魏氏梭菌引起獭兔急性坏死性胃肠炎的临床病例及救治

依据病兔的临床表现、病理解剖、血液和黏膜涂片检查分析,镜下检查见成对或链状短粗棒状杆排列、有卵圆形芽孢和荚膜的革兰氏阳性菌,拟定为魏氏梭菌引起獭兔急性坏死性胃肠炎。患病兔经肌肉注射5%氟苯尼考注射液,每千克体重0.3 mL,每日1次,连用3 d,隔离治疗;同时对本场健康兔采取饮水预防措施并取得满意效果。     

鹿快疫病原分离及基于α毒素的PCR诊断

从送检鹿病料中分离出一株致病菌,对病原体进行了生物学特性鉴定,并利用设计的特异性引物进行了PCR诊断,最终确定为鹿快疫.本文为腐败梭菌病的快速诊断、流行病学调查建立了方法.     

D 型产气荚膜梭菌贵州分离株ε毒素的 DNA 序列测定与分析

为研究产气荚膜梭菌ε毒素的结构与功能,以及对该病的防治提供基础依据,利用 D 型产气荚膜梭菌贵州分离株（CP02株）,根据 GenBank 中登记的产气荚膜梭菌ε毒素基因设计特异性引物进行PCR 扩增,扩增产物纯化后测定核苷酸序列并与 NCBI 登录的参考毒株进行相似性比较。结果表明：D 型产气荚膜梭菌 CP02株基因片段大小为456 bp,与 CWD_CN_409、NCTC_8533、NCTN_6121、ETX21512、KurCpD1、CtrCpD2、IVRI_Vac1和 IVRI_49菌株的核苷酸序列相似性在98．5％～100％,推导的氨基酸序列相似性在98．4％～100％。碱基突变以 A-G、C-T 间的转换为主,也发生低频率的 A-C、G-T 间颠换,但突变均为点突变。     

A型魏氏梭菌α毒素Asp-56基因定点突变与结构分析

为探索A型魏氏梭菌α毒素第56位天冬氨酸(Asp-56)对其生物学活性的影响,将α毒素Asp-56密码子(GAT)定点突变成甘氨酸(Gly-56)密码子(GGT),构建了含α毒素D56G突变基因表达质粒的重组菌株BL21(DE3)(pM-D56G),该突变体蛋白表达量约为22.48%。α毒素和α毒素D56G突变体蛋白的二级结构主要由α螺旋和无规则卷曲组成,两者的圆二色(CD)光谱检测有微小变化。生物学活性检测结果表明,α毒素D56G突变体蛋白失去了α毒素的磷脂酶C活性,且α毒素D56G突变体蛋白免疫的小鼠能够保护1MLD的A型魏氏梭菌标准株C57-1毒素攻击。此研究为进一步研究α毒素分子结构与生物学功能的关系奠定了基础。     

Infectious Agents Detected in the Feces of Diarrheic Foals: A Retrospective Study of 233 Cases (2003–2008)

Background: Diarrhea is common in foals but there are no studies investigating the relative prevalence of common infectious agents in a population of hospitalized diarrheic foals.
Objectives: To determine the frequency of detection of infectious agents in a population of hospitalized foals with diarrhea and to determine if detection of specific pathogens is associated with age, outcome, or clinicopathologic data.
Animals: Two hundred and thirty-three foals ≤ 10 months of age with diarrhea examined at a referral institution.
Methods: Retrospective case series. Each foal was examined for Salmonella spp., viruses, Clostridium difficile toxins, Clostridium perfringens culture, C. perfringens enterotoxin, Cryptosporidium spp., and metazoan parasites in feces collected at admission or at the onset of diarrhea.
Results: At least 1 infectious agent was detected in 122 foals (55%). Rotavirus was most frequently detected (20%) followed by C. perfringens (18%), Salmonella spp. (12%), and C. difficile (5%). Foals < 1 month of age were significantly more likely to be positive for C. perfringens (odds ratio [OR] = 15, 95% confidence interval [CI] = 3.5–66) or to have negative fecal diagnostic results (OR = 3.0, 95% CI = 1.7–5.2) than older foals. Foals > 1 month of age were significantly more likely to have Salmonella spp. (OR = 2.6, 95% CI = 1.2–6.0), rotavirus (OR = 13.3, 95% CI = 5.3–33), and parasites (OR = 23, 95% CI = 3.1–185) detected compared with younger foals. Overall 191 of the 223 foals (87%) survived. The type of infectious agent identified in the feces or bacteremia was not significantly associated with survival.
Conclusions and Clinical Importance: In the population studied, foals with diarrhea had a good prognosis regardless of which infectious agent was identified in the feces.     

Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis

The present study examines the responses of broiler chickens to oral administration of Clostridium perfringens freshly isolated from field cases of necrotic enteritis (NE). The challenge studies included long-term exposure and short-term exposure, factored in with dietary and management variables including high levels of dietary components such as fish meal, meat meal, abrupt change of feed, and fasting. In the long-term exposure trials, the birds were orally inoculated daily, with 1 ml (1.0 or 2 x 10(8) CFU/ml) of an overnight culture of C. perfringens for 7 days. Short-term exposure trials involved challenge with 1 ml (3 x 10(10) CFU/ml) administered as a single dose. The responses of broilers to orally administered C. perfingens under laboratory controlled conditions are presented and discussed in the context of authentic field cases of necrotic enteritis. None of the challenge trials produced overt clinical signs of NE and there were no mortalities associated with oral exposure to high doses of C. perfringens. However, many of the challenged birds showed distinctly pronounced pathological changes in the intestinal tissue. On gross examination the responses in birds challenged orally with C. perfringens could be placed into two categories: (1) no apparent pathological changes in the intestinal tissue and (2) sub-clinical inflammatory responses with focal, multi-focal, locally extensive, or disseminated distribution throughout various sections of duodenum, jejunum, ileum, and ceca. In birds that responded with intestinal lesions, hyperemia and occasional hemorrhages were the main gross changes. In some birds, the mucosa was covered with a brownish material, but typically, the mucosa was lined by yellow or greenish, loosely adherent material. Mild gross changes were seen in some control birds, but both qualitatively and quantitatively, the lesions were distinctly more pronounced in the challenged birds. Upon histological examination, none of the experimentally exposed birds showed overt mucosal necrosis typical of field cases of NE, but typically the lamina propria was hyperemic and infiltrated with numerous inflammatory cells. Most significant changes were seen at the interface of the basal domain of enterocytes and lamina propria. Multifocally, these areas were extensively edematous, allowing for the substantial disturbance of the structural integrity between the lamina propria and the enterocytes. The lesions observed in the present study were consistently reproduced in all of our challenge trials, hence these responses may signify newly emerging patterns of sub-clinical enteric disorders in contemporary strains of poultry. The pathological changes observed in broilers challenged orally with C. perfringens in the present study, differ significantly from those reported previously, and must be clearly differentiated from those described in cases of NE or ulcerative enteritis. Although no overt necrosis of the intestinal mucosa typical of field cases of NE were observed in the present study, the birds challenged with C. perfringens showed strong inflammatory reaction to the introduced pathogens. The distinct features of the microscopic lesions were changes involving apparently normal enterocytes at the interface of the basal domain of villar epithelia and lamina propria. Although the pathological changes in the intestinal tissues observed in our trials appear to be rather subtle when compared to field cases of NE, the nature of these lesions suggest a significant negative effect on the digestive physiology of intestinal mucosa.     

D型产气荚膜梭菌青海分离株ε毒素遗传变异分析

研究D型产气荚膜梭菌(Clostridium perfringens)青海分离株ε毒素基因的遗传变异特点,试验设计特异性引物扩增ε毒素基因,目的基因片段大小为888 bp.建立PCR反应体系并设置反应条件,扩增产物进行电泳检测、纯化、测定核苷酸序列,与参考序列进行同源比对分析.结果表明,目的基因扩增良好,分离菌株WC-epsilon-FL与菌株C60-3、NCTC 8346、Mukteshwar、AJ250956的核苷酸序列的同源性依次为99.9％、99.8％、98.9％、99.4％.     

