Duck hepatitis A virus type 3 (DHAV-3) is an infectious virus that is highly fatal to ducklings and causes significant economic losses in the duck industry worldwide. Biosecurity and vaccination are required to control the pathogen. In the present study, we attenuated a lowly pathogenic DHAV-3 clinical isolate, named as HB, by serial passaging in duck embryos, and followed by several adaptive proliferations in specific-pathogen-free (SPF) chicken embryos. The virulence of DHAV-3 at different passages was assessed by infecting 3-day-old ducklings. We found that the HB strain lost pathogenicity to ducklings from the 55th passage onwards. The 80th passage strain (HB80), which achieved good growth capacity in duck embryos with a viral titer of 108.17 50% egg lethal dose per milliliter (ELD50/mL), was selected as a live attenuated vaccine candidate. The HB80 strain did not induce clinical symptoms or pathological lesions in 3-day-old ducklings and showed no virulence reversion after 5 rounds of in vivo back-passage. The minimum effective dose of HB80 was determined to be 104.5 ELD50 by hypodermic inoculation of the neck. Importantly, a single dose of HB80 elicited good immune responses and provided complete protection against challenge with the lethal DHAV-3 strain. Compared with the genomic sequence of the parental HB strain, HB80 had 7 amino acid substitutions, two of them are in the hypervariable region of the VP1 and polymerase-encoding 3D regions, which may play a role in virulence attenuation. Our data suggest that the attenuated HB80 strain is a promising vaccine candidate for the prevention of DHAV-3 infections in China. HB80 has been registered as a New Veterinary Drug Registration Certificate by the Chinese Ministry of Agriculture and Rural Affairs (MARA), and is the first live attenuated DHAV-3 vaccine strain to be officially licensed in China.

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3′s typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4–10 and temperatures of 30–40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3′s specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera.

Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV. A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 ℃ and allowed for visual interpretation without requiring sophisticated equipment. The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100

Florfenicol resistance genes (FRGs) are widely present in livestock farms. The aim of this study was to evaluate the removal efficiencies of FRGs as well as the relationships between FRGs, mobile genetic elements (MGEs) and bacterial communities during the natural drying (ND) and anaerobic digestion (AD) processes of manure treatment in swine farms by combining bacterial isolation, quantitative PCR and metagenomic approaches. Solid manure showed a higher abundance of FRGs than fresh manure and was the main contamination source of fexA and fexB in ND farms, whilst biogas slurry displayed a lower abundance of FRGs than the wastewater in AD farms. Moreover, fresh manure and wastewater showed a high abundance of optrA , and wastewater was the main contamination source of cfr in both ND and AD farms. Both optrA / fexA -positive enterococci and cfr / fexA -positive staphylococci were mainly isolated along the farms ' treatment processes. The cfr -positive staphylococci were highly prevalent in wastewater (57.14 % - 100 %) and may be associated with nasal -derived cfr -positive porcine staphylococci. An increased abundance of Enterococcus , Jeotgalibaca and Vagococcus in the bacterial community structures may account for the high optrA abundance in wastewater and Jeotgalibaca may be another potential host of optrA . Furthermore, the abundance of FRG -related MGEs increased by 22.63 % after the ND process and decreased by 66.96 % in AD farms. A significant correlation was observed between cfr and IS Enfa4 , whereas no significance was found between optrA and IS 1216E , although IS 1216E is the predominant insertion sequence involved in the transfer of optrA . In conclusion, manure and wastewater represented independent pollution sources of FRGs in swine farms. Associated MGEs might play a key role in the transfer and persistence of FRGs. The AD process was more efficient in the removal of FRGs than the ND method, nevertheless a longer storage of slurry may be required for a complete removal.

