2006年至2010年山东省鸡传染性支气管炎病毒分子变异的研究

为研究山东省鸡传染性支气管炎病毒(IBV)的遗传变异规律,本研究2006年～2010年从山东省发病的商品鸡中分离鉴定了17株IBV,并对其S1基因、N基因和M基因分别进行RT-PCR扩增、测序及遗传进化分析.序列分析结果表明:与疫苗株H120相比,17个分离株S1蛋白的变异程度较大,存在广泛的基因突变和氨基酸替代,多数病毒株还存在氨基酸的插入;N蛋白无碱基的缺失和插入,仅存在核苷酸的突变和氨基酸的替代;M蛋白除病毒株CK/CH/SD09/005插入3个碱基外,其它16个分离株仅存在少数的碱基突变和氨基酸替代.S1基因、N基因和M基因的系统进化分析结果表明多数分离株的3个基因在进化上相对平行,与国内分离株LX4同属一个进化分支,同源性较高;分离株SDYT0605的3个基因与疫苗株H120同源性较高,可能是免疫压力下变异的疫苗株;分离株SDTA06111、SDWF0608和CK/CH/SD09/005的S1基因、N基因和M基因分属于不同的进化分支,可能发生了基因重组.本研究结果显示基因突变、插入和不同基因之间的重组是免疫压力下IBV变异的主要方式.     

鸡传染性支气管炎病毒广西流行株3种主要结构蛋白基因的遗传变异分析

为了解鸡传染性支气管炎病毒(IBV)广西流行株的遗传变异情况,应用RT-PCR方法对2004-2007年的7株广西IBV分离株的纤突蛋白S1基因、核(N)蛋白和膜(M)蛋白基因进行扩增、克隆、测序和同源性比较及系统进化分析。结果显示广西IBV分离株S1基因存在广泛的基因点突变,部分毒株出现基因插入和缺失,分离株之间氨基酸同源性为74.2%～98.7%;N基因无插入和缺失,但存在基因点突变,分离株之间氨基酸同源性为91.7%～99.3%;M基因存在点突变和插入现象,分离株之间氨基酸同源性为90.7%～98.2%。以疫苗株H120为参照,广西IBV分离株的S1、N和M基因都出现了变异,其中S1基因变异程度最大。7株广西IBV在S1、N和M基因氨基酸序列系统进化树中分别集中在2、3和3个基因群中,其中4株的S1、N和M基因分型结果不一致。结果表明广西IBV分离株的S1、N和M基因已发生变异,广西IBV存在广泛的基因突变、缺失或插入现象。研究的结果提示流行株的遗传变异可能是目前影响疫苗免疫效果的主要原因。     

4株鸡肾型IBV陕西分离株3种主要结构蛋白基因的遗传变异分析

采用RT—PCR方法对近年来本实验室分离的4株肾型IBV陕西分离株的纤突蛋白S1基因、膜蛋白基因（M）和核蛋白基因（N）分别进行扩增,测序后进行遗传变异分析。结果显示：与肾型疫苗株w93相比,各分离株S1基因均存在广泛的点突变,并且都存在基因插入现象,分离株之间氨基酸同源性为75．8％～99．4％;M基因除了存在点突变外,W09和WNl2在其5’端还存在9个核苷酸的缺失,分离株之间氨基酸同源性为91．0％～99．6％;N基N无插入和缺失,但存在基因点突变,分离株之间氨基酸同源性为99．3％～99．5％。4株IBV分离株在S1、M和N基因氨基酸系统进化树上分属于不同的进化群,且都与较早的肾型IBV陕西分离株w118遗传距离较远。结果表明,4株鸡肾型IBV流行毒株的s1、M和N基因均存在不同程度变异,这可能是免疫鸡群肾型鸡传染性支气管炎长期流行的主要原因．     

鸡传染性支气管炎病毒AH1/15株S1基因的克隆与序列分析

2015年1月从安徽省某疑似患鸡传染性支气管炎鸡场中分离到一株鸡传染性支气管炎病毒,命名为AH1/15株,使用特异性引物对AH1/15分离株S1基因进行扩增、克隆及序列测定,并将其与国内外流行株及参考毒株进行比对。结果显示,IBV AH1/15株S1基因全长为1638 bp;同源性分析结果发现,AH1/15株与国内外疫苗毒株核苷酸同源性为57.0%~59.6%;遗传进化分析结果发现,AH1/15株与国内外主要疫苗株以及流行的LX4型毒株亲缘关系较远,与近几年国内分离的CK/CH/GX/NN11-4、CK/CH/GD/CG11、CK/CH/SD09/005等毒株亲缘关系最近。结果表明,AH1/15是一株不同于疫苗株和流行株的变异株。     

