猪场大肠杆菌Ⅰ型整合子及其基因盒的分子流行病学研究

对某猪场大肠杆菌Ⅰ型整合子及其基因盒的分子流行病学进行了研究。结果表明:猪场大肠杆菌Ⅰ型整合子流行普遍,192株分离菌中105株携带Ⅰ型整合子,检出率达54.7%。共检出6种Ⅰ型整合子,它们以8种组合形式在猪场大肠杆菌中流行。猪群、饲养员及饲养环境分离菌株Ⅰ型整合子检出种类、检出率相近,但同耐药谱菌株Ⅰ型整合子携带无一致性。各种Ⅰ型整合子整合不同种类、不同数目的耐药基因盒,其中100%整合有氨基糖腺苷转移酶基因(aadA),77%整合有二氢叶酸还原酶基因(dhfr),仅少数整合有链丝菌素乙酰转移酶基因(sat)、β-内酰胺酶基因(bla)及红霉素酯酶基因(ereA)。Ⅰ型整合子携带菌株均表现多重耐药,其中100%耐受复方新诺明,90%耐受链霉素,75%耐受大观霉素。该猪场长期使用磺胺类、氨基糖苷类抗菌药物,尤其是将它们用作抗菌促长剂,造成了携带dhfr及aadA两种基因盒的Ⅰ型整合子在猪场大肠杆菌中广泛流行。     

猪源大肠杆菌耐药性与I型整合子关系研究

目的为了解猪源大肠杆菌中I型整合子的流行情况,探讨I型整合子与大肠杆菌耐药表型的相关性。方法采用微量肉汤稀释法测定119株猪源大肠杆菌对8类14种抗菌药物的耐药性,并采用PCR法检测猪源大肠杆菌I型整合酶基因（int11）并扩增其可变区,对PCR产物进行酶切分析,测序分析整合子可变区携带的耐药基因盒。结果119株大肠杆菌耐药现象十分严重,对四环素、磺胺异恶唑、新诺明全部耐药,所有菌株均呈多重耐药。119株猪源大肠杆菌中有92株含I型整合子,检出率77．31％。扩增出7类大小不同的基因盒插入区片段,范围为1008bp-3149bp。7类I型整合子在119株猪源大肠杆菌中存在13种流行组合。78．15％大肠杆菌菌株的I型整合子携带2种或2种以上的基因盒,其中以携带编码氨基糖苷类药物耐药基因（aadA1、aadA2、aadA5、aadA22、aadB）最多,其次为编码磺胺类药物耐药基因（dfrA1、dfrA12、dfrA17、dfrA27）,此外还携带编码利福平、林可霉素和氯霉素的基因1nuF、cm1A6、aar-3、off.结论I型整合子普遍存在于大肠杆菌中,且呈流行上升趋势;I型整合子参与耐药及多重耐药,但单株细菌携带的耐药基因盒与其耐药表型无对应关系。     

奶牛乳房炎大肠杆菌整合子与耐药表型的相关性研究

为研究自乳房炎奶牛分离的大肠杆菌整合子携带情况与其耐药表型之间的相关性，在获得菌株对20种抗菌药物耐药谱的基础上，设计Ⅰ、Ⅱ、Ⅲ类整合子简并引物扩增菌株的整合酶基因，并结合内切酶限制性片段长度多态性分析，调查菌株携带的各类整合子，然后进一步扩增菌株整合子的基因盒插入区，测序分析其携带的基因盒。结果发现，80株自奶牛乳房炎病例分离的大肠杆菌中有37株检出Ⅰ类整合子（检出率46．25％），未检出Ⅱ类和Ⅲ类整合子。Ⅰ类整合子阳性菌中31株扩增出3种不同大小的基因盒插入区片段，插入基因盒主要是二氢叶酸还原酶基因（dfr）和氨基糖苷类抗生素耐药基因（aadA），最主要的基因盒排列为dfrA17-aadA5。分析表明，Ⅰ类整合子集中分布在耐受7种以上抗菌药物的菌株中，与菌株的多重耐药性高度相关，而菌株Ⅰ类整合子携带的耐药基因盒与其耐药谱缺乏对应关系。     