OBJECTIVES:To investigate the population structure and antimicrobial resistance (AMR) of avian Pasteurella multocida in China. METHODS:Utilizing WGS analysis, we explored the phylogeny using a dataset of 546 genomes, comprising avian P. multocida isolates from China (n = 121), the USA (n = 165), Australia(n = 153), Bangladesh (n = 3) and isolates of other hosts from China (n = 104). We examined the integrative and conjugative element (ICE) structures and the distribution of their components carrying resistance genes, and reconstructed the evolutionary history of A:L1:ST129 (n = 110). RESULTS:The population structure of avian P. multocida in China was dominated by the A:L1:ST129 clone with limited genetic diversity. A:L1:ST129 isolates possessed a broader spectrum of resistance genes at comparatively higher frequencies than those from other hosts and countries. The novel putative ICEs harboured complex resistant clusters that were prevalent in A:L1:ST129. Bayesian analysis predicted that the A:L1:ST129 clone emerged around 1923, and evolved slowly. CONCLUSIONS:A:L1:ST129 appears to possess a host predilection towards avian species in China, posing a potential health threat to other animals. The complex AMR determinants coupled with high frequencies may strengthen the population dominance of A:L1:ST129. The extensive antimicrobial utilization in poultry farming and the mixed rearing practices could have accelerated AMR accumulation in A:L1:ST129. ICEs, together with their resistant clusters, significantly contribute to resistance gene transfer and facilitate the adaptation of A:L1:ST129 to ecological niches. Despite the genetic stability and slow evolution rate, A:L1:ST129 deserves continued monitoring due to its propensity to retain resistance genes, warranting global attention to preclude substantial economic losses.

本研究旨在分析猪源ST9型耐甲氧西林金黄色葡萄球菌(Staphylococcusaureus,MRSA)中前噬菌体的流行情况、结构特点和转导能力,探究前噬菌体在猪源MRSA流行克隆形成中的作用.基于全基因组信息,分析了近年来从我国多省分离的131株ST9型MRSA中前噬菌体的流行率、分型、亲缘关系和结构特征;选取含不同分型前噬菌体的菌株进行诱导,对诱导获得的噬菌体颗粒进行转导,测定转导子的耐药表型及体外适应性.研究结果显示:猪源ST9型MRSA的前噬菌体携带率为78.6％(103/131株),其中,63株携带完整前噬菌体序列,所有前噬菌体序列均不含耐药基因,仅2.9％(3/103株)的前噬菌体序列含毒力基因;前噬菌体谱型丰富,其整合酶分型主要为Sa2int和Sa4int;各型别前噬菌体结构同源性较高,完整前噬菌体可被诱导为长尾噬菌体;噬菌体颗粒可包装供体菌的aadD、tet(L)耐药基因并转导至受体菌中;转导子可获得卡那霉素、四环素耐药表型,体外生长能力与受体菌株无明显差异(P＞0.05).研究结果表明:猪源ST9型MRSA的前噬菌体携带率较高,谱型丰富,不携带耐药基因,部分噬菌体可包装供体菌的耐药基因转导至受体菌,产生的适应性代价小.

为建立快速检测鸡血清多杀性巴氏杆菌(Pm)抗体的胶体金免疫层析技术,本研究采用胶体金标记兔抗鸡IgG,制备金标垫,将重组脂蛋白B(rPlpB)和羊抗兔IgG抗体分别包被于硝酸纤维膜作为检测线和质控线,优化条件后组装成胶体金试纸条(CGTS).结果显示,胶体金标记兔抗鸡IgG的最佳pH为8.5,最佳标记浓度为7 μg/mL;rPlpB和羊抗兔IgG抗体的最佳使用浓度均为0.2 mg/mL.利用该方法检测SPF鸡血清,鸡Pm、鸡大肠杆菌、鸡沙门菌、鸡新城疫病毒和鸡传染性支气管炎病毒阳性血清,结果显示仅鸡Pm阳性血清检测为阳性,其他均为阴性,表明CGTS的特异性强.将琼脂扩散(AGP)效价为1∶16的鸡Pm阳性血清2倍倍比稀释后以CGTS检测,结果显示1∶320倍稀释仍检测为阳性,表明CGTS的敏感性较高.检测鸡Pm阳性血清和SPF鸡血清时,同批次不同试纸条之间、不同批次之间均无差异,表明该方法的重复性好.对89份鸡血清采用CGTS和间接ELISA检测,结果显示,二者阳性符合率为91.5%,阴性符合率为100%,总符合率为94.4%.禽霍乱灭活疫苗免疫后14 d,66.7%的鸡采用该方法能够检出抗体阳性,免疫后21 d,所有鸡均检出抗体阳性.本研究首次基于rPlpB建立了检测鸡血清Pm抗体的CGTS方法,该方法操作简单、特异性强、敏感性高、重复性好,适合在基层推广,为评价禽霍乱疫苗的免疫效果提供了可行技术.

