产气荚膜梭菌（Clostridium perfringens）是一种革兰氏阳性、杆状、产芽孢厌氧菌，是一种重要的条件性致病菌，可引起人和动物多种疾病，包括气性坏疽、食物中毒、非食源性腹泻等。C. perfringens可产生大量的毒力因子，包括肠毒素（enterotoxin，cpe）、α毒素（cpa）、β毒素（cpb）、β2毒素（cpb2）ε毒素（etx）、ι毒素（iap/ibp）、坏死性肠炎β样毒素（necrotic enteritis B-like toxin，NetB）、TpeL毒素（Toxin C. perfringens large cytotoxin，tpeL）、微生物胶原酶等20余种毒素及酶类。根据其产生的不同毒素组合可将C. perfringens菌株分为七种类型（A-G）。由C. perfringens感染诱发的鸡坏死性肠炎（necrotic enteritis，NE）是一种常见的禽类肠道疾病，每年可在全球造成约60亿美金的经济损失。目前认为能引发NE的C. perfringens菌株通常为G型，产生NetB毒素。过去NE的防控依赖于抗生素的使用，但随着中国、欧盟、美国、加拿大等国家和地区的“禁抗”、“限抗”等政策的实施，曾经被控制较好的NE已重新流行并对家禽养殖业造成较大影响。而相比于国外对鸡源C. perfringens主要流行型别及其毒素特征的较为系统的研究，我国仍较为缺乏对鸡源C. perfringens毒素型分布数据的掌握。高饲养密度、高蛋白饲料、鸡球虫感染等多种因素均可引起NE，而鸡球虫病是NE的核心诱因。因此，鸡球虫常被用于构建NE实验室模型以探究NE的致病机制及防治策略，但鸡球虫与C. perfringens共感染NE实验室模型仍需优化。此外，在NE替抗策略研发中，多种疫苗已被测试，但其免疫保护功效未达预期，开发有效的疫苗已成为迫切需要。基于此，本论文旨在进一步明确我国鸡源C. perfringens的流行特征，为NE防控提供本土化病原学依据；优化巨型艾美耳球虫（Eimeria maxima）与C. perfringens共感染NE模型，为NE致病机制研究和替抗策略研发提供稳定、可重复的标准化工具；针对单一病原疫苗效果不足的问题，开发新型联合免疫策略，将鸡球虫抗原与产气荚膜梭菌关键抗原结合，构建多价重组亚单位疫苗与DNA疫苗，评估其免疫保护功效。具体研究内容如下：

我国部分地区鸡源C. perfringens的分离鉴定

从四川、安徽、新疆、广东、江苏部分地市肉鸡养殖场采集疑似NE病鸡的肠道内容物等样品。采用胰胨-亚硫酸盐-环丝氨酸琼脂（TSC）选择培养基从样品中分离纯化C. perfringens，并对分离菌株进行形态观察、生化鉴定和16S rDNA分子鉴定；再通过聚合酶链式反应（PCR）技术测定C. perfringens的毒力基因，并根据检测结果和现有毒素分型系统进行归类分型。结果表明，从383份样品中分离出297株分离菌，其形态特征、生化鉴定和16S rDNA分子鉴定均符合C. perfringens特性；PCR结果显示分离出A型C. perfringens 291株，阳性率为97.98%（291/297），其中仅cpa阳性菌株有208株，cpa和cpb2阳性的A型菌株有83株；G型C. perfringens（cpa和netB阳性）6株，阳性率为2.02%。此外，cpb、etx、iap、cpe、tpeL五种毒力基因未被检测到。

