the gut of B.dubia, including 1 species of Enterococcus, 2 species of Bacillus, 2 species of Serratia, and 2 species of Citrobacter. Ten strains capable of producing digestive enzymes were screened out from the obtained 20 strains. Among them, Bacillus strains D6, D12, and D20 can produce all the 4 digestive enzymes. Serratia strains D3, D7, D9, D11, and D15 had the ability to produce 3 digestive enzymes: cellulase, protease, and lipase. Citrobacter strain D5 and Enterococcus strain D17 can produce cellulase and protease, respectively.[Conclusion] A variety of bacteria in the gut of B.dubia can produce digestive enzymes to help degrade macromolecular nutrients, which may affect the health of host by assisting food digestion. Strains D12, D7, and D11 have the strongest ability to produce cellulase, protease, and lipase, respectively, and are the gut functional strain resources worthy of further development and utilization.

Abstract:[Background] Drug-resistant Escherichia coli is a major pathogen that causes the death of poultry in large-scale breeding. Virulent phages have broad application prospects in preventing and treating drug-resistant bacterial infections. [Objective] To isolate a phage from the environmental samples of chicken farms, identify the biological characteristics, and evaluate its therapeutic effect on colibacillosis in white feather broilers.[Methods] Phage vB_EcoM-E33 (E33) was isolated via double-layer plate method. Its morphological characteristics were observed under a transmission electron microscope. After cloning of the genome sequence, the genomic features of the phage were analyzed. The host range, multiplicity of infection (MOI), stability, and one-step growth curve of phage E33 were determined by double-layer plate method. The colibacillosis model was established with white feather broilers to test the therapeutic effect of phage E33. [Results] A bacteriophage E33 of the family Straboviridae was isolated from a chicken feces sample, with a host range of 35.4% (34/96). The full-length genome of phage E33 was 170 625 bp, including 271 open reading frames (ORFs), two tRNAs, and no virulence or resistance gene. E.coli E32 was used as the host strain for the proliferation of E33, which showed an incubation period of 10 min and a burst size of 60 PFU/cell. When the MOI was 0.001, phage E33 had the highest titer of 1.93×10⁹ PFU/mL. Phage E33 kept stable below 50 ℃ and within the range of pH 3.0-11.0, while it was sensitive to ultraviolet light. The colibacillosis model of white feather broilers was established by oral administration with 10⁸ CFU/mL E.coli O78, and intramuscular injection of 10⁸ PFU/mL phage E33 demonstrated good therapeutic effect on the model. [Conclusion] Phage E33 has a broad host range, high lysis ability, and good tolerance to physical and chemical factors and does not carry harmful genes. It demonstrates a good development value and is expected to be used as an antibiotic alternative for the prevention and control of colibacillosis in chicken farms.

Abstract:[Background] Pseudorabies virus (PRV) has been mutating since 2011 and the classical vaccine strain can no longer completely resist the infections of PRV variants. There have been outbreaks of pseudorabies in many domestic pig farms and the PRV variants have become prevalent in China. [Objective] To isolate a PRV variant, analyze its genetic evolution and pathogenicity, and provide experimental data for the epidemiological investigation and vaccine development of PRV. [Methods] The brain tissue samples infected with PRV were collected from a pig farm in Heilongjiang province. The primers were designed according to gE and gB conserved sequences of PRV in GenBank for PCR identification. The gE and gC genes were sequenced for phylogenetic analysis. The virus was isolated from BHK-21 cells by plaque purification method and further identified by electron microscopy and indirect immunofluorescence. The growth curve was established and the pathogenicity was studied. [Results] The isolate was identified as a PRV epidemic strain by PCR and sequencing and named HLJ-01. The phylogenetic tree showed that the isolate was in the same clade with the epidemic variants isolated in recent years in China. The results of amino acid sequence analysis showed that gE and gC of the isolate had the characteristic sequences of domestic epidemic variants, indicating that the isolate was an epidemic variant. The growth curve showed that the titer of HLJ-01 was the highest (10^8.5 TCID₅₀/mL) 48 h after infection. The virus particle of HLJ-01 had the diameter of about 150 nm, and it was a sphere with envelope and radial spikes outside, presenting typical characteristics of PRV. The animal infection experiments showed that the mortality rates of 10^7.0, 10^6.0, and 10^5.0 TCID₅₀ infection groups were 100%, 80%, and 60%, respectively. After inoculation of HLJ-01, all the piglets showed typical symptoms and pathological changes of PRV infection, which confirmed that HLJ-01 had strong pathogenicity to piglets. [Conclusion] An epidemic variant of PRV with strong pathogenicity was isolated and identified, which laid a foundation for the epidemiological analysis of PRV and the screening of candidate vaccine strains.

Abstract:[Background] Pigeon Newcastle disease (ND), caused by pigeon paramyxovirus type Ⅰ (PPMV-1), is a severe infectious disease. However, there is no effective method to control this disease. [Objective] To elucidate the phylogenetic analysis of BJ-C and provide a scientific basis for the prevention and control of pigeon ND. [Methods] Six pairs of specific primers overlapped at the beginning and ending were designed. The genomic cDNA sequence of BJ-C strain was used as the template for amplification and sequencing. The phylogenetic tree of BJ-C strain was established by sequence alignment with the published PPMV-1 and NDV strains on the NCBI website. [Results] The genome length of BJ-C strain was 15 192 nt. BJ-C strain had the closest genetic relationship with PPMV-1/BJ-01/CH strain based on the whole genome analysis, with nucleotide similarity of 99.96% and amino acid similarity of 100%. BJ-C strain shared the same branch with PPMV-1/BJ-01/CH strain, whereas it was far from other ND virus strains such as the LaSota vaccine strain. The phylogenetic tree built based on F gene showed that BJ-C strain was in the same branch with BJP2013 strain isolated in China. The sequence alignment of the hypervariable region (47-420 nt) of F gene showed that the Ⅵb subtype strains including Pigeon/Anhui/2369/2012, Pigeon/Guangdong/GZ288/2013, BJP13, Pigeon/ Zhejiang/2036/2012, PPMV-1/Belgium/11-09620/2011, and BJ-C were in the same clade. [Conclusion] The full-length genome sequence of BJ-C strain was obtained and the strain was grouped to the class Ⅱ Ⅵb type based on phylogenetic analysis. Our results are of theoretical significance for the development of pigeon ND vaccines and other prevention and control methods.

