重组C型肉毒梭菌毒素的表达与免疫保护性分析

本研究旨在获得重组C型肉毒梭菌毒素蛋白,并评价其免疫保护性。将麦芽糖蛋白（MBP）和C型肉毒梭菌毒素重链C末端（CH_C）的编码基因序列进行优化和串联,获得基因片段GMBPCH_C。将GMBPCH_C克隆至pET28a-（＋）后转化大肠杆菌BL21（DE3）感受态细胞,分别在15和37℃两种温度条件下诱导表达。利用Ni-IDA亲和层析方法对可溶性表达的目的蛋白进行纯化,从而获得重组蛋白rMBPCH_C。将rMBPCH_C与Montanide ISA 201佐剂混合制备成疫苗,免疫4只家兔,剂量为100 μg/只。根据《中华人民共和国兽药典》（2015年版）规定的方法检测一免后21 d及二免后14 d家兔血清的中和抗体效价。同时,在二免后14 d对家兔进行攻毒。结果表明,rMBPCH_C在37℃的诱导温度下,主要以包涵体的形式表达;在15℃的诱导温度下,可溶性表达的比例可达50%。一次免疫后,免疫组4只家兔血清对C型肉毒梭菌天然毒素（简称天然毒素）的中和效价均可达到1（0.1 mL血清中和1个小鼠最小致死量（MLD）的天然毒素）。二免后,家兔血清的中和抗体效价可达到4~8。用10个家兔MLD的天然毒素攻毒后,免疫组家兔得到了100%（4/4）的保护,而用1个家兔MLD的天然毒素攻毒后,对照组家兔100%（2/2）死亡。以上结果说明,rMBPCH_C具有良好的免疫原性,从而为C型肉毒梭菌病基因工程亚单位疫苗的研制提供了重要的试验数据。     

Corynebacterium pseudotuberculosis infection in goats. VIII. The effect of vaccination against experimental infection

The effect of an inactivated vaccine against C. pseudotuberculosis infection was tested on castrated male kids from a herd free from caseous lymphadenitis. The animals were divided into 3 groups with 8 animals in each. Group 1 was immunized with crude filtrated C. pseudotuberculosis toxoid and whole killed organisms, while Group 2 in addition was given levamisole. The kids were vaccinated twice at an interval of 4 weeks. Group 3 consisted of unvaccinated animals. All groups were challenged subcutaneously with live bacteria 4 weeks after the last vaccination. Unvaccinated animals showed the most severe course of illness after challenge. Development of abscesses in the regional lymph nodes (Inn. subiliaci) was significantly more common in unvaccinated than in vaccinated kids at necropsy 2 months after challenge. There was, however, no such difference between the vaccinated groups, and there was no difference between any of the groups as regards abscess formation at the inoculation site. In each of the 2 vaccinated groups, there was a titre rise following vaccination in the hemolysis inhibition test, whereas no such rise was seen in the bacterial agglutination test. The titre values in both tests increased significantly after challenge in all the groups, the increase being most rapid in the vaccinated animals. The present investigation indicates that development of caseous lesions in lymph nodes in goats, following subcutaneous inoculation with C. pseudotuberculosis, can be reduced by an inactivated vaccine containing whole organisms and crude toxin.     

Campylobacter-induced interleukin-8 responses in human intestinal epithelial cells and primary intestinal chick cells

Campylobacter (C.) jejuni and C. coli can cause gastrointestinal disorders in humans characterized by acute inflammation. Inflammatory signals are initiated during interaction between these pathogens and human intestinal cells, but nothing is known about the stimulation of avian intestinal cells by Campylobacter. Interleukin-8 (IL-8) as a proinflammatory chemokine plays an important role in mobilizing cellular defence mechanism. IL-8 mRNA expression in both human intestinal cells (INT 407) and primary intestinal chick cells (PIC) was determined by quantitative real-time RT-PCR. The secretion of IL-8 protein by INT407 was measured using ELISA. Although C. jejuni and C. coli are considered to be harmless commensals in the gut of birds, the avian Campylobacter isolates investigated were able to induce the proinflammatory IL-8 in PIC as well as in INT407. In an in vitro system, C. jejuni as well as C. coli were able to induce IL-8 mRNA in PIC. Relation between the virulence properties like toxin production, the ability to invade and to survive in Caco-2 cells and the level of IL-8 mRNA produced by INT 407 and PIC after infection with Campylobacter strains was also investigated.     