为建立快速检测鸭血清多杀性巴氏杆菌抗体的方法,将兔抗鸭 IgG 标记胶体金,喷涂金标垫,用浓度为 0.2 mg/mL的荚膜和 0.2 mg/mL羊抗兔IgG抗体分别作为检测线和质控线,制备了检测鸭多杀性巴氏杆菌抗体的胶体金试纸条(D-CGTS).特异性试验显示,仅鸭抗多杀性巴氏杆菌血清检测为阳性,阴性血清、鸭抗大肠埃希氏菌、鸭抗鸭疫里默氏菌、鸭抗沙门氏菌和鸭抗金黄色葡萄球菌血清的检测结果均为阴性,表明D-CGTS的特异性良好.敏感性试验结果显示,对效价 1∶16(琼脂扩散试验)的鸭抗多杀性巴氏杆菌血清 1∶320 倍稀释后采用制备的试纸条检测结果仍为阳性,表明 D-CGTS 的敏感性较高.重复性试验结果显示,同批次的不同试纸条和不同批次的试纸条,检测阴性血清和鸭抗多杀性巴氏杆菌血清的结果相同,表明D-CGTS的重复性好.对 92 份鸭血清采用D-CGTS和ELISA进行检测,结果显示,阳性符合率为94.03％,阴性符合率为 100％,总符合率为 95.65％.结果表明,制备的D-CGTS操作简单,具有较强的特异性、较高的敏感性和较好的重复性,适合在兽医临床推广应用.

[目的] 建立一种可同时检测并鉴别鸭圆环病毒和鹅圆环病毒的PCR方法.[方法] 根据已发布的鸭圆环病毒和鹅圆环病毒基因组分子特征设计3条引物,应用3条引物建立了一种在一个反应管中可检测并鉴别鸭圆环病毒和鹅圆环病毒的三引物PCR方法.[结果] 该方法可特异性扩增鸭圆环病毒和鹅圆环病毒,并根据扩增片段大小鉴别病毒种类,检测最低限分别为110 、80 pg·μL?1.应用该方法对临床疑似病例样品进行检测,其结果与用2种病毒对应的单病毒PCR检测方法一致.[结论] 建立的三引物PCR方法特异性强,敏感性高,丰富了水禽圆环病毒病的临床病例的诊断及流行病学监测的技术手段.

为探究跛行番鸭的发病原因,采集关节内的脓性渗出物进行细菌分离,PCR鉴定细菌种属,琼脂扩散试验鉴定其血清型,纸片法分析其药物敏感性.结果,分离鉴定到 1 株血清 1 型鸭疫里默氏菌.该菌敏感的药物有阿米卡星、庆大霉素、新霉素、氟苯尼考和头孢氨苄等.此结果为该鸭场防治鸭疫里默氏菌病疫苗选用和临床用药提供了科学理论依据.

