In recent years, the advancement of nanomedicine has revolutionized the field of vaccine development. Numerous nanoparticle-based nano vaccine candidates have been continuously developed. Herein, we describe a universal avian influenza virus (AIV) vaccine candidate based on ferritin self-assembling nanoparticles (NPs). The ferritin fused with 3M2e was delivered by an recombinant Salmonella vector, resulting in the formation of a novel recombinant strain, rSC0130(pYA3342-3M2eNPs). The 3M2e-ferritin fusion protein was efficiently expressed and self-assembled into NPs within the recombinant strain. The delivery of the recombinant strain rSC0130(pYA3342-3M2eNPs), which carries the 3M2e-ferritin fusion NPs, induced robust specific cellular and humoral immune responses against AIV in vivo, ultimately providing chickens with effective cross-protection against challenges from H9N2 and H7N9 avian influenza viruses. Our study underscores the potential of the Salmonella platform for delivering protein-based NPs and offers new insights into the development of universal vaccines for AIVs.

Salmonella enterica serovar Typhimurium (S. Typhimurium) vectors, which induce broad cellular and humoral immune responses, are excellent candidates for delivering foreign antigens. However, S. Typhimurium strains display limitations, including low levels of antigen protein expression when delivering viral antigens. In this study, we found that replacing the hemagglutinin (HA) precursor sequence of H9N2 AIV (avian influenza virus) with that from H7N9 AIV significantly improved HA protein expression. Building on this, we combined the H9N2 HA leader sequence with a tissue plasminogen activator (tPA) signal peptide and delayed lysis Salmonella mRNA interferase regulation vector (SIRV) system previously developed by our team. This novel approach markedly enhanced the expression of viral antigens delivered by Salmonella vectors. Our results demonstrate that both the H9N2 HA leader sequence and the tissue plasminogen activator (tPA) signal peptide significantly increased H7N9 AIV HA protein expression and substantially improved the protective efficacy of the attenuated S. Typhimurium vector delivering the H7N9 HA protein vaccine against H7N9 AIV challenge. These findings offer valuable insights for developing more effective attenuated Salmonella-based recombinant H7N9 AIV vaccines and provide a valuable reference for vaccine strategies against other infectious diseases.

Avian reovirus (ARV) is the primary pathogen responsible for viral arthritis. In this study, 2340 samples with suspected viral arthritis were collected from 2019 to 2020 in 16 provinces of China to investigate the prevalence of ARV in China and to characterize the molecular genetic evolution of epidemic strains. From 113 samples analyzed by RT-PCR, 46 strains of avian reovirus were successfully isolated and identified. The genetic evolution of the σC gene showed that 46 strains were distributed in 1–5 branches, with the largest number of strains in branches 1 and 2. The σC gene homology among the strains was low, with approximately 62% homology in branches 4 and 5 and about 55% in the remaining branches. The strains circulating during the ARV epidemic in different provinces were distributed in different branches. The SPF chickens were immunized with inactivated vaccines containing strains from branches 1 and 4 to analyze the cross-immune protection elicited by different branches of ARV strains. A challenge protection test was performed using strains in branches 1, 2, 4, and 5. Our results showed that inactivated vaccines containing strains from branches 1 and 4 could fully protect from strains in branches 1, 4, and 5. The results of this study revealed the genetic diversity among the endemic strains of ARV in China from 2019 to 2020. Each genotype strain elicited partial cross-protection, providing a scientific basis for the prevention and control of ARV.

2020年在某鸡场做跟踪服务时,发现该场两批蛋鸡初开产就产软壳蛋、破壳蛋,并且无产蛋高峰.分析以往的生产记录和检测数据,认为雏鸡在小日龄感染QX型传染性支气管炎(IBV),青年鸡阶段又发生了滑液囊支原体(MS),是导致该问题发生的主要原因.现将该案例的发生发展情况,综合分析及针对性防控建议汇总如下,希望对养殖生产有所借鉴和帮助.

为了解我国猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的流行及遗传变异情况,2020年对来自7个省的570份疑似PRRSV感染样品进行RT-PCR检测,并对60个PRRSV阳性样品进行ORF5基因测序及分析比较.RT-PCR检测结果显示,2020年送检样品中,PRRSV阳性检出率为23.68％(135/570),其中保育猪病料的阳性检出率最高,为32.24％(108/335).同源性对比及遗传进化分析结果显示,60个PRRSV均为PRRSV2(美洲株),主要属于谱系1和谱系8,占比分别为51.67％和43.33％.氨基酸分析结果显示,60个PRRSV的GP5蛋白氨基酸以点突变为主,其中10个PRRSV的GP5蛋白氨基酸出现缺失突变,且非中和表位、中和表位、潜在毒力位点及N-糖基化位点均发生不同程度的变异.结果表明,保育猪群为PRRSV高发病群体,谱系1 PRRSV或已成为国内流行优势毒株,且PRRSV不断发生变异,这或许会影响现有疫苗的免疫保护效果.因此,针对PRRS需要综合防控,除选用安全有效的PRRSV弱毒活疫苗进行免疫外,加强猪场生物安全及饲养管理也极其重要.