动物源大肠杆菌整合子携带氨基糖苷类耐药基因盒的研究

收集540株不同动物源大肠杆菌,采用微量肉汤稀释法检测其对22种药物的耐药性;利用多重PCR方法检测菌株携带3型整合子的情况;采用χ2检验分析1型整合子阳性菌及阴性菌的耐药性;采用PCR—DNA测序法对整合子阳性菌株携带的氨基糖苷类耐药基因盒进行分析。结果显示,540株菌对链霉素等20种药物耐药率超过37％,全部菌株均呈多重耐药;1型整合子检出率为86．48％,未检出2及3型整合子;整合子阳性菌株对头孢唑啉等13种药物耐药率显著高于整合子阴性菌;随机选择的67株1型整合子阳性菌株中,有92．54％菌株携带有编码氨基糖苷核苷转移酶和乙酰转移酶基因（aadA2、aadA5、aadA22、aacA4）,共有5种类型基因盒,其中以介导对甲氧苄啶、链霉素和大观霉素耐药的dfrA17＋aadA5组合最为常见;在2株茵中分别发现介导对阿米卡星、氯霉素及甲氧嘧啶耐药的aacA4＋catB3＋dfrA1组合,和介导链霉素、大观霉素耐药的aadA22基因盒,是在四川分离菌中首次发现这2种基因盒。结果表明,1型整合子广泛存在于大肠杆菌临床菌株中,大肠杆菌耐药性与整合子相关,菌株中广泛携带氨基糖苷类及甲氧苄胺嘧啶耐药基因盒。     

猪源大肠杆菌耐药性与Ⅰ型整合子关系研究

目的为了解猪源大肠杆菌中Ⅰ型整合子的流行情况,探讨Ⅰ型整合子与大肠杆菌耐药表型的相关性。方法采用微量肉汤稀释法测定119株猪源大肠杆菌对8类14种抗菌药物的耐药性,并采用PCR法检测猪源大肠杆菌Ⅰ型整合酶基因(int I1)并扩增其可变区,对PCR产物进行酶切分析,测序分析整合子可变区携带的耐药基因盒。结果 119株大肠杆菌耐药现象十分严重,对四环素、磺胺异恶唑、新诺明全部耐药,所有菌株均呈多重耐药。119株猪源大肠杆菌中有92株含Ⅰ型整合子,检出率77.31%。扩增出7类大小不同的基因盒插入区片段,范围为1008bp～3149bp。7类Ⅰ型整合子在119株猪源大肠杆菌中存在13种流行组合。78.15%大肠杆菌菌株的Ⅰ型整合子携带2种或2种以上的基因盒,其中以携带编码氨基糖苷类药物耐药基因(aadA1、aadA2、aadA5、aadA22、aadB)最多,其次为编码磺胺类药物耐药基因(dfrA1、dfrA12、dfrA17、dfrA27),此外还携带编码利福平、林可霉素和氯霉素的基因lnuF、cmlA6、aar-3、orf。结论Ⅰ型整合子普遍存在于大肠杆菌中,且呈流行上升趋势;Ⅰ型整合子参与耐药及多重耐药,但单株细菌携带的耐药基因盒与其耐药表型无对应关系。     

东北地区鹿源多重耐药大肠杆菌整合子的检测

为了解东北三省(黑龙江省、辽宁省、吉林省)不同养殖场分离的鹿源大肠杆菌菌株的耐药性及Ⅰ型整合子在流行株中的携带情况,试验在检测耐药性的基础上,采用PCR方法对流行株中Ⅰ型整合子进行检测和测序,并对检测结果和耐药基因盒与GenBank数据库的序列进行比对和分析。结果表明:分离菌株对氨苄西林、头孢菌素和四环素的耐药率达50%以上。Ⅰ型整合子和Ⅱ型整合子的检出率分别为51.18%和4.12%,Ⅲ型整合子未检出。基因盒的检出率为56.32%,序列分析得出共包含6种携带不同基因盒的整合子,分别编码对甲氧苄啶耐药的二氢叶酸还原酶(dfrA1、dfrA15、dfrA12和dfrA17)和对氨基糖苷类耐药的乙酰转移酶(aadA1、aadA2和aadA5)。基因盒阵列aadA1-aadA2-dfrA1-dfrA12在这些大肠杆菌中最为普遍。说明Ⅰ型整合子在细菌耐药的传播过程中发挥着至关重要的作用。     