Abstract:[Background] Endometritis, one of the common reproductive tract diseases of sows in large-scale breeding farms, causes severe economic losses to the breeding industry. Bacterial infection is one of the common causes of endometritis, while the specific pathogens and pathogenesis remain to be studied. [Objective] To explore the microbiota in birth canal on sow endometritis.[Methods] The third-generation bacterial 16S rRNA gene high-throughput sequencing was employed to compare the microbiota in the birth canal of sows with endometritis and healthy sows. According to the sequencing results, bacteria were isolated from the vaginal secretions of the sows with endometritis. Furthermore, quantitative real-time PCR (qPCR) was conducted to determine the number of Porphyromonas in the birth canal. [Results] The richness and diversity of the microbiota in the birth canal showed significant differences between health sows and those suffering from endometritis. Compared with the healthy sows, those suffering from endometritis had increased relative abundance of Proteobacteria and Bacteroidetes (P<0.05). The dominant genera in the birth canal of healthy sows were Chryseobacterium, Bacillus, and Lachnospira, which showed declined abundance in the sows with endometritis. Escherichia, Rodentibacter, and Porphyromonas were the dominant genera in the birth canal of the sows suffering from endometritis. Porphyromonas somerae is a major bacterial species in the birth canal of sows with endometritis. According to the results of microbiota analysis, a strain of Porphyromonas was isolated from the vaginal secretion of sows with endometritis, which shared the 16S rRNA gene homology of 99.04% with P.somerae DSM 23386 strain JCM 13867 (NR_113090.1). In addition, the number of Porphyromonas in the birth canal of the diseased sows was higher than that in the healthy sows (P<0.05). [Conclusion] Porphyromonas may be related to the endometritis in this batch of sows. This study lays a foundation for deciphering the etiology and pathogenesis of sow endometritis as well as a theoretical basis for treating endometritis in sows.

Abstract:[Background] In June 2021, a sick piglet in Maoming, Guangdong, which had pustules, swollen joints of limbs, and pus in the joints, was detected. [Objective] To identify the pathogen for the piglet, analyze its antimicrobial susceptibility, and thus to guide the clinical medication. Moreover, to analyze the whole genome sequence of the isolated strain, explore its virulence factors and antimicrobial resistance genes, thereby revealing its molecular mechanism of pathogenicity and antimicrobial resistance. [Methods] Bacteria were isolated from the joint pus. The bacterial species was identified by Gram staining, 16S rRNA gene and whole genome sequencing. The hemolysis activity, coagulase activity, and growth characteristics of the isolated strain were determined based on hemolysis test, coagulase test, and growth curve, respectively. The pathogenicity of the strain was evaluated with a mouse model. The antimicrobial susceptibility of the strain was determined by disk diffusion test. Whole genome sequence analysis was performed to explore virulence factors and antimicrobial resistance genes of the strain. [Results] The isolated strain was identified as Staphylococcus hyicus. The strain was non-hemolytic, displayed no coagulase activity, and grew well in Tryptic Soy Broth at 37℃ under shaking conditions (120 r/min). The strain exhibited high pathogenicity, and was sensitive to seven antimicrobials including oxacillin and spectinomycin and resistant to nine antimicrobials including penicillin G and erythromycin. It carried a number of virulence factors and antimicrobial resistance genes. [Conclusion] A S. hyicus strain was isolated from the joint pus of a diseased piglet. Antimicrobials such as oxacillin and spectinomycin can be used for the prevention and control of the infection caused by this strain. Furthermore, whole genome sequencing analysis was performed for this strain, which laid a foundation for further research on its molecular mechanism of pathogenicity and antimicrobial resistance.

Abstract:[Background] Hafnia alvei, a Gram-negative bacillus, an opportunistic pathogen, a saprophytic bacterium, is commonly detected in human and animal intestines, sewage, soil, and dairy products and can cause septicemia in human and animal, showing the potential to result in diarrhoea. [Objective] To isolate, identify and analyze the biological characteristics of potential pathogenic bacteria in the death of a barking deer (Muntjac) in Kunming Jiaozi Snow Mountain Nature Reserve. [Methods] A part of the intestinal tissue of the dead barking deer was collected aseptically for bacterial isolation and identification, and the isolate was subjected to drug sensitivity test and animal regression test. [Results] The isolate was identified as Hafnia alvei and named KMJZXS0312. It was resistant to 7 antibiotics including penicillin and cefthiophene, intermediately resistant to florfenicol, kanamycin, furazolidone, and amoxicillin, and sensitive to 13 antibiotics such as enrofloxacin and compound sulfamethoxazole. The animal regression test showed that the strain caused flatulence in the stomach and intestinal tract, thin and translucent intestine, spot hemorrhage in the liver, pinpoint hemorrhage in the lung. In addition, the strain caused degeneration and swelling of hepatocytes, as well as loose and lightly staining of the cytoplasm. Finally, it led to the death of mice.[Conclusion] A pathogenic H. alvei strain was isolated from a barking deer intestinal tissue. The biological characterization and drug sensitivity test in this study provide new biological information of the strain, which has important public health significance.