为了解鸡传染性支气管炎病毒(IBV)在我国的流行规律,通过RT-PCR方法,对2019—2021年从我国29个省级行政区送检的38442份疑似IBV感染样品进行病原检测和鉴定,对检出的阳性样品进行S1基因测序分型,然后对检测结果进行时间、空间和群间统计分析.结果显示:共检出6436份IBV阳性样本,阳性检出率为16.7％.2019—2021年的IBV阳性检出率分别为12.5％、16.4％、21.0％,呈逐年增长趋势:阳性检出率呈现一定的季节性变化特点,7—9月检出率(11.3％～15.4％)较低,而3—6月(17.3％～19.3％)和10—12月(17.5％～19.0％)较高.全国29个省级行政区中有27个检出阳性样品,其中东部和中部家禽养殖密集区阳性检出率较高,而西部和北部地区较低.白羽肉鸡阳性检出率最高(28.9％),肉种鸡阳性检出率最低(5.0％),0～6周龄阶段IBV感染较为严重.共检出5种基因型,其中QX型(66.5％)、GVI型(27.7％)占比较高,为当前流行的优势基因型;不同品种鸡群中流行的基因型存在一定差异,白羽肉鸡群中以QX和GVI型为主,而其他鸡群中流行的基因型较为复杂;QX型0～3周龄(33.2％)、GVI型3～6周龄(66.4％)阳性检出数量占比较高,6周龄以后占比均较低(<14.0％).结果表明:我国鸡群中IBV感染普遍,尤其是家禽养殖密集区,且流行呈逐年加重趋势;冬春或秋冬季节多发,小日龄鸡群感染严重;流行基因型复杂,以QX型和GVI型多见.结果提示,我国的IB防控面临较大压力与挑战,需要加强优势基因型疫苗的研发,重点做好小日龄鸡群的IB防控.

不同的鸡滑液囊支原体(MS)分离株对鸡体的侵袭部位和侵袭力不同,根据侵袭部位可分为呼吸型、滑膜囊炎型和生殖型.近几-生殖型滑液囊支原体的发病率较高,给养殖业带来了较大的影响和经济损失,希望引起大家关注.

近年来,禽呼肠孤病毒(ARV)在我国白羽肉鸡群中的感染率呈上升趋势,流行范围较广,给我国肉鸡养殖企业带来了较大经济损失.ARV易变异重组,因此全面了解其分子流行病学特征具有十分重要的意义.为了解我国白羽肉鸡群中ARV的遗传变异特点,2020—2021年从山东、河北、福建、安徽、辽宁、江苏等6个省份收集疑似ARV感染样品,采用RT-PCR方法筛选阳性样本,然后对阳性样本进行σC基因(1088 bp)测序,根据测序结果分析ARV不同基因型分布规律及占比.结果显示:2020—2021年,从6个省份中,经RT-PCR检测,检出ARV阳性样品157份,总阳性检出率为62.1％,不同省份的阳性检出率为44.4％～77.3％.157份阳性样品可分为6个基因型,其中基因1型61份(38.85％)、基因2型41份(26.11％)、基因3型15份(9.56％)、基因4型9份(5.73％),基因5型30份(19.11％),基因6型1份(0.64％);61份基因1型样品中,38份与标准ARV疫苗株(S1133)属于同一亚分支,另外23份与4599参考株处于单独的另一亚分支.结果表明:我国肉鸡群中存在一定程度的ARV流行;流行的ARV基因型较为复杂,且存在基因1型变异毒株,当前的疫苗株已不能对其提供足够的保护.结果提示,今后需要进一步开展ARV感染监测,评估其流行及变异情况,调整免疫策略,加快新疫苗等防疫技术及产品研发.本研究初步了解了我国部分省份的ARV分子流行病学特征,对我国ARV感染的防控具有重要意义,也为变异株新型疫苗研发提供了依据.

