江西省部分地区鸡白痢沙门氏菌噬菌体分型,药敏性及质粒谱分析

３３株鸡白痢沙门氏菌大致分为３个噬菌体型，即２０株为２２００型，６株为３０００型，７株为００００型。依其来源，有的鸡场可能为单一例，有的为多种型，２２００型１１株存在７条质粒带，小质粒带分子量分别为１．９、２．０、２．８、５．２、８．２Ｍｄ；大质粒带，ＨＧ源株为３０、５０Ｍｄ，Ｇｑ与ＪＺ源株为３６．４４Ｍｄ。３０００型４株在４条质粒带，小质粒带分子量分别为１．９、２．０、２．８Ｍｄ；大质粒带，ＪＳ     

鼠伤寒沙门氏菌和福氏志贺氏菌流行株耐药质粒的研究

对分离到的９株鼠伤寒沙门氏菌和５株福氏志贺氏菌进行药敏试验、质粒提取和电泳分析，各菌株均分离出２－４条粒带。将含有３．２７×１０＾４，３．０１×１０＾４和６．０１×１０＾４ｂｐ的持粒转化给大肠埃希氏菌ＨＢ１０１，使ＨＢ１０１获得了相应菌的部分耐药性，用转化有３．２７×１０＾４ｂｐ的ＨＢ１０１注射小白鼠，１５ｄ后用相应的原始鼠伤寒沙上氏菌强毒株攻击，结果使小白鼠获得了保护；用含有３．２７×１０＾４ｂ     

仔猪大肠杆菌病流行株抗性质粒的研究

对分离的２７株猪源性大肠杆菌进行抗性试验，选取１０株双抗性菌株提取质粒ＤＮＡ，电泳分析表明各菌株均存在２～４条质粒ＤＮＡ带。将２１．２２７ｋｂ和５．１４８ｋｂ质粒转化大肠杆菌ＭＣ１０６１／Ｐ３，使其获得了相应菌株的抗性，转化菌纤毛蛋白与Ｋ８８、Ｋ９９高免血清琼扩试验为阴性，证明２１．２２７ｋｂ和５．１４８ｋｂ质粒未携带Ｋ８８、Ｋ９９基因。用２１．２２７ｋｂ转化菌注射小鼠，则小鼠死亡，说明２１．２２７ｋｂ携带毒力基因。     

禽源致病性大肠杆菌分离株R质粒的研究

从本室临床分离到的２０株禽源大肠杆菌，经药敏试验筛选到四株抗性菌株提取质粒ＤＮＡ，电泳分析表明菌株各存在数量不等（１－４个）的质粒ＤＮＡ条带。分别用试剂盒回收出５种大小不同的Ｒ质粒，分别转化大肠杆菌ＤＨ５α；但只有２０．５ｋｇ、９．４ｋｂ两种质粒转化成功，并获得了相应的抗药性，用含９．４ｋｂ质粒转化的大肠杆菌攻毒，可致死小鼠，说明该质粒可能携带有毒性基因。     

北京地区奶牛乳汁中的真菌及其与隐性乳房炎的关系

通过对北京地区１２８头奶牛的混合乳样进行真菌分离，共分离出真菌４２株，占３２．８％。其中假丝酵母１７株，占４０．５％；曲霉菌４株，占９．５％；隐坏菌３株，占７．１％；毛霉菌４株，占９．５％；羊毛状小孢子菌１４株，占３３．３％。真菌感染与隐性乳房炎监测（Ｂ．Ｍ．Ｔ．法）的关系是：“－”占２４．４０％；“±”占２０．８％；“＋”占４５．２％；“＋＋”占３６％；“＋＋＋”占１００％。说明随着真菌感染数的增加，隐性乳房炎的阳性率和严重程度也随着增加，故认为，在奶牛生产中，隐性乳房炎的发生与真菌感染有着密切关系。     

山西12市五种肉品沙门氏菌检验情况

对山西１２城市农贸市场五种肉品沙门氏菌污染及血清型分布情况进行检验，猪肉污染率２６．４％，白条鸡２１．７％，白条狗１６．０％，菜蛇３．５％，黄鳝９．３％。共检出３个亚种、１２个血清型、４３９株沙门氏菌。检出国内未报道血清型７株；国际尚未报告血清型３株。     

从牛尿分离钩端螺旋体的研究

从４７８份现场牛尿样中分离钩端螺旋体，结果表明，稀释法接种优于过滤后接种。第１、２、３稀释管污染率分别为４１．１％、２４．４％和１７．４％；培养阳性率分别为１．２％。３．３％和０％。可见第２管结果最佳。来自江西、湖南和浙江的４７８份牛尿样中分离出１５株钩端螺旋体，阳性率为３．１％。其中江西上高县牛尿培养阳率最高，达６．２％。１３株钩体鉴定了血清群。５Ｊｃ００４，５Ｊｃ０９８，５Ｊｃ１４８，５Ｊｃ１     