E. maxima与C. perfringens共感染NE模型的建立与评估

将200只14日龄的肉鸡分为10组，分别为Control组（空白对照组）、C. perfringens（CP）组（1.0×10⁹ CFUs/羽）、E. maxima（EM）1组（5.0×10³卵囊/羽）、EM2组（1.0×10⁴卵囊/羽）、EM3组（2.0×10⁴卵囊/羽）、EM4组（5.0×10⁴卵囊/羽）、EMCP1组（5.0×10³卵囊/羽+1.0×10⁹ CFUs/羽）、EMCP2组（1.0×10⁴卵囊/羽+1.0×10⁹ CFUs/羽）、EMCP3组（2.0×10⁴卵囊/羽+1.0×10⁹ CFUs/羽）和EMCP4组（5.0×10⁴卵囊/羽+1.0×10⁹ CFUs/羽）。在14日龄时，使用不同剂量E. maxima孢子化卵囊感染。在感染后4天使用C. perfringens攻毒，在20日龄时终止试验，剖杀鸡只，记录攻虫前、20日龄体重及存活率并进行肠道病变记分。结果显示，随着E. maxima感染剂量的增加，单一E. maxima感染组的增重逐渐减缓，但在EMCP共感染组未见该趋势，EMCP1组中增重率显著高于其余EMCP共感染组，EMCP2组、EMCP3组、EMCP4组间未见显著差异。肠道病变记分呈现与体重增长率较为相似的趋势，EMCP1组肠道病变程度较其他EMCP共感染组较轻，且EMCP2组、EMCP3组、EMCP4组间未见显著区别。在EMCP共感染组中，EMCP2组的存活率为85%，而EMCP3组和EMCP4组存活率偏低，分别为80%和70%。综合增重情况、肠道病变记分、存活率三个指标进行判断，EMCP2组的攻虫攻毒剂量（1.0×10⁴卵囊/羽+1.0×10⁹ CFUs/羽）最适用于E. maxima诱发NE实验室模型的构建。

NE重组亚单位疫苗与DNA疫苗制备及其免疫保护效果研究

克隆了E. maxima延伸因子（Elongation factor）1α（EmEF-1α）、C. perfringens胶原蛋白粘附分子（Collagen adhesion protein，CpCna），C. perfringens嵌合分子CpFZ【靶向果糖-1,6-二磷酸醛缩酶（Fructose-1,6-bisphosphate aldolase，FBA）和锌金属蛋白酶（Zinc metalloprotease，Zm）】、C. perfringens嵌合分子CpNA（靶向NetB和α毒素），构建了其原核表达质粒和真核表达质粒，制备了重组亚单位疫苗和DNA疫苗，并将7日龄肉鸡分为13组。重组亚单疫苗免疫保护试验共分7组（试验1），分别为：rEmEF-1α免疫组（rEM）、将rCpCna、rCpNA和rCpFZ混合的C. perfringens重组蛋白免疫组（rCP）、使用rEmEF-1α、rCpCna、rCpNA和rCpFZ的联合免疫组（rEMCP）、未感染未免疫对照组（NCON1）、感染未免疫对照组（CCON1）、佐剂对照组（ACON）和pET-32a标签蛋白对照组（pET-32a）。DNA疫苗免疫保护试验共分6组（试验2），分别为：pEmEF-1α免疫组（pEM）、将pCpCna、pCpNA和pCpFZ混合的免疫组（pCP）、将pEmEF-1α、pCpCna、p-CpNA和pCpFZ混合的联合免疫组（pEMCP）、未感染未免疫对照组（NCON2）、感染未免疫对照组（CCON2）和pVAX1空载体对照组（pVAX1）。两项试验于7日龄一免，17日龄二免，采用E. maxima/C. perfringens共感染NE实验室模型，通过增重率、肠道病变记分、存活率和特异性IgY水平的动力学变化，评估其保护效力。结果显示，与EM免疫组（rEM或pEM）、CP免疫组（rCP或pCP）和对照组相比，rEMCP免疫组和pEMCP免疫组均表现为增重显著改善、零死亡率和最轻的肠道病变，二免后血清特异性IgY抗体显著升高并维持在较高水平。以上研究结果进一步证实加入抗球虫免疫可降低NE致病程度，且针对两种病原的联合免疫策略具有一定潜力。