为了解我国不同生长阶段猪群猪瘟(classical swine fever,CSF)、猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)及猪伪狂犬病(pseudorabies,PR)的免疫情况,从而为制定合理有效的免疫程序提供理论依据,进而更好地防疫此3种疫病,本试验应用ELISA方法对2018年收集的14580份猪血清样本进行了CSF、PRRS及PR-GE抗体检测.结果 显示,2018年,我国猪群CSF、PRRS、PR-GE抗体阳性率分别为78.84％,78.23％和27.47％;不同阶段猪群同种疫病抗体阳性率差异较大,其中,CSF抗体阳性率公猪最高(91.67％)、保育猪最低(57.00％),PRRS抗体阳性率育肥猪最高(91.31％)、保育猪最低(50.02％),PR-GE抗体阳性率公猪最高(33.98％)、后备母猪最低(19.17％);不同阶段猪群同种抗体离散度也存在较大差异.

自2000年以来,我国不同地区经人工授精的蛋种鸡和肉种鸡在开产后出现产蛋下降的情况逐年增加.为确定引起该病的病原,本研究从山东、辽宁、河南地区的蛋种鸡和肉种鸡场采集发病样品66份,进行病毒和细菌的分离鉴定.结果 显示,所有样品中均未发现疑似病毒和支原体,而鸭源鸡杆菌检出率高达100％.对分离株16S rRNA基因序列进行核苷酸比对和遗传进化分析,结果表明所有分离株16S rRNA基因核苷酸高度同源,与已报道的鸭源鸡杆菌同源性达99.8％以上,处于同一遗传进化分支.动物回归试验结果表明,135日龄开产母鸡经生殖道2次接种鸭源鸡杆菌5d后开始减料、产蛋下降,个别鸡精神沉郁,但无死亡现象,剖检主要表现为输卵管囊肿,卵泡破裂、变形以及肝脏黄色坏死,与自然发病症状一致.由此确定,引起人工授精种鸡产蛋下降的病原为鸭源鸡杆菌.

The H9N2 avian influenza virus is not only an important zoonotic pathogen, it can also easily recombine with other subtypes to generate novel reassortments, such as the H7N9 virus. Although H9N2 live attenuated vaccines can provide good multiple immunities, including humoral, cellular, and mucosal immunity, the risk of reassortment between the vaccine strain and wild-type virus is still a concern. Here, we successfully rescued an H9N2 live attenuated strain [rTX-NS1-128 (mut)] that can interdict reassortment, which was developed by exchanging the mutual packaging signals of HA and truncated NS1 genes and confirmed by RT-PCR and sequencing. The dynamic growth results showed that rTX-NS1-128 (mut) replication ability in chick embryos was not significantly affected by our construction strategy compared to the parent virus rTX strain. Moreover, rTX-NS1-128 (mut) had good genetic stability after 15 generations and possessed low pathogenicity and no contact transmission characteristics in chickens. Furthermore, chickens were intranasally immunized by rTX-NS1-128 (mut) with a single dose, and the results showed that the hemagglutination inhibition (HI) titers peaked at 3 weeks after vaccination and lasted at least until 11 weeks. The cellular immunity (IL-6 and IL-12) and mucosal immunity (IgA and IgG) in the nasal and trachea samples were significantly increased compared to inactivated rTX. Recombinant virus provided a good cross-protection against homologous TX strain (100%) and heterologous F98 strain (80%) challenge. Collectively, these data indicated that rTX-NS1-128(mut) lost the ability for independent reassortment of HA and NS1-128 and will be expected to be used as a potential live attenuated vaccine against H9N2 subtype avian influenza.

Salmonella enterica serovar Typhimurium utilizes a series of strategies to evade host innate immune defenses, including the serum complement system. Many microbial pathogens have evolved the ability to bind the complement regulatory protein factor H (FH) through their surface factor H-binding proteins (FHBPs) to circumvent the complement-mediated bactericidal effect. However, the roles of FHBPs in Salmonella pathogenesis are not well understood. In this study, we demonstrated that the survival of S. Typhimurium in human serum was decreased in a time and concentration dependent manner. Pre-incubation with FH attenuated the sensitivity of S. Typhimurium strain χ3761 to complement-mediated serum killing, suggesting FH binding enhance survival in serum. We aimed to identify novel S. Typhimurium FHBPs and characterize their biological functions. Here, six potential FHBPs were identified by two-dimensional (2D)-Far-western blot, and three of them were further confirmed to bind FH by Far-western blot and dot blot. We found that deletion of ompC (ΔompC) significantly inhibited the survival of S. Typhimurium strain χ3761 in human serum. Our results indicated that the ompC mutation does not affect χ3761 adhesion to HeLa cells. Furthermore, a mice infection model showed that deletion of ompC had no significant effect on the histopathological lesions or viability compared with the wild-type strain χ3761. In summary, these results suggested that OmpC is an important FHBP, but not a critical virulence factor of S. Typhimurium.