2株鸡传染性支气管炎病毒的分离鉴定与S1、M和N基因序列分析

为跟踪了解广东地区鸡传染性支气管炎病流行情况,2019年从广东省清远、惠州两地疑似鸡传染性支气管炎发病鸡群中采集分离到2株传染性支气管炎病毒,并对其进行了鉴定和分析。结果显示,在鸡胚上盲传3代后均出现鸡胚的典型病变,死亡鸡胚全身出血明显,未死亡鸡胚发育受阻,呈明显的侏儒胚、蜷缩。将2个分离株分别命名为CK/CH/GD/QY1119和CK/CH/GD/HZ1225,分别测定其半数感染量(EID_(50)),扩增其S1、M和N基因核苷酸序列,获得相应的推导氨基酸序列并对其分析。分离株CK/CH/GD/QY1119和CK/CH/GD/HZ1225的EID_(50)分别为10~(-7.5)/0.1 mL和10~(-6.75)/0.1 mL,2个分离株S1基因与Mass型等疫苗株同源性为76.2%～82.1%,同源性均较低。与H120疫苗株相比,2个分离株的S1、M和N基因存在若干氨基酸的差异,2个分离株S1基因均存在氨基酸的插入,分离株CK/CH/GD/QY1119的M基因存在氨基酸的缺失,分离株CK/CH/GD/HZ1225的M基因存在氨基酸的插入。进化树分析显示,2个分离株分别属于LDT3型分支和QX型分支,均在S1、M和N基因进化树上的分属上存在差异。结果表明本次分离得到的2个分离株的S1、M和N基因均存在不同程度变异,在临床防控中应引起重视。本试验为鸡传染性支气管炎的有效防控提供了参考依据。     

两株鸡传染性支气管炎病毒的分离鉴定及S1基因序列分析

为调查广东地区鸡传染性支气管炎病毒(IBV)的流行及其遗传变异情况,本研究通过病料SPF鸡胚接种和鸡胚尿囊液的RT-PCR鉴定,于2013年从广东湛江地区不同发病鸡场分离到两株IBV,分别命名为CK/CH/GD/ZJ10/2013和CK/CH/GD/ZJ11/2013,并对这两株IBV的S1基因进行序列分析。结果显示,CK/CH/GD/ZJ10/2013株S1基因全长1 626 bp,编码542个氨基酸,其裂解位点为NRFRR,属于基因型Ⅲ,并推测其为基因型Ⅲ毒株(CK/CH/GX/NN11-3)与基因型Ⅰ毒株(GX-NN-6)在S1基因处发生重组而产生的新毒株;CK/CH/GD/ZJ11/2013株S1基因全长1620 bp,编码540个氨基酸,其裂解位点为HRRRR,属于基因型Ⅰ(类QX型);两株IBV的S1基因间核苷酸序列及其推导的氨基酸序列同源性较低,与位于同一基因型的参考毒株间同源性较高,而与中国使用的Mass型常规疫苗H120和H52之间的同源性最低,仅为75.7%~76.3%和77.1%~77.9%。本研究可为广东省IBV的流行病学调查和分子生物学研究提供参考。     

山东地区鸡传染性支气管炎病毒抗原相关性和血清分型

为进一步明确山东地区鸡传染性支气管炎病毒(IBV)的流行病学,对IBV分离株之间以及与疫苗株之间的抗原相关性进行了分析。分别制备了3个疫苗株(4/91、H120和Ma5)和18个分离株的单因子阳性血清,通过鸡胚交叉中和试验测定了阳性血清对同源病毒和异源病毒的中和效价,计算了不同毒株之间的抗原相关系数(R值),并根据R值进行血清分型。结果显示,3个疫苗株和18个分离株可分为5个血清型。疫苗株4/91属于血清Ⅰ型,H120、Ma5与2个分离株(SDWF0608和SDLY0701)属于同一血清型,分离株SDZB0808与SDLY0702、CK/CH/SD08/007和CK/CH/SD09/001等15个毒株为同一血清型,CK/CH/SD09/006和CK/CH/SD10/001分别与其他3和6个分离株组成两个血清型。进一步分析发现,部分IBV毒株之间表现单向中和反应性,属于同一血清型的IBV毒株之间抗原相关性可能存在明显差异。本研究结果一方面表明IBV毒株之间复杂的抗原相关性,另一方面显示IBV流行株与3个疫苗株之间抗原性存在明显差异。     