Abstract:[Background] Bovine pasteurellosis, caused by serotypes A, B, and E of Pasteurella multocida (Pm), is an acute infectious disease of cattle. Polymerase chain reaction (PCR) is an effective means to diagnose and control this disease. [Objective] To establish a multiplex PCR assay for rapid detection of serotypes A, B, and E of Pm and provide technical support for the rapid and accurate clinical diagnosis of bovine pasteurellosis. [Methods] According to the conserved regions of hyaD-hyaC, bcbD, and ecbJ genes of Pm, we designed three pairs of specific primers for PCR and determined the appropriate annealing temperature (T_m) by temperature-gradient PCR assay. Chessboard test was employed to optimize the primer concentration and then a multiplex PCR assay was established. The recombinant plasmid standard and positive strain were used to determine the sensitivity (limit of detection) of the established assay. The nucleic acid samples of 8 common bovine pathogens (Mannheimia hemolytica C1655, Escherichia coli C237, Listeria monocytogenes C1597, Staphylococcus aureus C3053, Salmonella dublin C79351, Mycobacterium paratuberculosis C1625, bovine infectious rhinotracheitis virus CAV1546, and Mycoplasma bovis C65-1) were used to determine the specificity of the multiplex PCR assay. Three batches of diagnostic reagents were prepared to perform inter-batch and intra-batch tests on sensitive and specific samples to determine the repeatability of the assay. Three different models of PCR instruments were used to detect sensitive and specific samples with the established method to determine the applicability of the assay. The performance of the assay in clinical application was evaluated by detection of clinical samples and simulated infection samples. [Results] The optimal multiplex PCR assay was established under the following conditions: T_m of 55 ℃ and the three pairs of primers at the concentrations of 0.25, 0.30, and 0.20 μmol/L, respectively. The established method could simultaneously detect Pm serotypes A (821 bp), B (203 bp), and E (363 bp). The multiplex PCR assay had high sensitivity. It showed the limits of detection of 43.080, 3.710, and 4.350 copies/μL for recombinant plasmid standards pMD-A, pMD-B, and pMD-E, respectively, as well as the limit of detection of 10² CFU for the positive bacterial liquid. Moreover, the established assay had strong specificity as it only produced bands for the serotypes A, B, and E of Pm and no bands for other pathogens. The consistent results of the inter-batch and intra-batch tests indicated good repeatability of the assay. The detection results of clinical samples and simulated infection samples showed a 100% coincidence rate with the pathogen isolation and identification. [Conclusion] The multiplex PCR assay for the detection of serotypes A, B, and E of Pm was successfully established, which provided technical support for the identification and epidemiological investigation of Pm.

Abstract:[Background] Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type III (PCV3), and swine influenza virus (SIV), the major respiratory pathogens threatening pig health, lead to great economic loss in swine industry. Therefore, it is an urgent task to develop efficient and rapid detection assay for clarifying the prevalence of these pathogens in China. [Objective] We aim to establish a rapid and accurate triplex RT-PCR method for simultaneous detection of PRRSV, PCV3, and SIVand facilitate epidemiological investigation and disease surveillance. [Methods] Three pairs of specific primers were designed according to the gene sequences of PRRSV, PCV3 and SIV and the M and N genes (436 bp) of PRRSV, Cap gene (619 bp) of PCV3, and M gene (199 bp) of SIV were amplified. The annealing temperature and primer concentration were optimized and the specificity, sensitivity and reproducibility of the triplex RT-PCR were tested. [Results] The triplex RT-PCR can rapidly detect PRRSV, PCV3 and SIV, but there was no amplification for classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine circovirus type II (PCV2), porcine parvovirus II (PPV2), porcine parvovirus III (PPV3) and torque teno sus virus I (TTSuV1). Thus, it was highly specific. The lower limit of detection for PRRSV, PCV3 and SIV was 100 copies/μL and the inter-batch and intra-batch test results were consistent. The method was applied to detect the 67 clinical samples in some farms in Heilongjiang Province. The result indicated that the detection rate of PRRSV, PCV3 and SIV was 16.5%, 10.5% and 10.5% respectively, and suggested mixed infection. [Conclusion] The triplex RT-PCR is highly sensitive and specific, which can be used for the detection of clinical samples and prediction of disease prevalence.

Abstract:This article has been retracted at the request of the authors because of some problems in the calculation method of clinical detection rate in clinical sample detection.

Abstract:[Background] Streptococcus suis (SS) and Pasteurella multocida (Pm) are zoonotic pathogens that can cause severe diseases in humans, which are easily confused with swine fever and swine erysipelas in clinical diagnosis because of mixed infections. [Objective] To establish a multiplex real-time fluorescent quantitative PCR assay for simultaneous detection of SS and Pm, so as to realize early diagnosis and treatment. [Methods] Two pairs of specific primers and TaqMan were designed based on the gdh gene of SS and the plpE gene of Pm. The universal primers and probe were designed based on the sequence of bacterial 16S rRNA gene. After optimization of the reaction conditions, a multiplex real-time fluorescence quantitative PCR method was established for the simultaneous detection of SS and Pm.[Results] This method can specifically detect SS and Pm, and the results was entirely consistent with the sequencing results after isolation of bacteria. The lower limits of detection for recombinant plasmid standards were 4.53×10² copies/μL and 3.97×10² copies/μL, respectively. The results of repeated experiments showed that the intra- and inter-group coefficients of variation were less than 3%. [Conclusion] The method established in this study was simple, accurate, and reliable. It can be used for the simultaneous detection of SS and Pm and provides an effective detection tool for the prevention and treatment of the diseases caused by SS and Pm.

Abstract:[Background] Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV) are the four main pathogens that cause viral diarrhea in pigs and often occur in mixed infections. [Objective] To establish a method for simultaneous detection of the four viruses in clinical practice. [Methods] The specific primers were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of RoRV, and the corresponding recombinant plasmids were constructed. By optimizing the PCR conditions, a multiplex RT-PCR assay for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRV was successfully established. Further, the sensitivity, specificity, and reproducibility of the established method were evaluated. [Results] The sensitivity test showed that the multiplex RT-PCR assay had the lower limits of detection of 1.75×10², 1.5×10³, 1.6×10² and 1.6×10² copies/μL for PEDV-M, TGEV-N, PDCoV-N, and PoRV-VP7 recombinant plasmid standards, respectively. The results of specificity test showed that only the four target viruses in this study could be detected, while other common porcine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine pseudorabies virus (PRV) could not be detected. The five replicate tests with 10⁸, 10⁶ and 10⁴ copies/μL of recombinant plasmids as templates and other conditions unchanged all produced clear and uniform bands. The 52 clinical diarrhea samples from various regions in Shandong Province were tested by the established quadruple RT-PCR assay. The results showed that the positive rates of PEDV, TGEV, PDCoV, and PoRV were 37% (19 samples), 6% (3 samples), 10% (5 samples) and 25% (13 samples), respectively. 2 (4%), 2 (4%), and 1 (2%) samples showed mixed infections of PEDV and PoRV, mixed infections of PEDV and TGEV, and mixed infections of PEDV and PDCoV, respectively. Further, the results of the multiplex RT-PCR assay were validated by monoplex RT-PCR, which showed 100% compliance. Finally, five positive clinical samples were randomly selected and sent for sequencing to verify the results, and all of them were the gene fragments of the corresponding viruses. [Conclusion] In this study, a quadruple RT-PCR assay for the simultaneous detection of PEDV, TGEV, PDCoV and PoRV was established, and the results provide a technical tool for the differential diagnosis and epidemiological investigation of four clinical porcine diarrhea virus diseases.

