鸭肠炎病毒核衣壳蛋白质互作蛋白质基因荧光定量PCR检测方法的建立

为进一步阐明鸭肠炎病毒(DEV)的致病机理,本试验建立检测鸭肠炎病毒核衣壳蛋白质(NP)的互作蛋白质(PKCI)基因荧光定量PCR方法。针对PKCI基因设计特异性引物,经PCR扩增目的基因构建重组质粒p MD18-T-PKCI,将其作为阳性标准品构建荧光定量PCR标准曲线,并对建立的荧光定量PCR方法进行重复性、特异性和敏感性试验。结果显示:标准曲线的线性关系为Y=-3.31x+42.00,相关系数为-1.00,扩增效率为100%,熔解曲线仅出现单特异峰,对H5亚型、H7亚型和H9亚型禽流感病毒及新城疫病毒、鸭肝炎病毒均未检测到荧光信号。表明本试验建立的方法具有良好的稳定性、特异性和灵敏性。     

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.     

Evaluation of clinical, macroscopic, and histopathologic response to treatment in nonhypoproteinemic dogs with lymphocytic-plasmacytic enteritis

BACKGROUND: Lymphocytic-plasmacytic enteritis (LPE) is a common cause of chronic vomiting and diarrhea in dogs. However, little information is available about endoscopic or histopathologic improvement after therapy in dogs with LPE. HYPOTHESIS: The objective was to study the clinical, endoscopic, and histopathologic evolution of LPE during and after immunosuppressive treatment with prednisone and metronidazole. Most dogs also were treated symptomatically with metoclopramide and cimetidine. ANIMALS: Sixteen dogs with LPE and normal serum protein concentrations diagnosed at the Veterinary Medical Teaching Hospital of the Complutense University of Madrid were monitored during and after drug treatment. The control group consisted of 9 dogs that had no gastrointestinal signs for the preceding 12 months. METHODS: In this prospective clinical treatment trial, clinical, endoscopic, and histopathologic scores were evaluated to describe disease evolution during conventional therapy. Dogs with LPE were monitored for 120 days from the start of treatment. Re-evaluation was performed on post-treatment days 30, 60, 90 (end of treatment), and 120. RESULTS: The average disease activity index observed in our study fell progressively from its initial value, and the decrease between consecutive re-evaluations was statistically significant until day 60 (P = .04). Our results indicate that 75% of the animals revealed improvement of endoscopic gastric lesions (defined as a reduction of the endoscopic score) after treatment, and 75% exhibited improvement of endoscopic duodenal lesions. Statistical analysis of the data revealed significant differences between pre- and post-treatment gastric and duodenal macroscopic endoscopic lesions (P < .05). On the other hand, treatment did not lead to any significant changes in the severity of the gastric and duodenal histopathologic lesions of the affected dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Treatment of nonhypoproteinemic dogs with LPE led to clinical and endoscopic improvement, but histopathologic lesions were unchanged during therapy.     

Three cases of idiopathic eosinophilic enteritis with chronic obstinate diarrhea in Japanese Black fattening cattle

Eosinophilic enteritis (EOE) is a type of inflammatory bowel disease and is characterized clinically by chronic obstinate diarrhea. Three Japanese Black (JB) fattening cattle (2 males and 1 female) on different cattle farms presented with chronic episodic diarrhea without fever or dehydration. Soft reddish spherical carneous tissues (1−3 cm) were occasionally excreted within the diarrheic feces. Administration of antibiotics, antidiarrheal drugs and vermicides had no therapeutic effect, but dexamethasone improved the fecal characteristics. The symptoms persisted until the animals were slaughtered at 27–30 months of age. Histopathological examination of the intestines revealed marked eosinophilic infiltration in the lamina propria and submucosa. From these findings, we diagnosed these cattle as the first cases of EOE in JB cattle.     