The effect of administration of a single dose of T-2 toxin on blood coagulation in the rabbit.

The single intravenous administration of T-2 toxin to rabbits at a dosage of 0.5 mg per kg body weight produced an alteration in several blood coagulation parameters. The activities of factors VII, VIII, IX, X and XI were decreased by approximately 40% six hours after T-2 toxin administration. Plasma fibrinogen concentration became elevated within 24 hours after T-2 toxin exposure. Circulating platelet numbers were unaffected by T-2 toxin administration. The similarity of coagulation parameter changes induced by T-2 toxin in animals receiving daily subcutaneous injections of vitamin K (0.5 mg per kg body weight) and those in nonvitamin K supplemented animals suggests that T-2 toxin does not function as a vitamin K antagonist.     

Implication of coprophagy in pathogenesis of chicken botulism

Oral administration of 1 x 10(7) viable spores of Clostridium botulinum type C killed the chickens kept on a board floor to allow them coprophagy, whereas the same dose of the spores failed to develop symptoms in those kept on a wire-net floor not to allow them coprophagy. Type C toxin was detected in the cecal droppings of the chickens of both the groups after feeding the spores and also in serum of symptomatic as well as asymptomatic chickens kept on a board floor. Thus, coprophagy, by which chickens ingest type C toxin (C1 L toxin) and the bacterial cells, seems to be a prerequisite for development of chicken botulism.     

鸡肠内菌发酵党参转化多糖实验研究

用最小二乘法模拟制作了肠内混合菌C8GF20、双歧杆菌C4BF12、乳杆菌C4M50F8在含药培养基中的生长数学模型,以单因子14水平拟合并验证了数学模型的准确性,通过发酵液粗多糖水平变化评估3种菌发酵党参转化多糖的活性作用。当培养基中药物添加量在12.5%-15%时,细菌增长最佳;3种菌实验数学模型经拟合验证,对应的RSD分别为5.45%、4.17%和4.19%;党参发酵液中粗多糖增量在驯化7代后趋于稳定并达最高值,C4M50F8的最高值为124.68%、C4BF12的最高值为114.29%、C8GF20的最高值为119.36%。结果提示,党参加入浓度为12.5%时效果最佳,实验菌发酵党参后多糖增加量均在100%以上,其中乳杆菌C4M50F8效果最优。     

Effect of fusarium T-2 toxin on hematological and biochemical parameters in the rabbit.

The single intravenous administration of purified T-2 toxin to rabbits to 0.5 mg per kg body weight produced a decrease in hematocrit, while blood cell count, and serum alkaline phosphatase activity. The plasma clotting time, as measured by the activated partial thromboplastin time assay, was prolonged after intravenous T-2 toxin administration. In contrast, the administration of T-2 toxin to rabbits at 2.0 mg per kg body weight by gastric intubation produced oral lesions, diarrhea and anorexia in the animals but did not cause significant alteration in hematological and biochemical parameters. The results suggest that the rabbit may be a suitable model for further examination of the biochemical mechanisms involved in the cytotoxic action of T-2 toxin.     

Effects of T-2 mycotoxin on tumor susceptibility in mice

The effect of Fusarium-produced T-2 toxin on tumor growth was evaluated in ICR, CFW, and C57B6/6 mice inoculated with murine sarcoma, Ehrlich ascites carcinoma, or B16F1 melanoma tumor cell lines. Mice were given T-2 toxin intragastrically either at the rate of 2 mg of toxin/kg of body weight daily for 5 days or a single dosage of 4 mg of toxin/kg and were inoculated SC with tumor cells 1 or 2 days after administration of toxin. Tumor growth was assessed 15 to 41 days after tumor challenge by determining the frequency of tumor development and tumor weights. Significant increases in the frequency of development of murine sarcoma (P less than 0.005), Ehrlich ascites carcinoma (P less than 0.01), and B16F1 melanoma tumors (P less than 0.05) were detected in toxin-treated mice, compared with control mice. Murine sarcoma and B16F1 melanoma tumor weights also were significantly (P less than 0.01) higher in toxin-treated mice. The effect of T-2 toxin on tumor growth was more marked after 5 daily treatments than after a single dose.     