Abstract:[Background] Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) has been proven to be an efficient and powerful third-generation gene editing tool and has achieved great progress in the discovery of functional genes. However, few studies have reported about using this method to screen the host genes associated with porcine epidemic diarrhea virus (PEDV). [Objective] To screen out the genes related to PEDV replication in the whole genome by using the CRISPR/Cas9 system, and to perform preliminary verification of candidate genes, so as to provide scientific reference for breeding PEDV-resistant pigs. [Methods] A genome-wide knockout library of human hepatoma cell line (Huh-7) was constructed via CRISPR/Cas9 technology, and the Huh-7 library cells were infected with PEDV. Then, the key host factors affecting PEDV replication were screened by high-throughput sequencing. The interference and detection experiments of virus replication were performed to preliminarily verify the candidate genes that affected PEDV replication. [Results] A CRISPR/Cas9 system was successfully constructed to screen the genes related to PEDV replication in the whole genome. The genes with top enrichment degree, including those encoding integrin α11 (ITGA11), replication protein A2 (RPA2), kinesin family member 2A (KIF2A), induced myeloid leukemia cell differentiation protein 1 (MCL1), poly (ADP-ribose) polymerase 1 (PARP1), and solute carrier family 18 member A1 (vesicular monoamine transporter, SLC18A1), were verified. Compared with the control group, the interference of ITGA11 via siRNA significantly down-regulated the mRNA and protein levels of PEDV-N and reduced the virus titer in the IPEC-J2 cells infected with PEDV. [Conclusion] The genome-wide knockout library based on the CRISPR/Cas9 system can be used as an effective tool for screening PEDV replication-related functional genes. ITGA11 may be a potential target gene for the breeding of PEDV-resistant pigs.

Abstract:[Background] Campylobacter is a major zoonotic bacterial pathogen which can colonize the intestinal tract of a variety of animals. However, adhesion and invasion to intestinal epithelial cells and chicken intestine colonization of Campylobacter strains from different hosts are unclear. [Objective] To compare the adhesion and invasion to intestinal epithelial cells and the chicken intestine colonization of Campylobacter strains from different hosts. [Methods] Five Campylobacter strains isolated from different hosts, including human-, chicken-, duck-, and bovine-derived Campylobacter jejuni strains and a porcine-derived C.coli strain, were studied. The strains were identified by multiplex PCR and tested for motility and biofilm formation ability. The adhesion of each strain to human intestinal epithelial cell line Caco-2, porcine intestinal epithelial cell line IPEC-J2, and rat intestinal epithelial cell line IEC-6 was determined respectively. The invasion of each strain to intestinal epithelial cells was determined by gentamicin protection test. The differences in adhesion and invasion were statistically analyzed. Further, chickens were challenged orally with the five strains respectively. We collected the cecal samples on different days post inoculation (DPI) to determine the chicken intestine colonization ability of Campylobacter. [Results] The motility of human-derived C.jejuni was significantly higher than that of other four animal-derived Campylobacter strains, and the biofilm formation ability of bovine- and porcine-derived strains was significantly higher than that of other strains. The human-derived C.jejuni showcased stronger adhesion and weaker invasion to Caco-2 cells than animal-derived strains. The adhesion ability of duck- and bovine-derived strains to IPEC-J2 cells was significantly lower than that of other strains, and the invasion ability of the duck-derived strain was significantly lower than that of other strains. There was no significant difference in the adhesion ability of the five strains to IEC-6 cells, while the invasion ability of chicken-derived strain was significantly lower than that of other strains. The animal-derived strains showed higher colonization levels than the human-derived strain at 1, 3 and 6 DPI, while only the bovine-derived strain had significantly higher colonization level than the human-derived strain at 10 and 15 DPI. All the strains reached a stable colonization level of 8-10 Log₁₀ (CFU/g) at 15 DPI. [Conclusion] Campylobacter strains from different hosts can adhere to and invade the intestinal epithelial cells of various animal origins and colonize chicken intestine, which suggest that Campylobacter can transmit among and colonize different animals. The results are helpful for developing targeted prevention and control measures against Campylobacter.

Abstract:[Background] The response to environmental stimuli is important for the survival, proliferation, and infection of bacteria. Fully revealing the molecular mechanism of Brucella in coping with environmental stimuli can provide a theoretical basis for the prevention and control of brucellosis. The available studies have demonstrated that ycjX is highly expressed in the bacteria under heat stress and may be regulated by σ³² and the expression of ycjF rises significantly during bacterial sepsis. The two genes may constitute an operon in Gram-negative bacteria. [Objective] To explore the roles of ycjX and ycjF in the response of Brucella to heat stress. [Methods] Heat stress test was performed on Brucella suis S2, △ycjXF, and C△ycjXF and the survival rates of the three strains were calculated. The operon modes of ycjX and ycjF were identified via RT-PCR method. The activity of β-galactosidase was determined to measure the promoter region activity. The chromatin immunoprecipitation (ChIP) assay was employed to explore the endogenous regulation of ycjX and ycjF by σ³². The pGEX-4T-1-σ³² recombinant protein was expressed and purified. The electrophoretic mobility shift assay (EMSA) was employed to analyze the binding relationship of σ³² with ycjX and ycjF. [Results] The growth rate of △ycjXF slowed down compared with B.suis S2 and C△ycjXF under heat stress. The co-transcriptional analysis showed that ycjX and ycjF were in the one transcript of Brucella. The promoter region of the two genes was active. The ChIP assay showed that σ³² can be enriched in the promoter region of ycjXF, and the EMSA determined that σ³² can directly bind to ycjXF in vitro. [Conclusion] Under the regulation of σ³², ycjX and ycjF play a role in the response of Brucella to heat stress.