胶东地区耐药猪源大肠杆菌I型整合子-基因盒流行状况分析

在胶东地区119株多重耐药大肠杆菌中,共92株菌有I型整合子整合酶基因,检出率达77．31％。92株I型整合子携带大肠杆菌共扩增出7类不同大小的基因盒插入区片段,大小分别为1008bp、1318bp、1586bp、1663bp、1913bp、1945bp、3149bp,其中以大小为1008bp的插入区片段的检出率最高,为31．67％。而检出率最低的为长度为1318bp的基因盒插入区。将基因盒插入区扩增片段的测序结果与Genebank中的相关序列进行比对,得出I型整合子携带的耐药基因盒种类分别为aadA1、aadA2、aadA5、aadA22、aadB、dfrA1、dfrA12、dfrA17、dfrA27、1nuF、cmLA6、aar-3、orff分别编码对相应药物的耐药性。     

食品动物源大肠杆菌多重耐药菌株整合子-基因盒的分子特征

为研究分离自四川省食品动物源大肠杆菌多重耐药菌株整合子-基因盒分布及分子特征,采用多重PCR方法检测327株多重耐药菌株中I、Ⅱ、Ⅲ型整合酶基因,用PCR-测序法对整合子阳性菌进行基因盒序列分析.结果表明327株Escherichia coli中294株(89.91%)检出有I型整合子,未检出Ⅱ、Ⅲ型整合子.在随机选取的81株l型整合子阳性菌中,67株(82.72%)能扩增出800～3000 bp的耐药基因盒插入区.基因盒以编码对甲氧磺胺嘧啶类和氨基糖苷类药物耐药的dfrA、dhfrI和aadA基因家族为主,尤其以dfrA17+aadA5为优势基因盒.分别在2株菌中检出aacA4+catB3+dfrA1和aadA22基因盒.aadA22是四川分离菌株中首次报道,介导氨基糖苷类耐药.结果表明I型整合子普遍存在于大肠杆菌临床菌株中.尽管菌株携带的整合子-基因盒与其多重耐药谱间不存在对应关系,但不同来源菌株中检测出相同的耐药基因盒片段,说明了通过整合机制耐药基因可在不同菌株问水平传递.     

健康畜禽肠道大肠杆菌耐药性及整合子-基因盒检测

采用微量肉汤稀释法检测455株大肠杆菌对24种抗菌药物的敏感性;多重PCR检测菌株Ⅰ、Ⅱ、Ⅲ型整合酶基因;采用PCR-RFLP和PCR-测序法对整合子-基因盒进行序列分析。结果显示,455株菌除对头孢唑啉、头孢曲松、头抱噻呋和阿米卡星耐药率低于40％外,对其余药物表现出高耐药率,99．23％菌株呈多重耐药性;455株菌Ⅰ型整合子阳性率87．69％,Ⅱ型整合子1．98％,未检出Ⅲ型整合子;随机选取的204株整合子阳性菌中174株（85．29％）扩增出了基因盒可变区。基因盒主要以编码氨基糖苷腺苷基转移酶和二氢叶酸还原酶的aadA、dfrA基因为主,耐药基因排列成Ⅰ型整合子8种基因盒,Ⅱ型整合子3种基因盒。Ⅰ型整合子以dfrA1-aadA1（32．76％）基因盒为主,其次是aadA22（24．14％）和dfrA17-aadA5（24．14％）。Ⅱ型整合子以d厂rA1-sat2-aadA1（66．67％）为主,Ⅱ型整合子阳性菌含有I型整合子-基因盒;菌株含整合子-基因盒越复杂,其多重耐药性越严重。结果表明,本次分离的健康动物肠道大肠杆菌耐药非常严重,整合子-基因盒分布广泛,已成为耐药基因储库,对耐药基因扩散起重要作用。     

胶东地区耐药猪源大肠杆菌Ⅰ型整合子-基因盒流行状况分析

在胶东地区119株多重耐药大肠杆菌中,共92株菌有I型整合子整合酶基因,检出率达77.31%。92株I型整合子携带大肠杆菌共扩增出7类不同大小的基因盒插入区片段,大小分别为1008bp、1318bp、1586bp、1663bp、1913bp、1945bp、3149bp,其中以大小为1008bp的插入区片段的检出率最高,为31.67%。而检出率最低的为长度为1318bp的基因盒插入区。将基因盒插入区扩增片段的测序结果与Genebank中的相关序列进行比对,得出Ⅰ型整合子携带的耐药基因盒种类分别为aadA1、aadA2、aadA5、aadA22、aadB、dfrA1、dfrA12、dfrA17、dfrA27、lnuF、cmLA6、aar-3、orfF分别编码对相应药物的耐药性。     

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.     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -     

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.     

Chemotaxis of macrophage in inflammation

Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.     

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