Products to alleviate the effects of necrotic enteritis and aflatoxin on growth performance,lesion scores,and mortality in young broilers

This study investigated the effects of necrotic enteritis challenge or a necrotic enteritis challenge with an additional challenge from dietary aflatoxin B₁ compared to broilers that were unchallenged. Also tested were products designed to help alleviate those effects. Lesion scores were higher in broilers challenged to induce necrotic enteritis, but this did not increase with added aflatoxin. The presence of dietary aflatoxin during a necrotic enteritis challenge significantly increased the negative effects of the necrotic enteritis challenge. During the necrotic enteritis challenge virginiamycin, Calibrin-Z, or a blend of Calibrin-Z, an organic acid, and yucca, helped to decrease the effects of the challenge. When birds are challenged with necrotic enteritis and dietary aflatoxin is present, Calibrin-Z had an advantage over all other tested products.     

Laboratory procedures for the diagnosis of gastrointestinal tract diseases of dogs and cats

An increasing number of laboratory tests are available for diagnosis of gastrointestinal tract diseases in dogs and cats. Use of these tests can lead to more accurate and rapid diagnoses. This review discusses laboratory tests, both new and old, and the role they currently play in the evaluation of animals presented with gastrointestinal problems. A minimum database helps assess the severity of the disorder, detect extra-gastrointestinal causes of problems and assists in formulating diagnostic and therapeutic plans.

Faecal examination remains one of the most important diagnostic procedures in the investigation of gastrointestinal problems. Zinc sulphate faecal flotation is an excellent routine screening technique for helminth and protozoal infections, including giardiasis. Rectal cytology can assist in the diagnosis of large bowel disorders. Interpretation of faecal immunodiagnostic tests is hampered by insufficient knowledge of test sensitivities and specificities. Routine faecal cultures are not warranted and faecal occult blood tests are rarely indicated.

Serum tests for gastric inflammation are now under development. The serum trypsin-like immunoreactivity test remains the gold standard technique for the diagnosis of exocrine pancreatic insufficiency. Breath hydrogen tests can be helpful in assessing the functional relevance of mild abnormalities in small-bowel biopsy specimens. Subnormal concentrations of serum cobalamin appear to be more specific indicators of gastrointestinal disease in cats than in dogs. Tests for small intestinal bacterial overgrowth remain controversial and assessment of gastrointestinal permeability has yet to prove its value in the diagnostic assessment of companion animals with gastrointestinal problems. Faecal alpha₁-protease inhibitor (α₁-PI) shows promise for the diagnosis of protein-losing enteropathy.     

酸化和渗透压不同处理顺序对肠炎沙门菌失活的影响

为探讨酸化和渗透压不同处理顺序对肠炎沙门菌失活的影响,运用统计学模型模拟了最优处理条件下,肠炎沙门菌在不同初始接菌量下的失活过程。实验结果表明:在25℃和10 h内,先15%Na Cl再p H值4.5处理使肠炎沙门菌失活的效果大于相反顺序的处理、同时处理或者单独处理的效果。模拟结果表明:在最优处理条件下的各个阶段中,随着初始接菌量的增加,肠炎沙门菌分别达到0.986 4和0.993 9失活率时对应的失活时间的变异性逐渐降低。对于大量群体细菌,用确定性失活动力学一级模型拟合的D值是单一的定值;而对少量群体细菌,由于个体细胞本身的变异性,D值表现出较高的变异性,并且可用概率分布的形式表示。酸化和渗透压不同处理顺序的研究能为食品工业生产控制致病菌提供借鉴,且应用的统计学建模方法有助于从个体细胞的角度理解群体失活过程,提高风险评估的准确性。     

Differences between intestinal segments and soybean meal–induced changes in intestinal mucus composition of common carp Cyprinus carpio L.