Clostridium perfringens is a Gram-positive, rod-shaped, spore-forming anaerobic bacterium and an important opportunistic pathogen that can cause various intestinal diseases in humans and animals, including gas gangrene, food poisoning, and non-foodborne diarrhea. C. perfringens produces a wide range of virulence factors, including over 20 toxins and enzymes such as enterotoxins, alpha toxin, beta toxin, epsilon toxin, iota toxin, Necrotic Enteritis B-like toxin (NetB), and microbial collagenase. Based on the different combinations of toxins produced, C. perfringens strains can be classified into seven types (A-G). Necrotic enteritis (NE) in chickens, induced by C. perfringens infection, is a common avian intestinal disease that causes global economic losses of approximately $6 billion annually. Currently, it is believed that C. perfringens strains capable of causing NE are typically type G, which produce NetB toxin. In the past, prevention and control of NE relied heavily on antibiotics. However, with the implementation of antibiotic-free and antibiotic-restriction policies in China, the European Union, the United States, Canada, and other regions, NE, which was once well-controlled, has re-emerged and significantly impacted the poultry industry. Compared to the relatively systematic research on the predominant types and toxin characteristics of chicken-derived C. perfringens in other countries, China still lacks comprehensive data on the distribution of toxin types in chicken-derived C. perfringens. Additionally, multiple factors such as high stocking density, high-protein feed, and chicken coccidiosis infection contribute to NE, with chicken coccidiosis being a major predisposing factor. Therefore, Eimeria spp. are often used to establish laboratory models of NE to investigate its pathogenic mechanisms and prevention strategies. However, the co-infection laboratory model of Eimeria spp. and C. perfringens for NE still requires optimization. Furthermore, in the development of antibiotic-alternative strategies for NE, various vaccines have been tested, but their protective efficacy has not met expectations. Developing effective vaccines has become an urgent need. Based on this, this study aims to further clarify the epidemiological characteristics of chicken-derived C. perfringens in China, providing localized etiological evidence for NE prevention and control; to optimize the co-infection NE model of Eimeria maxima and C. perfringens, providing a stable, reproducible, and standardized tool for NE pathogenesis research and antibiotic-alternative strategy development and to address the insufficient efficacy of single-pathogen vaccines by developing a novel combined immunization strategy. This strategy integrates Eimeria antigens with key C. perfringens antigens to construct multivalent recombinant subunit vaccines and DNA vaccines and evaluate their immune protection efficacy. The specific research contents are as follows:

1. Isolation and identification of chicken-derived C. perfringens isolates in some regions of China

Intestinal contents and other samples were collected from broiler farms in parts of Sichuan, Anhui, Xinjiang, Guangdong, and Jiangsu provinces from chickens suspected of having NE. C. perfringens were isolated and purified from the samples using TSC selective medium, and the isolates were subjected to morphological observation, biochemical identification, and 16S rDNA molecular identification. Polymerase chain reaction was then used to detect virulence genes of C. perfringens, and the isolates were classified based on the results and existing toxin typing systems. The results showed that 297 isolates were obtained from 383 samples, all of which met the characteristics of C. perfringens in terms of morphology, biochemical identification, and 16S rDNA molecular identification. PCR results revealed that 291 isolates were C. perfringens type A, with a positivity rate of 97.98% (291/297), including 208 strains positive only for cpa and 83 type A strains positive for both cpa and cpb2. Six isolates were C. perfringens type G (cpa and netB positive), with a positivity rate of 2.02%. Additionally, the five virulence genes cpb, etx, iap, cpe, and tpeL were not detected.

