猪胸膜肺炎放线杆菌、肺炎支原体和多杀性巴氏杆菌联合检测芯片的研制及应用

本研究旨在建立联合检测胸膜肺炎放线杆菌、猪肺炎支原体和多杀性巴氏杆菌的DNA芯片.用7个从3种病菌基因组中扩增出的不同特异性靶DNA制作基因芯片,并对芯片的靶DNA和探针浓度、杂交温度、重复性、特异性和灵敏度进行了研究.结果表明,检测芯片的特异性强,能与测试的李氏放线杆菌、猪鼻支原体和副猪嗜血杆菌等9种病原区分;灵敏度高,在50μL标记反应体系中,能检测到10～50 pg基组DNA,芯片可重复利用.用芯片对44株目标菌的不同型标准菌株、分离株和疫苗株进行了检测.其信号值≥1 000,信号噪音比(SNR)≥6.用芯片对45头病猪和97头健康猪的临床样品选择培养物进行了检测,其检出率分别为多杀性巴氏杆菌71.1%和49.5%、胸膜肺炎放线杆菌42.2%和26.8%、猪肺炎支原体20%和22.7%,混合感染率分别为42.2%和24.7%.在检测临床样品时,芯片法与PCR的符合率为97.8%～100%,与分离鉴定法的符合率为87.6%～95.6%.研究表明,研制的芯片特异性强、敏感性高、可重复使用,是一种能有效用于胸膜肺炎放线杆菌、猪多杀性巴氏杆菌和猪肺炎支原体鉴定和联合检测的新工具.     

猪肺炎支原体S株的分离与鉴定

从典型猪肺炎支原体病变肺组织传代分离到一株支原体,经培养特性、血清学鉴定、生化鉴定、PCR检测及测序分析证明其为猪肺炎支原体,纯化后命名为S株。将S株P46基因和P97基因R1区的氨基酸序列与其他菌种的相应序列进行同源性分析,该菌株P46基因氨基酸序列与其他菌种同源高达99%以上,P97基因R1区的氨基酸重复数为11个,不同于其他菌株;免疫原性试验结果表明该菌株具有良好的免疫原性。     

一株猪肺炎支原体的分离鉴定

从全国部分猪场采集到疑似猪支原体肺炎肺组织病料12份,提取DNA进行猪肺炎支原体PCR和多重PCR检测,将病料研磨后分离猪肺炎支原体,最终分离到1株疑似猪肺炎支原体;通过测序分析、形态观察、生化试验、血清学试验证实其为猪肺炎支原体。该菌株能适应人工培养基的培养,且传代生长良好,液体培养基中培养活菌滴度达109CCU/m L;菌株有一定的致病性,免疫原性好,可作为疫苗备用菌株,该菌株的分离鉴定为研制猪支原体肺炎疫苗奠定了基础。     

一株猪肺炎支原体HN0613株的分离鉴定

采集疑似猪支原体肺炎病理变化的肺脏,经Friis液体、固体培养基培养、纯化,PCR、测序分析、生化鉴定、生长抑制、代谢抑制和致病性试验证实分离获得一株猪肺炎支原体菌株,命名为HN0613.该菌株能适应人工培养基的培养,且传代生长良好,液体培养基中培养达108 CCU/mL;菌株有较强的毒力,可作为疫苗候选株进一步研究.该菌株的分离鉴定为研制猪支原体肺炎灭活疫苗奠定了基础.     

猪肺炎支原体YN200901株的分离鉴定

采集疑似猪肺炎支原体病理变化的肺脏,接种到改良牛心培养基、猪胃消化液培养基进行培养,经瑞特染色、镜检,菌落狄氏染色、PCR鉴定及序列比对、生化试验鉴定,抗猪肺炎支原体血清生长抑制试验,分离获得一株猪肺炎支原体云南地方流行菌株,命名为YN200901。试验结果为云南本地株猪肺炎支原体的后续研究提供了物质基础。     