为分离能裂解鸭源大肠杆菌的噬菌体,采用富集法从鸭场污水中分离噬菌体,透射电镜观察其形态,结果显示,分离到1 株噬菌体,电镜显示该噬菌体属长尾病毒科,命名为vB_EcoS214,头部直径约 40 nm,尾部长约 220 nm,宽约 10 nm.该噬菌体有较宽的裂解谱,裂解力较强,可成为噬菌体鸡尾酒制剂的候选噬菌体.能裂解鸭源大肠杆菌噬菌体的成功分离,为鸭源大肠杆菌病的噬菌体疗法提供参考.

The LA-MRSA-CC398 lineage is known for its low host specificity and frequent multiresistance to antimicrobial agents. Colonized swine and their related surroundings represent a considerable risk of LA-MRSA-CC398 colonization or infection for occupationally exposed people through which such isolates might be further disseminated within the human community.

【Objective】 This study was aimed to screen some broad-spectrum Riemerella anatipestifer phages as candidates for therapeutic phage combinations.【Method】 The host range of 35 phages were determined by spot tests with different contents of phages.The methods of transmission electron microscope observation, thermal sensitivity and pH stability evaluation, determination of the best multiplicity of infection（MOI） in the plural, one-step growth curve drawing and sterilization experiment were used to analyze the morphological features and some biological characteristics of phage.【Result】 Phage vB＿RanS＿202 had the broadest cleavage spectrum among 35 phages, which could cleave 42%（42/100） of Riemerella anatipestifer strains including 6 serotypes of 1,2,6,10,11 and 13.The phage had a hexagonal head with approximately 60 nm in diameter and a long tail with 220 nm in length, which was not heat-resistant.The titer of phage decreased from 1.0×10~7 PFU/mL to 1.8×10~5-2.6×10~5 PFU/mL after 80 min of 50 ℃ water bath or 20 min of 60 ℃.The phage could maintain its activity at pH 5.0 to pH 9.0.Using RAf488 as host bacteria, the optimal MOI of the phage was 0.01.One-step growth curve indicated that the latent period of phage was 60 min, the outbreak period was 75 min, and the outbreak quantity was 150 PFU/cell.The results of bactericidal experiment against RAf488 showed that phage had good germicidal efficacy when MOI was 0.004 to 0.016.【Conclusion】 Phage vB＿RanS＿202 had a wide lysis spectrum and the ability of bactericidal in liquid.The results of this study provided a reference for the subsequent screening of phage therapeutic strains.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen, often multi-resistant to antimicrobial agents. Among swine, LA-MRSA of clonal complex (CC) 398 dominates in Europe, Australia and the Americas, while LA-MRSA-CC9 is the main epidemic lineage in Asia. Here, we comparatively investigated the metabolic properties of rare and widespread porcine LA-MRSA isolates from Germany and China using Biolog Phenotype MicroArray technology to evaluate if metabolic variations could have played a role in the development of two different epidemic LA-MRSA clones in swine. Overall, we were able to characterize the isolates' metabolic profiles and show their tolerance to varying environmental conditions. Sparse partial least squares discriminant analysis (sPLS-DA) supported the detection of the most informative substrates and/or conditions that revealed metabolic differences between the LA-MRSA lineages. The Chinese LA-MRSA-CC9 isolates displayed unique characteristics, such as a consistently delayed onset of cellular respiration, and increased, reduced or absent usage of several nutrients. These possibly unfavorable metabolic properties might promote the ongoing gradual replacement of the current epidemic LA-MRSA-CC9 clone in China with the emerging LA-MRSA-CC398 lineage through livestock trade and occupational exposure. Due to the enhanced pathogenicity of the LA-MRSA-CC398 clone, the public health risk posed by LA-MRSA from swine might increase further.