禽流感二价抗原油乳剂灭活疫苗的研制

以禽流感病毒（ＡＩＶ）Ｈ５Ｎ４株、Ｈ７Ｎ３株为抗原研制了Ｈ５Ｎ４－Ｈ７Ｎ３二价油乳剂灭活疫苗,并对其物理性状、安全性、免疫效力、保存期及抗体消长规律进行了检测。结果表明,试验鸡疫苗在免疫后３周到９个月内对ＡＩＶ－Ｈ５Ｎ４的攻击均获全部保护。疫菌４℃保存１５个月,其免疫效力没有下降。     

禽巴氏杆菌病B26—T1200弱毒疫苗对鸡的免疫产生期和免疫期试验

以禽巴氏杆菌病Ｂ２６－Ｔ１２００弱毒疫苗免疫后８小时即能产生免疫力（１／５鸡得到保护）；２－３天有３／５鸡获得保护；４－５天后能产生坚强免疫力（４／５－５／５保护），免疫后２，３，４和５个月的总保护率为８３．３％（８０／９６）；免疫后５个月，６批苗的平均保护率为７５％（１８／２４）。     

鸡新城疫病毒东北地区分离株融合蛋白基因的克隆与鉴定

以 Ｎ Ｄ Ｖ 东北地区分离株 Ｄ Ｂ３ 、 Ｄ Ｂ５ 的基因组 Ｒ Ｎ Ａ 为模板, 通过 Ｒ Ｔ Ｐ Ｃ Ｒ 技术扩增其 Ｆ 基因的ｃ Ｄ Ｎ Ａ,并将其克隆到质粒ｐ Ｕ Ｃ１９ 中, 转化感受态大肠杆菌 Ｄ Ｈ５α, 经分子量比较、酶切分析、 Ｐ Ｃ Ｒ 等鉴定方法证明, 我们分别获得了含有 Ｎ Ｄ Ｖ Ｄ Ｂ３ 、 Ｄ Ｂ５ 株 Ｆ基因的阳性重组质粒。     

Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.     

断奶仔猪源大肠杆菌F18菌毛的分子流行病学研究

利用F18菌毛a因子单克降抗体以及已建立的鉴定F18菌毛及其亚型的双重PCR法,对来自断奶仔猪水肿病和／或腹泻病例的60株VTEC、24株VTEC／ETEC以及24株ETEC的进行了F18菌毛检测,以了解F18ab^＋和F18ac^＋大肠杆菌在江苏省断奶仔猪群的分子流行病学。结果表明：通过F18菌毛a因子单克隆抗体,可检测出52株大肠杆菌为F18^＋,检出率为48．15％;而通过双重PCIL方法,共检测出63株大肠杆菌为F18^＋,检出率为58．33％,其中53株（49．07％）为F18ab^＋10株（92．6％）为F18ac^＋。另外还发现：在VTEC、VTEC／ETEC以及ETEC的菌株之间,这2种F18菌毛亚型的分子流行病学是不同的。在VTEC中,F18ab^＋,菌株37株（61．67％）,未发现F18ac^＋菌株;在VTEC／ETEC中,F18ab^＋菌株15株（62．50%）,F18ac^＋菌株8株（33．33％）;而在ETEC中F18ab^＋菌株只有1株（4．17％）,F18ac^＋菌株只有2株（8.33％）。以上数据表明：④PCR法检测F18菌毛优于单抗法;②F18菌毛是VTEC／ETEC、VTEC的重要致病因子,而在ETEC中则明显低于VTEC／ETEC和VTEC;⑧F18ab^＋菌株一般为SLT-IIe^＋,而F8ac^＋菌株一般为STI^＋。     

Virulence factors in Escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in Japan

A total of 567 strains of Escherichia coli were isolated from piglets with neonatal diarrhea (ND) or post-weaning diarrhea (PWD) in Japan. They were investigated for enterotoxigenicity and possession of adhesins and O antigens. There were clear differences between the strains of ND origin and those of PWD origin in the occurrence of enterotoxigenic E. coli (ETEC) strains, type of enterotoxin and frequency of adhesins: ETEC was found in 77 (25.7%) of 300 strains of ND origin and in 137 (51.3%) of 267 strains of PWD origin. ETEC strains producing heat-labile enterotoxin (LT) or heat-stable enterotoxin (STa), alone or in combination were evenly distributed among the strains of PWD origin. In contrast most of the ETEC strains of ND origin produced LT alone. Adhesins appeared in 42 (54.5%) of 77 ETEC strains of ND origin and in 36 (26.3%) of 137 ETEC strains of PWD origin. Adhesins were less common in ETEC strains of PWD origin than in those of ND origin. Some K99-positive ETEC strains of PWD origin produced both LT and STa. There was a similarity in the distribution of O antigens, particularly O149 and O157, between the strains of ND origin and those of PWD origin.     