Abstract:[Background] Haemophilus parasuis can cause a variety of inflammatory reactions and high mortality, while the inflammatory mechanism remains unclear.[Objective] To study the activation of caspase-11 and NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome in RAW264.7 cells by H.parasuis outer membrane vesicles (OMVs) and the key role of caspase-11 in the OMVs-induced expression of inflammatory cytokines. [Methods] The RAW264.7 cells were infected with H.parasuis OMVs and collected 6, 12, and 24 h post infection. RT-PCR was employed to determine the mRNA levels of caspase-11, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1. Western blotting was employed to determine the protein levels of caspase-11, NLRP3, ASC, and caspase-1 48 h post infection. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the levels of interleukin-1β (IL-1β) and IL-18 in the cell supernatants 6, 12, 24, 48, and 72 h post infection. After the RAW264.7 cells were stimulated with different concentrations of OMVs (0, 2.5, 10, 25, 50, and 100 μg/mL) for 24 h, the cell supernatants were collected for the measurement of IL-1β and IL-18 levels by ELISA. The RAW264.7 cells with the silencing of caspase-11 were established and then infected with OMVs, and the supernatants were collected for the measurement of IL-1β and IL-18 levels 12, 24 and 48 h post infection. [Results] The mRNA level of caspase-11 was up-regulated in the RAW264.7 cells 6, 12 and 24 h post infection with OMVs (P<0.01). The mRNA level of NLRP3 was higher than that in the control group 6 and 24 h post infection (P<0.01). The mRNA level of ASC was significantly lower than that in the control group 12 and 24 h post infection (P<0.05). The mRNA level of caspase-1 was significantly higher than that in the control group 6, 12, and 24 h post infection (P<0.05). Western blotting showed that the expression levels of caspase-11, NLRP3, ASC, and caspase-1 were up-regulated after infection with OMVs. The level of IL-1β elevated in a time-dependent manner after the cells were stimulated with OMVs for 12, 24, 48 and 72 h and was higher than that in the control group (P<0.01), and the level of IL-18 was higher than that in the control group at the time points of 6, 12, 24, 48 and 72 h (P<0.05). When the cells were stimulated with different concentrations of OMVs, the inflammatory effect increased in a dose-dependent manner. The level of IL-1β in the caspase-11-silenced cells stimulated with OMVs was lower than that in the non-silenced group at the time points of 12, 24 and 48 h (P<0.05), and the level of IL-18 showed the same trend as that of IL-1β at the time points of 24 and 48 h (P<0.05). [Conclusion] The OMVs of H.parasuis play an important role in the inflammatory response induced by H. parasuis. OMVs can induce the activation of non-canonical NLRP3 inflammasome signaling pathway mediated by caspase-11 in RAW264.7 cells.

Abstract:[Background] The digestive tracts of humans and animals harbor complex microbiota composed of diverse species which inhabit different parts of the intestine and perform specific functions. In recent years, butyrate-producing bacteria have gradually become a research hotspot in microbiology. Butyrate-producing bacteria are mainly spore-forming Gram-positive anaerobic bacteria, which are of great significance to intestinal health.[Objective] To screen butyrate-producing bacteria from the rumen of ruminants and study its growth characteristics, and further optimize its culture conditions to improve the butyrate production. [Methods] Butyrate-producing bacteria were screened from sheep rumen contents by dilution coating method and identified via morphological observation and 16S rRNA gene sequence analysis. Single factor test and Box-Behnken design were employed to optimize the acid producing conditions of the screened strain in reinforced Clostridium medium (RCM). [Results] The strain screened and identified was Clostridium beijerinckii and was named C.beijerinckii R8. The butyrate production of C.beijerinckii R8 reached 2.48 g/L under the conditions of 1.22% inoculation amount, 38.45 ℃, and pH 6.08 for 64.67 h. [Conclusion] A strain of C.beijerinckii R8 was screened, which grows and produces butyrate in RCM, demonstrating a high application value.

Abstract:[Background] Pulmonary flora is closely associated with host health and respiratory diseases. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a common opportunistic pathogen in clinical practice, while its impact on pulmonary flora after infection remains unclear. [Objective] To explore the perturbation of carbapenem-resistant K.pneumoniae CRKP2 on the pulmonary flora of C57BL/6 mice. [Methods] The C57BL/6 mice were randomly assigned into 3 groups which were intranasally inoculated with CRKP2, carbapenem-sensitive K.pneumoniae KP2044, and sterile PBS solution, respectively. The structure of pulmonary flora was analyzed via 16S rRNA high-throughput sequencing. [Results] Compared with those in healthy mice, the alpha diversity and beta diversity of the pulmonary flora in the mice after KP2044 and CRKP2 strain infection significantly changed. Specifically, the infection with strain CRKP2 and KP2044 significantly increased the relative abundance of Proteobacteria and decreased that of Lactobacillus. Compared with KP2044, CRKP2 showed decreased biofilm formation and caused low mortality of infected mice, which indicated that CRKP2 infection led to weaker alteration of pulmonary flora than KP2044 infection.[Conclusion] Although K.pneumoniae is an opportunistic pathogen, high-dose carbapenem-resistant K.pneumoniae CRKP2 significantly affects the pulmonary flora of healthy mice. Although stain CRKP2 has multi-drug resistance, it leads to lower disturbance on the pulmonary flora than strain KP2044. Therefore, we hypothesize that the degree of disturbance of the pulmonary flora by K.pneumoniae infection may be related to strain virulence.