2. Establishment and evaluation of the E. maxima and C. perfringens Co-Infection Model of NE

A total of 200 14-day-old broilers were divided into 10 groups: the Control group (blank control), C. perfringens (CP) group (1.0×10⁹ CFUs/bird), E. maxima (EM) 1 group (5.0×10³ oocysts/bird), EM2 group (1.0×10⁴ oocysts/bird), EM3 group (2.0×10⁴ oocysts/bird), EM4 group (5.0×10⁴ oocysts/bird), EMCP1 group (5.0×10³ oocysts/bird + 1.0×10⁹ CFUs/bird), EMCP2 group (1.0×10⁴ oocysts/bird + 1.0×10⁹ CFUs/bird), EMCP3 group (2.0×10⁴ oocysts/bird + 1.0×10⁹ CFUs/bird), and EMCP4 group (5.0×10⁴ oocysts/bird + 1.0×10⁹ CFUs/bird). At 14 days of age, the birds were challenged with different doses of E. maxima sporulated oocysts. Four days post-infection, they were challenged with C. perfringens type G. The experiment was terminated at 20 days of age, and the birds were euthanized for necropsy. Body weight before challenge and weight at 20 days, survival rate, and intestinal lesion scores were recorded. The results showed that as the dose of E. maxima infection increased, weight gain gradually slowed in the single E. maxima infection groups, but this trend was not observed in the EMCP co-infection groups. The  body weight rate in EMCP1 was significantly higher than in the other EMCP co-infection groups, while no significant differences were observed among EMCP2, EM/CP3, and EMCP4 groups. Intestinal lesion scores followed a trend similar to weight gain, with EMCP1 exhibiting milder lesions compared to the other EMCP co-infection groups, while no significant differences were observed among EMCP2, EM/CP3, and EMCP4 groups. Among the EMCP co-infection groups, EMCP2 had a survival rate of 85%, while EMCP3 and 4 had lower survival rates of 80% and 70%, respectively. Based on weight gain, intestinal lesions, and survival rate, the challenge dose of EMCP2 (1.0×10⁴ oocysts/bird + 1.0×10⁹ CFUs/bird) was determined to be the most suitable for constructing an E. maxima-induced NE laboratory model.

3. Preparation and immune protection efficacy study of NE recombinant subunit vaccine and DNA vaccine

The elongation factor 1α (EmEF-1α) of E. maxima, the collagen adhesion protein (CpCna) of C. perfringens, and the chimeric molecules CpFZ [targeting fructose-1,6-bisphosphate aldolase (FBA) and zinc metalloprotease (Zm)] and CpNA (targeting NetB and alpha toxin) of C. perfringens were cloned. Their prokaryotic and eukaryotic expression plasmids were constructed, and recombinant subunit vaccines and DNA vaccines were prepared. Seven-day-old broilers were divided into 13 groups. The recombinant subunit vaccine immune protection experiment (Trial 1) consisted of 7 groups: rEmEF-1α immunization group (rEM), C. perfringens recombinant protein immunization group (rCP, a mixture of rCpCna, rCpNA, and rCpFZ), combined immunization group (rEMCP, using rEmEF-1α, rCpCna, rCpNA, and rCpFZ), uninfected and unimmunized control group (NCON1), infected and unimmunized control group (CCON1), adjuvant control group (ACON), and pET-32a tag protein control group (pET-32a). The DNA vaccine immune protection experiment (Trial 2) consisted of 6 groups: pEmEF-1α immunization group (pEM), mixed immunization group (pCP, a mixture of pCpCna, pCpNA, and pCpFZ), combined immunization group (pEMCP, a mixture of pEmEF-1α, pCpCna, pCpNA, and pCpFZ), uninfected and unimmunized control group (NCON2), infected and unimmunized control group (CCON2), and pVAX1 empty vector control group (pVAX1). In both trials, the first immunization was administered at 7 days of age, and the second immunization was administered at 17 days of age. The E. maxima/C. perfringens co-infection model of NE was used to evaluate protective efficacy of vaccine preparations through body weight gain, intestinal lesion scores, survival rate, and kinetic changes in specific IgY levels. The results showed that compared to the EM immunization groups (rEM or pEM), CP immunization groups (rCP or pCP), and control groups, the rEMCP and pEMCP immunization groups exhibited significant improvement in weight gain, no mortality, and the mildest intestinal lesions. Additionally, serum-specific IgY antibody levels significantly increased after the second immunization and remained at high levels. These findings further confirm that incorporating anti-Eimeria immunity reduces the severity of NE and demonstrate the potential of a combined immunization strategy targeting both pathogens.

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