为建立一种方便、快捷的鸭源鸡杆菌血清抗体诊断方法,通过用鸭源鸡杆菌YT-1株制备抗原,然后免疫健康家兔制备阴阳性血清,建立了鸭源鸡杆菌平板凝集检测方法,并进行特异性试验,确定抗原最佳工作浓度,同时用该方法进行临床样品检测.结果显示:鸭源鸡杆菌染色抗原与鸡源阳性血清产生明显凝集,而与鸡源阴性血清以及鸡大肠杆菌、鸡沙门氏菌、副鸡嗜血杆菌、禽流感病毒、鸡新城疫病毒、鸡传染性支气管炎病毒和禽腺病毒等病原的阳性血清均不凝集;当抗原浓度为5×109 CFU/mL时,抗原与血清凝集最佳.结果表明,建立的鸭源鸡杆菌血清抗体平板凝集试验方法具有良好的特异性,可用于开产前后鸡群鸭源鸡杆菌的检测评估及流行病学调查.

为探究2020年底在南方部分地区出现的肉种鸡群以产蛋下降为主要特征的传染性疾病,本研究通过分子生物学、病毒分离、血清学、动物回归试验,并对分离到的坦布苏病毒E基因进行测序分析,结果显示,从样本中检测到坦布苏病毒,并通过接种鸡胚和细胞分离到该病毒;攻毒试验显示该病毒可以对SPF蛋鸡造成严重的产蛋下降,与临床发病症状一致;血清学检测分析显示发病鸡群坦布苏病毒抗体血清学变化显著;E基因测序分析发现该坦布苏病毒区别于目前国内水禽流行毒株,与泰国坦布苏病毒的同源性较高.以上研究结果分析表明造成近期肉种鸡群产蛋下降的病因为一种新型坦布苏病毒.该研究为家禽产蛋下降案例的分析与防控提供了方向与思路,为不同易感宿主坦布苏病毒的分离鉴定及其遗传变异分析提供了理论基础.

近几年,在鸡群中出现一种以肝破裂血水为主要特征的疾病,简称为肝破裂血水综合征,给养殖单位造成较大经济损失.为探究鸡肝破裂血水综合征发生原因,采集11省区市9种品系鸡的458份临床肝破裂血水样本,开展病原分离以及分子生物学检测、病理学诊断、内毒素检测和动物试验,并结合流行病学调查进行病因探讨.病原分离结果显示:检测到大肠杆菌14份,占比2.8％;沙门氏菌7份,占比1.5％;未检测到弯曲杆菌以及未分离到相关病毒.PCR、RT-PCR结果显示:检测到禽腺病毒(FAdV)3份,占比0.66％;禽戊型肝炎病毒(HEV)1份,占比0.22％;未检测到鸡传染性贫血病毒(CIAV)、马立克病毒(MDV)、禽白血病病毒(ALV).Random PCR检测未发现RNA和DNA病毒;内毒素检测发现15份样本超标,占比3.3％.动物试验结果显示,腹腔攻毒不能复制出临床肝破裂血水表征.选取11省区市99份有代表性的肝脏样本,固定后病理学诊断发现:无疑似髓样白血病/白血病病理变化;疑似细菌造成的坏死性肝炎4份,占比4.04％;疑似禽网状内皮细胞增生病并发血管瘤病理变化1份,占比2.02％;淀粉样变94份,占比93.94％.流行病学调查发现:该病在全国各地均有发病,发病鸡品种和日龄广泛,无明显季节性;同一鸡场不同品种,在饲料、免疫和管理都一致的情况下,有的品种肝破裂血水,有的品种正常.以上研究表明,近几年鸡群中出现的肝破裂血水综合征病例与常见的可引起鸡肝破裂出血的因素无明显相关性,初步分析与鸡体本身存在遗传自身免疫性缺陷因素有关,机体反应过度,出现细胞因子风暴综合征导致鸡肝脏淀粉样变出血,从而出现肝脏破裂血水的临床症状.

为了解我国鸡滑液囊支原体(Mycoplasmasynoviae,MS)感染的最新流行情况,本研究于2016-2019年从江苏、安徽、山东、河南、河北、宁夏、黑龙江、湖北等8个省份疑似发生MS感染的鸡场采集样品,进行MS菌株的分离鉴定,结果共获得48株MS分离株.对这些分离株的vlhA基因片段测序和分析显示,其中46株均属于K基因型,另外2株分别属于A基因型和E基因型.研究结果表明,当前国内流行的MS优势基因型是K基因型,与其他国家和地区流行的基因型存在显著性差异.本研究结果为制定适合我国鸡群MS控制与净化的策略提供了必要的流行病学依据.