Abstract:[Background] Klebsiella pneumoniae, one of the common opportunistic pathogens after Escherichia coli, can lead to hemorrhagic enteritis and systemic sepsis in giant pandas (Ailuropoda melanoleuca). [Objective] To identify the biological characteristics of K.pneumoniae from giant pandas and provide scientific guidance for the prevention and control of the disease. [Methods] The biofilm formation, high viscosity, drug resistance, and 15 common virulence genes of 46 strains of K.pneumoniae from A.melanoleuca were studied via crystal violet staining, string test, K-B disk diffusion method, and PCR, respectively. Further, we selected an isolate (pneumoniae-X-5) that may be pathogenic according to the above biological characteristics to study its pathogenicity in mice. [Results] All the 46 strains of K.pneumoniae could form capsule, among which 12 strains showed the phenotype of high viscosity and 65% (30/46) of the strains could form biofilm. Among the strains, 58% (27/46) were multi-drug resistant and 100% were resistant to ampicillin, oxacillin, penicillin, and vancomycin. Of the virulence genes, ureA had the highest detection rate of 91.30% (42/46). The LD₅₀ of pneumoniae-X-5 in mice was 8.9×10⁴ CFU/mL. The mice challenged with this strain had thickened alveolar septa, inflammatory cell infiltration, hepatocyte degeneration and necrosis, splenic congestion, separation of duodenal mucosal epithelium and lamina propria, and partial cell necrosis in the lamina propria. The spleen contained the highest amount of bacteria in the dead mice, followed by the liver. [Conclusion] This study elucidates the multi-drug resistance, biofilm formation, high viscosity and other pathogenic characteristics of K.pneumoniae from A.melanoleuca, providing a scientific basis for the prevention, control, and clinical treatment of K.pneumoniae disease in A.melanoleuca.

Abstract:[Background] Major histocompatibility complex (MHC) is highly polymorphic, thus influencing the outcome of biomedical experiments. Particularly, specific MHC-B alleles are closely related to the development of various diseases. Macaca fascicularis (Mafa) is an important animal model for conducting biomedical research. Currently, Mafa-B alleles in Mafa have not been comprehensively characterized. [Objective] To explore the comprehensive information on Mafa-B alleles in Mafa and to identify co-expression and evolution of Mafa-B alleles. [Methods] Through third-generation sequencing, the genomic information of MHC-B of Mafa was obtained. Then, we designed specific primers to amplify Mafa-B alleles in a cohort of 33 Vietnamese Mafa individuals and characterized Mafa-B alleles with multiple bioinformatics methods. [Results] On the basis of information of 92 Mafa-B alleles, we identified 65 novel Mafa-B alleles. Among them, 8 alleles were identical to sequences previously reported in Mafa of other geographical origins and 32 were also found in macaque populations. In addition, we identified 7 high-frequency Mafa-B lineages and 7 pairs of co-expressed Mafa-B alleles, and detected one potential recombination event. Evolutionary analysis revealed the high similarity of Mafa-B sequences in Mafa from different geographical origins.[Conclusion] These co-expressed Mafa-B alleles in Vietnamese populations have undergone selection pressure by certain antigens, and Mafa populations of different geographical origins may fine-tune the Mafa-B sequences in response to pathogens. This study lays a foundation for elucidating the genetic background of MHC in Mafa.

Abstract:[Background] Sedentary behavior, which is common among patients with type 2 diabetes, blocks the blood glucose control. However, its specific mechanism is not fully clarified. [Objective] To simulate sedentary behavior by limiting the activity of mice, to elucidate the mechanism for the aggravation of glucolipid metabolism disorder in type 2 diabetes mice by this behavior, and thus to lay a theoretical basis for relevant health education and intervention. [Methods] Male C57BL/6J mice were used in this study. Mice were randomized into the normal (CON) group, type 2 diabetes model (MOD) group and type 2 diabetes with activity restriction (SED) group. Model mice were used in the MOD group and SED group, and for the SED group, the time for the activity of mice was controlled to simulate the sedentary behavior for 8 weeks. The metabolic disorder of glucose and lipids in mice was determined by kits and morphological observation. The damage of ileum was observed based on hematoxylin and eosin (HE) and alcian blue-periodic acid-Schiff (AB-PAS) staining. Quantitative reverse transcription PCR (RT-qPCR) was used to determine the changes of fecal microbes in mice, and TBA kit to measure the content of total bile acid in serum and liver of mice. The mRNA and protein expression of farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) in ileum and liver of mice were detected by qPCR and Western blotting, respectively. [Results] Compared with the CON group, the MOD group showed in increase in blood glucose and lipids, insulin resistance, oral glucose tolerance disorder, and obvious intestinal pathological changes. Restriction of mouse activity enhanced the disorder of glucose and lipid metabolism in diabetes mice (P<0.05). The diabetes mice displayed imbalance in intestinal flora, as manifested by the reduction in beneficial bacteria and increase in harmful bacteria at phylum and genus levels, which was exacerbated by the restriction of mouse activity. The total bile acid content in serum and liver was higher in the MOD group than that in the CON group, and higher in the SED group than in the MOD group (P<0.05). The expression of FXR and TGR5 in the MOD group was lower than that in the CON group (P<0.01), and the expression of these receptors was further inhibited by activity restriction. Spearman’s correlation analysis also showed that glucolipid metabolism was in significant correlation with intestinal flora and bile acid, a metabolite of intestinal flora. [Conclusion] Activity restriction aggravated the disorder of glucose and lipid metabolism in T2DM mice, which may be related to the imbalance of intestinal flora and bile acid metabolism, and the further down-regulation of bile acid receptor.

Abstract:[Background] The coronavirus disease 2019 (COVID-19) pandemic has lasted for nearly three years in the globe, which has not only caused serious harm to humans but also affected companion animals. The COVID-19 vaccines for human have been used globally, while those for animals are rarely reported. [Objective] To develop a bivalent vaccine against both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rabies virus (RABV) for animal use.[Methods] We cloned the S and S1 genes of SARS-CoV-2 into the region between G and L genes of the attenuated RABV vaccine strain rHEP-Flury to construct the recombinant plasmids pHEP-nCOV-S and pHEP-nCOV-S1, respectively. The two plasmids were respectively co-transfected into BHK-21 cells with the helper plasmids and finally the recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 were rescued. The recombinant viruses were confirmed by RT-PCR and direct fluorescent antibody staining against RABV N protein. Western blotting was employed to detect the expression of S and S1 proteins in the cells infected with the recombinant viruses. The growth curves, pathogenicity, and immunogenicity of recombinant viruses were confirmed in NA cells and mice. [Results] The rescued recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 respectively carrying the S and S1 genes of SARS-CoV-2 were confirmed by direct fluorescent antibody assay based on the green fluorescence from the supernatants 7 days post infection. rHEP-nCOV-S1 rather than rHEP-nCOV-S showed stronger proliferation and diffusion abilities than the parental virus rHEP-Flury in NA cells. The specific bands at 72 kDa and 144 kDa in the Western blotting confirmed the efficient expression of S and S1 in the recombinant viruses, respectively. The mice vaccinated with the recombinant viruses did not show significant changes in the body weight compared with those vaccinated with rHEP-Flury, and the recombinant viruses induced the production of neutralizing antibody against RABV in mice. [Conclusion] The production of the recombinant RABV carrying the S/S1 gene of SARS-CoV-2 provides a foundation for the development of the bivalent vaccine against both SARS-CoV-2 and rabies virus for animal use.