Prevalence,molecular fingerprinting and drug resistance profile of enterovirulent <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from free-ranging yaks of Tawang district,Arunachal Pradesh,India

Of 273 samples (rectal swab) collected from free-ranging yaks of Tawang district, Arunachal Pradesh, 42 Shiga toxin-producing Escherichia coli (STEC), six enteropathogenic E. coli (EPEC) and 27 enterotoxigenic E. coli (ETEC) strains were isolated. All the STEC and EPEC strains were further investigated for respective stx variants (for STEC only) and additional putative virulence factors. The 27 ETEC strains were also screened for characteristic enterotoxin gene(s) and colonization factors. Occurrence of ETEC was significantly (p < 0.05) higher in the diarrheic yaks and yaks of less than 1 year of age. Majority of enterovirulent E. coli isolates were resistant to amikacin, azithromycin, chloramphenicol, colistin, doxycycline, furazolidone, nalidixic acid, nitrofurantoin, streptomycin and tetracycline. Dendrogram, constructed with molecular fingerprinting profiles obtained from RAPD (Randomly Amplified Polymorphic DNA) and ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR, placed the isolates in different clusters irrespective of their serotypes, virulence gene and drug resistance pattern. Collectively, the study indicates that yaks, being a potential reservoir of multidrug resistant STEC and EPEC, may represent significant risk to public health in this region. Higher recovery of ETEC isolates from yaks with diarrhea points out that ETEC may be a major determinant for repeated occurrence of diarrhea in yaks.     

Replicon typing of F18 fimbriae encoding plasmids of enterotoxigenic and verotoxigenic Escherichia coli strains from porcine postweaning diarrhoea and oedema disease

The presence of fimbrial adhesin F18 is frequently found in enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) strains responsible for diarrhoea and oedema disease of weaned pigs. The F18 adhesin occurs in two antigenic variants: F18ab is characteristic of VTEC while F18ac is more typical for ETEC. F18 encoding plasmids of 17 phenotypically characterized porcine E. coli isolates (10 ETEC, 6 VTEC and 1 ETEC/VTEC) were tested with a DNA probe for F18 fimbrial adhesin and with replicon probes for the RepFIa, RepFIb and for the RepFIc family of basic replicons. In all the cases, the F18 probe hybridized to only one plasmid band of size higher than 42MDa. All F18 plasmids were determined to be unireplicon plasmids belonging to the RepFIc replicon family of the F incompatibility complex. There was no difference between F18ac plasmids of ETEC and F18ab plasmids of VTEC strains in terms of replicon type or subtype. However, the size of F18ab plasmids of the VTEC strains varied between 42 and 98MDa, in contrast to F18ac plasmids of ETEC strains (constantly approximately 98MDa).     

A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves

We investigated the clonal relationships among 41 enterotoxigenic (ETEC) or non-enterotoxigenic (NETEC) Escherichia coli strains producing the F17 a fimbriae isolated from diarrheic calves in France or Belgium in the early 1980s. Twenty-three of the 26 ETEC strains were highly clonally related, most of them with a O101:K32:H9-serotype. The NETEC strains were also divided in clonal subgroups, most of them with O101:H-serotype. The F17 a positive ETEC strains are no longer isolated from diarrheic calves in these countries. It is postulated that the use of a vaccine including O101, K32 and H9 antigens in addition to K99 (F5) explains the strongly reduced isolation of the O101:K32:H9, K99 (F5) E. coli clone.     

Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli

The goal of this study was to isolate and characterize phages that might be used in prevention and treatment of porcine post-weaning diarrhea due to O149 enterotoxigenic E. coli (ETEC). Serotype O149:H10:F4 was especially targeted because this is the dominant ETEC serotype. Mixtures of 10 strains of O149:H10:F4 ETEC and of 10 O149:H43:F4 ETEC were used as hosts for isolation of phages in sewage from 38 Ontario pig farms. Six phages (GJ1-GJ6) that lysed O149:H10:F4 ETEC and three (GJ7-GJ9) that lysed O149:H43:F4 ETEC were isolated. All phages produced large, clear plaques. All nine phages had necks and contractile tails and therefore belonged to the Myoviridae. Their estimated genome sizes were 48.3-50.7kb and their restriction enzyme fragments suggested that they were closely related. Phages GJ1-GJ6 lysed 99-100% of 85 O149:H10:F4 ETEC, 0-12% of 42 O149:H43:F4 ETEC, 3-35% of 37 non-O149 porcine ETEC, and 6-68% of the 72 strains of the ECOR collection. Phages GJ7-GJ9 lysed 86-98% of the O149:H43:F4 ETEC, 2-53% of the O149:H10:F4 ETEC, and 24-41% of the non-O149 porcine ETEC. Titres of the nine phages were unaffected by exposure for 16h to pH 5-9. Among phages GJ1-GJ6, resistance of O149:H10:F4 ETEC to one phage was generally not accompanied by resistance to other phages. It is concluded that the nine phages are suitable candidates for prophylaxis and therapy of porcine post-weaning diarrhea due to O149 ETEC.     

Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.     

Persistence of Escherichia coli O157:H7 in experimentally infected swine

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.     

Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains.

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein (uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non-E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.