七株鸡传染性支气管炎病毒S1和N基因序列分析

本研究于2020年从广东省部分地区疑似鸡传染性支气管炎病毒(IBV)感染的病料中分离到7株病毒,对分离的病毒进行S1及N基因序列分析,结果显示:这7株分离株S1基因与我国常用的MASS型疫苗株H120之间核苷酸相似性为77.3%～81.9%,氨基酸序列相似性为75.2%～80.2%;N基因与4/91、H120疫苗株核苷酸相似性均为85.9%～89.1%。上述结果表明,流行株与经典疫苗株之间的序列差异较大。S1与N基因遗传进化分析显示:7株IBV中5株的S1基因属于GI-19(QX-type),2株分别属于GI-22(CK/CH/LSC/99-type)和GI-28(LDT3-type);6株N基因属于LX4-type,1株属于CK/CH/LSC/99-type。这7株IBV主要以QX-type为流行株,其中有3株发生了重组。这说明目前广东省主要流行QX-type毒株,但基因重组发生频率高,本研究为加强广东省鸡传染性支气管炎的免疫防控提供了参考。     

传染性支气管炎病毒分离株的生物学特性鉴定及其遗传变异分析

通过鸡胚矮小试验、鸡胚气管环组织培养试验、干扰新城疫病毒复制试验和动物回归试验,将2006-2007年在江苏省分离的7株鸡传染性支气管炎病毒（IBV）鉴定为嗜肾型毒株。采用RT-PCR扩增IBV分离株的S1基因,并进行克隆和序列分析。结果显示7个IBV分离株S1基因的核苷酸同源性为94．6％-99．4％,处于同一个群,分别属于3个亚群。这些毒株与大多数国内近年分离株的同源性较高,而与Massachusetts、T、4／91和793B血清型毒株（包括H120和H52疫苗毒株）的同源性较低。IBV流行毒株和疫苗毒株的差异是造成免疫鸡群发生传染性支气管炎的重要原因。     

中国2005年～2006年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

本研究应用RT—PCR方法扩增到我国2005年-2006年8个省市12株IBV病毒核蛋白基因,并将其进行了克隆、序列测定及分析。以疫苗株H120作为参考毒株,对12株IBV分离株核蛋白基因序列进行分析。结果发现我国分离株的N基因及其局部功能区序列存在广泛的氨基酸替代现象。同时还发现毒株CK／CH／LSD／05I与疫苗株H120同源性最高（93.4％）。与GenBank中的11个IBV参考毒株核蛋白基因序列进行比较和分析,发现本研究分离的大部分毒株（9株）分布于第1群中,可见我国主要的IBV分离株在N基因进化关系上形成了自己独立的进化群。通过与S1基因系统发育进化树比较发现,毒株CK／CH／LSD／05I存在基因重组现象。以上结果表明我国2005年-2006年IBV毒株存在基因突变和基因重组现象。     

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.     

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     

Evaluation of the Microbiological Status of Milk and Various Structures in Mammary Glands from Naturally Infected Dairy Cows

A knowledge of the microbiological status of milk and of the different structures in the mammary glands has great importance in elucidating the pathogenesis of mammary gland infections. The objective of this study was to evaluate the microbiological status of various structures in the mammary glands from naturally infected dairy cows following slaughter. A total of 94 samples of milk, 184 samples of mammary parenchyma, 168 samples of gland cisterns, and 168 samples of teat cisterns were collected for microbiological examination. Microorganisms were detected in 59.9% of all samples, 67.0% of the milk samples, 70.1% of the mammary parenchymas, 55.9% of the gland cisterns and 48.8% of the teat cistern samples. When all samples were considered, coagulase-negative Staphylococcus were the most prevalent (35.7%) followed by coagulase-positive Staphylococcus (12.2%), Corynebacterium bovis (2.4%), Prototheca sp. (1.9%), and Streptococcus dysgalactiae (1.5%). There was a significantly higher occurrence of microorganisms in the milk and mammary parenchyma compared to the gland cisterns and teat cisterns. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development and survival of free-living stages of gastrointestinal nematodes of sheep and goats on pasture in the Nigerian derived savanna

Four grass plots were sequentially contaminated with goat faeces containing known numbers of unembryonated eggs of predominantly Haemonchus contortus and Trichostrongylus spp. between October 1982 and April 1983. Four other plots were similarly contaminated with sheep faeces between February and May 1987. An additional plot was repeatedly contaminated with sheep faeces from February to April 1987. Populations of free-living stages in faeces and of infective larvae (L₃) in the herbage were subsequently monitored until the end of April and June of 1983 and 1987 respectively. During February and May 1987 two control cultures of sheep faeces were incubated in the laboratory at 25°C–30°C and at a constant temperature of 50°C and the free-living development was also monitored. L₃ developed very readily in the faeces cultured at 25°C–30°C and in those spread on a grass plot in October, at the end of the wet season, but developed less on the plot contaminated in May at the start of the wet season. Worm eggs in faeces deposited on plots during the hot dry season (December to April) or incubated at 50°C died and disintegrated after 24–48 h exposure to the high environmental temperatures. The results indicate that it is unlikely that gastrointestinal nematodes of sheep and goats can develop or survive on open pasture during the dry season in the Nigerian derived savanna zone.