Suspected swainsonine poisoning in a Belgian horse

The case of a horse with acute symptoms of excitement, exaggerated fright reactions and trembling is presented. In addition to these cerebral symptoms, mild cerebellar ataxia and a renal tubular lesion were diagnosed. Suspected swainsonine poisoning was confirmed by the presence of the toxin (154 ng/ml) in the serum sample taken immediately after admission. A good recovery was seen after fluid therapy with supplementation of potassium, a dopamine drip and administration of diazepam. The effects of the toxin, by inhibiting the lysosomal enzyme α‐mannosidase and mimicking the genetic mannosidosis, are discussed.     

红豆草黑腐病病原鉴定及毒素组分研究

为明确红豆草(Onobrychis viciaefolia Scop.)黑腐病的病原及其病菌的主要制病机理,对甘肃省兰州、定西等地红豆草黑腐病进行调查,通过对病叶进行组织分离得到病菌,经纯化培养、致病性测定后进行病原鉴定;病菌用PSK培养液在适宜条件下培养,经乙酸乙酯萃取,获得粗毒素液,进行生物测定后,经过浓缩、纯化,得到较纯的毒素样品,利用气相色谱-质谱联用仪(HP6890-5973I)测定了该毒素组分。结果表明:该病菌属半知菌亚门极细链格孢菌(Alternaria tenuis Nees.),病菌粗毒素液对红豆草种子根伸长、离体叶片和叶圆片具有较强的毒性;红豆草细链格孢菌毒素的主要组成成分为乙酸-N-羟基琥珀酰亚胺酯(C₆H₇NO₄),相对含量为67.02%。     

The Early Effects of Clostridium perfringens Type D Epsilon Toxin in Ligated Intestinal Loops of Goats and Sheep

Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals. The disease is caused by C. perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine. In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine. Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species. Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin. Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin. Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment; these changes were more marked in goats than in sheep. No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin. These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms.     

The role of procaine in adverse reactions to procaine penicillin in horses

Procaine penicillin is a commonly used antibiotic in equine medicine but its use is associated with a substantial incidence of adverse reactions. Soluble procaine concentrations were determined by HPLC in several commercially available procaine penicillin preparations, including some that were involved in adverse reactions. The mean (+/- SEM) soluble procaine concentrations in the veterinary preparations was 20.18 +/- 5.07 mg/ml, which was higher than the concentration in the only procaine penicillin preparation for use in humans in Australia of 7.3 mg/ml. Heating the veterinary procaine penicillin preparations to 50 degrees C for 1 day led to a significant (P less than 0.01) increase in the amount of soluble procaine. Heating to 50 degrees C for 7 days also produced a significant (P less than 0.02) increase. Soluble procaine tended to return to baseline concentrations when veterinary procaine penicillin preparations were heated to 50 degrees C for 2 days then stored for 7 days at room temperature. Administration of procaine HCl intravenously (IV) at 2, 5, and 10 mg/kg produced behavioural, locomotor and vascular reactions, which were clinically similar to those reported in adverse reactions to procaine penicillin. The more severe reactions occurred at higher doses, although different horses responded variably at the same dose. Some adverse reactions lead to recumbency but none were fatal. The blood procaine concentrations 1 min after IV administration averaged 19.0 +/- 12.6 and 25.3 +/- 16 micrograms/ml at 2.5 mg/kg and 5 mg/kg, respectively. Ten min after administration, blood procaine concentrations were significantly higher (P less than 0.001) in the 5 mg/kg group than in the 2.5 mg/kg group. Intramuscular (IM) procaine HCl at 5 mg/kg produced significantly lower (P less than 0.001) blood concentrations than similar IV doses, and, in contrast to the IV doses, the amount of procaine in the blood was significantly higher 5 and 10 min after administration than it was after 1 min. Mild excitatory reactions in 4/5 horses were noted 5 to 10 min after IM administration. Administration of diazepam 20 s before procaine HCl prevented the excitatory adverse reaction in 2/2 horses, but administration after the procaine did not influence the outcome.     

Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice

Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia.     

Susceptibility of mink to Clostridium botulinum type E toxin

Experiments aiming at elucidation of the toxicity of Clostridium botulinum type E for mink are described. The observations indicate that amounts in the order of 2 x10^s intraperitoneal MLD (mice) or approximately 200 MLD per g of type E toxin will kill a mink after oral administration. The symptoms observed in the animals were atypical as there was an unusually short period between administration of the toxin and the onset of symptoms and deaths of the animals. Similar results were obtained when Clostridium botulinum type E toxin was fed to Swiss mice. When mice were protected by subcutaneous injections of type E antitoxin prior to feeding the animals survived without showing any symptoms.Subcutaneous injection of type E toxin in amounts of the order of 2 x10^s intraperitoneal MLD (mice) killed mink, and typical symptoms of botulism were observed. This quantity corresponds to ap-proximately 2 intraperitoneal MLD (mice) per g.Comparison is made with previous observations obtained in similar experiments made with Clostridium botulinum type C toxin. It is shown that mink arc substantially less susceptible to type E than to type C toxin when the toxins are given by mouth. On this basis previous results in reports on outbreaks of botulism in mink caused by Clostridium botulinum type E may be regarded as questionable.     

The distribution and density of Clostridium difficile toxin receptors on the intestinal mucosa of neonatal pigs

Clostridium difficile is an enteric pathogen affecting a variety of mammals, but it has only recently been diagnosed as a cause of neonatal typhlocolitis in pigs. The most important virulence factors of C. difficile are 2 large exotoxins, toxin A (TcdA) and toxin B (TcdB). TcdA is a potent enterotoxin with effects on host tissues that are dependent upon receptor-mediated endocytosis of the intact toxin. TcdB is an effective cytotoxin, but it apparently does not bind receptors on intact mucosal epithelium. TcdB is much less toxic in vivo unless there is underlying damage to the mucosa, and it is not essential for the virulence of C. difficile. One hypothesis to explain the resistance of most species as neonates (e.g., humans and hamsters) is that they may lack significant numbers of TcdA receptors. The susceptibility of neonatal pigs suggests cells of the gastrointestinal mucosa express sufficient numbers of toxin receptors for lesion development. Immunohistochemical (IHC) assays documented specific binding of TcdA, but not TcdB, to the epithelium of the small and large intestine. The carbohydrate Galalpha1-3beta1-4GlcNAc-R has been described as an important receptor for TcdA. However, IHC indicated a distribution on cell surfaces much different from that of TcdA binding, suggesting a specific interaction of toxin with an alternative receptor.     

Distribution of enrofloxacin in intestinal tissue and contents of healthy pigs after oral and intramuscular administrations

The concentration of enrofloxacin in plasma, intestinal tissue, lymph nodes and intestinal contents was investigated in healthy pigs after oral (p.o.) and intramuscular (i.m.) administration of a single dose of 2.5 mg/kg bw. Tissue and content samples were collected from jejunum, ileum, caecum and colon from pigs killed at 2, 3 and 6 h after dosing. Intramuscular administration resulted in significantly higher concentrations in plasma, intestinal tissue and lymph nodes at 2 h but not at 3 or 6 h compared with p.o. administration. The absorption and distribution phase was longer after oral administration, and maximum concentrations in tissue and plasma were determined later than after i.m. administration. No difference between route of administration was observed in the intestinal content. Enrofloxacin concentrations in faeces during a 5-day dosing regimen with i.m. and p.o. administration were determined by both HPLC and bio-assay. Higher concentrations were found after i.m. administration during the first day, but the difference was not significant after 2 days. The biologically active concentrations determined by bio-assay constituted 48-75% of the total concentrations determined by HPLC. On the basis of these results it was concluded that in order to ensure an immediate high concentration of enrofloxacin, and thereby avoid an initial selection for resistant mutants, the intramuscular route seems to be preferable to the oral route.     