Abstract:[Background] Enteritis is one of the clinical signs of pigs infected with porcine circovirus type 2 (PCV2), and it has an impact on pig performance. It can lead to reproductive failure. The cecum is an essential digestive organ in monogastric animals, and the impact of PCV2 infection on it should be investigated. [Objective] The purpose of this study was to look into the immunological activity and flora of the cecum after PCV2 infection. [Methods] Twelve healthy weaned piglets were randomly assigned to the control (n=6) or infection (n=6) groups. Piglets in the infection group received the virus via oral delivery (5 mL) and intramuscular injection (5 mL), for a total of 10 mL/piglet. The control group was similarly injected with PK15 cell culture. The viral nucleic acid load and antibody dynamics were identified 56 days post-infection (dpi), and micromorphology, viral antigen distribution, immune function, and microbial ecology in pig cecum contents were also examined at 21 and 56 dpi. [Results] PCV2 antigen signal was greater at 21 dpi than at 56 dpi, and viral nucleic acid load and antibody in serum were both at high levels. PCV2 antigen was mostly dispersed in the epithelial cells and lamina propria of the cecum mucosa. While the ability of T cells to proliferate was greatly increased, the amount of SIgA in cecal secretions was significantly reduced. Pigs with the infection experienced significant cecal epithelial cell loss and intestinal gland atrophy. Diversity and quantity of the cisternal vegetation were greatly diminished. Beneficial bacteria like Butyrivibrio and Ruminococcaceae-NK4A214-group were noticeably reduced, whereas conditional harmful bacteria like Alloprevotella were noticeably elevated. The viral nucleic acid load decreased to 7 dpi at 56 dpi, whereas the antibody level remained high. Other indicators essentially reverted to control group levels. [Conclusion] PCV2 infection causes immune dysfunction in the piglets’ cecum, which damages the cecum mucosa and results in an increase in conditionally pathogenic bacteria and a decrease in beneficial bacteria. These changes are connected to the viral content.

Abstract:[Background] The three strains, CVCC 68201, CVCC 68202, and CVCC 68203, are usually used to produce purified avian tuberculin in China, while their biological characteristics and pathogenicity to guinea pigs remain unclear. [Objective] To explore the biological characteristics of Mycobacterium avium and its pathogenicity to animals, so as to provide technical support for the prevention and control of avian tuberculosis and bovine tuberculosis. [Methods] The genomes of the three strains of M.avium were sequenced and the nucleotide similarity was determined. Guinea pigs were respectively infected with the three strains of M.avium. After that, we observed the clinical symptoms, pathological changes, and intradermal allergy and measured the weight gain and organ indexes to evaluate the virulence of the three strains to guinea pigs. [Results] CVCC 68201, CVCC 68202, and CVCC 68203 were all identified as M.avium, and their genomes had the highest similarity to that of M.avium subsp. avium FDAARGOS_1608. The infection with M.avium affected the weight gain of guinea pigs, which was mainly manifested as growth retardation. The average weight of the guinea pigs five weeks post infection with CVCC 68201 or CVCC 68202 was significantly lower than that of the uninfected guinea pigs. The results of the intradermal allergy challenge showed that the area of skin redness and swelling of the guinea pigs infected with CVCC 68201 was significantly larger than that of the other two infection groups, which indicated that CVCC 68201 caused more intense delayed-type allergy. The spleen and lungs of the guinea pigs had different degrees of swelling and hemorrhage after the infection, and the lung index of the guinea pigs infected with CVCC 68201 was different from that of the uninfected group (P<0.01). Different degrees of alveolar changes were observed in the lungs of the infected guinea pigs, which was severer in the CVCC 68201 infection group, as demonstrated by enlargement and slight bleeding. The acid-fast staining of lung and spleen tissue sections of infected guinea pigs showed scattered infiltration of red mycobacteria. [Conclusion] All the three strains are pathogenic to guinea pigs and can cause local lesions. This study provides a basis for the preparation and identification of M.avium and lays a foundation for the research on the method for differential diagnosis of bovine tuberculosis.

Abstract:[Background] Porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive disorders in pregnant sows and respiratory diseases in piglets. In recent years, NADC30-like strains of PRRSV have become dominant in China. [Objective] To develop a virus-like particle (VLP) vaccine against NADC30-like strains of PRRSV. [Methods] We linked the encoding GP5 protein open reading frame 5 (ORF5) and ORF6 (encoding M protein) genes of NADC30-like strain to the downstream polyclonal sites of P₁₀ and P_H promoters in pFastBac^TM dual vector to obtain the shuttle plasmids pFB-30-ORF5 and pFB-30-ORF6, respectively. After identification by restriction endonuclease digestion, the ORF6 gene was inserted into the downstream region of P_H promoter in the shuttle plasmid pFB-30-ORF5 to construct the shuttle plasmid pFB-30-ORF5-OPF6. The above three shuttle plasmids were respectively transformed into DH10Bac competent Escherichia coli cells, and the recombinant plasmids were identified by blue-white screening and PCR. The recombinant plasmids were then transfected into SF9 insect cells. After the appearance of cytopathic changes, the virus was harvested and passed on blindly for three generations. Whether there were virus-like particles was observed under a transmission electron microscope. After the SF9 cells were infected with the third-generation virus, the GP5, His-tag, and Flag-tag antibodies were used as the primary antibodies for the recombinant protein identification by indirect immunofluorescence assay (IFA) and Western blotting. [Results] Three shuttle plasmids pFB-30-ORF5, pFB-30-ORF6, and pFB-30-ORF5-OPF6 were successfully constructed and identified by restriction endonuclease digestion. After blue-white screening and PCR verification, the recombinant rod particles were obtained and named Bacmind-30-ORF5, Bacmind-30-ORF6, and Bacmind-30-ORF5-ORF6, respectively. Obvious cytopathic effect was observed in SF9 cells 120 h post infection with recombinant rod particles. After harvesting of the virus suspension, the spherical VLPs with the diameter of about 50 nm were observed under a transmission electron microscope. The binding of colloidal gold particles around VLPs was observed via immunoelectron microscopy, and the results of immunofluorescence assay showed obvious green specific fluorescent foci in the experimental group. Western blotting showed that all the three kinds of VLPs generated specific bands with the expected size. [Conclusion] Three kinds of virus-like particles of NADC30-like PRRSV were prepared, which laid a foundation for the research and development of vaccines against the new prevalent strains of PRRSV.