山东疫区魏氏梭菌血清型的多重PCR检测

采用多重PCR对从流行过魏氏梭菌病的牧场粪便样品中分离魏氏梭菌的α、β、ε、ι 毒素基因进行了检测,确定了血清型。电泳成像显示,仅魏氏梭菌扩增出了特异性条带,其他对 照菌株无,说明该方法具有很高的特异性。通过对山东德州、枣庄、泰安、临沂、青岛等地养殖场 的418个样品分离的62个菌株的检测,检出率为14．8％,均为A型。研究确认,多重PCR是 魏氏梭菌血清型鉴别的一种快速、简便的研究方法,山东省流行型别与国外报道不完全一致。     

饲粮中添加丁酸梭菌和丁酸钠对产蛋鸡生产性能和蛋品质的影响

试验旨在研究在产蛋鸡饲粮中添加丁酸梭菌和丁酸钠对不同品种产蛋鸡产蛋率、鸡蛋雀斑、暗斑以及蛋品质的影响。选择270日龄狼山鸡、芦花鸡、北京油鸡各320只,随机分成2组,每组8个重复,每个重复20只鸡,对照组（CON）饲喂基础饲粮,试验组（EXP）饲粮中添加100 mg/kg丁酸梭菌＋500 mg/kg丁酸钠。预饲期3 d,试验期为5周。试验结果表明,与对照组相比,①试验组狼山鸡、芦花鸡、北京油鸡产蛋率分别提高12.5%、12.0%和24.9%（P<0.01）;②试验组狼山鸡鸡蛋雀斑3级率下降34.2%（P<0.05）,芦花鸡和北京油鸡鸡蛋各级雀斑率均无显著差异（P>0.05）,狼山鸡、芦花鸡、北京油鸡鸡蛋各级暗斑率均无显著差异（P>0.05）;③试验组狼山鸡蛋重降低1.7%（P<0.05）,蛋形指数增加2.3%（P<0.05）;芦花鸡蛋重增加1.5%（P<0.05）;北京油鸡蛋白高度增加13.9%（P<0.05）,哈氏单位增加4.7%（P<0.05）。综上,在产蛋鸡基础饲粮中添加一定量的丁酸梭菌和丁酸钠,可以提高狼山鸡、芦花鸡、北京油鸡产蛋率,对改善狼山鸡鸡蛋的雀斑也有一定的作用,且有助于提高芦花鸡、北京油鸡的蛋品质。     

重组产气荚膜梭菌β毒素突变体的表达与免疫保护性分析

旨在获得产气荚膜梭菌β毒素（CPB）的重组突变体,并评价其毒力及免疫保护性。对已知的产气荚膜梭菌CPB编码基因进行优化设计,同时引入4个氨基酸突变位点,分别是第212位的精氨酸突变为谷氨酸,第268位的亮氨酸突变为甘氨酸,266位的酪氨酸和275位的色氨酸突变为丙氨酸。此外,在该基因5'端添加Th细胞表位（T）和鞭毛蛋白（flagellin）N末端的编码序列,经人工合成获得重组基因片段（GTF_NCPB_m4）。将GTF_NCPB_m4克隆至原核表达载体pET-30a（＋）中进行表达与纯化,获得重组蛋白rTF_NCPB_m4。利用Western blot方法检测rTF_NCPB_m4与C型产气荚膜梭菌毒素抗血清的反应性,并检测rTF_NCPB_m4对小鼠的毒力。随后,以rTF_NCPB_m4免疫家兔,按照《中华人民共和国兽药典》（2015年版）规定的方法检测家兔血清对C型产气荚膜梭菌毒素的中和抗体效价。结果表明,rTF_NCPB_m4主要以包涵体的形式表达且能与C型产气荚膜梭菌毒素抗血清反应。小鼠安全性试验显示,50 μg的rTF_NCPB_m4对小鼠仍无致死性;免疫rTF_NCPB_m4后,每毫升家兔二免抗血清可中和10~20个小鼠最小致死量（MLD）的C型产气荚膜梭菌毒素;1个家兔MLD的C型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了100%（4/4）的保护。以上结果说明,rTF_NCPB_m4在丧失毒力的同时保留了良好的免疫原性,从而为C型产气荚膜梭菌病基因工程疫苗的研制提供了重要的数据。