产气荚膜梭菌α毒素基因工程菌表达产物免疫原性研究

已构建的能表达产气荚膜梭菌α毒素保护性抗原基因工程菌株Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）经动物试验证实没有毒性。从ＩＰＴＧ诱导后的工程菌中提取包涵体,再辅以氢氧化铝胶制成抗原,免疫小鼠３０ｄ后,用产气荚膜梭菌强毒株培养物上清及培养菌体攻击,结果免疫鼠能抵抗至少２ＬＤ１００的攻击,证明Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）（ｐＸＥＴＡ１）工程菌株表达产物具有良好的免疫原性。     

Corynebacterium pseudotuberculosis exotoxin: fatal hemolytic anemia induced in gnotobiotic neonatal small ruminants by parenteral administration of preparations containing exotoxin

Inoculation of live Corynebacterium pseudotuberculosis, culture supernatant, ammonium sulfate-fractionated crude exotoxin, or chromatographically purified exotoxin preparations into gnotobiotic small ruminants (n = 13) caused death of the ruminants within 48 hours. Characteristic changes observed in animals living greater than or equal to 2 hours after inoculation included hemorrhage and edema at the site of injection, severe hemolytic anemia and hemoglobinuria, dark red fluid in body cavities, lung edema, and icterus. The crude exotoxin preparation caused a syndrome of acute shock in 2 lambs that died within 15 minutes after inoculation. Clinical and pathologic responses of animals inoculated with culture supernatant and purified toxin were similar. Histopathologic evidence indicated that the exotoxin caused necrotic changes in the proximal convoluted tubules of the kidneys. Inoculation with live organisms caused multiple foci of suppurative inflammation in skeletal muscle and adjacent adipose tissue, whereas such changes were not observed in animals administered exotoxin preparations. Although C pseudotuberculosis exotoxin induced a hemolytic anemia in the experimental animals, it did not lead to in vitro lysis of ovine, caprine, or bovine erythrocytes, unless they had been sensitized with Rhodococcus (Corynebacterium) equi filtrate. The toxic sphingomyelin-specific phospholipase D from C pseudotuberculosis had a molecular weight of 31,000 daltons and an isoelectric point of approximately 9.6. The elution profile of exotoxin on a carboxymethyl Sephadex column was studied and the majority of the enzymatic activity was eluted by a NaCl gradient (0.25M to 0.7M) with a maximum at 0.35M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatal placental hemorrhage in pregnant CD-1 mice following one oral dose of T-2 toxin.

Forty-eight hours after oral administration of a single dose (3.0 mg/kg BW) of T-2 toxin to mice on days 7, 8, 10, 11 and 12 of pregnancy, 17% maternal mortality following vaginal hemorrhage was encountered. Necropsy examination of the dead females revealed that massive hemorrhages originating from the placental regions had occurred into the reproductive tract. This observation supports the studies in which hemorrhagic disease has been described as characteristic for intoxications with T-2 toxin. The results suggest that fatal hemorrhage during pregnancy can occur in hemochorial and hemoendotheliochorial placental mammals as a result of T-2 toxin administration.     

Susceptibility of Foxes to Clostridium Botulinum Type C and E Toxins

Investigations were performed to determine the exact susceptibility of foxes to Clostridium botulinum type C and E toxins.Doses of 5 mill. MLD type C toxin mixed with the feed did not cause symptoms of botulism in either cubs or adult foxes. Subcutaneous injections of 300,100(0 MLD or more were fatal to cubs, while 750,000 MLD caused the death of all adults.Regarding type E toxin, doses of 1 mill. MLD affected neither cubs nor adults on oral administration. Subcutaneously injected doses of 5,000 MLD or more killed all cubs, while 10,000 MLD was required to produce lethal effect on adult animals.The conclusion made is that foxes are highly resistant to both type C and E Clostridium botulinum toxins following oral application. It is further revealed that foxes are 60–70 times more susceptible to type E than to type C toxin when injected subcutaneously.