The chloramphenicol-florfenicol resistance gene cfr, also called as a multiresistance gene, not only confers phenicols and streptogramin A but also lincosamides, oxazolidinones and pleuromutilins antibiltics.1 The cfr gene has been detected in isolates of Gram-positive and -negative genera such as Staphylococcus, Streptococcus, Enterococcus, Bacillus, Jeotgalicoccus, Macrococcus, Mammaliicoccus, Escherichia, Proteus, Morganella, Pasteurella, Providencia, Vibrio and Leclercia.2–4 The cfr gene is not only found on the chromosomes, but also found on the plasmids. Studies have shown those plasmids can play a vital role in the dissemination of cfr in both Gram-negative and Gram-positive genera.5 Recently, an ∼40-kb conjugative plasmid pFJ6683 carrying cfr gene was identified by our group.6 Here, we report the complete nucleotide sequence of this plasmid. A unique plasmid pFJ6683 was extracted from the Pasteurella multocida FJ6683 with Qiagen plasmid extraction kits (Qiagen, Hilden, Germany). Sequencing was performed using the PacBio RSII sequencer, and data were assembled by Canu. Gene prediction and annotation were performed with the RAST tool.7 The sequence comparison and mapping were performed using BLAST (http://blast.ncbi.nlm.nih.gov) and Easyfig v.2.1.8

Objectives: To reconstruct the evolutionary history and genomic epidemiology of Staphylococcus aureus ST9 in China. Methods: Using WGS analysis, we described the phylogeny of 131 S. aureus ST9 isolates collected between 2002 and 2016 from 11 provinces in China, including six clinical samples from Taiwan. We also investigated the complex structure and distribution of the lsa(E)-carrying multiresistance gene cluster, and genotyped prophages in the genomes of the ST9 isolates. Results: ST9 was subdivided into one major (n = 122) and one minor (n = 9) clade. Bayesian phylogeny predicted the divergence of ST9 isolates in pig farming in China as early as 1987, which then evolved rapidly in the following three decades. ST9 isolates shared similar multiresistance properties, which were Likely acquired before the ST9 emergence in China. The accessory genome is highly conserved, and ST9 harboured similar sets of phages, but Lacked certain virulence genes. Conclusions: Host exchange and regional transmission of ST9 have occurred between pigs and humans. Pig rearing and trading might have favoured gene exchanges between ST9 isolates. Resistance genes, obtained from the environment and other isolates, were stably integrated into the chromosomal DNA. The abundance of resistance genes among ST9 is Likely attributed to the extensive use of antimicrobial agents in Livestock. Phages are present in the genomes of ST9 and may play a role in the rapid evolution of this ST. Although human ST9 infections are rare, ST9 isolates may constitute a potential risk to public health as a repository of antimicrobial resistance genes.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have been reported in various countries worldwide. However, although China is one of the biggest pig and pork producers, large-scale studies on pig-associated LA-MRSA from China are scarce. The aims of this study were to analyze 2420 non-duplicate samples collected from pigs at swine farms and slaughterhouses in different regions in China during 2014 for the prevalence of pig-associated MRSA and to determine the antimicrobial resistance pheno- and genotypes of the respective isolates. MRSA isolates were identified in 270 (11.2%) samples. The isolates were characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, pulsed-field gel electrophoresis (PFGE) and screening for resistance genes. All MRSA isolates belonged to the clonal complex 9 and spa type t899, but showed variable PFGE patterns. All isolates were non-susceptible to oxacillin, cefoxitin, clindamycin, chloramphenicol, florfenicol, ciprofloxacin, and valnemulin. High rates of resistance were also observed for tetracycline (99.6%), erythromycin (97.0%), quinupristin-dalfopristin (97.0%), and gentamicin (80.4%). Three linezolid-non-susceptible isolates containing the multi-resistance gene cfr and nine rifampicin-non-susceptible isolates with mutations in rpoB were detected. Resistance to β-lactams was exclusively associated with mecA, while phenicol resistance was mainly attributable to fexA, except in the three cfr-positive isolates. The pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) was identified in all MRSA isolates, and no other pleuromutilin resistance genes, except cfr in three isolates, were detected. Pigs are the most important hosts of LA-MRSA in China. Screening for pig-associated MRSA is necessary to monitor changes in epidemiology and characteristics of these important pathogens.