2017年10月—2019年5月,本实验室从广东、四川、河北、山东、安徽、辽宁等广大养鹅地区采集了67份典型雏鹅痛风样品进行了实验室检测以及病原的分离鉴定,检测结果表明:所有样品中均检测到了鹅星状病毒,并且约有94.03％ 的样品为两个不同种的鹅星状病毒混合感染.病原分离结果表明:鹅星状病毒FLX株的变异株病毒(命名为SCCD)可以在鹅胚上稳定增殖,并且能稳定致死10日龄鹅胚,致病力较鹅星状病毒FLX增强;新型鹅星状病毒株(命名为SDPD)不能在鹅胚和SPF鸡胚上稳定增殖.病毒的基因组测序结果表明:鹅星状病毒FLX株与鹅星状病毒FLX的变异株病毒SCCD株、新型鹅星状病毒SDPD株全基因组核苷酸相似性分别为89.7％、58.1％,ORF1b基因氨基酸相似性分别为98.4％、61.0％,ORF2基因氨基酸相似性分别为81.0％、42.5％.将新分离的鹅星状病毒株接种1日龄健康雏鹅,结果表明,鹅星状病毒SCCD株和鹅星状病毒SDPD株虽然能使雏鹅发生死亡,引起肝炎、肾肿胀和增重减少等变化,但雏鹅均无典型痛风(脏器尿酸盐沉积)表现,而两种鹅星状病毒株混合攻毒能使44％ 的雏鹅发生典型痛风,并与临床发病症状一致.因此,临床上引起雏鹅痛风的病原可能是两种鹅星状病毒,即新型鹅星状病毒和鹅星状病毒FLX的变异株病毒.

Background Recombinant Salmonella enterica serotype Choleraesuis ( S . Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S . Choleraesuis vaccine candidate strain rSC0011 (ΔP crp527 ::TT araC P BAD crp Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 , Δ, deletion, TT, terminator) delivering SaoA, a conserved surface protein in most of S . suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein, we described another recombinant attenuated S. Choleraesuis vector, rSC0012 (ΔP fur88 :: TT araC P BAD fur Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 ) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results The strain rSC0012 strain with the ΔP fur88 ::TT araC P BAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔP crp527 ::TT araC P BAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD 50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S . Choleraesuis challenge in BALB/C mice. Conclusions The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S . Choleraesuis and S. suis .

Streptococcus suis, a major zoonotic pathogen in swine, can be classified into 35 serotypes. However, no universal vaccine against the multiple serotypes of S. suis is available, though some studies have shown homologous protection. Hence, developing an effective universal vaccine to protect pigs against multiple S. suis serotypes is necessary, or at the very least, to protect pigs against diseases caused by the dominant pathogenic serotypes. Enolase, a highly conserved surface protein, is present in all of the described S. suis serotypes. rSC0016 is an improved recombinant attenuated S. Choleraesuis vaccine vector, combining a sopB mutation with regulated delayed systems, achieving an adequate balance between host safety and immunogenicity. In order to develop a universal vaccine against the multiple serotypes of S. suis, a novel recombinant vaccine strain rSC0016 that carries a heterologous antigen enolase was developed in this study. According, it was found that the recombinant vaccine strain rSC0016(pS-Enolase) exhibited better colonization compared to the vaccine control strain rSC0018(pYA3493). In addition, a mouse model immunized with the strain rSC0016(pS-Enolase) elicited significant IgG antibody responses against both enolase and Salmonella antigens, while inducing good mucosal, humoral, and cellular immune responses against enolase. Finally, immunization with rSC0016(pS-Enolase) was shown to confer 100%, 80%, and 100% protection against the serotypes of SS2, SS7, and SS9, respectively, and significantly reduced histopathological lesions in mice. Overall, this study provides a promising universal vaccine candidate for use against the multiple serotypes of S. suis.

猪瘟E2基因工程亚单位疫苗能诱导机体产生抗CSFV中和抗体,被认为是最具应用前景的新型猪瘟疫苗.本研究利用稳定表达重组CSFV E2蛋白的细胞系HEK-293T-E2,制备E2亚单位疫苗并在动物体内检测其免疫原性.结果 显示,制备的E2亚单位疫苗能成功诱导试验兔和仔猪产生抗CSFV抗体,相比对照组,试验动物能够抵抗猪瘟标准强毒石门株的攻击,并且没有产生体温升高及其他不良反应,证实了该疫苗的有效性和安全性.本研究为新型猪瘟亚单位疫苗的研发提供了重要线索.