Abstract:Signal transduction pathways enable cells to respond to different complex external environmental stimulation in time, thus producing biological effects in response to infection by various pathogens. Mitogen-activated protein kinase (MAPK) and its downstream targets are among the most critical signaling modules that can transform environmental stress into many cellular processes. They are most common in mammalian cells and are almost involved in the physiological and pathological responses of all cells. MAPK regulates the host’s immune response to various environmental stress, including bacterial infection and inflammasomes. Recent studies have shown that pathogens may release specific effectors or toxins to hijack the MAPK signaling pathway during infection in two ways. One is to degrade essential proteins to affect signal transduction, and the other is to influence host cell post-translational modifications, such as phosphorylation and ubiquitination, to regulate many cellular processes. This review discussed the regulation and activation of MAPK in innate immunity and explored the complex mechanism of pathogens in manipulating MAPK activation to enhance infection and the potential role of MAPK regulated by pathogens as a novel target against pathogen infection.

Abstract:Blood-brain barrier (BBB), one of the natural structural and functional barriers of the central nervous system (CNS), can prevent the invasion of pathogenic bacteria. However, pathogenic bacteria can interact with brain endothelial cells via their virulence factors to induce host immune response and massive secretion of cytokines and chemokines and destroy tight junction proteins to penetrate the blood brain barrier, causing bacterial meningitis and irreversible nervous system damage. Streptococcus is a major pathogen causing bacterial meningitis. In recent years, significant progress has been achieved in the research on the molecular mechanism of Streptococcus penetrating the blood-brain barrier. This article reviews the progress in the mechanism of main Streptococcus species including Streptococcus pneumoniae, Streptococcus suis, group B Streptococcus, and Streptococcus equi in passing through the blood-brain barrier.

Abstract:Persistent infection and drug resistance caused by the biofilm of Staphylococcus spp. have always been a difficult problem in clinical treatment. The research on the molecular mechanism of biofilm formation has become the key to the prevention and treatment of infections associated with the biofilm of Staphylococcus spp.. The establishment of animal models of infection by Staphylococcus spp. is conducive to the study of biofilm formation, diffusion, and pathogenesis and the in vivo evaluation of the anti-biofilm effect of drugs. However, there are many influencing factors of biofilm formation in animals, such as animal species, implantation material, inoculation site, infection dose, observation time, and evaluation method. This study systematically summarized the animal models of biofilm infection by Staphylococcus spp. in the past 40 years, focusing on the establishment methods, application scope, advantages, and disadvantages of animal models, thereby providing a theoretical basis for the prevention and treatment of biofilm infection by Staphylococcus spp..

Abstract:The cGAS-STING signaling pathway is an intracellular DNA sensor that recognizes self-lesioned or externally entered double-stranded DNA in the cytoplasm. It not only is associated with tumors, viral and bacterial infections, and autoimmune diseases but also plays a role in the nonspecific immune system. The available studies of cGAS-STING signaling pathway mainly focus on mammalian tumor-related diseases and the diseases related to the innate immune system. Considering the significant regulatory role of CGAS-STING signaling pathway, we comprehensively expound the role of this pathway in pathogen infection of livestock and poultry, with an view to provide a theoretical basis for the prevention and control of livestock and poultry diseases.

Abstract:Pathogens and their drug resistance are key issues of global public health. Numerous zoonotic pathogens can be transmitted to humans through the food industry chain. Drug resistance makes infections more difficult to treat, increasing the risk of disease transmission and death. Studying the variation patterns, virulence, and pathogenic mechanisms of pathogens at the molecular level will help us to find new drug targets and developing new drugs. DNA polymerase IV (Pol IV), an important member of the γ family polymerases, is ubiquitous in the three life domains (prokaryotes, eukaryotes, and archaea). This enzyme is involved in the translesion DNA synthesis and plays a critical role in bacteria. It not only responds to DNA damage under SOS response and RpoS regulation but also is involved in the acquisition of antibiotic resistance and adaptation. This paper reviewed the recent studies related to bacterial Pol IV, including the genetic and structural features, expression regulation, and effects on bacterial adaptation, and discussed the feasibility of Pol IV as a potential drug target.

Abstract:Iron is an essential nutrient for the survival of most bacteria. However, excessive iron will cause damage to bacteria through the reactive oxygen species produced by Fenton reaction. Bacteria usually maintain the iron homeostasis via a variety of mechanisms including uptake, regulation, chelation, and efflux. Riemerella anatipestifer is a Gram-negative bacterium newly categorized into Riemerella, a genus of Weeksellaceae. Because R. anatipestifer mainly infects poultry, the genes involved in the iron metabolism of this bacterium have unique characteristics. In this review, the research progress of iron metabolism in this bacterium was systematically summarized and elaborated. It includes that the roles of TonB system, TonB-dependent receptors, and Fur and Dps proteins in the import, regulation, and chelation of iron, as well as in the pathogenesis of R. anatipestifer. It will provide valuable information for fully understanding the mechanisms of iron homeostasis and for further research of the iron metabolism of R. anatipestifer.

Abstract:Autophagy is a cellular process that removes unnecessary or dysfunctional components through a lysosome-dependent regulated mechanism. It occurs in the presence of stimuli such as virus to maintain homeostasis. Autophagy can help defend against viral invasion and may promote viral replication. Porcine deltacoronavirus (PDCoV) can induce autophagy. In this paper, we summarize the types and process of autophagy, interaction of autophagy with PDCoV, and related detection methods, to elucidate how autophagy resists the infection and supports the replication of PDCoV. The findings are expected to serve as a reference for further research on the pathogenic mechanism of PDCoV and prevention and control of this virus.