The alimentary tract is a possible site where pathogens and toxins can enter. The alimentary tract is protected, amongst others, by mucus. In this study, tissue samples and crude mucus preparations from different parts of the intestinal tract of Cyprinus carpio (from intestinal bulb onto the hindgut) were examined using histological, histochemical and biochemical techniques. Furthermore, the response of the intestinal mucosal layer and intestinal mucus of C. carpio to acute soybean meal (SBM)‐induced enteritis was investigated. In the present study, an indication for a different protein core of mucus high molecular weight glycoproteins (HMGs) for first and second segment could not be found. However, differences in mucus glycosylation could be found. Along the gut axis, the size of the major protein peaks were not similar, which can be caused by a different glycosylation. Also, differences in staining for the antimicrobial peptide beta‐defensin 2 were found. Furthermore, changes in HMGs upon SBM diet were found similar to those found in inflammatory bowel disease (IBD) in humans. Initial changes include: changes in mucin composition, the presence of BD3 and of bacteria in internal organs. After the initial changes, all values measured returned back to the initial pre‐SBM diet values.     

检测石蜡切片中鸭病毒性肠炎病毒间接原位PCR方法的建立

 【目的】建立能对石蜡切片中鸭病毒性肠炎病毒（DEV）核酸进行定位的原位PCR方法,为鸭病毒性肠炎（DVE）存档蜡块的回顾性诊断、致病机理研究等提供有效的实验手段。【方法】据DEV的UL30-UL31基因序列设计PCR引物和寡核苷酸探针,以DEV感染死亡鸭肝脏组织石蜡标本制作切片,经蛋白酶K消化、原位PCR扩增和生物素标记的寡核苷酸探针原位杂交,建立了检测石蜡标本中DEV的间接原位PCR方法并应用于人工感染DEV不同时间的鸭肝脏、DVE发病鸭的存档蜡块和临床病料检测。【结果】间接原位PCR对DVE死亡鸭肝脏的石蜡标本检测结果为阳性,而鸭病毒性肝炎、鸭疫里默氏杆菌病、鸭多杀性巴氏杆菌病、鸭沙门氏菌病和鸭大肠杆菌病死亡鸭肝脏的石蜡标本检测结果为阴性;间接原位PCR对人工感染DEV后2、4、6、12、24、48和72 h不同时间的鸭肝脏检测结果均为阳性,阳性细胞有肝细胞、窦皮细胞和枯否氏细胞,阳性信号多出现于坏死细胞的碎片中或细胞坏死后形成的空泡内及空泡边缘;对存档蜡块、临床病料的检测与病毒分离鉴定吻合率为100%。【结论】本研究建立的间接原位PCR方法具有直观、敏感、特异性强的优点,在显示核酸阳性信号的同时,还能判别含有靶序列的细胞类型以及组织细胞的形态结构特征与病理变化。可用于DVE的诊断、分子流行病学调查、存档蜡块的回顾性诊断和致病机理的研究。     

Granulomatous enteritis in rainbow trout (Oncorhynchus mykiss) associated with soya bean meal regardless of water dissolved oxygen level

This study investigated morphological changes associated with soya bean meal‐induced enteritis (SBMIE) in distal intestine (DI) of rainbow trout (Oncorhynchus mykiss) fed a soya bean meal (SBM)‐based diet and exposed to normoxia or hypoxia created by optimal and low water flow rates, respectively. A 28‐day adaption period was followed by a 42‐day challenge period where 600 fish were subjected to dietary challenge and/or hypoxia. Twelve tanks each containing 50 juvenile trout were assigned randomly in triplicate to each treatment. Histopathological and immunohistochemical evaluation revealed pathological features that have not previously been described in association with SBMIE. Vacuolar degeneration of epithelial cells mainly at the base of mucosal folds, epithelial cysts, epithelial dysplasia, necrosis, shedding of necrotic cells, and granulomatous inflammation including infiltration of enlarged, sometimes finely vacuolated or “foamy” macrophages, multinucleated giant cells and increased proliferation of fibroblasts were observed. Acid‐fast bacteria were not detected in enlarged macrophages; however, these cells contained AB‐PAS‐ and sometimes cytokeratin‐positive material, which was interpreted to be of epithelial/goblet cell origin. Hypoxia did not affect the morphological changes in DI. These results suggest that SBM was associated with a granulomatous form of enteritis in DI of rainbow trout regardless of water oxygen level.