猪肺炎支原体DJ-166株的分离鉴定

从山西某猪场采集疑似猪支原体肺炎肺组织病料,接种CH培养基培养,克隆纯化获得一株菌株。该菌株经PCR、培养特性、生化特性和血清学特性试验鉴定为猪肺炎支原体,命名为DJ-166株。该分离菌株在人工合成培养基上传8代稳定后,活菌计数达到108CCU/m L;菌液培养物气管注射猪后,致病性较弱;免疫原性试验证明该分离菌株具有良好的免疫原性,此结论为猪支原体肺炎疫苗的研究提供了必要条件。     

猪肺炎支原体JM株的分离鉴定及主要抗原蛋白基因序列分析

为了对从广东地区若干个屠宰场采集的164份疑似猪肺炎支原体感染的猪肺脏组织进行猪肺炎支原体(Mycoplasma hyopneumoniae,MHp)的分离鉴定,并了解其主要抗原蛋白基因的遗传变异特点,试验先对采集的肺脏组织进行16S rRNA基因PCR检测,阳性样本进行病原的分离培养、形态学观察、双重PCR鉴定、分离株主要抗原蛋白膜蛋白P46全基因与黏附蛋白P97基因R1R2区序列分析及效价的测定。结果表明：164份临床样本经16S rRNA鉴定后102份为MHp阳性,阳性率为62.2%;阳性样本接种江苏二号(KM₂)液体培养基生长良好,颜色呈规律性变黄,在固体培养基上均能呈现特异性菌落形态;各代次分离菌株经16S rRNA双重PCR鉴定均能获得3个大小约1 000 bp的目的条带,测序结果与国内外MHp各参考株核苷酸相似性为96.42%～98.21%,说明3株分离株均属于MHp,且可稳定遗传,分别命名为JM-24、JM-39、JM-44,分离株生长效价可达1×10^5.67 CCU/mL;3株分离株主要抗原蛋白P46全基因和P97基因R1R...     

种猪场猪支原体肺炎流行病学调研

猪支原体肺炎在我国广泛流行,为查清该病在山西省流行情况,对部分种猪场和散养户进行了流行病学调研,证实猪群咳嗽、气喘现象较为普遍;对疑似病猪、死猪采集病料,用PCR试剂盒检测猪肺炎支原体病原阳性率为26％以上;对非免疫猪群抽检血样,用ELISA试剂盒检测猪肺炎支原体血清抗体阳性率为36．7％以上。以上调研证明,山西种猪场猪支原体肺炎感染较为严重,与临床调查结果基本相符。可能与山西昼夜温差大,养猪户只重视保温、不重视通风,绝大多数养猪户不接种猪肺炎支原体疫苗有一定关系。为今后山西省防治该病提供了重要依据。     

用PCR检测长沙市猪肺炎支原体和猪鼻支原体的感染情况

为对长沙市猪支原体肺炎病原流行病学进行初步调查,用PCR方法对从湖南省长沙市5个县（市、区）205头猪中收集的326份病料（205份猪肺和121份猪鼻拭子）分别进行了猪肺炎支原体和猪鼻支原体检测,并对部分基因进行了序列分析。结果发现,猪肺炎支原体检出率为24.4%（50/205）,猪鼻支原体检出率为22.3%（27/121）;长沙市每个县（市、区）的生猪中均存在这两种支原体,其中长沙县生猪阳性率最高（42.4%和31.5%）;序列分析表明,检测出的基因序列与国内外相关序列有97%～100%的同源性。本试验证实了猪肺炎支原体和猪鼻支原体在长沙市猪场的存在。     

猪喘气病的诊断治疗与净化

猪喘气病也叫猪地方流行性肺炎或猪支原体肺炎,是由猪肺炎支原体引起的一种传染性肺炎，发病率很高。主要症状为咳嗽和气喘,病变特征是融合性支气管炎。病猪初、中期能吃能喝，但生长缓慢，生长速度降低16％，饲料利用率降低22％;在规模化猪场多易继发猪胸膜肺炎以及放线杆菌、多杀性巴氏杆菌、猪副嗜血杆菌、[第一段]     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in